Home

AutoQuant 8.0.2 Suite User's Manual

1. Remove Selected Framels Add Frames Dimensions te Pike ae i Preview Data Selected Frame fi is Aae Emen View Selected Frame 1e 3 Movie Parameters Set Movie Orientations from within the 3D Viewer Choose Frame Range All Frames Start Frame fo C Frame Range End Frame fo Rotating Morph None fixed angle Full Rotation Movie CO Full Rotation Time Point F Rock Time Stamp Format O None C YYYYIMM DD HH MM 55 M5 absolute O HH iMM 55 MS relative C DD HH MM 55 relative Caption es _ Normalize Frame Intensities Preview Movie Example 4 Generate Movie Generate Movie Cancel Help March 2004 Page 173 AutoQuant Imaging Inc Manual and Tutorials When this dialog box is first opened the table will be empty Datasets may be added via the Add Frames button Datasets may be removed by highlighting one or more entries in the table and clicking Remove Selected Frame s The Add Frames button will be disabled once frames have been added and the Remove Selected Frame s button will be disabled when the table is empty Adding Datasets Clicking the Add Frames button will open the Import Time Lapse Data dialog box ioi xi Channels To Import C Single O Multiple Y Enable Red Enable Green Enable Blue Composite m Importing Data Importing Channel Red C Green Blue Data File Set mage
2. 3 From the View menu select Triple View The XY ZY and XZ Optical Slice Viewers are displayed in the Triple View box Click on the Show Crosshair button The red lines indicate where each slice is in relative position to the others in the dataset To remove the red lines click Hide Crosshair 4 To Close the Triple View click on the X in the upper right hand corner of the Triple View box Note Do not close TLBview tif XYMin Projection Please continue on to the next section Zoom This feature allows you to increase or decrease the size of the currently active view by a percent age based scale either by selecting a Zoom factor from the View menu or by selecting a Zoom factor from the Zoom box on the toolbar You have three choices for changing the size of the Image 1 Under the View menu select Zoom and then select 200 The TLBview tif XY Min Pro jection will be zoomed up to 200 of its original size 2 Move the cursor to the corner of the view When the cursor changes from a X toa ry click the left mouse button down and hold it down while dragging the corner until the image is the desired size The default setting for locking the Image Aspect Ratio Retain Aspect Ratio is on therefore the image will remain proportionally correct 3 Change the size of the image by using the Zoom drop down box on the Toolbar Close all datasets before continuing on to the next section March 2004 Page 68 AutoQuant Im
3. Dark Current Bias and Flatfield Corrections The data correction utility is a component in AutoDeblur This provides features for automatic data correction including automatic bias and flatfield correction This component corrects data from video rate and cooled CCD cameras Processing Many Datasets AutoDeblur allows the sequential processing of many datasets by using its Batch Processing feature Basic Principles Underlying the AutoDeblur System The basic mathematical and conceptual principles underlying the AutoDeblur system have been widely published in scholarly journals and conference proceedings worldwide Constrained Maximum Likelihood Estimation may be thought of in two ways First consider a 3D fluorescent sample We may think of the true 3D probe concentration as an unknown random quantity In an intuitive sense the image reconstruction algorithm is searching for the 3D fluores cent probe concentration which is the most likely one to have caused the raw optically sec tioned image dataset taking advantage of any available constraints In a mathematical sense the algorithm is based on the formulation of a mathematical function called the likelihood function This likelihood function depends on both the unknown 3D fluorescent probe concentration and the 3D PSF The algorithm iteratively solves for the 3D image that maximizes this likelihood function In other words the likelihood function is solved for the correct probe c
4. Dye Kg for Ca Fura 2 224 nM Quin 2 115 nM Indo 1 250 nM Ca Green 190 nM March 2004 Page 184 AutoQuant Imaging Inc Manual and Tutorials Viscosity This is the Viscosity factor which is the effect that the viscosity will have on the spectra of the dyes Viscosity is defined as the measure of the resistance to flow that a fluid exerts If the viscos ity factor is not known it is recommended that 0 7 0 85 be entered into this field PostProcessing This section allows you to clean up noisy images during processing This will result in a much more accurate analyses of the datasets Remove Spot Noise This option lets the user remove spot noise caused by a few bright pixels that may be generated during the ratio process Gaussian Smoothing This feature removes noise by using a Gaussian filter thus creating a more accurate image Calculate Clicking this button will run the Ratiometrics process and create a new image Statistics Clicking this button will open a statistics window displaying a histogram and statistics of the intensities For more information on the Statistics window see page 200 Close This button will close the Ratiometrics dialog Help This button will open the Online Help opened to the Ratiometrics topic Ratiometrics Tutorial 1 Navigate to the Ratiometrics folder then open HighCal 001 HighCa2 001 LowCal 001 and LowCa2 001 For each file it will ask which pattern deno
5. 1 Make the Pollen deb Max Projection the active view 2 From the Visualization menu select Best Focus The Best Focus projection of the dataset will be displayed Again you may adjust the Min and the Max intensities by opening Image Enhancement from the View menu Please close all views before going on to the next tutorial Slice Viewer This function generates slices through the image optically and then displays the face of each slice in the Slice Viewer The thickness of each slice corresponds to the Z spacing or step size Viewing in the Slice Viewer is a good way of examining the degree of optical sectioning in the dataset You are advised to examine your dataset in a Slice Viewer because it allows you to see subtle and not so subtle changes from slice to slice Make the Pollen deb XY Max Projection dataset active by clicking within its view 1 From the Visualization menu select Slice Viewer to generate an optical slices projection of the dataset from the XY perspective Note When a projection operation is selected the resultant view formed depends upon the cur rently active view For example in the previous step the XY Max projection was the active view Thus when the Slice Viewer menu item was selected optical slices were created from an XY per spective 2 To step through the optical slices simply move the slider at the bottom of the view This can be accomplished either by clicking on the button and
6. 2 From the PreProcessing menu select Background Equalization The Background Equal ization box will appear Adjust the Background by typing in the Background Equalization box or by scrolling the bar until the Maximal Equalization value equals 45 3 Check the Remove Negative Intensities box to enable it When this box is checked Auto Visualize will remove any background points that have negative intensity values These negative pixel intensity values can come from the autoscaling function in the software or from artifacts in the dataset itself Click OK The background adjusted dataset will be displayed 4 Close the Background Equalization dataset Untitled before going on to the next section Note Do not close SmallHip avz Please continue on to the next section Invert Data This function inverts the intensity scale of the entire stack of optical sections This is different from the Invert View function under the View menu item which only inverts the intensity scale of the image in the current view 1 From the PreProcessing menu select Invert Data The SmallHip avz dataset will be pro cessed automatically and displayed upon completion of the inversion March 2004 Page 117 AutoQuant Imaging Inc Manual and Tutorials 2 You may close the Invert Data image Untitled dataset before going on to the next sec tion Note Do not close SmallHip avz Please continue on to the next section Image Alignment Slice to
7. B B S12 Sh2 Ratiometrics 184 Backlash 45 Bad Pixel Treatment 128 Settings 128 Bad Pixel Treatment Settings Median 128 Bandlimit 33 Basic Principles Underlying the AutoDeblur System 34 Batch 215 Cancel 163 Delete 163 Help 164 Best Focus 168 Bias 34 Bias Fields 126 Bias Frame 35 46 Bias Frames 45 March 2004 Page 230 AutoQuant Imaging Inc Manual and Tutorials Bin factor Isosurface 99 Bio Rad PIC pic 59 Bitmap File bmp 59 Black Level 40 41 Blind adaptive deconvolution 145 Blind Deconvolution Methods No PSF 33 Blue Channel 107 Blur Nonisotropic 31 Bottom of the Scan 36 Bottom Slice 36 Brightness 86 C Cascade 213 CCD Camera Shutter 45 Charge Integration Time 45 Cooled CCD Camera 45 Cooled CCD Cameras 45 Dark Current 45 Flatfield Non uniformity 45 Frame Averaging 40 RS 170 Cameras 46 Well Capacity 39 Center of Mass 88 89 92 Change Import multi channel data 60 Change Frame Delay 177 Change intensities Import multi channel data 61 Changing a Color Map 105 Channel menu All Channels 107 Blue Channel 107 Green Channel 107 Red Channel 107 Channel specification Import multi channel data 61 Channel to Channel Alignment 124 Channels Slice Viewer 107 Check for Update 214 CHOICE OF LAW 25 Clear List 163 Close 57 Manual Alignment 123 Close All 213 Collecting Optical Sections 36 Colocalization 186 Color Map 105 Color Map menu 79 Color Maps Tab 95 Color Selection 79 Color s
8. Captions Axes Tab This tab allows you to enter text to display at the top and bottom of the 3D Viewer Captions Header Text entered into this section will be displayed at the top of the 3D Viewer Footer Text entered into this section will be displayed at the bottom of the 3D Viewer Text Size This feature sets the size of the Header and Footer Type the desired font size into the text box Text Color This feature sets the text color of the Header and Footer The choices are White Black Red Green Blue Cyan Yellow Magenta Orange Custom Axes and Scale Bar Axes Checking this feature will display color coded axes to make it easier to determine the orientation of the object The X axis will be red the Y axis will be green and the Z axis will be blue Axis Labels Checking this feature will display the name of each axes above below or next to 1t depending on its orientation The default labels will be X Y and Z respectively however you can change the names by entering the new name in the corresponding text box Grid Checking this feature will display a grid around the object in order to give a perspective of the size Of the features within the object Spacing Labels Checking the Spacing Labels feature will display the spacings on the grid as well as the axes to assist in showing the actual size of the object and its features Length This feature allows you to enter the distance in between the spacing labels Fo
9. i played slices click the Subvolume button then left click on any plane of the orthogonal slice view The slice displayed will change as you drag the mouse The slice selected will be high lighted in yellow This will work in any view within the 3D Viewer 7 To zoom the dataset click the Zoom button a in the 3D Viewer Toolbar and left click and drag from the bottom right to the top left of the image Reverse the drag direction to shrink the image 8 From the View menu select Volume Projection Hardware if your video card supports it If it does not move on to step 9 below By default the Maximum Projection of the dataset is shown Rotate the dataset by clicking the Rotate button OF then left clicking and dragging on the display As the object rotates a low resolution version of the dataset is rendered When the March 2004 Page 72 AutoQuant Imaging Inc Manual and Tutorials mouse button is released a higher resolution version is rendered For maximum resolution with large datasets select Full Res View from the Options menu 9 From the View menu select Cube Surface From the Oblique menu select Display Slice 4 The Oblique Slice appears on the XY plane To rotate the Slice click the Oblique button E then left click on the object and move your mouse To move the Slice click on the Subvolume but ton left click on the slice and move your mouse To rotate the object while the Oblique button or any o
10. Cube Surface This feature displays a cube which has on each surface a painted projection of the dataset as would be seen from directly above that surface view The projection shown depends on the pro jection selected Isosurface This feature displays your dataset as a solid body with distinct surface and edge recognition and light reflection with quick rendering and rotation Height Map This feature displays a 3D image intensity height map of the rendered dataset Projection This feature allows you to specify a 2D projection The choices are Maximum Projection Minimum Projection Sum Projection Voxel Gradient Alpha Blending Best Focus Surface Slice Full Res This button allows the 3D Viewer to generate a full resolution view of the dataset This may take a few seconds to generate and only works for the Volume Projection view If you rotate the dataset the rendering returns to the lower resolution version Save Current This button allows you to generate a 2D view of the image that can be saved This function is sim ilar to taking a print screen or a snap shot of the image in the 3D Viewer window March 2004 Page 85 AutoQuant Imaging Inc Manual and Tutorials Image Appearance frame Render Range 0 100 Maximum and Minimum Rendering Thresholds This feature allows you to specify the maximum and minimum image rendering thresholds Inten sities above and below these respective percentages
11. Figure A A successful unsaturated image Figure B A saturated image of the same sample Note the obvious bright flat white back ground Note that the highlighted background features marked 1 in Figure A are erroneously March 2004 Page 38 AutoQuant Imaging Inc Manual and Tutorials eliminated in the saturated image Note that other features of the sample marked 2 are eroded in the saturated image A reliable method for detecting saturation is to compute and display the image intensity histo gram A straight vertical spike line at the end of the abscissa see illustration in Figure C is an indication of saturation which is to be avoided n o Q S o 5 5 o Q O Y o E S E 5 Z Gray values Minimum Maximum gray Maximum possible value in this frame possible gray gray value value Figure C Histogram of gray values in an image frame that has saturated pixels The maximum gray value of the frame and the maximum possible gray value coincide Typically though not always a spike will appear in the histogram as shown above If you observe saturated pixels lower the exposure time and repeat the grabbing and viewing pro cedure As a rule of thumb adjust the exposure time so that the brightest pixel is at approximately 75 of the well capacity of the CCD camera This condition is often detectable depending upon the sam ple again by using a histogram The 75 well capacity condition is
12. ment topic Data Correction The Data Correction function performs the Flat Field and Bias Field corrections and the correc tions for the camera and lamp flicker whose principles are described in the Guidelines for Col lecting 3D Image Data section of this manual The Data Correction function is for widefield datasets fluorescence or brightfield only Do not use it for confocal datasets It is recommended that you do a Data Correction on your dataset before processing a Widefield dataset fluorescence or brightfield Slow Scan Cooled CCD The Data Correction feature can correct for Slow Scan Cooled CCD cameras when charge inte gration time is selectable March 2004 Page 125 AutoQuant Imaging Inc Manual and Tutorials 1 Navigate to the TLB folder in the Tutorial Data directory Set the Files of type to All Files and select star 1 The File Input dialog box will appear shown below File Iput xj 2 4 sequence of files associated with the selected file is detected Do you wish to load the entire sequence Yes No Select Yes 2 From the PreProcessing menu select Data Correction and click on Slow Scan Cooled CCD The Slow Scan Cooled CCD Data Correction box will appear Within the Files tab you will need to locate and load the files for the Flat Field the Bias Field 1 and the Bias Field 2 If you have not collected these fields then their selection boxes should be unchecked The recommendation is to col
13. to 30 and click the Apply to All Slices button The dataset will be automatically segmented 3 Move your cursor over a white region of the segmented dataset The cursor will become a crosshair Click your left mouse button to select that region for Seed Fill automatic labelling The Label Object box will appear Click on the Apply button The Seed Fill region will be outlined in blue March 2004 Page 193 AutoQuant Imaging Inc Manual and Tutorials 4 Close the Label Object box and the Segmentation Parameters box Note Do not close the Labeled TobaMC tif XY SLICE VIEWER GREEN dataset Please con tinue on to the next section Undo Label This feature lets you remove the last Label placed on the dataset or the last set of Labels com bined To utilize this function select Undo Label from the Segmentation menu and AutoVisualize will remove the last placed label on the dataset Note Do not close the Labeled TobaMC tif XY SLICE VIEWER GREEN dataset Please con tinue on to the next section Highlight Labels This function colors the area within a Label with a pastel overlay The overlay helps in viewing the area within the Label There are 5 different color overlays that the system will cycle through depending on the Label Number The pastel color overlays appear in the following number order 1 Blue 2 Magenta 3 Green 4 Yellow 5 Red 1 From the Analysis menu select Segmentation and click on Highligh
14. Break only those guidelines for which your experimental design restrictions absolutely prevent you from March 2004 Page 51 AutoQuant Imaging Inc Manual and Tutorials following these guidelines In other words only break a guideline if you have no choice but to do SO Sampling Issues As arule of thumb the in plane sampling ought to be equal to the resolution element resel size or spot size of the image According to Webb et al 0 44 Ad lresel NA and Ad 1 44n 2 NA where Ad and Ad are the spot sizes in micrometers for the in plane XY and axial z dimen sions respectively and where A and NA are the wavelength and numerical aperture respectively The least critical deviation is to make Ad larger than that indicated by the above equation This is because most of the noticeable smearing in images is along the axial dimension which is not affected by making Ad larger The next least critical deviation from this rule is to make Ad smaller than indicated by the above equation In fact there are distinct advantages in doing so Generally speaking the finer the sampling is along z the better is the deblurring along z The above rule is considered the optimal trade off between this axial deblurring and the potential pho tobleaching and other problems such as extra disk space required that may be caused by having finer axial sampling The next least critical deviation is to make Ad larger than indicated
15. Color Deconvolution Parameters Image Info C All Channels A a hee Total Iterations eo Image C Red AE 30x 80 2 ee correction e E areen EN sen FC Blue 64 x 32 E Super Res Factor 1 m Noise Smoothing Low Medium High Other Value 2 a Within the 2D Deconvolution box set the parameters to the following values A For the Data to deblur selection click on the Region of Interest box B The Color selection should be All Channels C For the Deconvolution Parameters increase the Total Iterations number to 30 The PSF Correction Factor 2 and Super Res Factor 1 remain unchanged D The Image Info box settings remain unchanged The image size is displayed in pixels 1 e 256 x 256 E The Noise Smoothing setting should remain as Low because this image does not contain a great amount of background noise 3 The Expert tab the ROI tab and the Frames tab settings remain unchanged from their default values Process an Image 1 Click OK to begin the deconvolution process Note To interrupt the deconvolution press the ESC key and wait for the current iteration to fin ish 2 When the 2D Blind Deconvolution is complete a 2D Deconvolution Results box appears with three tabs Results Channels and Save March 2004 Page 147 AutoQuant Imaging Inc Manual and Tutorials 2D Deconvolution Results In the Results tab you are able to view the result of the
16. However in spite of this improvement over the widefield microscope the confocal microscope has its own limitations 1 While it indeed rejects most of the out of focus light it by no means rejects all of it and thereby retains an obvious haze even though this haze is much reduced from that of the widefield microscope 2 Another effect of this imperfect out of focus light rejection is that the image retains sub stantial axial smearing For an image which is not diffraction limited this smearing appears akin to a linear motion blur in the axial direction For an image which is diffraction limited as with empty magnification this smearing appears as a substantial nonisotropic blur or nonisotropic spatial resolution with most of the smearing along the axial direction 3 Owing to this light rejection far fewer photons are detected so a rather substantial quan tum photon noise component may be seen in the raw dataset This noise component routinely causes limitations in protocols where very fine structures need to be seen For instance it limits our ability to detect a void in the fluorescence concentration that is often useful to pinpoint impalement sites of electrophysiology probes Turner Szarowski et al 1991 and it limits our ability to determine if a dendritic spine is present It is difficult to judge whether a void in the image intensity is due to a fine structure that is really there or if it is due to erroneous random flu
17. arc lamp flicker instabilities in the power supply to the lamp instabilities in the power supply to the camera fluc tuation in the 110 Volt A C power that supplies the camera electronics microscope and other electronics in the microscope system Other remote possibilities are adverse laboratory condi tions temperature changes room lights being turned off and on and movement in the room caus ing shadows over the microscope Although the source of these flicker effects is difficult to pinpoint exactly it is certain that they occur and that they occur regularly 8 Close all views before continuing on to the next section Attenuation Correction The Attenuation Correction function corrects the attenuation as a function of depth into the sam ple Such attenuation is due to several factors Excitation light absorption Fluorescent emission light absorption Photobleaching Changes in the optical point spread function as a function of depth which in turn is fundamentally due to refractive index mismatches between the sample embedding medium coverslip and lens immersion medium A dataset that requires Attenuation Correction is recognized by noticing darkening within an image as you go from the top to the bottom of the view 1 From the File menu select Open Navigate to the Confocal folder and select RedNuc 0 2 A dialog box will appear with the question A sequence of files associated with the selected file is
18. as a general rule of thumb for optimal results we do not recommend making the in plane resolution any finer than as indicated in the above equation If you do so we recommend March 2004 Page 52 AutoQuant Imaging Inc Manual and Tutorials that special care be taken to be sure that your confocal microscope aperture is stopped down so that it is in the fully confocal position See the next two sections for recommended sampling when the aperture setting is other than fully confocal Confocal Aperture AutoDeblur runs on the mathematical assumption that you have a fully confocal microscope This condition is true so long as the confocal pinhole photodetector aperture is closed down to a size that is less than or equal to the waist of the scanning spot diameter of the Airy disk In the specimen plane for a 1 4 NA lens this spot size is around 0 25 micrometers and the recom mended confocal aperture diameter after projection onto the specimen plane is then 0 25 micrometers or less This number however does not represent the actual physical diameter of the aperture For example The Molecular Dynamics Sarastro confocal scanning system for a 60X 1 4 NA lens a spot size of 0 25 micrometers in the specimen plane is magnified to approximately 50 micrometers in the detector plane Thus the recommended true aperture size is 50 micrometers for this particular setup Please check the specifications of your confocal microscope to
19. the Intensity Profile Along Line will display colored lines red green and blue each of which represents its respective channel color For example the red line is the relative intensity values with respect to the Max and Min intensity of the image s Red Chan nel The orientation of the intensities is relative to the image not the start and endpoint of the line When in the Slice Viewer the Intensity Profile only displays intensities values for a single slice for the line being drawn At the bottom of the Intensity Profile Along Line box the Angle displays the angle in degrees between the last line drawn and the current line This works only in the Piece wise Line mode To disable the Line Length selection click on Done or click the Line Length icon on the Toolbar Note The distances displayed will be in microns per pixel This applies to the angles also March 2004 Page 199 AutoQuant Imaging Inc Manual and Tutorials Three Dimensional Line Lengths You may calculate line lengths and the angle between lines in all three dimensions by following this procedure 1 The image must be in a Slice Viewer 2 Left click and draw the first desired line Then left click again to start a new line You may then change the current slice by clicking the right or left arrow key on your keyboard causing the line to be drawn through several slices The Intensity Profile Along Line box will display the Line Distance under the histogra
20. you may then save the Untitled movie to a supported movie format Clicking the Cancel button will end the movie generation process and will revert the view to the first frame of the movie without the new movie view being generated Concatenate Movies 1 From the File menu select Open Movie 2 Navigate to the Movies folder and open Colrpollenrs avm 3 From the Visualization menu select Concatenate Movies March 2004 Page 176 AutoQuant Imaging Inc Manual and Tutorials 4 The Concatenate Movies box will appear Click the Add a Movie button and navigate to the directory where the movie files are stored Select the first movie to be added in the concatena tion Colrpollenrs avm and click the Open button The movie will be placed first in the Movie List 5 Click the Add a Movie button again and select the Hip avm to be opened Once the Open button is clicked Auto Visualize will add the SmHip avm to the Movie List in the second place If you make a mistake in selecting a movie the movie may be easily removed by selecting the movie and clicking the Remove button Auto Visualize will remove the highlighted movie from the Movie List For now leave both movies in the Movie List 6 Click the OK button and Auto Visualize will generate a new movie comprised of the Colr pollenrs avm and the SmHip avm movies This concatenated movie can then be saved and concat enated again with another movie if desired 7 Click th
21. 10 1 82 89 Richardson W H 1972 Baysian Based Iterative Method of Image Restoration Journal of the Optical Society of America 62 1 55 59 Roysam B H Ancin A K Bhattacharjya A Chisti R Seegal and J N Turner 1994 Algo rithms for Automated Characterization of Cell Populations in Thick Specimens from 3 D Confo cal Fluorescence Data Journal of Microscopy 173 2 115 126 Roysam B A K Bhattacharjya C Srinivas and J N Turner 1992 Unsupervised Noise Removal Algorithms for 3 D Confocal Fluorescence Microscopy Micron and Microscopica Acta 23 4 447 461 Shaw P J and D J Rawlins 1991 Three Dimensional Fluorescence Microscopy Progress in Biophysics and Molecular Biology 56 187 213 Shepp L A and Y Vardi 1982 Maximum Likelihood Reconstruction for Emission Tomogra phy IEEE Transactions on Medical Imaging 1 2 113 121 Sheppard C J R and M Gu 1994 3D Imaging in Brightfield Reflection and Transmission Microscopes 3D Image Processing in Microscopy Munich Society for 3D Imaging in Micros copy Snyder D L A M Hammoud and R L White 1993 Image Recovery from Data Acquired with a Charge Coupled Device Camera Journal of the Optical Society of America A 10 5 1014 1023 Snyder D L M I Miller L J Thomas and D G Politte 1987 Noise and Edge Artifacts in Maximum Likelihood Reconstructions for Emission Tomography IEEE Transactions on Med
22. 33 Confocal 128 512 64 0 07 0 07 0 15 March 2004 Page 220 AutoQuant Imaging Inc Manual and Tutorials Table 2 File Name Numerical Refrac Modality X Y and Z X Y and Z Spacing Aperture tive Dimensions Pixels Microns Index Time Series Data 1 32 1 4502 Widefield 256 256 23 32 32 4783 Test tif 256 256 1 Test_parameter set A file containing datasets parameters Triple label segment seg A file containing datasets labels Volume amp Surface Area text A text file of the measurements for Surface Area amp Volume of a dataset Toolbar Icons File Toolbar Icons L3 Open Ctro m Save As Ctrl 5 Eh Print Ctrl P March 2004 Page 221 AutoQuant Imaging Inc Manual and Tutorials Visualization Toolbar Icons My Max Projection x gt i Min Projection gt Sum Projection Ga Slice Viewer Bon Slice Synchronizer voxel Gradient Shading QE Alpha Blending Af Best Focus View Toolbar Icons XY xv XZ xz ZY zy Xy Triple view Montage D Stereo view g Retain Aspect Ratio E Stretch Aspect Ratio m Correct X2Z ZY Aspect Ratio r 3D Viewer Enhancement Invert View Horizontal et gp Vertical March 2004 Page 222 AutoQuant Imaging Inc Manual and Tutorials Analysis Toolbar Icons Rectangle T Ellipse E Polygon Fet Freehand Crop the Stack Slice by Slice Cropping 4 Crop Dimensions E Extend Slices 52 Resi
23. Chelo 120 Alignment Los Flema cti 120 Write Lois arias 120 Setfolder Patilla lied rl eet ets 120 Manual Alo Mtra A ita 121 TWO MIE WS ra A til 122 PEN a o ee TE 122 COM DING i e esol AAA tt A CA 122 PICKED vi A Ba A a A Rd dad 122 LOOMS Oti TO II A tee veka ude AEE AA A Ade A seks 122 PEO A NR OOO 122 COM A A AA Ote 123 RESIZE its nE E a EENE Eose Le aN ta edem ctesatabensetates 123 ROA AAA AA 123 REPOSO it A id 123 Slice Controla aca 123 Slice Status BOX initial 123 Apply Ae an AA A ici kalau eal ae 123 WO Vii ad a ad 123 Close ta tata 123 Channel to Mim RAN Si 124 Alignment Direction tupla ocre 124 Base SHCE ai A A een anu en es 124 March 2004 Page 7 AutoQuant Imaging Inc Manual and Tutorials Advanced SEO iii lidia 124 Noise Backsround ii A E E E 124 V id Regi n Fluid dci 124 Bd a a OO 124 Random ui ld ii bra 124 Itata A EAE O tran aude oficia 125 Alignment Log File cuina As 125 OK Lain T T E E 125 Cancel EOV E OLETA A AE An He eh E EEA ni teed 125 A 125 Data COn lo rr leia lod 125 Slow Scan Cooled OCD usvcisndiitia ri iii sida 125 Hish Speed Ca hes ate A 128 Back Sround SUDAN aia 129 TAS M ACEO oe eras ah secession eh A 130 Rotation Offset egg ogc cc e did a tete lead ne 131 Adding PILES iii di 132 Removina Files irrg ee iii era 132 Tutorials for Deconvolution Menu Items 0 000 133 Deconvolution Settings vaa did 133 Standard Settings usina ilatina ela
24. Combine Channels dialog so if the file is not visible try moving the dialog box to view what is behind it You may now save this file as a combined channel file in the stk seq tif 24bit or bmp format Data Properties This function allows you to view the properties of your dataset March 2004 Page 62 AutoQuant Imaging Inc Manual and Tutorials File Name This lists the name of the selected file File Size This lists the size of the selected file Bits Pixel This displays the bits per pixel for the selected file File Type This lists the type of file that is selected Dimensions X Y Z The Image Dimensions are the Width pixels Height pixels and Depth slices The Width Height and Depth will automatically be set for a dataset stored as TIFF tif AutoDeblur deb AutoVisualize avz BioRad Pic pic IPLab STK stk Bitmap bmp or PGM pgm formats The Dimensions will need to be entered by you the first time an 8 bit 12 bit 16 bit or 32 bit for mat dataset is loaded Spacing X Y Z The X Y and Z Spacing also known as voxel size is the size of 1 pixel in microns in the X Y and Z direction respectively The Z spacing is often referred to as the step size The Spacing is listed in the following order X Y then Z X Spacing The X Spacing is the width of one pixel in micrometers Guidelines The X Spacing can be calculated by dividing the spatial
25. Current Color Maps frame 95 Current Rotation frame 89 Angle 89 Round 90 Current View 80 D Damages 21 Dark Current 34 Darkness 86 Data Correction 124 126 Cooled CCD 128 Cooled CCD Data Correction 125 Data to Save box 151 Deblurring Oversampled 31 37 Deblurring Data Inverse Filtering 152 156 Nearest Neighbors or No Neighbors 156 Deblurring Subfields Cropping 108 Deconvolution Menu 1D Deblur 158 AutoDefault 153 Expert 137 Standard 133 Deconvolution menu 146 Process Current Slice 157 Deconvolution Parameters 147 Deconvolution Performance 136 Dendritic Spines 31 Depth Of Field 27 37 Depth of the Sample 37 DIC 118 Dichroic Mirror 41 46 Differential Interference Contrast 118 Direct Eye Viewing 41 Display Floor 81 87 Display light Isosurface 99 Display Range 86 Display Slice 76 Distance Tolerance Object Counting Tracking 207 DOF 37 Dye Pair s Foster Distance RO FRET 180 Dynamic SubVolumes 139 Edit Menu 164 Electrophysiology Probes 31 ENFORCEMENT OF TERMS 24 Excitation Shutter 41 Expert Settings Warning 136 Explanations of 3D Previewer Menus 73 Exposure 38 Exposure Time 38 Extend Depth 112 Extend Slices 112 F Feature Selection Object Counting Tracking 205 209 Feature Tolerance Object Counting Tracking 208 File Exit 66 Open 56 Print 65 File Menu Close 57 Open 56 64 106 117 File menu Save As 110 File Names Numeric Suffix 43 Filename Prefix box 151 Files Recent Files List 66
26. Cy3 570nm DsRed 583nm Rhodamine 590nm Cy3 5 596nm Propidium Iodide 617nm Texas Red 620nm Cy5 670nm Cy5 5 694nm and Cy7 767nm By using these dye selections a sam ple can be viewed as colored with the chosen dye wavelength March 2004 Page 79 AutoQuant Imaging Inc Manual and Tutorials Background This feature allows the user to set the color of the background of the 3D Viewer The choices are White 75 Gray 50 Gray 25 Gray Black Black to White and White to Black Black to White and White to Black create gradients from top to bottom Black to White would be black on top and fade to white on the bottom Additional options are available in the Color Maps tab in the Control Panel Reverse This feature reverses the order of the color map intensity scale of the selected color map Color map points that normally indicate high intensity areas will then indicate low intensity areas and vice versa Save Menu This feature is used to save your dataset as either a 2D image or a rotated volume Current View This feature saves the current view as a 2D image to the main viewing window You may save the 2D image permanently by selecting Save As from the File menu in the main window and saving the dataset Rotated Volume This feature saves the rotated image to the main viewing window You may save the Rotated Vol ume permanently by selecting Save As from the File menu in the main window and saving the d
27. E 106 Red Chana nario 106 Green Comet dt 106 Blue Chanel ainia plain 107 Viewing the Channels and Slices of a Multi channel Data Set o an 107 Tutorials for PreProcessing Menu Items cccconocaanorn 108 Select RESIGN rri a r by qsaasy deed a a a A a E E aaia 108 S E E E EEE T A E T 108 Crop the Stacki nearen O 108 A O 110 March 2004 Page 6 AutoQuant Imaging Inc Manual and Tutorials Apply to current slice Only oo eee eeeesecsseeeeeeeeeeseecaeecseceseeeseeeneesaeesaecsaeceeeeeeeseeeenaees 110 Apply tovallishices atkins e a A da Ad 110 Apply to given slice range osit beia i a a Ea a EE 111 Crop DIMENSIONS O 111 Extend Slices annone an a a a ye ales gots bash aga sada eon a RS 112 Resize oine oea sas tg e i spp eae a a i i a 112 Optical Density Come a a a a a as 113 Attenuation Correction ci pa 116 Backeroimd Gu ali Zain sepesi te t ea E E E E EAEE 117 nyert Dati ASAS AA 117 Image Ahenment is A ag tee ee eee te 118 Slice 10 SCC ae A GE BIN ee ls Lin E 118 Automatic Alignment ressonen pidand d ea a a aa Ea EEEE rs 118 Alonment AKIS nnan aa A AA AA a 119 AS E ai 119 Maidana aia roads 119 Advanced SEO iii biie 119 Image Walp cintia iii li dd a ts 119 Void Region Flia tit 119 Black calandra lolitas 119 Random tl iba ddr de 119 Wile E A SEE NEE dias 120 Noise Threshold iS R E AN E 120 Base Slice EE E E ES 120 Find Automatically vivia iint ceca E A REE A 120 Current slice italia damas 120 Base
28. Enter a value of 1 2 in the Gamma Correction field Notice that the horizontal line on the histogram moves up as well as a change in the image The Gamma can also be adjusted by click ing on the grey box on either side of the horizontal line on the histogram and dragging it up or down Note A Gamma value of 1 0 restores the image to its original state Values greater than 1 brighten the image whereas values below I darken the image Gamma values may be between 0 1 and 10 0 5 You may also adjust the darkness and brightness of the image in any of several ways Enter a new value in the Intensity text boxes The text box on the left is for the minimum and the text box on the right is for the maximum the Image Enhancement dialog will default to the minimum and maximum values present in the image The minimum must be less than the maximum March 2004 Page 103 AutoQuant Imaging Inc Manual and Tutorials Enter a new value in the Percentage boxes between 0 and 100 The text box on the left is for the minimum and the text box on the right is for the maximum The minimum must be less than the maximum Left click on the red box on top of the histogram and drag it to move the minimum inten sity indicator red line to the right Left click on the green box on top of the histogram and drag it to the left to move the maximum intensity indicator green line on the histogram The maximum intensity indicator can only be placed to the r
29. Expert Settings is enabled The Expert Settings will be explained in its own section on page 137 Output Settings The Output Settings allows you to select the file format in which you want the resultant file to be opened 9 Select TIFF 16 bit This is the same format as the Input File 10 Click OK this sets the Standard Settings for the FitcDapi_crop tif dataset Standard Settings Explanations Total Iterations The Total Iteration field under the Deconvolution Settings section allows you to choose the num ber of iterations performed during a deconvolution The higher this number the more times the dataset will be processed Lower iterative numbers will execute in less time whereas the higher iterative numbers will take more time and generally provide improved resolution Beyond a cer tain number depending upon the imaging modality widefield or confocal the deconvolution may provide unstable results The recommended number of Total Iterations is 30 If the total Iterations is set to a number above 60 that field will become yellow indicating it is outside the recommended range The algorithm will still run however If there is no entry or if zero is entered or the entry is greater than 5 000 then the field will be highlighted red indicating that it is outside of the acceptable range and the algorithm will not run until the field is corrected March 2004 Page 135 AutoQuant Imaging Inc Manual and Tutorials The Sav
30. Guardband Pixels The Guardband size defines the width of a border surrounding each subvolume This border is the region where the subvolumes are processed to seam them together This guardband prevents arti facts at the seams The possible values are integers from 0 to N 2 where N is the width or height of the XY field in pixels whichever is smaller Guidelines Generally the larger the guardband the fewer the artifacts and the better the image quality However deconvolution time increases with guardband size so a default value is set which minimizes deconvolution time and eliminates artifacts in most cases The default value is 10 This number may be increased if seaming artifacts appear If so first increase the number to 15 then 20 and then 25 until the seaming artifact is gone Z Guardband Pixels The Z Guardband specifies the number of slices that will be added at the top and bottom of the subvolume This Guardband prevents artifacts at the seams of these subvolumes The possible values are integers from 0 to N 2 where N is the depth of the XZ or YZ field March 2004 Page 139 AutoQuant Imaging Inc Manual and Tutorials Guidelines The Z Guardband should never be larger than the subvolume overlap region A value of 6 is adequate for most image stacks Intensity Correction Selecting this option will correct for differences in intensities between slices The default setting for this option is unchecked for Confocal images
31. In addition to placing the microscope on a rigid platform it is desirable to isolate the platform from external vibrations For this it is useful to be aware of the sources of vibrational energy around the microscope Also vibrations usually from cooling fans can be transmitted to the microscope via unavoidable accessories such as electrical cables While most of the vibrational disturbances are vertical it is possible to have disturbances that are along the horizontal direction Finally one must consider shock control in addition to vibration control Shocks often occur in a laboratory environment due to heavy footsteps nearby when buildings are being repaired or when heavy equipment is being moved Keep in mind that vibration isolation equipment must be considered not only for the microscope system itself but also for laboratory vibration sources such as computer and electronic systems containing fans and relays It is important to note that vibrations are best controlled at the source if at all possible Practical and Inexpensive Solutions Inexpensive vibration isolator components are available based on a variety of principles ranging from pneumatic through those based on the use of viscoelastic polymers see Edmund Scientific Barrington NJ catalog no A35 264 Sorbothane Vibration Mounts Such vibration mounts may be simply placed under a heavy pallet made of wood ceramic Plexiglas slate aluminum or some other convenien
32. Items ooonnnoncncnnnnnononnnas 167 Maximum Projection geo etic vada iia 167 Mimim m Pro econ dd bi 167 SUM Projection a e 167 Voxel Gradient Shading isis cn 168 Best FOCUS insistas 168 Shee Viewer se i a n O 169 Slice oy CHOTA Zer 5 e RT 170 Selects a VIEW aoe e E E E E ET E E E A E ts Buus 170 Scrolling Throws Slices koseni eneas a A a NS 171 AS 171 Removina Datasets ai tras 171 Mowe Maker as a as crac o oct e o cd 171 Time Series Movie Maker rrecn a a Geese r tedconaden shat ted a TES 173 aako tiota B Fei a E E E E ces cede EE E E E 174 Moyie Parameters rinitis ad 175 Choose Frame Rang8 ci A At ici 175 Rotating Morph imita item 175 Lime Stamp Format anna nds 175 CAP leds 175 a A ON 176 Generating the Time Lapse Movie ns 176 Contatenate MOVIES oil iii 176 Change Frame Delay tias tino bass ad daniel dad 177 March 2004 Page 10 AutoQuant Imaging Inc Manual and Tutorials Tutorials for Analysis Menu Options coooconancncconanannonnona 178 PRE P Wii AA A A A EA AA Dap E E AAA io Maal 178 Cofret PRE Cross Talk uta 178 ALS OPS tit ia 178 FRET Images and Calibration Images oooonnconnncnnnnnnnncnocononcconannnnnnn con nc nono nnnannncnnncnnnnos 178 PreProCessin iia ci occ aea ects eoe ae i e tev vie dete dde 179 Use Automatic Alignment 0 0 eee ceeceseeeseesseceseceeeeeeseaeesaeceaeceeeeseeeeeeeaaeed 179 Subtract Backsrourid s iced ete ei enna Blah ape eens 179 Sue PER elie tes eid a dine Se He adi e
33. Kd Ratiometrics 184 L Launch Imaris 58 LCD Glasses 84 Least Squares 33 License US Government 25 Lighting Isosurface 99 Likelihood Function 34 LIMITATION OF LIABILITY NOTICE 24 Limitation of Liability Notice 21 Line Mode 199 Load channels Import multi channel data 60 Load Settings 66 Loop Mode 78 M Magnetic Tape 16 Magneto Optical disks 16 MANUAL 22 Manual Alignment 121 Apply 123 Close 123 Combine 122 Controls 123 Flicker 122 Reposition 123 Resize 123 Rotation 123 Slice Control 123 Slice Status Box 123 Undo 123 Zoom 122 Zoom Step 122 Manual Input 89 Manual input 92 Manual Segmentation 192 Apply Label 192 Select Displayed Label 198 Maximum Likelihood 33 Maximum Projection 167 Measure 199 Median setting 128 Microscope Condenser 35 Condenser NA 36 Eyepiece 41 Fine Focus Knob 44 Fluorescence Excitation Lamp 35 Graduations 44 Horizontal Vibrations 51 Iris Diaphragm 36 Lamp Brightness Control 40 Micrometers Per Revolution 44 NosePiece Focusing Microscope 45 Objective NA 36 Relative Motion 50 Rigid Platform 51 Stage Focusing Microscope 45 Trans Illumination Lamp 35 Microscopes Confocal 30 Transmitted Light Brightfield 30 Widefield Epi Fluorescence 30 Microsphere 32 Mid Point Active 78 Minimum Projection 167 Modality Box 134 142 153 Modeling Assumptions 33 Montage View 69 Montage view 69 morph 175 Movie Time Series 173 Movie menu 77 movies rotating morph 175 N Nearest Neighbors 15
34. Navigate to the directory of the desired dataset then double click the dataset to load it Clear Classes Clicking the Clear Classes button will clear all classes that have already been loaded Save Data Clicking this button will allow the user to save the data for the object definitions This can save time for future experiments if defining the same type of objects Each object will be saved as a separate csv file Count Objects This frame contains the controls to count and save the segmented dataset either as an entire vol ume or single volumes at a time Separate Touching Objects Clicking this button will separate objects that segmenting did not fully separate It does this by stripping away layer by layer until the objects are disconnected in order to differentiate them then it rebuilds them to their original size Current Volume Clicking the Current Volume button will count all objects in the designated classes in the currently displayed volume All Volumes Clicking the All Volumes button will count all objects in the designated classes in all opened vol umes Save Current Volume Clicking the Save Current Volume button will save the current segmented and counted volume for later viewing A Save As dialog will open in which the filename and type can be entered to save the dataset Save All Volumes Clicking the Save All Volumes button will save all segmented and counted volumes for later view ing A Save As dialog will ope
35. SEQ seq This is the Image Pro file format It is a proprietary format of MediaCy bernetics Fluoview tif This is a 16 bit TIFF file It is a proprietary format of Olympus Instru ments Bio Rad PIC pic This file format is a proprietary format of Bio Rad IPLab This is a file format produced by Scanalytics Inc PGM pgm This is a commonly file type with UNIX computer systems It uses 8 bits per pixel Bitmap File bmp This is a Windows file format for images Note When you select Save As if your dataset is in a Slice Viewer Projection and you have changed either the gamma brightness or flip settings a dialog box will appear with the question Save data as shown No will discard the gamma brightness and flip adjustments Selecting Yes will keep the gamma brightness and flip adjustments Save Current View This function is similar to taking a print screen or a snap shot of the image in the viewing window When working with a dataset that was saved as Save Current View the XZ and ZY views are unselectable This is useful for use in publications or presentations March 2004 Page 59 AutoQuant Imaging Inc Manual and Tutorials 1 From the File menu select Save Current View Name the file BlueGreen2d stk The file will be saved as a 2 dimensional picture or an image made up of just one slice 2 Close all datasets before going on to the next section Import Multi Channel Data T
36. Select the Neuron deb dataset and click OK 4 In the Operations frame of the Image Macro Functions box select Crop as the first func tion and click the blue arrow This places the Crop function in the Operation Order frame Selecting the Crop operation causes the Crop Rectangular Dimensions box to appear in the lower left side of the Image Macro Functions box which contains field position boxes to set the Top Left Front Corner and the Lower Right Back Corner position of the dataset 5 In the Crop Rectangular Prism frame set the following Top Left Front Corner X 8 Y 8 Z 1 Lower Right Back Corner X 71 Y 71 Z 32 If you choose to perform an Operation like Crop Extend or Resize on any or all of the datasets the new dimensions will be displayed automatically in the New Dimensions frame for the cur rently highlighted file The Apply To box contains all of the files on which the Image Macro func tions will be performed on If you wish to remove a file click on the file and click the red X button in the lower right corner Note When Extend is chosen the Extend Depth box appears on the lower left side of the Image Macro Functions box In this box you would set the number of false slices you would like added to the top and bottom of your dataset When Resize is chosen the Resize Factors box appears with fields for resizing the X Y and Z dimensions of your image 6 Select OK Each dataset will be automatically cropped
37. Settings Paste Optics Settings Copy Deconvolution Settings Paste Deconvolution Settings volution tasks is a text file with the Batch Job Name and tst extension It AutoQuant product Copy PSF File Settings Paste PSF File Settings o Imaging Suite 9 1 Change Settings Change PSF File Image File Name ianti utoO uant Imaging Suite 39 1 Tutorial Dataidefield Pollen deb None see Leeus O L pese J __ Pme The batch will start in Cancel Timer March 2004 Page 155 AutoQuant Imaging Inc Manual and Tutorials When you click on the Change Operation tool a Change Operation dialog box will appear allowing you to choose between the 3D Deconvolution and the Inverse Filter Once the all desired tasks have been loaded in you can either begin the processing immediately by clicking the Start button or you can schedule the processing to begin at some point in the future by selecting a date and time from the Start Time box then clicking the Start button Once this is done a countdown will appear near the bottom of the dialog indicating when the batch will begin processing For more information on Batch Processing proceed to the Batch Processing section Operation ia Change Operation l x 3D Iterative Deconvolution C 3D Inverse Filter Cancel The output of the inverse filter operation will have the same file format as the raw input dataset Note Do
38. Since the image is specially colored only grayscale images can be viewed in anaglyph stereo mode Auto Threshold This feature automatically determines what is the best minimum threshold for that dataset and implements it Toolbar The 3D Viewer contains a toolbar for some of the more common actions performed on the dataset Rotate When the Rotate button is depressed use the left mouse but ton to click and drag on the object to rotate it along any axis Zoom A When the Zoom button is depressed hold down the left mouse button and move the mouse from left to right and back Dragging the mouse to the right will zoom out dragging the mouse to the left will zoom in Use the right mouse button to click and drag on the object to rotate it along any axis Subvolume When the Subvolume button is depressed left click on any plane of the orthogonal slice view The slice displayed will change as you drag the mouse The slice selected will be highlighted in yellow Use the right mouse button to click and drag on the object to rotate it along any axis i Oblique Rotate ES To activate the Oblique Rotate button select Display Slice from the Oblique Menu The Oblique Slice appears on the XY plane To rotate the Slice click the Oblique Rotate button then left click on the object and move your mouse Use the right mouse button to click and drag on the object to rotate it along any axis Threshold ata To increase or dec
39. a miniscule amount and not even just once Doing so by even a very small amount will ruin the scan due to backlash and hysteresis of the focusing system To compensate for erroneous hysteresis effects do the following before grabbing the first frame First focus into the specimen If you have a stage scanning microscope first move the stage down by 20 micrometers or more below the position where you wish to grab the first frame Then being careful to move the knob in only one direction as mentioned earlier once you are in this stage of operation never reverse the direction of the knob slowly and carefully move to the posi tion where you want to grab the first frame This operation counteracts a hysteresis that is inherent in the microscope s focusing mechanism Collecting Bias and Flatfield Frames Cooled CCD Cameras Bias and flatfield dataset frames are used by AutoDeblur to automatically calculate the cooled CCD camera s dark current distribution pre amplifier bias and flatfield non uniformity This pro cedure needs to be repeated every day or preferably for every dataset collection This process compensates for potential misalignments in the optics which may drift from day to day and to compensate for dust that may settle in the optical train which also changes from day to day Collect two bias frames and one flatfield frame The bias frames are taken with the camera shutter closed or with light to the camera blocked Sel
40. a prefix which will be used in the file name of each resultant image created by the experiment Use Cross Talk Coefficients This section allows you to enter cross talk values in lieu of using calibration images For each image donor and acceptor there are three values to be filled in These should be between 0 0 and 1 0 Ideally settings should be set between 0 0 and 0 5 Settings of 0 5 and 1 0 are acceptable but not ideal The only way to verify is to visually inspect the resultant images For this reason unless March 2004 Page 179 AutoQuant Imaging Inc Manual and Tutorials you know what these coefficients are from previous experiments it is ideal to have calibration images Calculate Clicking this button initiates the selected Correct FRET Cross Talk algorithm Clear Files Clicking this button will clear all file associations made in the FRET Images and Use Calibration Images sections There is no undo for this Close This button closes the FRET dialog box Any files created using the FRET dialog will remain open only the dialog box closes Help This button will launch the Online Help file opened to the FRET section Calculate FRET Efficiency Once the Cross Talk has been corrected the analysis of FRET can begin Data for FRET Efficiency Estimation This section allows you to select the images to run statistical analysis on Do this by clicking on the dropdown arrow then selecting the correct image based on
41. algorithm For more information on the differences between the algorithms go to www aqi com and download the FRET Application Note from the Support section Select the desired algorithm by clicking on its radio button FRET Images and Calibration Images This section makes associations between the images and excitation emission specimen set To optimize the use of the FRET feature the proper datasets must first be obtained Below is a list of headings and the images they correspond to that will allow for optimal use of the FRET feature when naming your datasets make sure you know which one corresponds to which excitation emission specimen set Headings and Corresponding Datasets DDf Donor excitation and Donor emission applied to the FRET specimen DAf Donor excitation and Acceptor emission applied to the FRET specimen AAf Acceptor excitation and Acceptor emission applied to the FRET specimen DDd Donor excitation and Donor emission applied to the Donor specimen DAd Donor excitation and Acceptor emission applied to the Donor specimen A Ad Acceptor excitation and Acceptor emission applied to the Donor specimen DDa Donor excitation and Donor emission applied to the Acceptor specimen DAa Donor excitation and Acceptor emission applied to the Acceptor specimen AAa Acceptor excitation and Acceptor emission applied to the Acceptor specimen Not necessary but can be used with the Gordon and Herman and AutoQuant Imaging Inc algori
42. and Counting Expert Settings 000 0 ce eeeesececeeeeeeeeeneeeneeeneeees 203 Select Object CIASSES cds dl sda s Geeas A old asin 204 No Traming CountAlD iii ida 204 Performance dios 204 Current Classes turcos adn dll E pl isis eai 204 Add Classes Define Classes Redefine Classes ccccceccsececceeeteceneceeeeeeeeeeteeenaees 205 Object Classes for Counting 0 eee ceseeeceeeeeseeenseceecesecseeeeaeeeaeenaecsaeeneeeaes 205 Load Classes ict iste nits ario 206 Clear Classes A Gotu dened dan Biles aia 206 COUNT ODIECKS viii aida ae presa 206 Separate Touching Object tira ati 206 Current Volmeri ii id aliado aSa 206 JAI VoM eS tee 206 Save Current Volume tii lio ee deceit 206 Save ATV OLUMECS ici aaa 206 View Current Statistics Mutant iia 206 View All Statistical iria a 207 Object Counting Results iii o 207 Change peinada A a eat 207 EXporpRe atures tdi in A connie ORRA 207 To Tracking gt gt seid panini oie ea andes dae een 207 Clos fst Sets a a A A A AR 207 Tracking a dai 207 o RE UN 207 AROMA UD ri AA A Aci 207 Auto Partial ai cascada data 208 Manta ModilY vidas lali bd 208 View Data ea nia 209 View Track Eanes iii as 209 Feature Selection si stencil sita ease hai nia aeons 209 Select Alli oe ee da 209 DeselectAll diia 209 Calculate Statistical a 209 2D Feat res tica cd es dni OD dai E atadas 210 SD FECALUTES xt sssnsevsssadssdoondhstenedesteoetesseredsvactannieesontes deedsebsaedeecusdisuvacossitsevdeesbue
43. and checked for Widefield and TLB images It works much like the Optical Density Correction feature in the PreProcessing menu Minimum Intensity Removal The Minimum Intensity Removal feature will subtract the minimum intensities from the image Thus with Minimum Intensity Removal selected an image that has intensities ranging from 11 245 will be adjusted to have intensities of 0 234 The default for Minimum Intensity Removal is Yes Accelerated SA Detection Selecting Accelerated this is selected by default will accelerate the process of detecting the spherical aberration of the dataset Accuracy is slightly better with acceleration not selected but the trade off of speed with the acceleration is greater than the accuracy compromise Pre Condition Imported PSF This option will pre condition an imported PSF Object First Guess The Object First Guess has three options Flat Sheet Smoothed Raw Image and Use a Wiener Fil ter The First Guess selects the initial estimate of the object that is used to initiate a blind deconvo lution process Flat Sheet Select this option to use a constant array as the object guess Smoothed Raw Image Select this option to use a smoothed version of the original image as the object first guess Inverse Filter Select this option to use an Inverse Filter as the object first guess Previous Result Select this option to use the previous result from an already performed deconvolution as the object first guess Su
44. at a specific threshold value 2 From the View menu open Image Enhancement This will allow you to modify the thresh old of the Voxel Gradient Shading projection 3 Enter 60 into the Minimum Percentage textbox the lower left box in the Thresholds frame or slide the bar until the percentage value becomes 60 Leave the Max projection at 100 The Voxel Gradient shading dataset will automatically update 4 Try changing the Threshold to other values to view the new results 5 Click the OK button on the Image Enhancement dialog and click the Close button on the Voxel Gradient Rendering box Note Do not close Pollen deb XY Max Projection or the Pollen deb XY Voxel Gradient dataset Please continue on to the next section Best Focus Note This section applies to users of the Auto Visualize software only This function takes parallel rays perpendicular to the viewing surface and casts them through the 3D image The voxel encountered along each ray with the greatest local contrast is selected for display in the resulting 2D image This produces an image constructed from the most in focus fea tures from the volume March 2004 Page 168 AutoQuant Imaging Inc Manual and Tutorials A Best Focus projection creates an image out of the regions within a volume that best stands out against their neighbors This is useful for observing more subtle features in an image which may be not as pronounced in other projections
45. be created 3 From the PreProcessing menu select Crop and choose Slice by Slice Cropping The Cropping Mode Selection box will appear 4 Press the Apply Region button An unchecked check box will appear to the left of the number 1 in the list box This represents the first region of interest selected 5 Click on the check box next to the number to select it A highlighted overlay will appear in the selected region You may now choose the Crop function or the Remove Region function The Crop function will crop out the previously selected region of interest and will display the cropped dataset as a new dataset The Remove Region function will remove the region of interest from the dataset and will display the removed region as black within the dataset The Remove Region function is useful for removing artifacts from datasets You may select from one of three following Slice by Slice crop options Apply to current slice only This function allows you to select a region of interest on the current slice Apply to all slices This function takes the selected region of interest on the current slice and applies it to all of the slices in the dataset A new volume is created from the cropped area of interest March 2004 Page 110 AutoQuant Imaging Inc Manual and Tutorials Apply to given slice range This function takes the selected region of interest and allows you to apply it to a specified range of slices The newly created c
46. be returned with all product materials included in the shipping including the soft ware media e g CD manual 1f shipped hardware key and any other product materials March 2004 Page 25 AutoQuant Imaging Inc Manual and Tutorials shipped The complete set of product materials must be returned for the 90 day warranty to be valid and for a refund to be entitled The 90 day period begins on the day of shipment of the soft ware to the end user from the Representative or from AutoQuant whoever ships the product to the end user No return or refund is entitled after the 90 day period has expired Upon such a valid return the dealer is obligated to refund the price of the product less shipping costs The Represen tative will return the product materials to AutoQuant and AutoQuant will refund the discounted price of the product less shipping costs to the Representative March 2004 Page 26 AutoQuant Imaging Inc Manual and Tutorials Introduction What is 3D Deconvolution Also known as deblurring or 3D image restoration deconvolution is a computational tech nique for removing out of focus haze from stacks of optical sections The out of focus haze can be mathematically modeled as a point spread function PSF Deconvolution methods can therefore be thought of as methods for inverting the unavoidable and natural blurring effect of the point spread function PSF For example suppose we focus a micros
47. be used however the Use Cross Talk Coefficients tab exists for when these images are not available 1 Select Gordon and Herman from the Algorithms section 2 Click on the Use Cross Talk Coefficients tab on the right side of the FRET Analysis dialog box 3 Enter the estimated Cross Talk Coefficients for each of the 6 entries on the tab 4 Enter the FRET Conversion Factor G into the text box If this value is not known the default setting of 1 can be used or an estimation between 0 and 1 can be entered G is defined as the ratio of acceptor that is excited due to FRET versus the donor that is quenched due to FRET 5 Click Apply The application will create 3 new files with the results Note If there are already results files opened clicking the Apply button will update those files automatically it will not create new files To create new files click the Calculate button 6 Changes can be made to the Cross Talk Coefficients settings Once the changes have been made to create new files click the Calculate button to update the existing files click the Apply button Calculate FRET Efficiency Tutorial Once the algorithm has run on the images and the new files are opened analyses can be run on the images 1 In the FRET Analysis dialog box click on the Calculate FRET Efficiency tab 2 In the Data for FRET Efficiency Estimation section assign the appropriate images to the Corrected FRET Corrected Donor and Corr
48. button will open the Segmentation and Counting Expert Settings dialog box The settings for segmentation can be changed here Segmentation and Counting Expert Settings Perform Background Equalization Checking this box will perform Background Equalization on the dataset before segmenting it For more information on Background Equalization see page 117 The default for this feature is checked Use Noise Removal Checking this box will perform Noise Removal on the dataset before and after segmenting it This consists of a preprocessing smoothing of the image followed by a postprocessing analysis and March 2004 Page 203 AutoQuant Imaging Inc Manual and Tutorials removal of particles determined to be too small to be valid objects The default for this feature is checked Use Wavelet PreProcessing Checking this box will perform Wavelet PreProcessing on the dataset before segmenting it This 1s strictly a preprocessing feature and will create a somewhat similar effect of the Noise Removal feature though not quite as accurate though much faster The default for this feature is unchecked Threshold The Threshold frame contains the controls to set the thresholding for the dataset Look For This feature determines what type of objects to look for in the dataset the options are Bright Objects Dark Objects and Gray Objects Each object has a default threshold setting Use Automatic Threshold Checking this box will set the U
49. calcium free and calcium saturated specimens Calibrate If the above mentioned set of calibration images are open click on the Calibrate button to assign these files to the proper setting Numerator Wavelength Assign the images associated with the numerator wavelength to this section The calcium free sample needs to be assigned to the Low Ion image and the saturated calcium sample needs to be assigned to the High Ion image Denominator Wavelength Assign the images associated with the denominator wavelength to this section The calcium free sample needs to be assigned to the Low Ion image and the saturated calcium sample needs to be assigned to the High Ion image OK This button will calibrate the images to fill in the necessary parameters in order to use the Ratio metrics feature Browse These buttons _ located to the right of each image textbox will open a Windows Explorer browser from which you can navigate to and click and drag files into the AutoDeblur AutoVisu alize workspace Cancel Clicking this button will cancel the calibration process and close the Calibration Images dialog Help Clicking this button will open the Online Help file opened to the topic relating to the Calibration dialog Kd This is the affinity of a fluorescent dye to calcium Different dyes have a different affinity Below is a table listing the most popular dyes used in measuring intracellular concentration Table 1 Kg
50. copy of the SOFTWARE for your own use or use at the facility to which the SOFTWARE is licensed You AGREE that i your use and pos session of such copies shall be solely under the terms and conditions of this Agreement and ii you shall place the same proprietary and copyright notices and legends on all such copies as March 2004 Page 22 AutoQuant Imaging Inc Manual and Tutorials included by AQI on the media containing the authorized copy of the SOFTWARE originally pro vided by AQI You may not copy or provide copies of the SOFTWARE in whole or in part to any other party 3 SOFTWARE USE RESTRICTIONS a Reverse engineering and tampering You may not reverse engineer de compile modify translate or disassemble the SOFTWARE Any tamper ing with the license protection system such as the license protection key that attaches to your printer parallel port or of the SOFTWARE is not permitted Violators will be prosecuted to the maximum extent permitted by applicable laws b Not a Medical Device This software is not intended for usage as a Medical Device The definition of a Medical Device appears in section 201 h of the FD amp C Act A Medical Device is an instrument apparatus implement machine contrivance or other related article including a component part or accessory which is 1 recog nized in the official National Formulary or the United States Pharmacopoeia or any supplement to them ii intended for use
51. correct fluctuations in the image intensity values across the depth of an image This only works on images with depth gt 1 The image intensity value often fluctuates erro neously because of random flicker from the camera shutter or the lamp instabilities Flicker occurs with nearly all widefield microscope systems It is due to several causes Most Cooled CCD cameras have a randomness in their shutter s speed This shutter speed fluctuation causes variations on the order of several milliseconds typically from one exposure to the next The effect can be seen best in a side view projection of a dataset March 2004 Page 113 AutoQuant Imaging Inc Manual and Tutorials To determine if your dataset needs Optical Density Correction look for abrupt fluctuations in image intensity summations from one depth to the next 1 From the File menu select Open Navigate to the TLB folder and click on star 1 In the Files of type field select 16 bit data if the folder appears empty 2 The first time you load a dataset that is a sequence of files a dialog box will appear with the question A sequence of files associated with the selected file is detected Do you wish to load the entire sequence Select Yes The dataset will be loaded 3 In order to appreciate fluctuations in image intensity an XZ projection needs to be gener ated From the Visualization Menu select Sum Projection Generate an XZ View by clicking on the XZ View icon
52. deblurring process at each iteration by use of the scroll bar in the View Iterations frame You have the option to display the PSF during the viewing process by checking the check box next to Display PSF You also have the option to alter nate between the current iteration number result and the original image by clicking on the Origi nal Current button Within the Adjust Deblur Parameters frame you may choose to do further iterations by clicking on the Further Processing button or you may click on Return to Setup to rerun the 2D Deconvolution with different parameters Within the Use Current Iteration Settings to Deblur frame you may choose to process the Full Image or Multiple Frames of the Full Image or a ROI Region Of Interest You may click the Use ROI box to enable it and then click the Full Image button to process that region of interest throughout the entire dataset 2D Deconvolution Results l x Results Channels Save View Iterations 5 of 5 al gt F Display PSF Original Current Adjust Deblur Parameters fi 0 es More Iterations Return to Setup m Use Current Iteration Settings to Deblur Full Image E Use RO M Current PSF Multiple Frames Start fi a End i a To Further Process a Data Set 1 With the 2D Deconvolution Results box open and the Channels tab active enable the Adjust All feature You may click on any one of the scroll bars and scroll back and forth With
53. determine the aperture settings needed to ensure fully confocal behavior This condition will differ among confocal manufactur ers It is well understood that often times this aperture size requirement may not be met Because of signal to noise photobleaching and other considerations the confocal aperture is often opened wider The main point to emphasize is that you should not do so casually and only if it is neces sary The dataset may still deblur fine but this will depend upon your sample and other experi mental conditions and is not extremely predictable Remember that the AutoDeblur system is very robust against noise It is generally more robust against noise than it is against improper con focal settings so if you have a choice between fully confocal behavior and noise for deblurring purposes it is better to sacrifice signal level noise for a proper confocal behavior Remember that AutoDeblur thinks that you have a fully confocal microscope so it will try to reconstruct a fully confocal PSF As you open the pinhole aperture your microscope behavior begins to lean towards widefield behavior and the true PSF begins to have widefield character such as a flaring hour glass like shape This behavior in a sense confuses the AutoDeblur software and it will still attempt to reconstruct a fully confocal PSF so you should avoid this condition if possible If this condition cannot be avoided AutoDeblur still has the robustness
54. digital Minichecker having resolutions of 0 1 micrometers should be used to verify the average step size Heidenhain sells a number of position gauges as well A resolution in the measurement of 0 1 micrometers is sufficient because it is the average of the step sizes that is most critical value One of the common malfunctions that can occur in dataset collection is gear slippage The slippage of the microscope gears or slippage of any linkage between the motor con troller and the microscope can be a critical error in dataset collection A position measurement device will check for this error If a positioning gauge is not available then the next best way of checking the positions is to record the position of the graduations on the fine focus controller Each graduation specifies a certain number of microns A careful recording of these graduation positions will allow you to identify slippage in the motor controller and in the linkage between the motor and the microscope but it will not allow you to determine if there is slippage in the micro scope gears themselves Check with your microscope s manufacturer to determine what type of gear mechanism is used and if this gearing may be prone to slippage Regardless of the design of this gearing any micro scope may have problems since any gearing mechanism may become stripped and easily so if it experiences harsh usage such as in educational environments Some microscope models have ball bearing fric
55. dimen sions Correct XZ ZY Aspect Ratio takes into account the ratio amongst the X Y and Z spacings when displaying the XZ and ZY image projections The difference occurs because of the difference between the camera resolution and the sectioning resolution Note The correct parameters must be entered into the Spacing found by selecting Settings Stan dard Settings from the Deconvolution menu For example a microsphere will look slightly oval in its XZ and ZY views unless the Correct XZ ZY Aspect Ratio is selected 1 Make the XZ view of the SmallHip avz dataset the active view From the View menu select Correct XZ ZY Aspect Ratio This will automatically correct the XZ and ZY ratios Note Please close all views before continuing on to the next section 3D Viewer Note This section is applicable only to users of the Auto Visualize software The 3D Viewer window displays a graphical representation of the dataset The different function alities of the 3D Viewer allow you to create oblique slices orthogonal slices movies apply differ ent color maps change projections adjust subvolume size rotate the dataset and stereo view the dataset in anaglyph mode Refer to page 16 for a list of video cards that support the functionalities used in the 3D Viewer The 3D Viewer menu items are View Projection Rotation Oblique Movie Color Map Save Options and Help March 2004 Page 71 AutoQuant Imaging Inc Manual and Tutor
56. dragging it to the left or right or by clicking either the left or right arrow key The current slice number is displayed to the left of the AUTO button 3 The Slice Viewer also has the ability to cycle through all the slices automatically To enable this feature click the button labeled AUTO to the left of the slider The AutoPlay feature will be engaged and the slices will begin being displayed sequentially 4 Clicking the button now labeled STOP will halt AutoPlay Note Do not close Pollen deb XY Max Projection Please continue on to the next section March 2004 Page 169 AutoQuant Imaging Inc Manual and Tutorials Slice Synchronizer This feature allows you to scroll through the slices of more than one image simultaneously In addition to allowing you to scroll through the slices of multiple images one at a time it also allows you to auto scroll through multiple images simultaneously Make sure that the Slice Viewer of the Pollen deb is still open From the Visualization menu select Slice Synchronizer The Slice Synchronizer dialog box will appear 1 Slice Synchronizer I x Dataset 1 1_Pollen deb 2 14_Pollen deb When this dialog box is first opened the table will contain all of the datasets that are open to an XY view Since it is including all open XY slice viewers they need not all have the same number of slices All datasets will be synchronized to the slice number of the first datase
57. filling in what appears to be empty spaces Decimation Frame Decimate This feature will perform an adaptive reduction of the triangles in the isosurface view In the text box labeled Remove enter the percentage by which to reduce the number of triangles then click the Decimate button In areas of low detail such as flat areas more triangles will be removed whereas in areas of higher detail less triangles will be removed but overall the percentage of tri angles entered in the text box will be removed cda reset button to return the number of triangles to the original amount based on the bin factor Lighting Frame The controls in this group control how light will be projected onto the dataset Display light This feature will display the beam of light which illuminates the dataset The beam can be rotated around the image by left clicking and dragging it to the desired perspective March 2004 Page 99 AutoQuant Imaging Inc Manual and Tutorials Shadow This feature will project a shadow of the dataset as the light is projected onto it X Y Z The X Y and Z controls determine the 3D coordinates of the light beam The ranges are 10 through 10 with 0 being directly above the plane It is best to drag the beam to the general desired position then fine tune its position using the up and down arrows next to the text box on each axis Thresholds Frame Thresholding can be performed on the dataset from this section
58. found in the File menu underneath Exit To open one of these most recent files select the file from the list by clicking on it Please close all views before going on to the next tutorial March 2004 Page 66 AutoQuant Imaging Inc Manual and Tutorials Tutorials for the View Menu ltems For this tutorial you may use your own data type Fluorescence Widefield Laser Scanning Confocal Brightfield Transmitted Light Spinning Disk Scanning Confocal Two Photon Fluorescence or you may follow along with the recommended dataset to use All 3D datasets can be viewed in a Single view All 3D datasets can be Zoomed Enhanced Inverted and its Aspect Ratio changed Choosing different views will display the image from different perspectives Single View This feature allows you to choose the view in which the dataset is presented The dataset can be viewed from three orthogonal perspectives The choices available are XY XZ and ZY where X represents the image width Y the image height and Z is the depth or may be thought of as the optical axis in microscopy applications When a choice is made the single view that is generated will be for the currently active projection For example if a Sum Projection is the active view selecting ZY will generate a ZY Sum Projection of the view XY This is the view of the face of the dataset or the front view of each slice XZ This is an edge like view of the dataset or of one of i
59. from PC1 and return it to PC 2 6 Click Transfer Into Computer to complete the transfer and discard the intermediate imprint files Updating the Dongle If you have purchased additional functionality or an extension to your maintenance or license agreement you will need to update your dongle to reflect the changes in you license 1 Click Start gt Programs gt AutoQuant gt AutoQuant Imaging Suite gt Dongle Update 2 You will be presented with the following screen March 2004 Page 18 AutoQuant Imaging Inc Manual and Tutorials AutoQuant Imaging Dongle Update xj User Registration ID boo Dongle ID 1636758174 End Maintenance Unimied License Termination Unimed Last Use 12 24 02 Dongle Update Cade Update Dongle Close 3 Contact AutoQuant Imaging Inc Provide the User Registration ID and Dongle ID shown in the Dongle Update Utility dialog By Email licenses aqi com By phone 518 276 2138 4 If the change that must be made has been approved you will receive an update code in the form of a long string of letters and numbers Enter this string into the Dongle Update Code text box and click Update Dongle 5 Upon successful update you will receive confirmation and the update will take effect the next time you run your AutoQuant product Starting the AutoQuant Software Windows Systems To start 1 Choose the Start button at the lower left hand corner of your screen an
60. in the dataset Q Why won t my images load into Photoshop A This is because you may have saved the images in a format that is not compatible with Photo shop Save the images as TIFF and when asked by the program if you want to export as multiple files answer Yes March 2004 Page 218 AutoQuant Imaging Inc Manual and Tutorials Appendices Table of Z Spacings Z Step Sizes Table 1 Numerical Step Size for Oil Step Size for Water Step Size for Air Aperture NA n 1 515 n 1 333 n 1 00 0 2 18 83 16 61 12 37 0 3 8 3 7 33 5 43 0 4 4 6 4 08 2 99 0 5 2 95 2 57 1 87 0 6 2 02 1 76 1 25 0 7 1 458 1 256 0 875 0 8 1 094 0 935 0 625 0 9 0 824 0 713 0 443 1 0 0 663 0 552 0 250 1 1 0 528 0 429 Undefined 1 2 0 424 0 330 Undefined 1 3 0 339 0 242 Undefined 1 4 0 267 Undefined Undefined 1 5 0 192 Undefined Undefined DOF Depth of Field AZslice DOF Wavelength A 0 50um A March 2004 Page 219 AutoQuant Imaging Inc Manual and Tutorials Table of Parameters Table 2 File Name Numerical Refrac Modality X Y and Z X Y and Z Spacing Aperture tive Dimensions Pixels Microns Index FitcDapi_crop tif 1 4 1 515 Widefield 90 120 50 0 07 0 07 0 15 FitcDap_crop2d tif 1 4 1 515 Widefield 90 120 1 0 07
61. installing this software on a computer YOU ARE AGREEING TO BE BOUND BY THE TERMS OF THIS AGREEMENT By doing so the user is acknowledging that he she has carefully read and considered all the provisions of this Software License Agreement and agrees they are fair and reasonably required to protect AQI s interests The end user thereby agrees to all of the terms spelled out in this Agreement IF YOU DO NOT AGREE TO THE TERMS OF THIS AGREEMENT RETURN THE UNOPENED MEDIA PACKAGE AND THE ACCOMPANYING DOCUMENTS TO AQI The following information is important and should be treated as valuable property Please save the following information for future use 1 GRANT OF LICENSE Subject to the terms of this Software License Agreement Auto Quant Imaging Inc AQP hereby grants to a single user Licensee non transferable except as expressly provided in Section 4 below and non exclusive license to use and execute the Auto Quant software SOFTWARE on a single computer or facility COMPUTER at any time during the period of validity of this license This software may only run on one computer If you have a multiple user license the SOFTWARE may be run simultaneously by no more persons than the number of users licensed 2 COPYING RESTRICTIONS In order to affect your license rights granted in Section 1 above you may install the SOFTWARE by copying it onto the hard disk drive of one computer You may make a full or partial backup or archival
62. needed to generate a movie You have two options to generate a movie 1 you may use the Quick Movies option under the Movie menu which allows you to generate a movie quickly by spinning the dataset about the X or Y axis by predetermined angles 30 45 60 90 or 180 degrees or 2 you may generate a movie by set ting your own Start Point Mid Point End Point and Step Angles For this tutorial we will start with the Quick Movies option 3 Within the 3D Viewer box click on the Movie menu and select Quick Movies Click on Rotate Y Axis and select the 60 Degree option This will generate a movie that rotates about the Y axis by 60 degrees in the Rock mode To stop the Movie at any time click on the Movie menu again and select Stop Movie To play the movie again you may click on Play Movie from the Movie menu 4 You may play the movie in Loop mode by selecting the Loop mode option under the Movie menu and then choosing Play Movie If you wish at any time to play the movie backwards you may do so by selecting Opposite Path from the Movie menu 5 Restore your original view by first stopping the movie and then selecting Original View from the Quick Movies options To generate a movie by selecting your own Start Point End Point and Step Angle do the follow ing 1 Choose your Start Point by either starting with the original view or by rotating your dataset with your mouse to a position that you want the movie to star
63. of deconvolution are usually most pronounced in the axial direction z axis as illus trated for a confocal image of a rat neuron below au eyma y PEPPER Sit i a Top View b Side View c Top View d Side View Figure 2 Confocal image of a CA3 hippocampal rat neuron Panels a and b show the projections of the dataset from the top and side respectively Panels c and d show the same dataset after deconvo lution The improved axial resolution becomes especially important when accurate morphometry is desired Figure 3 On the left is a rendering of the 3D image of a fluorescent dye filled micropipette imaged by a laser scanning confocal microscope On the right is the same object after deblurring The axis running from left to right corresponds to the microscope axial direction March 2004 Page 28 AutoQuant Imaging Inc Manual and Tutorials b c Figure 4 Showing the improved morphometry made possible by deblurring for the image of a known object fluorescent dye filled micropipette a Cross sec tions through the raw undeblurred dataset the sectioning positions are indicated by the white lines b Cross sections through the segmentation of the dataset at the same slices Panels c and d show cross sections through the deblurred dataset Note the symmetry of the latter result which agrees well with known reality The improved axial resolution due to deblurring is illustrated in Figures
64. plane to remain fixed relative to the screen when the object rotates This means that while the object rotates the oblique plane will be continually updated with the current view of the dataset on that fixed cutting plane Flip Plane The Oblique plane is set to remove one part of the object and it will retain everything below or behind the slice Flip plane reverses the part that is removed The XY XZ and ZY buttons allow you to automatically orient the object to the standard perspec tives of XY XZ or ZY Additionally the Flip button allows you to view the current orientation from the rear of the sample effectively rotating the object 180 degrees about the screen s Y axis XY XZ and ZY This feature slices through the object s XY XZ or ZY plane Parallel This feature orients the Oblique Slice the cutting plane so it is parallel to the 3D Viewer screen Movie Menu This feature allows you to generate a movie of your dataset Play Movie This feature starts a movie in playback Stop Movie This feature stops a movie in playback Quick Movies This feature allows you to generate a movie quickly by spinning the dataset about the X or Y axis using predetermined angles Rotate Y Axis This feature allows you to choose from 30 45 60 90 180 degrees for which the object will rotate about the Y Axis March 2004 Page 77 AutoQuant Imaging Inc Manual and Tutorials Rotate X Axis T
65. plane when the object rotates The oblique plane will be continually updated with the current view of the dataset on that fixed cutting plane while the object rotates Offset from Origin This feature allows you to choose a value to offset the Oblique Slice from the origin The slider moves the oblique slice back and forth along its normal axis The associated number indicates the offset in slices of the current oblique slice s position from the origin Preset Views frame XY XZ ZY The XY XZ and ZY buttons allows you to automatically orient the oblique plane to cut a straight section halfway along the optical axis from each of the standard perspectives of XY XZ or ZY Flip The Flip button allows you to reverse the direction of the cutting plane so that the visible por tion of the object becomes the cut portion and vice versa March 2004 Page 91 AutoQuant Imaging Inc Manual and Tutorials Origin This feature repositions the Oblique Slice to pass through the object s origin Parallel The Parallel button brings the Oblique plane parallel to the view screen Oblique Origin frame The controls in this group determine the object coordinates that serve as the oblique origin The oblique plane and parallel slice are calculated with respect to this origin X Y and Z Slice This feature indicates the X Y Z coordinates of the Oblique Origin Calculate Origin from Manual Input This feature allows you
66. put a black frame on the edge of the image where the aligned data has left a void This is recommended for Fluorescence and Darkfield datasets Random This will place a frame on the edge of the dataset made up of random pixel values where the aligned data has left a void This is recommended for datasets that will be March 2004 Page 124 AutoQuant Imaging Inc Manual and Tutorials deconvolved or for images that have obvious structures at the edges of the frame White This will put a white frame on the edge of the image where the aligned data has left a void This is recommended for Transmitted Light Brightfield datasets Alignment Log File The Alignment Log File allows you to name and save a log file which will detail the changes made to each slice Write Log Selecting this feature will prompt the Image Alignment feature to save a log of the changes made to each slice In the text box enter the name to call the log file Set folder path Click the set folder path button to open a windows browser to select the folder to which the log file will be saved OK Clicking the OK button will initiate the Channel to Channel Alignment feature and will create a new aligned image that will need to be saved Cancel Clicking Cancel will cancel and close the Channel to Channel alignment dialog No aligned image will be created or saved Help Clicking the Help button will open the Online Help file opened to the Channel to Channel Align
67. set of statistics appears in the Track Statistics dialog 4 Click the Export Features button in the Track Statistics dialog Name the file trackingfeatures then click Save This will save the statistics to a csv file Click Close to close the Track Statistics dialog 5 Click the Generate Movie button An abbreviated version of the Time Lapse Movie Gen erator dialog will open For more information about the Time Lapse Movie Generator 6 Leave all of the defaults as they are then click the Generate Movie button The movie may appear behind the 3D Viewer and the Time Lapse Movie Generator dialog try moving these two dialogs if the generated movie does not seem to appear 7 Click the Rock button 4 then click the Play button gt The movie will play show ing the objects moving 8 Click the Cancel button to close the Time Lapse Movie Generator dialog Click the Finish button in the Tracking dialog Click the X in the upper right hand corner of the Untitled O window to close the generated movie On the message that will pop up asking you to save the movie click Yes then name the movie trackingmovie 1 March 2004 Page 212 AutoQuant Imaging Inc Manual and Tutorials Windows Cascade This feature places all the open dataset views in overlapping and offset positions from top to bot tom of the viewing window Tile Horizontally This function stacks horizontally the open datasets in the window Closed or min
68. that it may provide a nice deblurred result but this will be dependent on your sample and other experimental setups and is less predictable than with fully confocal settings Consider the signal to noise requirements explained below Less Than Fully Confocal Conditions If you find it unavoidable to have the aperture setting opened wider than the fully confocal posi tion then we recommend likewise broadening your XY sample spacing by the same amount For March 2004 Page 53 AutoQuant Imaging Inc Manual and Tutorials instance 1f your confocal aperture is broadened to twice the diameter of its fully confocal posi tion then you should broaden your XY sample spacing microns per pixel to twice the above mentioned optimal sample spacing 0 5 micrometers for a 1 2 NA lens or higher Of course other experimental conditions may prevent you from doing this and this may be alright but as with other rules of thumb this is the so called recommended optimal setting Signal To Noise Considerations AutoDeblur will operate with a wide range of signal to noise levels The Expert Settings have a Noise Smoothing feature to handle different signal to noise levels Widefield datasets generally contain low levels of noise therefore the default setting is Low Confocal datasets generally con tain medium levels of noise therefore the default setting is Medium For Signal to Noise Ratio SNR levels of 10 or below a Noise Smoothing set
69. the Adjust All feature engaged all the channel scroll bars will slide simultaneously Notice how the fine details become clearer up to the last iteration of the deconvolution performed March 2004 Page 148 AutoQuant Imaging Inc Manual and Tutorials 2 To view a separate channel s processing disable the other channel s by removing the check mark in their respective boxes For this Lesson disable the Blue and Green Channels 3 Slide the Red Channel s bar back and forth note that the fine details becomes clearer to the last iteration but never reaches a point where the channel s clarity levels off or starts to become blurred This is a good indication that further processing is required 4 Click on the Results tab and click the Further Processing button AutoDeblur will process the dataset for 10 more iterations and display the 2D Deconvolution Results box alongside the processed results Return to the Channels tab and scroll the channels back and forth observing the increase in clarity and observe that around iteration 38 the image s clarity starts to stay the same This indicates that the image does not require further iterative processing 5 Slide the Red Channel s bar back and forth note that the fine details reach a point where an increase in clarity has stopped Leave the slider at that point 6 When you have deconvolved the Region of Interest to the desired level as above you may apply the parameter
70. the sample in place for a flat field we do not recommend doing so with fluorescence Doing so is prone to problems With fluorescence it is much more difficult and prone to errors to judge that the field is completely flat Also it is difficult and often impossible to collect an out of focus flat field picture that has sufficient signal level because the signal level decreases as the sample is taken out of focus This signal level problem may be compensated for by increasing your video gain or by increasing your exposure time doing so is prone to errors and is best avoided To summarize For a flat field brightfield image remove the sample For a fluorescence flat field image use a drop of dye with a coverslip Only if doing so is not possible due to experimental constraints try taking the sample well out of focus until you have a completely flat field Table 1 However be especially careful that there is no out of focus remnant of the sample and make cer tain that for fluorescence your exposure times and gains are set so that you have enough signal level in the flat field image In selecting the camera gain or gain on the frame grabber illumination intensity or neutral den sity filter refer to the discussion given earlier under Setting the Exposure Gain and Offset When using transmitted light brightfield first try the same gain that was used for collecting the optical sections during the axial scan Adjust the gain to ensure
71. the distortion actually present within the dataset rather than when the PSF was measured Q Can I process 2D images and time lapse movies A Yes AutoDeblur contains an iterative blind deconvolution for use on 2D images It is for researchers who do not have a Z motor or who wish to improve resolving power of their standard 2D images or who want to improve resolving power in time lapse movies Q Can I process many images as a batch A AutoDeblur contains functions that permit you to set up a batch of images that will then be sequentially processed After setting up the parameters for your deconvolution you may choose to place the image into a queue that you launch at a later time This is useful for laboratories who do a lot of imaging and for researchers performing time lapsed observations of 3D volumes These laboratories can set up their images to process a batch of images overnight and have them all ready for analysis in the morning Q How can I easily inspect my images before and after deconvolution A Not only does AutoDeblur contain five different types of deconvolution adaptive 3D blind nearest neighbors no neighbors inverse filtering and 2D deconvolution but it also provides many different ways to visualize the raw and processed images AutoDeblur enables a 3D dataset to be visualized using several different projections and orientations Each volume can be viewed from XY XZ and ZY orientations as a maximum minimum
72. the position of your dichroic mirror filter set Next adjust the off set or black level position s on your camera and or frame grabber card If possible to keep mat ters simple adjust only the setting of the camera and leave the rest of the system alone Make this adjustment so that you see a spike in the gray level histogram right at the zero left most value of the abscissa as shown in Figure E Avoid a completely saturated histogram as shown in Figure F This is identified by having only a single spike at the abscissa value of zero and having no spike of finite width 3 Set the gain As mentioned you may have essentially three independent ways to control the gain camera frame grabber and light intensity neutral density filter To keep matters simple try adjusting only one of the gains If fluorescence is being used with an intensified camera we recommend adjusting only the camera gain keeping the excitation light as low as possible If transmitted light brightfield is being used with an ordinary video rate CCD camera such as an RS 170 camera we recommend adjusting only the illumination light level Open the light to the camera Adjust the gain or illumination level setting until the highest gray level abscissa on the histogram which has a non zero value ordinate is at about 75 of the range of the abscissa as shown in Figure D Avoid minimize saturated pixels using the procedures described earlier in this section Marc
73. this field becomes editable and you must enter in the Gaussian Width Otherwise if you select Low Medium or High then the Gaussian Width automatically defaults to a set number This feature will be used to smooth the resultant image Noise Level Select the noise level that best represents your dataset If the Low Medium High options are not sufficient then select Other and then enter in your own Gaussian Width in the Gaussian Width section above Start 3D Deconvolution Note This section applies to users of AutoDeblur Blind Deconvolution is considered to be the most accurate algorithm available with AutoDeblur Blind Deconvolution does not require the calibration and measurement of the Point Spread Func tion PSF Blind Deconvolution can be used with Fluorescence Widefield Laser Scanning Confocal Brightfield Transmitted Light Spinning Disk Scanning Confocal or Two Photon Fluorescence 3D Blind Deconvolution is an iterative and constrained algorithm It is iterative in the sense that it repeats the same computational operations many times while converging to the enhanced image solution It is constrained in the sense that it only accepts deconvolved images that have the cor rect mathematical properties of nonnegativities that is it does not allow the tracer concentration to have negative values and smoothness suppresses snowy like noise due to low light levels 3D Blind Deconvolution is a method of deconvolution that ada
74. to set the object s rotational center manually Whole Volume This feature gives you a direct way to restore the dataset to its full dimensions after 1t has been cropped from a subvolume Subvolume This feature allows you to set the object s rotational center to be the geometric center of the cur rently selected subvolume Center of Mass This feature repositions the oblique slice to pass through the center of mass of the dataset Ortho Slices This feature resets the oblique origin to the last selected orthogonal slice intersection point Object Center This feature resets the oblique origin to the object s center of rotation March 2004 Page 92 AutoQuant Imaging Inc Manual and Tutorials Apply This feature sets the oblique origin to the coordinates presently indicated in the X Y and Z slice edit boxes Equation Ax By Cz D gt 0 Note This is for advanced users This feature contains the edit boxes used to specify an exact oblique plane by specifying its planar equation Movies Tab Quick Movie frame This frame allows you to quickly create a simple rotational movie using a few often used movie parameters Movies created from this group rotate about a single axis from one angular offset to another Rotate current view about This feature allows you to decide whether to constrain the Movie to the Object or the Screen the axis X Y Z which the object will be rotated about and the starting p
75. to your previously set proportions and saved in the same folder as the original dataset The new dataset will be named as follows if the original dataset was named pollen deb then the new dataset will be pollen_operation performed_1 deb March 2004 Page 130 AutoQuant Imaging Inc Manual and Tutorials Rotation Offset The feature selects a rectangular region from an image and applies a cropping based on that region across multiple volumes It also allows the specification of a rotation angle at which to crop Once cropped the volumes are reoriented to the opposite angle at which the crop occurred which causes them to appear level From the PreProcessing menu select Image Macro The Image Macro Functions dialog box will appear xj Dpesstiorni Order Top Ocours Feet a x Crop R ctangda Dimensions Apply To Crop Pesctsngubsr Prism Top Lett Fiort Comer fa ela 2h Lower Flight Beck Correr e foo ae 100 E 55 Rotation Olset E degiess When you choose the Crop operation from the dialog the prompt for cropping appears in the lower left region of the dialog You may then select a region from the image by dragging out a bounding box on top of the image When the region is selected 1 e when the mouse is released the numeric boundaries of the bounding box will be entered into the crop boundary edit boxes in the Image Macro Functions dialog box Once the regio
76. width of the image frame in micrometers by its width in pixels X Spacing is entered in micrometers and ordinarily includes a decimal point An accuracy of 3 or better is required Y Spacing The Y Spacing is the height of one pixel in micrometers Guidelines The Y Spacing can be calculated by dividing the spatial height of the image frame in micrometers by its height in pixels Y Spacing is entered in micrometers and ordinarily includes a decimal point An accuracy of 3 or better is required Z Spacing The Z Spacing is the sampling distance in micrometers between adjacent optical sections March 2004 Page 63 AutoQuant Imaging Inc Manual and Tutorials Guidelines Typically the Z Spacing step size between optical sections is selected by the user collecting the dataset The Z Spacing can be determined by dividing the axial depth of the dataset in micrometers by the number of optical sections collected IMPORTANT It is critical to have the correct Z Spacing step size within a 3 margin of error This should be measured with a position gauge e g Heidenhain or Mitutoyo For this tutorial you may use your own data type Confocal Transmitted Light Brightfield Spinning Disks Confocal or 2 Dimensional images or you may follow along with the Tutorial recommended dataset 1 From the File menu select Open or from the Toolbar click on the Open File icon 2 From the Tutorial Data directory select the Widefield folder and
77. 0 07 0 15 Blue_Green stk 1 4 1 515 Widefield 90 120 50 0 07 0 07 0 15 FitcDapi decon tif 1 4 1 515 Widefield 90 120 50 0 07 0 07 0 15 Malaria_Red tif 1 3 1 518 Widefield 202 243 60 0 1299 0 1299 0 3043 Malaria_Green tif 1 3 1 518 Widefield 202 243 60 0 1299 0 1299 0 3043 Malaria_Blue tif 1 3 1 518 Widefield 202 243 60 0 1299 0 1299 0 3043 Pollen deb 0 7 1 515 Widefield 90 80 110 0 357 0 3636 0 4 Colensc tif 1 4 1 5 Widefield 256 256 1 2D dataset Brain avz MRI 256 256 64 Hip avz CT scan 512 512 90 0 9 0 9 4 8 SmallHip avz CT scan 128 128 120 3 6 3 6 3 6 RotatedHip avz CT scan 128 128 120 3 6 3 6 3 6 Vicera avz CT scan 128 128 120 3 6 3 6 3 6 Skinless avz CT scan 128 128 120 3 6 3 6 3 6 HipMovie avm Movie CT scan 128 128 120 3 6 3 6 3 6 VoxelTLB avm Movie Brightfield 130 130 41 0 279 0 279 1 0 TLB view 1 3 1 515 Brightfield 198 208 64 0 653 0 0653 0 4 Star 0 0 25 1 0 Brightfield 494 348 46 0 6897 0 6897 8 0 Star 1t0 0 25 1 0 Brightfield 494 348 1 0 6897 0 6897 8 0 Star bs1 0 25 1 0 Brightfield 494 348 1 0 6897 0 6897 8 0 Star bs2 0 25 1 0 Brightfield 494 348 1 0 6897 0 6897 8 0 Starfish_crop2 deb 0 25 1 0 Brightfield 128 128 54 0 6897 0 6897 8 0 TobaMC tif 1 4 1 515 Confocal 128 128 32 0 23 0 23 0 4 Neuron deb 0 8 1 515 Confocal 384 512 64 0 94 0 94 1 0 Neuron _crop1 deb 0 8 1 515 Confocal 128 128 64 0 94 0 94 1 0 RedNuc 1 3 1
78. 133 Deconvolution Methods nitro tt 133 Optics Settings arica ld dde tra atras 134 Calctlate Spacin gs 100 A id 134 Deconvolution Settings iii A e 135 Output SEIS nt it 135 Standard Settings Explanations it A adi 135 Total Iterations sici n taa atada 135 The Save Inter Val mrri onen aea Aa aa 136 Performanc oenen aid its ibele 136 Nolse Esveli a AA Aine a 136 Nolse Wallis IA RO il 136 Use Recommended Expert Settings 0 cee ceeceeeeseeeseceeceneeeeeeseeesaecaeceeeeeeeeseeenaees 136 O tp t Sotnas ima ada adi S 136 EXPRESE a A A A RR 137 SUDVOl ME SECO ai 137 Pre processidg SECO iria io ia irc 138 PSP Orta dt a 138 Expert Settings Explanations 0 cece ssceeseceseceeeeeseeeseceaeceseeeeeseaeesaeceaeceeeeseeeeeeeaaees 138 FANALE BOs ia E aiii 138 XY M ntage AA A EAN 139 Le MONA niiina ei n E EE SR A a E s 139 Dynamic SUDVOLUMES oo eee eeceeceeeeeseeesecsseceseeeeesceeesaecsaececseeeseeeeaeesaaeeaeenes 139 Subvolume overlap Pixels ccceccecssccessecneececeeceeececeeeecseeeaeceeaeeeeaeeceeeeenaees 139 XY Guardband Pixels ia 139 March 2004 Page 8 AutoQuant Imaging Inc Manual and Tutorials Z Guardband Pixels iii idiotas caba a 139 Intensity COME A ee a 140 Minimum Intensity Removal oo eee eeeceseceseeseeeeeeescecaececeeeeseeeeseeeaaeeaeeees 140 Accelerated SA Detection estara enn a ca 140 Pre Condition Imported PSF oooooccnnccocccocanoncnononancnnnocononaconnconccnnncnnncnn cora ccn naco
79. 138 How are saturated pixels handled 217 How do I know the restored features are real 217 How many iterations 216 How to Use the Manual 15 Hysteresis 45 Image Algebra 189 Image Alignment Projection 122 Two Views 122 Image Appearance Group 84 Image Aspect Retain Aspect Ratio Stretch Aspect Ra tio 70 Image Info 147 Image Macro 130 Image Macro Operation Order 130 Image Size 113 Imaris 58 Immersion Medium 37 Impalement Sites 31 Import channel Import multi channel data 60 Import multi channel data Create 2D dataset 61 Create 3D dataset 61 Generate datasets 61 Remove 61 Import multi channel dataChange intensities 61 Import Multi channel data Add 60 Import multi channel data Available data sets 60 Change 60 Channel specification 61 Import channel 60 Load channels 60 Select all 60 Select data 60 Important Features of the AutoDeblur System 33 Importing Multi Channel Data 60 improved morphometry 29 Intensified CCD 33 Intensity Profile Along Line box 200 Introduction 27 3D Deconvolution 27 Inverse Filter 152 Deconvolution Menu 153 Inverse Filtering 152 Nearest Neighbors or No Neighbors 152 Invert Data 117 Invert View 104 IPLab 59 Isosurface 74 99 Bin factor 99 Display light 99 Lighting 99 March 2004 Page 233 AutoQuant Imaging Inc Manual and Tutorials Reset 99 Shadow 100 Thresholds 100 Wire frame 99 Iteration s to Save box 151 Iterative Method 33 J Jansson Van Cittert 33 K
80. 2 4 Leave the default names of New 1 for the Label As and the Category fields Click on the Apply button Auto Visualize will apply the New 1 label to the region of interest on slices 17 through 32 5 Click on the Slice Viewer and draw out an ellipsoid region in the lower right hand corner of the image Make sure this region does not touch the previously selected region Again in the Label Object box select Apply to given slice range and set the From slice to 1 and set the to slice to 16 6 Verify that the name of the new label is New 2 for the Label As change if necessary and the Category fields Click on the Apply button Auto Visualize will apply the New 2 label to the region of interest on slices 1 through 16 7 Scroll through all the slices in the Slice Viewer to view the two Labels that were applied Note You may also choose to apply a label to all the slices in the stack by selecting the Apply to all slices button 8 Close the Label Object box Note Do not close the TobaMC tif XY Slice Viewer Please continue on to the next section Apply Seed Fill Label This function allows you to do automatic labelling to a single channel of a dataset as well as a grayscale image 1 Select Green Channel from the View Menu Select Apply Seed Fill Label from the Seg mentation menu under the Analysis menu The Segmentation Parameters box will appear 2 Set the Minimum Threshold Value
81. 3 pollen deb 4 etc Open Movie 1 From the File menu select Open Movie Set the Files of type to avm 2 From the Movies directory select Colrpollenrs avm and click Open Note The avm file extension indicates an Auto Visualize movie header file This header file con tains the necessary information to playback the movie By selecting this file the entire movie will be loaded 3 The first frame of the movie is then displayed Click the Play button gt to play the movie The movie will play once through then automatically stop 4 To stop a movie that is currently playing click the Stop button m 5 Click the Loop Mode button to put the movie playback into Looping Play Click the Play button and the movie will continuously play in a Looping motion until the Stop button is clicked 6 Click the Rock Mode button 52 to put the movie playback into Rocking Play Click the Play button and the movie will continuously play in a Rocking motion until the Stop button is clicked Close This function closes the current active view To close the dataset make the image the active view and select Close from the File menu With the focus on Colrpollenrs avm select Close from the File menu to close the movie March 2004 Page 57 AutoQuant Imaging Inc Manual and Tutorials Launch Imaris If you have Imaris installed on your machine you will have a Launch Imaris option on your file menu as well as an icon E to l
82. 3 and 4 for the case of a known object a dye filled micropipette Deconvolution can often reveal fine details that are not directly observable Figure 5 shows an example of this fact March 2004 Page 29 AutoQuant Imaging Inc Manual and Tutorials Figure 5 Example of improved detail revealed by deconvolution The upper panel shows the top and side projectional views of a portion of a neuron The lower panel shows the same views after deconvolution Notice how the spines of the neuron become observable Application of the AutoDeblur Deconvolution Package The AutoDeblur software package can be used to perform 3D deconvolution in the context of the following forms of microscopy 1 Transmitted Light Brightfield Microscopy TLB 2 Widefield Epi Fluorescence Microscopy WE 3 Confocal Pin Hole Laser Scanned Epi Fluorescence Microscopy CLSM 4 Spinning Disk Scanning Confocal and 5 Two Photon Fluorescence For the confocal fluorescence microscope images of adjacent planes are sequentially digitized and optically sectioned in a manner that is generally similar to the widefield case except that each frame is collected by way of a raster scanned laser spot and a photomultiplier pinhole detector Each confocal optical section is by itself already relatively deblurred in the sense that the confo March 2004 Page 30 AutoQuant Imaging Inc Manual and Tutorials cal optics rejects most of the out of focus light
83. 4 142 153 Remove Import multi channel data 61 Remove Label 196 Render Range Maximum Minimum 86 Rendering 65 84 Reposition Manual Alignment 123 Reset Isosurface 99 Resize Manual Alignment 123 Resize Factor 113 Resize Methods 113 Resizing an Image 112 Resolution 33 Resolution Improvement 33 Results Dialog box 148 Reverse 80 Reverse Document Contrast 117 Different from Invert 117 Rmax Ratiometrics 183 Rmin Ratiometrics 184 Rock Mode 78 95 Rotate 76 89 Rotate Object frame 90 Axis 90 Rotate X Axis 78 Rotate Y Axis 77 Rotated Volume 80 rotating morph 175 Rotation Manual Alignment 123 Rotation Tab 89 Roughness Penalty 137 RS 170 Cameras 40 46 S Sample Depth 37 Nonhomogeneity 32 Refractive index 32 Saturated Image 39 Saturated Pixels 41 saturated portions 216 Saturated Regions 38 Saturation Detection 39 Save All Labels For Backup 198 Save As 58 Save Current 85 Save Current Settings As 66 Save Current View 59 Save menu 80 Save Segmentation Selected Labels as an Image 199 Saving a Processed Data Set 150 Saving Individual Channels As Separate Stacks 58 Scan Actual Axial Distance of 36 Reversing Direction 45 Thickness of the Scan 37 Top and Bottom 36 Screen 76 Search for Help On 214 Segment 190 Select all Import multi channel data 60 Select data Import multi channel data 60 Select Region 108 Selecting a Region Of Interest 146 Separate Touching Objects Object Counting Tracking 206 Set End Point 78 Se
84. 6 Nearest Neighbors Method 33 Neutral Density Excitation Filter 40 New Dimension 112 Noise 33 Noise Reduction 33 Noise Threshold Automatic Alignment 120 Noisy 216 Nonnegative Image Values 33 Number of Columns 69 Number of Rows 69 Number of Slices 37 Numerical Aperture 37 134 142 153 O Object Center 92 March 2004 Page 234 AutoQuant Imaging Inc Manual and Tutorials Object Center frame 88 Object Counting Tracking 202 Automatic Segmentation and Counting 202 Count Objects 206 Distance Tolerance 207 Feature Selection 205 209 Feature Tolerance 208 Object Information 205 Object Selection 205 Separate Touching Objects 206 Tracking 207 Training 204 Wavelet Processing 204 XY Brush Size 203 Object Information Object Counting Tracking 205 Object Selection Object Counting Tracking 205 Object Tab Dynamic SubVolumes 138 XY Montaging 137 Z Montaging 138 Oblique menu 76 Oblique Slice frame 91 Display Oblique Slice 91 Oblique Tab 91 Off 82 Offset 38 40 41 Open 145 Open an Image one optical slice warning 146 Opening an Image 145 Operation Settings Files 65 Operations frame of the Image Macro 130 Opposite Path 79 Optical Density Correction 113 Optical Density Correction Also known as Shutter Lamp Flicker Correction 113 Adverse laboratory condition 116 Arc Lamp Flicker 116 Instabilities in Power Supply 116 Optical Sections 27 Optical Slices 217 Optics Drift 45 46 Dust 45 Misalignments in t
85. 767nm In this way a sample can be viewed as colored with the chosen wavelength Wavelength This feature allows you to generate a color map based on a wavelength in nanometers In this way the sample can be viewed as colored with the chosen dye wavelength Background This feature sets the background color of the 3D Viewer The Top and Bottom selections allow you to create a gradient color for the background by selecting one color for the Top and another color for the Bottom A gradient will be formed between the two colors Alternatively the same color can be chosen for the top and bottom and the background will be a solid color The color choices are White 75 Gray 50 Gray 25 Gray Black Red Green Blue Cyan Yellow Magenta Orange and custom Apply This feature will apply the current color map settings to the 3D Viewer display March 2004 Page 96 AutoQuant Imaging Inc Manual and Tutorials Settings Stereo Tab Pixel Spacing frame X Y and Z Spacing This feature allows you to specify the X Y and Z pixel scale sizes in micrometers Aspect Ratio This feature allows you to specify the dataset s ratio between the Z and X dimensions Apply This button applies the selected settings Auto Calculate Ratio This feature when checked automatically calculates and displays the dataset in the resulting ratio of the Z spacing to the X spacing When unchecked you may modify the visible aspect ratio without
86. 7cm on the video screen The resulting calculations are illustrated above AutoDeblur or Auto Visualize can be used to measure the x y locations of pixels on a screen using a ROI box the width of the box will be displayed on the Status bar of the main window If the number of pixels between graduations along the x and y directions is n and n respectively then March 2004 Page 49 AutoQuant Imaging Inc Manual and Tutorials the number of micrometers per pixel along the x and y directions denoted Ax and Ay respec tively is given by es no Ax micrometers graduation t gt AA pumicrome CEP EY n pixels graduation Ay micrometers graduation Ay micrometers pixel _ _ _ _ _ _ _mue DEEN n pixels graduation If you do not have a stage micrometer available then you can use the following method to approx imate the pixel spacing From the manual for your CCD camera determine the size of the each pixel e g 6 10 microns is typical Divide this number by the lens magnification and the camera zoom you are using This will approximate the pixel spacing in your image For example a 40x lens with a 1 5x camera zoom and 6 micron CCD pixels will give you a pixel spacing for the image of 0 lum pixel The equation is camera pixel size lens magnification camera zoom 6um 40x 1 5x 0 1um pixel Vibration Control To maximize the quality of the dataset acquisition it is recommended tha
87. An Iterative Technique for the Rectification of Observed Distributions The Astronomical Journal 79 6 745 765 Macias Garza F K R Diller A C Bovik S J Aggarwal and J K Aggarwal 1989 Improvement in the Resolution of Three Dimensional Data Sets Collected Using Optical Serial Sectioning Journal of Microscopy 153 2 205 221 Martin L C and B K Johnson 1931 Practical Microscopy Blackie and Son London March 2004 Page 226 AutoQuant Imaging Inc Manual and Tutorials Miller M I and B Roysam Bayesian Image Reconstruction for Emission Tomography Incor porating Good s Roughness Prior on Massively Parallel Processors Proceedings of the National Academy of Sciences Vol 88 No 8 pp 3223 3227 April 1991 Miller M I and D L Snyder 1987 The Role of Likelihood and Entropy in Incomplete Data Problems Applications to Estimating Point Process Intensities and Toeplitz Constrained Covari ances Proceedings of the IEEE 75 892 907 Oppenheim A V and R W Schafer 1975 Digital Signal Processing Prentice Hall Engle wood Cliffs NJ Poenie M 1990 Alteration of intracellular Fura 2 fluorescence by viscosity a simple correc tion Cell Calcium 11 2 3 85 91 Politte D G and D L Snyder 1991 Corrections for Accidental Coincidences and Attenuation in Maximum Likelihood Image Reconstruction for Positron Emission Tomography IEEE Trans actions on Medical Imaging
88. AutoQuant Imaging Inc Manual and Tutorials AUTOQUANT IMAGING INC User Manual Version 9 3 Engineering Excellence by AutoQuant Imaging March 2004 Page 1 AutoQuant Imaging Inc Manual and Tutorials Table of Contents How to Use This Manual ooccnnocnconcnconcnnononnononcnnonnncnno nenas 15 Installation Transferring a License Uninstalling and Hard Ware Requirements ai ctvicccsecieiecsccescmeccnccccdicesesiecccececderctcencesess 16 Sales ION AMOR SIA ida 16 Technical Support 1 A A eee ease 16 Software and Hardware Requirements Windows Systems coocoooccnocccoonononcnonnnonaconnncnnno 16 Software Installation nati 17 Dongle Installation and Requesting a Permanent License ooooonoccnocaconcconancnnncconcnancnnnn cons 17 Installing Version 9 0 and Subsequent Releases ooconoccnnnconocccoccnnncncnoncnonccnnncnancnos 17 Transferring Moving a License 20 i c 4 ccd ii dt is laa 18 Updating th Dongle saio a e last 18 Starting the AutoQuant Software Windows Systems coooocoocococcconcnononononcnoncnnnncona cono ncnnos 19 A PE ie ede eee ee a 19 Uninstalling the SOM Ware asia sita i meds aacie oda ta toes E Leceg aha eee 20 Limitation Of Liability oooocccconnncccnncnncncnnannononanncnnnnannnnnnnas 21 Limitation of Liability Notice tii ia 21 Copyright NOCE ui A A E E ete E RA ES 22 Software License Apreciado dd 22 INTOQUCHON ainia cid cass acenaceescecntsceecescensdiscesetacunaacnannen 27 Wh
89. Cali brated XY Spacings Either tab can be used to calculate the spacings of the image In the Theoret ical XY Spacings tab you will enter your microscope parameters which will then be used to calculate your image spacings In the Calibrated XY Spacings tab you will need to take a calibra tion image measuring the spatial calibration To learn how to do this see page 48 Enter the result March 2004 Page 134 AutoQuant Imaging Inc Manual and Tutorials ant measurements in the Calibrated XY Spacings tab After either tab has been populated click the Transfer Spacings button The spacings will now be transferred into the Image Spacing text boxes Deconvolution Settings The Third Section of the Standard Settings is Deconvolution Settings which prompts you for the deconvolution parameters The Deconvolution Settings section has the following input fields Total Iterations Save Interval Noise Level Noise Value Performance Use Recommended Expert Settings and Go to Expert Settings 8 In the Total Iterations field enter a value of 30 In the Save Interval field enter a value of 15 This will perform 30 iterations of deblurring saving the results through the 15 amp 30 iterations Verify that Low is selected for the Noise Level Based on the Noise Level chosen the Noise Value is automatically populated except when Other is chosen in which case you must enter a value in the Noise Value field Verify that the Use Recommended
90. E EEEE 63 File Type sion aii 63 DIMENSIONS CA Lt E E A E E E E E 63 Spacing X YZ da 63 XM SPACING E E E A EE a 63 Y Spacing a da raaka 63 Z Span D aaa ici 63 N merical Apertures A E 64 Scope Modens csan a eaa ii 64 Channel Wavelens thai 65 Page SetU iia ii 65 E 65 O A thse ee ER eee ees ee ee ace 65 A A a au E pes els 65 A O 65 Load Settings pis ns a de 66 Save Current Sern es AS ia 66 E coax cleave aces a edi tia le ela Ye cp cil hls cons Coie td tte taeda 66 Recent Peles Sa 66 Tutorials for the View Menu Items ooooonnnnnnnnnnnncnnnnnnnonanas 67 e O NT 67 o o a e Be 67 Pd ti dal 67 E O O 67 Triple View ii Ane en oda 68 TR A A ES AO 68 Montage WWA ai 69 ICO VIE E 70 Image Aspect Retain Aspect Ratio Stretch Aspect Ratio ooooocnoccniocanocnnooncnonccannnnnnnons 70 Correct XZ ZY Aspect Ratio cuina ii dende s 71 A scence asda gases Sass a sangha aa atts Gel aca ep thay daa a ate 71 Useful Key amp Mouse Combinations for the 3D Viewer ooooonoccccnoccconocccinnncnnnnnnnnns 73 Explanation of 3D Viewer MEUS A ton 73 VIEW IED la tas aren lcd 73 Volume Projection Software and Hardware ooooonnoccnonccnoncnononcnonnnononocnna conan ccnnnccnnnan ns 73 Orthogonal Slices in tad 74 Cube Surtace A UE d 74 ISOSUTAC AA A E EE AOS RN 74 Adjust Minimum Threshold oooccnncnncnnoconacconcconnnancnanacnoconocnn nono nona crono ro nannncnnno 74 Hersh Map assi asii 74 March 2004 Page 4 AutoQuant Imaging
91. EFAULT IS A BREACH OF A FUNDAMENTAL CONDITION OR TERM OF A FUNDAMENTAL March 2004 Page 24 AutoQuant Imaging Inc Manual and Tutorials BREACH SOME STATES COUNTRIES DO NOT ALLOW THE EXCLUSION OR LIMITA TION OF LIABILITY FOR CONSEQUENTIAL OR INCIDENTAL DAMAGES SO THE ABOVE LIMITATION MAY NOT APPLY TO YOU Information furnished by AQI in the SOFTWARE manual is believed to be accurate and reliable However no responsibility is assumed by AQI for its use nor for any infringements of intellec tual property rights or other rights of third parties that may result from its use No license is granted by implication or otherwise under any patent rights of AQI 9 CHOICE OF LAW This notice will be governed by and interpreted in accordance with the laws of the State of New York in The United States of America 10 US GOVERNMENT RESTRICTED RIGHTS Use duplication or disclosure by the US Government is subject to restrictions as set forth in subparagraph c 1 ii of the Rights in Technical Data and Computer Software clause at DFARS 252 227 7013 or subparagraphs c 1 and 2 of the Commercial Computer Software Restricted Rights at 48 CFR 52 227 19 as appli cable Manufacturer of this SOFTWARE is AutoQuant Imaging Inc 877 25h Street Watervliet NY 12189 Federal ID Number 14 1772880 11 COPYRIGHT NOTICE O Copyright 2003 by AutoQuant Imaging Inc The SOFTWARE package is protected by United States copyright laws and internation
92. First Guess 140 Flat Sheet 140 Fix plane 77 Fix to Screen 91 Flat Field 126 Flat Field Non Uniformity 46 Flat Sheet 140 Flatfield Correction 34 Flatfield Frame 35 46 Flatfield Frames 45 Flatfield Non Uniformity 46 Flatfield Slide 46 Flicker Manual Alignment 122 Flip Horizontal Vertical 105 March 2004 Page 232 AutoQuant Imaging Inc Manual and Tutorials Flip Plane 77 Fluorescence 41 Fluorescent Microspheres 32 Fluoview tif 59 Foster Distance 180 Frame Averaging 43 Frame Grabber Cards 40 Frame Grabbing 38 Frames sec 94 FRET 178 Acceptor Quantum Yield Qa 179 Dye Pair s Foster Distance RO 180 FRET Conversion Factor G 179 Specified Cross Talk 179 Subtract Background 179 FRET Conversion Factor G FRET 179 Further Processing of a Data Set 149 G Gain 38 40 41 Adjustment 40 Gamma 86 Gaussian Smoothing Ratiometrics 185 GENERAL 25 Generate datasets Import multi channel data 61 Generating Different Views and Projections 67 167 Go to Origin 76 Go to Point 78 Go To View 76 GOVERNING LAWS 24 GRANT OF LICENSE 22 Grayscale box 150 Green Channel 106 107 Grynkiewicz Equation for Ion Concentration Ratiometrics 183 Guidelines for Collecting 3 D Image Data 35 H Haze 27 Height Map 74 Help 214 Help Topics 214 Highlight Labels 194 Histogram 39 41 Histogram of Data Intensity 200 Histogram of ROI of Current View 200 histogram stretching intensity filter 104 Hourglass Central Radius
93. From the Edit menu select Change Settings The 3D Blind Non Blind Deconvolution box will appear Change the Save Interval setting to 20 and click Update Batch This will update the settings in the Batch for the PollenPA deb file Change PSF File This feature allows you to change the PSF file used in a Fixed PSF Non blind deconvolution 1 To use this feature select the PollenPA_psf deb file in the Batch file 2 From the Edit menu select Change PSF File An Open box will appear Navigate to the Batch folder and select Pollen_psf deb Click the Open button The file will automatically replace the PollenPA_psf deb Change Operation This feature allows you to change the operation to be performed on the dataset 1 To use this feature select Task 1 in the Batch Processing dialog box 2 From the Edit menu select Change Operation A Change Operation dialog will appear Select the desired operation Help Menu The Help item from the Help menu will open the Online Help system directly to the Batch Pro cess section Cancel This function closes the Batch function without starting the Batch process The cancellation will only apply to the file currently being processed and will not effect the previously processed files or the files yet to be processed Clear List This function allows you to remove all the files in the Batch queue Delete This function allows you to remove the selected file from the Batch queue To use this feature
94. From the File menu select Open or from the Toolbar click on the Open File icon Click on the Widefield folder and open it 2 From the Widefield folder select the colencsc 1 tif dataset and click Open The Files of type field must be set to All Files or TIFF tif tiff March 2004 Page 145 AutoQuant Imaging Inc Manual and Tutorials 3 A dialog box will appear with the statement Dataset only contains one optical slice the orthogonal projection along XZ and YZ will be inactive Click OK File Open xj Data set only contains one optical slice the orthogonal projection along X2 and YZ will be inactive 4 AutoDeblur will now load the colencsc 1 tif dataset and display it in the XY Max Projec tion view Setting Up a 2D Blind Deconvolution Select a Region of Interest 1 Click and hold the left mouse button down while dragging out a Region of Interest ROD similar to the one shown below 9 colencsc 1 tif 100 2 From the Deconvolution menu select 2D Blind Deconvolution The 2D Deconvolution box will appear with the Standard tab as the active tab A 2D Deconvolution Results box which will show the result of the deconvolution will also appear March 2004 Page 146 AutoQuant Imaging Inc Manual and Tutorials 2D Deconvolution x Standard Expert ROI Frames Data to deblur Background e Max Projection C Multiple Frames Black IV Region of Interest C White
95. Inc Manual and Tutorials Projection MENU otitis radares 75 Surface Sice it atea 75 Rotation N a N A a eaen ies 75 TT 75 ODE Ci AA AA AA ee 75 A tt NA 76 BEANS O a 76 DS O TO 76 CONVIENE aaa 76 Rotate 90 A Gy att cote nines oth eoaceaneauseadsdeaa auch ine kek 76 Oblique Menu ninio dit 76 Display Slee i183 e ea taa 76 COLOM ia een as ide Sa eter aad 76 Fix Pline nen a YP Es 71 Blip Planea nia 77 SSI BY Sp COR CE a EE 77 Parallel A R PEATA TEA A TAP EE EO A TEE T ET TE 71 Movie Mentira is 77 Play Movie sicc ece ieee iia bhi ae ich dean iain Aan den n aaa aiae 77 Stop Movie sie heel nena A eee ee ds La needs 11 QUICK MOVIES iii A T A ENAS 77 Rota AMAS datan TT Rotate X AXIS ias tia o a a a 78 Original Vi Wii A Ae hee ed ass 78 Set Start POMO A I I RE 78 Set Mid POM EA ld vs Sees Sat ae ees PIO casa 78 Mid POCA CIVE ai 78 Set EndiPOMt A ad O UA dees 78 Set Step Angle a A AI oe 78 COLO POMAR E AE EA anes at 78 loop Mode ici A ta 78 Rock Mode dada 78 Opposit Path id A A A aS 79 Crate MOVIE uti A EEE CLEA E aS 79 Color Map ME cio do 79 Color aa A a a a A A E A AECA DIE 79 Look Up Table iaa iaa 79 Waveleneth ii A daa 79 Proba A dat cl oh N E T 79 Backsrounid ii AA AA 80 REVISE dardo 80 Save MIU ists Pes OO 80 Current View ts cts teases ea cess eo BE BELO E AAEM eee 80 Rotated Volume tit ii 80 Lo Clipboard tii dias 80 Save Default View A A A ito 80 Reset Default VIEW rrarena an RIEL EEE EPI 80 Save CNS nad
96. Intuitively this may be explained in that the microscope represents a bandlimited system which means that any good light signal will lie only inside the bandlimit Much of the undesirable noise energy lies outside of the bandlimit and the algorithm inherently recognizes this and automatically rejects the out of band noise while prop erly constraining the deblurred image to have only nonnegative values This advantage is espe cially important for confocal microscopy where images often have a large noise component Important Features of AutoDeblur Resolution Improvement and Noise Reduction AutoDeblur improves the resolution of the microscope in the x y and z dimensions Additionally datasets from the microscope system may be especially noisy as is often the case with confocal datasets and with fluorescent widefield datasets using an intensified CCD camera AutoDeblur greatly reduces this noise Statistical Optimality Because AutoDeblur is based on Constrained Maximum Likelihood Esti mation theory it is in this sense statistically optimal Intuitively the algorithm searches for the most likely 3D dye concentration which caused the collected image dataset Correct Modeling Assumptions and Constraints The AutoDeblur design is based on carefully justified mathematical modeling of the optical system including Poisson statistical modeling of the noise component in the detector electronics The Poisson model not only works well with low p
97. Notice the horizontal lines in the dataset 4 From the PreProcessing menu select Optical Density Correction The Optical Density Correction box will appear Note If the image is multi channel the summation data will be provided for each channel in the Summation Data frame of the Optical Density Correction box Optical Density Ca Summation Data Min 386 Max 1087 Sum 6 006813E 09 Avg 759 5914 Order of Polynomial 3 Change Cancel Help The order of the Polynomial is set at 3 This is the default value that can be used to correct for optical density You have the choice to use the default value of 3 by clicking on Continue you may change the order of the polynomial by clicking on Change or you may Cancel March 2004 Page 114 AutoQuant Imaging Inc Manual and Tutorials Note Increasing the order of the Polynomial will increase the correction in the image intensity to a certain point after which possible aberrations will be observed depending on the dataset 5 Click on the Change button The Change Order of Polynomial box will appear ig Change Order of Polynomial x Change Order of Polynomial fi El Select Channel z C Red C Green Blue the average intensity profile before optical density correction the polynomial fitted in the raw averaged intensity profile pare Minimum Summation Y alue 1 237238E 08 Maximum Summation Va
98. Opposite Path option will actually turn the object 330 degrees Create Movie This feature when clicked generates a finalized version of the specified movie and will create a new movie document which may then be saved to an AVI or similar format movie file Display Movie on Desktop Message Checking this box will once the movie is created cause a message to appear alerting you to the fact that the 3D Viewer may need to be moved in order to view the created movie Click OK then move the 3D Viewer if necessary Checking the Do not display this message again box will ensure that this message does not get displayed again Color Maps Tab Current Color Maps frame Display This feature allows you for a multichannel image to select whether or not a channel will be dis played The checkboxes next to each channel label allows you to turn on or off the display of that channel If more than one channel is displayed the option of changing the Color Map may not be used each channel is represented as one of the Color Maps red green or blue Select This feature is active when only one channel is active and it indicates that a new color map may be selected for that channel Color Map This feature displays the current color map for the associated channel The choices are Gray Red Green Blue Cyan Yellow Magenta Orange and Custom Reverse This feature reverses the intensity scale of the selected color map The color map points th
99. Slice Automatic Alignment Note This feature is only available as an added plug in Contact your dealer for purchase infor mation This feature corrects for X amp Y translation and warp nonlinear distortion between frames Mis alignment and warp can be caused by stage motion movement of the sample during live imaging and other causes 1 Open the Unaligned avz file from the Ocular folder in the Tutorial Data directory 2 From the PreProcessing menu select Image Alignment the select Automatic The Image Alignment box will appear Click the Advanced Settings button and then the dialog will appear as it does below Image Alignment Alignment Axis Noise Threshold 20 Iv x Set Threshold Vv Y 0 ae OK Cancel Help Image Warp Base Slice OX Find automatically 1 4 1 I 1 p Eurent slice None Severe Corenusiice r Void Region Fill Base Charmel Pandom C Channel 1 Channel 3 andom v Channel 2 Automatic m Alignment Log File Write Log Align txt set folder path March 2004 Page 118 AutoQuant Imaging Inc Manual and Tutorials Alignment Axis This features allows the alignment to be restricted to the X axis the Y axis or both axes The default setting is Both X and Y Axes X This setting will only align an image horizontally Y This setting will only align an image vertically Note Selecting both X and
100. UE io ease em EREET 159 Slice SClSCt OM evita tadas 159 Image Characteristics onosi Ke ie E lida een les i hee eden oe 159 Shear Angles aia lisis 159 Image NOE ii A A Wa et ae 159 Channel to Decon VO VE aiii belon ini EEE ida 160 Advanced Settings 00 At a 160 Batch Procesal alii ts a ina 160 O 160 A E 161 DO a Ton 161 Edt Mi A a a 161 Copy Optics Set Sari aia 161 Paste Optics Sete oi A ia 161 March 2004 Page 9 AutoQuant Imaging Inc Manual and Tutorials Copy Deconvolution Settings ei ceeceeeeseeeseeeeceeceeeseeeeeaeceaecaeseeeseeceeeeeeeeeeseaaees 161 Paste Deconvolution Settings ccc eeceeessceeseesseceseceeeeeeeeeaeceaecaeeeeeeeeeesaessaeceseaaees 161 Copy PSF Pile Setter Ada ele 162 Paste PSF File Setas iaa EA 162 COPA aiii ia io ceabe dai 162 Paste Al A e e stadt OO OSO 162 Change Settings is AAA A 163 Change PSF Pileta 163 Change Operation cin ieee ln ve eed Aaa 163 Help Menu cti as 163 Can el ts ts cto cea dl ld e led de tr ant 163 Clear t 163 Delete td A A A Le ala Dee 163 O IG ss tee ca ular bunt is ceode ten sls s E T 164 Addins Files tothe Batoh sar 28 dia 164 Sav Max Projection esiisa a yhed e o aiaa it 164 Scheduling a Batch sail nna dad a EA G AN 164 Cancel TIMET ceinen e Eea E E EA A ren nee eres En SS 164 Cancelling Pausing and Resuming a Batch Process oooconoccnnocinocononnnonnnonnncnnncnnno 165 Show Deconvolution PEE Wi AAA 166 Tutorials for Visualization Menu
101. Use only with Confocal datasets Also known as 2D Slice deconvolution this is a form of blind deconvolution that is especially customized for processing a single XZ or ZY slice slices that are parallel to the optical axis It is used for applications where a quick result is necessary and an XZ or ZY cross sectional view is sufficient and the accuracy afforded by the blind deconvolution is also necessary A typical March 2004 Page 158 AutoQuant Imaging Inc Manual and Tutorials processing time is under 8 seconds for a single XZ slice with a 256x256x32 dataset Pentium III 450 Mhz 1 Click on the Neuron_crop1 tif Max projection view to make it the active view 2 Generate the XZ view of the image 3 Generate the XZ Slice Viewer of the dataset 4 From the Deconvolution menu select XZ ZY Slice Deconvolution The XZ ZY Slice Deconvolution box will appear click Start 5 When the XZ ZY Slice Deconvolution has come to completion the deblurred image will appear This image is not a fully three dimensional stack of optical sections It represents the 2D Slice deconvolution of the selected slice in the XZ optical view DIC Restoration Use with Differential Interference Contrast datasets only The DIC Restoration feature converts a DIC image into an image that represents the optical thick ness of the specimen Restoration Method This section allows you to select between an Iterative and an Inverse Filter process t
102. Where n is the refractive index of the lens and NA is the numerical aperture This 1s the formula for the axial width of a confocal PSF For 2 photon microscopy use the excitation wavelength in this formula Assuming a 1 4 NA lens this distance is approximately 800 nanome ters For more substantial deblurring along z experiment with even finer sampling For example 400 or 200 nanometers or finer Keep in mind that the above rules are only rules of thumb for ensuring that deconvolution will show substantial deblurring If on the other hand the experimental objectives do not call for an improved x y resolution but they do call for an improved z resolution then follow the Sam pling Criterion for Z and sample as you otherwise normally would do along x y If the experi mental objectives do not call for an improved z resolution but they do call for an improved x y resolution then follow the Sampling Criterion for X Y and sample as you otherwise normally would do along z The modality setting of AutoDeblur which is found under the menu item Deconvolution Deconvolution Settings Standard Settings on the right side of the Optics Settings Microscope Modality should be set to Two Photon Fluorescence This is because the optical PSF of the typical 2 photon microscope theoretically assuming the detector aperture is wide open is equivalent to that of a confocal PSF except that it is determined by the excitation wavelength rather than the emi
103. Y will align the image on both its horizontal and vertical axes 3 Leave the default setting of Both X and Y axes enabled Advanced Settings Clicking the Advanced Settings will display additional Automatic Image Alignment Settings These settings are already set based on the image information however if you feel that these are not as accurate as you would like them you can fine tune them Image Warp This feature corrects for warp due to motion between frames The more serious the warp the longer the processing time You may slide the pointer to any setting on the scale the far left being no warp the far right being extreme warp 4 Verify that the Image Warp setting is None this is the default Void Region Fill This function will put a frame like cover over the areas that no longer contain data After the data are aligned gaps and spaces are created at the edges This function chooses how those areas are filled in Note The Frame may have straight scalloped or irregular edges to it Black This will put a black frame on the edge of the image where the aligned data has left a void This is recommended for Fluorescence and Darkfield datasets Random This will place a frame on the edge of the dataset made up of random pixel values where the aligned data has left a void This is recommended for datasets that will be March 2004 Page 119 AutoQuant Imaging Inc Manual and Tutorials deconvolved or for images tha
104. _T001_S001_2001_C01 tif Browse First Data File mage_T003_S001_2001_C01 tif Last Data File mage_T003_S001_2010_C01 tif Slices per Time Point 10 Total Time Points 3 Total Slices 30 OK Cancel Help In this dialog box you may choose to import either a single channel grayscale image or a multi channel color image consisting of up to three channels When Multiple is selected in the Channels to Import frame you may choose which channels to enable or disable In the Importing Data frame for each channel enabled you will need to specify a sequence of files to import If multiple channels are to be imported you would first select the radio button for the channel of interest Red Green Blue and then click the Browse button to choose the files If Single or Composite is selected in the Channels to Import group then the checkboxes relating to individual channels will not be displayed Clicking the Browse button will open a standard File Open dialog Browse to the Time Lapse folder in the tutorial folder Select Image_T001_S001_Z001_C0O1 tif The Select Pattern dialog will appear asking for the location of the time sequence number Select the file where comes directly after Image_T March 2004 Page 174 AutoQuant Imaging Inc Manual and Tutorials Once a time lapse dataset has been loaded the Import Time Lapse Data dialog will provide infor mation about the time series that has been loaded Spe
105. a This feature allows you to layer an image over a height map You must first create a sequence containing the slices you wish to use All of the slices must be the same size You may display up to two height maps and two images so the sequence cannot be greater than four images In a four slice sequence slices one and two will be displayed as a height map and slices three and four will be displayed as an image on top of the height map s Height Source This controls which slice s is displayed as the height map Select either Slice 1 only slice 1 will be displayed as a height map Slice 2 only slice 2 will be displayed as a height map or Slices 1 amp 2 both slices will be displayed as a height map Image Source This controls which slice s is displayed as the image on top of the height map s If slice 1 is cho sen for the Height Source then your only options for Image Source will be Height Data which will overlay no image on top of slice 1 or Slice 2 which will overlay the image of slice 2 over the height map of slice 1 If slice 2 or slices 1 and 2 are chosen for the Height Source then you can select Height Data which will use no image on top of either slice Or you can choose Slice 3 which will overlay slice 3 as an image on top of both slice land 2 Lastly you can chose Slices 3 amp 4 which will overlay slice 3 onto slice 1 and slice 4 onto slice 2 March 2004 Page 101 AutoQuant Imaging Inc Manual and Tutorials
106. abled Verify that the Use Recommended Expert Settings is enabled In the Output Settings frame In the Output File Format verify that the setting is on TIFF 16 bit This is the same format as the Input File Verify the Base File Name for Deconvolved Data Click the Save the PSF Files box This will create a PSF file that can be loaded for later use Click the box next to the Spherical Aberrations section then click Detect in the section that will appear AutoDeblur will automatically detect the level of spherical aberration in your dataset This should only take a few minutes 5 Once the spherical aberrations have been detected click Start 6 The 3D Blind Deconvolution will start The results will be displayed in the main viewing window March 2004 Page 143 AutoQuant Imaging Inc Manual and Tutorials 13D Deconvolution FitcDapi_crop tif x Deconvolution Methods Adaptive PSF Blind C Fixed PSF Non Blind a Optics Settings Select Previous Settings y Change m Microscope Settings and Image PSF Dimensions Modality Widefield Fluorescence Lens NA 0 8 Refractive Index 1 ESE Pixels Spacing Image Pixels Spacing Microns Probe FITC Wavelength nm Width 90 0 3 27 Channel 520 Height 120 0 3 36 Channel 2 Depth 150 0 4 60 Channel 3 Load PSF from File Theoretical PSF p Spherical Aberrations 3 Deconvolution Settings Default A
107. ages 1 Deterministic and Maximum Likelihood Reconstructions Applied Optics 29 26 3795 3804 Cooper J A S Bhattacharyya J N Turner and T J Holmes 1993 Three Dimensional Transmitted Light Brightfield Imaging Pragmatic Data Collection and Preprocessing Consider ations MSA Annual Meeting Cincinnati San Francisco Press 51 276 277 Deitch J S K L Smith J W Swann and J N Turner 1991 Ultrastructural Investigation of Neurons Identified and Localized Using the Confocal Scanning Laser Microscope Journal of Electron Microscopy Technique 18 82 90 Dempster A P N M Laird and D B Rubin 1977 Maximum Likelihood from Incomplete Data via the EM Algorithm Journal of the Royal Statistical Society B 39 1 37 Elangovan M Wallrabe H Chen Y Day R Barroso M and Periasamy A 2003 Characteriza tion of one and two photon excitation fluorescence resonance energy transfer microscopy Meth ods 29 58 73 Erhardt A G Zinser D Komitowski and J Bille 1985 Reconstructing 3 D Light Micro scopic Images by Digital Image Processing Applied Optics 24 2 194 200 March 2004 Page 224 AutoQuant Imaging Inc Manual and Tutorials Fay F S W Carrington and K E Fogarty 1989 Three Dimensional Molecular Distribution in Single Cells Analysed using the Digital Imaging Microscope Journal of Microscopy 153 2 133 149 Gerchberg R W and W O Saxton 1974 Super Resol
108. aging Inc Manual and Tutorials Montage View Note This section is applicable only to users of the Auto Visualize software This feature generates a series of Optical Slices and allows you to view them in a Montage frame box In the Montage view you are able to specify the number of rows and columns and select the number of slices you would like to skip 1 e if you select 2 for the number of slices to skip Auto Visualize will display the 1 4 7 10 13 and so forth in the Montage frame 1 Select Open from the File menu Navigate to the Brain folder Select the dataset brain avz and click Open 2 The Image Dimensions dialog box now appears because a header file hasn t been associ ated with this dataset Enter a value of 256 for the Width 256 for the Height and 64 for the Depth Click OK to continue 3 Once the brain dataset is loaded and the default XY Max projection is displayed from the View menu choose Montage View 4 A Multi Slice Dialog box prompts you for the Number of Columns and Number of Rows used in displaying the multiple slices The default size of the viewing window is 5 columns by 3 rows for a total of 15 slices 5 To change the number of views displayed in the Montage frame enter a 6 for the Number of Columns and enter a 4 for the Number of Rows Enter a 1 for the Starting Slice number This will show the first slice of the dataset in the upper left corner of the Montage frame Enter a 1 for
109. ak Show Deconvolution Preview Clicking on the Pause button will pause the batch and the Pause button will change to a Resume button Clicking on the Resume button will then resume the batch Clicking on the Cancel button will launch the following dialog box Cancel Batch Process S 1 Do you wantto cancel all the remaining batch processes or justthe current deconvolution that is running Y Cancel Current Deconvolution Continue Clicking the Cancel Entire Batch button will cancel the entire batch Clicking the Cancel Current Deconvolution will cancel the current job without creating a deconvolved dataset for that job and move to the next Clicking Continue will not cancel any of the jobs and will resume the batch March 2004 Page 165 AutoQuant Imaging Inc Manual and Tutorials Show Deconvolution Preview In the lower left hand corner of the Performing 3D Deconvolution progress dialog there is a checkbox for Show Deconvolution Preview Clicking on this box will display a small preview of the image and you will be able to view the improvement in the image with each iteration The default setting for this is unchecked Please close all datasets before moving on to the next section March 2004 Page 166 AutoQuant Imaging Inc Manual and Tutorials Tutorials for Visualization Menu ltems Maximum Projection This function takes parallel rays perpendicular to the viewing surface and casts
110. al treaty provisions It is illegal to copy the software except as specifically allowed in this Software License Agreement Any person found storing or using an unlicensed copy of this software is lia ble to AQI to the maximum extent permissible under applicable US and International laws and International Treaty Provisions Should you have any questions to this Agreement please contact AutoQuant Imaging Inc 877 25 Street Suite 2008AB Watervliet NY 12189 518 276 2138 12 TERM This Software License Agreement shall remain in effect only for so long as you are in compliance with the terms and conditions of this Agreement This Agreement will termi nate if you fail to comply with any of its terms or conditions You AGREE upon termination to destroy all copies of the SOFTWARE 13 GENERAL This Software License Agreement is the entire agreement between us super seding any other agreement or discussions oral or written and may not be changed except by a signed agreement If any provision of this Software License Agreement is declared by a Court of competent jurisdiction to be invalid illegal or unenforceable such a provision shall be severed from the Software License Agreement and the other provisions shall remain in full force and effect 14 WARRANTY AutoQuant offers a 90 day warranty of the software product For this 90 day period the software may be returned by the end user to the representative for reasonable cause and must
111. ally the AutoQuant software is not guaranteed to work on every 3D microscopy image even if it is of a type that the AutoQuant software is stated to be intended for in this manual Information furnished by AutoQuant Imaging Inc in this manual is believed to be accurate and reliable however AutoQuant Imaging Inc assumes no responsibility for its use nor for any infringements of patents or other rights of third parties which may result from its use No license is granted by implication or otherwise under any patent rights of AutoQuant Imaging Inc AutoQuant Imaging Inc shall not be liable in any event for incidental or consequential damages in connection with or arising out of the use of the furnishing performance or use of this docu mentation the AutoQuant software or any additional software provided for use with the Auto Quant software To the maximum extent permitted by applicable law in no event shall AutoQuant Imaging Inc or its dealers be liable for any damages whatsoever including without limitation damages for loss of business profits business interruption academic prestige academic funding loss of busi ness information or any pecuniary or other loss arising from the use or inability to use this Auto Quant Imaging Inc product even if AutoQuant Imaging Inc has been advised of the possibility of such damages Because some states jurisdictions do not allow the exclusion or limitation of lia bility for conse
112. along the corresponding axis Click the Apply button to make the changes take effect In addition the three checkboxes in this group allow you to display any combination of the planes by checking or unchecking them Click the Apply button to make the changes take effect Manual Input This feature allows you to set the object s rotational center manually using the X Y and Z Slice boxes Whole Volume This feature allows you to set the objects rotational center to be the geometric center of the dataset s full volume This is the default center Subvolume This feature allows you to set the object s rotational center to be the geometric center of the cur rently selected subvolume Center of Mass This feature allows you to determine the coordinates of the dataset s center of mass and then set the object s rotational center to these coordinates Object Center This feature allows you to determine the coordinates of the object s center of mass and then set the object s rotational center to these coordinates Note When in orthogonal slices mode the orthogonal planes may also be selected in the main display by holding CTRL and left clicking and dragging any one of the slices displayed Object Center frame This feature allows you to specify the rotation center of the object being displayed You may spec ify the exact coordinates of the center of rotation using the edit boxes The values may range from 1 to the numbe
113. altering the spacings Correct Aspect Ratio This feature when checked allows the visible dimensions of the object displayed in the 3D Viewer to be adjusted to take into account the pixel spacings in addition to the number of pixels in the X Y and Z dimensions Stereo View frame The controls in this group specify how a stereo view will be created and whether it is toggled on or not Mode This feature allows you to select between the available stereo view modes Anaglyph This feature allows you to split the image into two differently colored components to produce a 3 dimensional effect This is the familiar Red Blue 3D effect though other color pairs may be selected see Filter Only grayscale images can be viewed in anaglyph stereo mode Stereo Goggles Note This option will only be available if you have the hardware necessary to support it This feature allows you to split the image into two differently flickering components to produce a 3 dimensional effect that is visible through special stereo goggles This feature is for systems with OpenGL enable Stereo Viewing capability e g StereoGraphics CrystalEyes LCD glasses Since this method does not require special coloring color images as well as grayscale images may be viewed in this mode March 2004 Page 97 AutoQuant Imaging Inc Manual and Tutorials Filter This feature allows you to select image filters for the stereo view Primarily this allows the
114. ando 81 Load Settings ia Sn ein ea ed ie ed ola de ee eee 81 March 2004 Page 5 AutoQuant Imaging Inc Manual and Tutorials Options Menu unicidad cubedede n sued exsecduncanestad ea e a a nesin 81 Control Panel atacada abide tei gl bade dois 81 Whole Vole micosis ill ind 81 Full Res Vi A cea eae a ae sl Correct ASPEC sinnini a rao aA EA lane dE arabe laa dad 81 AUO ROE ts series ance SEa AEEA o O A O TORA E ANES 81 Display FIOO miii AEA ee ATE AEE 81 Display AXES Troe eea e ai aaaeei iaa 82 Display Grid ii AA AAA 82 Display Scale Brita a AR cae 82 LOBO id A a A cia 82 Perspective VIC Wienerne e a E A E E E 82 Stereo Mode aaa edd eee eee ind ee ia eee a 82 Of et A a ale a ate See aan a ah eerie alah ale 82 Anas YDD usina alla cora 82 LCD Glasses rne lila lisa 82 ARPA COLS Ed 83 Auto Threshold ii a es ata RIA ag Oe haat 83 A se tie Aitet ined eed nll tp Rica dade aloha R 83 Control Peli AA ag a 84 Display Daba Ei a 84 SUDVOLUME Vadis 87 Rotation Tab uti tl tb 89 Oblique Tab A Sa ee 91 Movies Tabs A da 93 Color Maps Tab cirrosi ee a cit 95 Settines Stereo Tab ninia ata iia lic 97 Isosurtace Tabo a aaa 99 Height Map Tab ita 100 Captions Axes Tabs irs siete a ta 102 Help Tabs nese dete cinta a ee ce one ie ele aed 103 Image Enhancement messi shacades siepi erica es leebgaaapuaden van ddeesseesantelens 103 A A moneda aces ies outdid A eaten E 104 Flip Horizontal eat 105 COEM A AAN 105 CAMEO OA
115. ant Value number 4 Select Subtraction from the Operation drop down menu and click Calculate Auto Visualize will subtract the Pollen deb dataset from the PollenPlus5 deb dataset creating a new file March 2004 Page 189 AutoQuant Imaging Inc Manual and Tutorials 5 The resultant dataset Untitled avz may now be saved as PollenPlus4 deb 6 Please close all views before going on to the next section Segmentation Note This section applies to users of Auto Visualize only Segmentation is a method by which a region of interest within a dataset is identified for a subse quent operation such as measurements of surface areas volumes or intensity statistics The Segmentation function allows you to specify a criterion by which certain pixels from your original dataset are selected as a region of interest and represented as a binary file There are two methods of segmentation Automatic and Manual Automatic Segmentation utilizes one of two methods Fixed Threshold or Adaptive Threshold to select specific pixels of the dataset Manual segmentation utilizes labeling techniques to select specific pixels of the dataset Automatic Segmentation 1 From the Multi Channel folder open the TobaMC tif dataset A dialog box will appear with the question The program has detected properties that indicate this dataset MAY contain an old AutoQuant Proprietary Multi channel TIFF Is this true Select Yes 2 From the Anal
116. anual and Tutorials Feature Tolerance This slider sets the threshold for the amount that the selected features can change between datapoints If an object changes more than the set threshold it will be considered a new object and will not be included within that track Auto Partial The Auto Partial button allows the user to select which objects to track Once this button is clicked the Choose Objects to Track dialog will open up Before clicking this button ensure that all objects that need to be counted are being displayed If not in the View Data frame select As Time Point which will allow you to scroll through the time points to a frame that contains the desired objects Choose Objects to Track The Choose Objects to Track dialog is launched from the Auto Partial button In it are the con trols to select which objects to track View Classified This option displays the dataset as a segmented dataset View Original This option displays the dataset in its original non seg mented display Distance Tolerance This slider sets the threshold for the distance an object can move and remain in a track It is based on a percentage of the length of the object s radius Thus if the threshold is set to 300 then anything that moves more than 300 the length of its radius will be overlooked as an object within that track Feature Tolerance This slider sets the threshold for the amount that the selected features
117. ar to one shown above in View 2 If you use the Polygon Select Region tool every time you would like to change the direction of the line you must click within the view to anchor the line at a pivot point From the Segmentation menu select Apply Label Set the Apply to given slice range from 8 through 24 Click Apply 3 Scroll to slice 24 Using the Rectangle Select Region tool draw a Rectangle similar to the one shown above in the bottom right hand corner of View 3 From the Segmentation menu select Apply Label Select the Apply to given slice range and set the slice range from 16 through 32 Click Apply 4 Scroll to slice 16 From the Segmentation menu select Combine Labels The Label Object box will close automatically and the Combine Labels box will appear The Combine field March 2004 Page 195 AutoQuant Imaging Inc Manual and Tutorials will usually contain the lower numbered Label in this case number 1 The With field will contain the second Label number The Category value can be designated by you The default value for Category is 1 Combine With Reo H Cancel Category hoo log 5 Click OK and Auto Visualize will connect the first two overlapping labels 6 Select Combine Labels again and Auto Visualize will now connect the first combined Labels with the third Label the Rectangle The Combine Labels dialog box will have the same number values as displayed above The resulting triple combined Label w
118. arameters tab Within the Parameters tabs you will need to enter the Exposure times for the Average the Flat Field the Bias Field 1 and the Bias Field 2 These numbers should have been recorded and archived when the image stacks were collected The Average exposure time is the exposure time of the CCD camera that was set while the 3D dataset was collected To learn how to obtain these numbers refer to the operator s manual of your camera check the documentation of the software used to collect the image stacks or contact the camera and data collection software manufacturer Fl Slow Scan Cooled CCD Data Correction a xl r Exposure Times Average 0 03 sec Bias Filed 1 feo sec Flat Field 9 03 sec Bias Field 2 120 sec m Bad Pixels Treatment None Median Filter Wiener Regularization OK Cancel Help 8 The Exposure Times in seconds of the fields should be specified as illustrated above 9 The Bad Pixels Treatment setting should have the default value of None March 2004 Page 127 AutoQuant Imaging Inc Manual and Tutorials Bad pixels occur when the dark current of a pixel is so severe that it causes a bright spot in the picture Most image stacks do not require any treatment of bad pixels Therefore the recom mended default selection is None If the selection of None causes the corrected pictures to become black this is an indication that the dataset requires the treatme
119. asacres ia dia east REA 188 CLOSE ni A As 188 1a A REE E 188 Imate Alsebia A a a A SES 189 ESMERO o EET 190 ARO AE OA a aaa 190 E A ss ccdasstecsatadghactoavedentanmebaneadeecetasnseauveases 192 A E woh sald E KEAS oe ESS SaS 192 Apply Seed Fill Label iio a aaa 193 Und aber a E ES UE e ake el E DOS 194 Highlight LAbelS rt soneaeas Seach N EES ESS 194 Combine Labels A a A O A OR Be Rtas 194 Remove Label util tada 196 Combine Label with Automatic Segmentation ooooccnnnnccconoccnoncncnononononacnnnnnncnnnnncos 197 A 198 TO 198 Save Segmentation Selected La eee 199 A TO 199 Line Length ii Rs iia 199 Three Dimensional Line Lengths orion 200 SIBUSTIOS ietsie n R D R E N R E EE AERE 200 Three Dimensional Statistics cccceccceesceeeeceesseeececeececeeeeeceececaeceaeceeaeeeeaeeceteeeeaees 201 S rface Area and A ones n ar E ES EE a ESR 201 March 2004 Page 12 AutoQuant Imaging Inc Manual and Tutorials Object Counting Tracking icini inin eubsdedans coedavaesdandensadausacetens 202 Automatic Segmentation and Counting eooooococcnocnnonnnonnccnnnonanonan ccoo nono ncnnnnnona nono ncnns 202 RI REI 202 Current Volu Me eariad td da A a labo 202 All Volumes no Ai 202 Manta aabt 202 Manual Segmentation occ esesssesseceseceeeesseesseceseceseseneeeaeesaecsaeceeesseeeeeesaaeed 202 Use Recommended Expert Settings 0 cece ceeceeeeseesseceseceseeeeeeeeeesaecaecneeeeeeseeeeaaees 203 Go To Expert Settings scientist A ias 203 Segmentation
120. at is 3 DICO sosi naon ar ea ea uae aca aie ee eee 27 Application of the AutoDeblur Deconvolution Package ooononccnccnnnnnnocononnnoncnonncnnncnnos 30 What is Blind Deconvolution e 32 Important Features of AutoDeblur ciencia iaa 33 Basic Principles Underlying the AutoDeblur System ooooonoccnoncnnonanonnconncnoncnnnncnnn ccoo anno 34 Guidelines for Collecting 3D Image Datasets 35 DVL TSW id cad ya O PO cs dali Sit ap le aie Ves anion eta da lt a dave eed echelon anes 35 March 2004 Page 2 AutoQuant Imaging Inc Manual and Tutorials Optics Alignment amina tias 35 Collecting Optical Sections asses take a e ea dobra bdo 36 Setting the Top and Bottom of the Sample and of the Scan oes eeeeeeeee 36 Setting the Top and Bottom of the Scan iii cee We eee 37 Setting the Exposure Gain and Offset nicotina dis india dnd din ains 38 Cooled CCD Cameras sr ia 38 RS 170 Cameras tl 40 Petforming the Axial Scan Tios 43 Avoiding Backlash and Hysteresis iii wknd aae 45 Collecting Bias and Flatfield Frames Raid 45 Cooled CCD Cameras iia idad 45 RSATO Cameras e loe eds el le 46 Spatial Calibration 1 rose 48 ii A A aa cate 50 Practical and Inexpensive Solutions 20 0 ce cescecsseseeeseecsceeseceseceseeeaeeeaeceaeenseeeeeesaes 51 Collecting Confocal Datasets osn di 51 Sampling Issue O 52 Contocal Aperture soseer ae nr i 53 Less Than Fully Confocal Conditions essseseeeesesreseesrssresrsrrssttsssrresreresrissesresre
121. at nor mally indicate high intensity areas will then indicate low intensity areas and vice versa The Reverse checkbox is only available when only one channel is being displayed March 2004 Page 95 AutoQuant Imaging Inc Manual and Tutorials Monochrome This feature is for multi channel images Checking this box will cause the color image to be dis played as grayscale Activating this option allows a color image to be rendered in a stereo view using split color anaglyphs Select Color Map frame Color This feature allows you to select a color map based on a single color The associated drop down list offers a standard group of colors as well as the choice to select a custom color The Color choices are White Red Green Blue Cyan Yellow Magenta Orange and Custom Look Up Table This feature allows you to select a color map based on multiple colors The associated drop down list offers several maps that vary in color as intensity changes The preset colors or color spec trums are as follows Gray Red Fire Green Fire Blue Fire Black Body Copper Cool Jet and Spectrum Fluorescent Probe This feature allows you to choose a color map based on colors of popular fluorescent dyes Dapi 456nm Cy2 506 Fluorescein 519nm Fite 520nm Lucifer Yellow 528nm GFP 540nm Cy3 570nm DsRed 583nm Rhodamine 590nm Cy3 5 596nm Propidium Iodide 617nm Texas Red 620nm Cy5 670nm Cy5 5 694nm and Cy7
122. at some point in the future you can select a date and time from the Start Time text box The text box will not allow you to chose a time that has already passed for example if it is currently 4 17 03 14 26 it will not allow you to select 4 17 03 14 00 nor will it allow you to chose a date before the current date Once all of the desired jobs have been loaded and the start time has been selected click the Start button Once this is done a countdown will begin beneath the buttons on the bottom of the Batch Process dialog box showing the time remaining until the batch process will begin Cancel Timer Clicking the Cancel Timer button in the Batch Process dialog will cancel the timer It will not delete the jobs that have been loaded into the Batch Process dialog and the timer can be restarted by clicking the Start button March 2004 Page 164 AutoQuant Imaging Inc Manual and Tutorials Cancelling Pausing and Resuming a Batch Process Once a batch process has begun you can pause or cancel the batch While the batch is running the following dialog will appear Performing 3D Deconvolution Processing Channel 1 of 1 Iteration J of 10 Processing the sub yolume 1 of 1 1 minute remaining for the current channel 4 ccelerating Object Estimate Batch Job N ame Batch Processing Batch Job 1 of 1 Batch Log File C Program Files 4 utoG want ukol uant imagina Suite 9 15 Tutonal Osta Confocal B atch t
123. ataset To Clipboard This feature allows you to copy the 3D Viewer screen to a clipboard which can then be pasted into an image editing application such as Photoshop Save Default View This feature allows you to save the settings for the 3D Viewer so that whenever the 3D Viewer is opened it will open in whatever view it is saved in with whatever options are turned on and off Reset Default View This feature will reset the default view the view in which the 3D Viewer opens to the original software defaults March 2004 Page 80 AutoQuant Imaging Inc Manual and Tutorials Save Settings This feature allows you to save the settings that you have created and subsequently apply them to different datasets later Load Settings This feature allows you to load previously saved settings and apply them to the current dataset Options Menu This feature allows you to access general options that apply to all of the functionalities of the 3D Viewer Control Panel This feature opens up the Control Panel See Control Panel below for further details Whole Volume This feature gives you a direct way to restore the dataset to its full dimensions after it has been cropped from a subvolume Full Res View This feature allows the 3D Viewer to generate a full resolution view of the dataset This may take a few seconds to generate and only works for the Volume Projection view If you rotate the dataset the rendering return
124. atasets are supported Misalignment and warp can be caused by stage motion movement of the sample during live imaging and other March 2004 Page 121 AutoQuant Imaging Inc Manual and Tutorials causes Upon opening the Manual Alignment dialogue two images will open The image on the left will be slice 1 and the image on the right will be slice 2 Two Views The Two Views icon er which is depressed by default places two adjacent frames side by side in the Image Alignment workspace The image on the right is always the slice following the slice on the left e g if the image on the left is slice 5 the image on the right will be slice 6 In the Two Views mode the image on the right will be the one that is adjusted Projection To the right of the Two Views icon is the projection selection drop down menu This menu con tains four options Max Displays the Max projection of the images Min Displays the Min projection of the images Avg Displays the Average of the intensities of the images Sub Displays the difference between the images Combine The Combine icon when depressed will overlay the object image the image on the right on top of the base image the image on the left Flicker The Flicker icon when depressed will alternate the image on the left between the base image and the object image e g if the image on the left is on slice 1 pressing the Flicker icon will cause the image to altern
125. ate between slice and slice 2 When the Flicker icon is depressed it will change to a Stop icon MW The image will continue to alternate until the Stop icon is depressed Zoom Step The Zoom Step drop down menu located to the right of the Flicker icon determines the percentage by which the image is magnified when selected to do so 10 will increase the image by 10 each time you magnify the image Zoom The Zoom icon a allows you to magnify the images by a determined amount To zoom in on an image select the step size from the Zoom Step drop down menu Next click the Zoom icon and finally click the image you desire to zoom in on March 2004 Page 122 AutoQuant Imaging Inc Manual and Tutorials Controls The image controls located beneath the images will manipulate the object image This section will assume that you are operating in the Two Views mode Resize The Resize controls allow you to resize the image This is used to correct for stretching contract ing of the specimen Checking the Keep Aspect checkbox will adjust both the height and width simultaneously maintaining proportion Unchecking the Keep Aspect box will allow you to adjust the height and width independently Rotation The Rotation control allows you to rotate the image to correct for movement during acquisition To use the rotation control first locate the crosshairs on the object image Place the cursor over the crosshairs until the cursor becomes a
126. ates your candid comments regarding its products We welcome Information about your applications and your successes using this software Copies reprints of any publications reporting work performed using this software Comments regarding the usefulness and usability of the software Suggestions for improving this product Reports of any software bugs or problems that you may have encountered We will make every effort to address your comments and feedback in future product updates AutoQuant Imaging Inc 877 25 Street Watervliet New York 12189 U S A Phone 1 518 276 2138 Fax 1 518 276 3069 E mail sales aqi com support aqi com Web http www aqi com Copyright 1994 2003 AutoQuant Imaging Inc All Rights Reserved Windows Windows 95 Windows 98 and Windows 2000 are registered trademarks of Microsoft Corporation Pentium is a trademark of Intel Corporation Silicon Graphics and IRIX are trademarks of Silicon Graphics Inc R4600 is a trademark of MIPS March 2004 Page 229 AutoQuant Imaging Inc Manual and Tutorials Numerics 12 bit data 59 12 bit swapped for UNIX 59 16 bit data 59 16 bit swapped 59 1D Deblur XY YZ Deconvolution 158 2D Deconvolution 145 2D Deconvolution Box 146 2D Deconvolution Results box 147 2D images 215 2D Slice Deconvolution 158 XZ Slice 158 2 Photon Data 55 Sampling Criterion 55 32 bit float 59 3 D Data Collection Avo
127. atically calculated and displayed Note Do not close Pollen deb XY Max Projection Please continue on to the next section Sum Projection This function takes all the voxel values along each parallel ray perpendicular to the viewing sur face and sums their intensity values This creates a projection of all the summed values Sum Pro jections provide volumetric representations of the dataset in which more information from the dataset is considered than with the Maximum projection Background noise will also be included in this type of projection March 2004 Page 167 AutoQuant Imaging Inc Manual and Tutorials 1 To generate a Sum Projection for the Pollen deb dataset either click on the Sum Projec tion icon or select Sum Projection from the Visualization menu Note Do not close Pollen deb XY Max Projection Please continue on to the next section Voxel Gradient Shading This function finds the first voxel that is above the intensity threshold for each parallel ray per pendicular to the viewing surface then computes the dot product of the gradient at this point These dot product values make up the pixel intensity values in the 2 D projection This function is ideal for examining the surfaces of objects Click on the XY Max projection view of the Pollen deb dataset to make it the active view 1 From the Visualization menu select Voxel Gradient Shading Note The Voxel projection creates a shaded isosurface for a volume
128. aunch Imaris If you do not have Imaris installed on your machine you will not have these options If you have these options load a dataset into the AutoDeblur AutoVisualize workspace that you want to load into Imaris Either click the Launch Imaris icon or select Launch Imaris from the File menu You will be prompted to save the dataset as an ids file Once the dataset is saved as an ids file Imaris will launch and the dataset will be opened in the Imaris workspace If you select not to save the dataset as an ids file then you will be returned to the AutoDeblur Auto Visualize workspace and Imaris will not be launched Save As This function saves an image or sequence of images as a specified file type If the FitcDapi_crop tif file is not still open from the Open tutorial open it now 1 Select Save As from the File menu and type BlueGreen as the File name Click on the Save as type drop down arrow and select STK FILE stk 2 Click the Save button This will save the file in the stk format Note Do not close BlueGreen stk Please continue on to the next section Guidelines for naming your file s Note AutoDeblur and Auto Visualize follow the same naming conventions as standard Windows applications To move an AutoDeblur or Auto Visualize file into and out of a UNIX computer follow the file naming conventions of the UNIX operating system For example using an underscore for file names greater than 8 characters and
129. b is turned It is important to have a reliable axial scanning mechanism with some way of verifying the step sizes The accuracy of the average step size is critical This should be within 5 of the specified step size The accuracy of individual step sizes is not nearly as critical As a rule of thumb these ought to be within 20 of the specified step size but their average value to emphasize the point needs to be within 5 For example suppose that you specify that the step sizes are 0 2 microme ters and that the scan will be 50 planes deep Then by using a position gauge on the stage or by monitoring the focus knob graduations you should record and verify that the stage moved within 5 of the 10 micrometers between 9 5 and 10 5 micrometers With a high resolution gauge hav ing a resolution of 0 01 micrometers you may be able to verify the individual step sizes However with lesser expensive gauges having a resolution of 0 1 micrometers 1t will be sufficient to verify the larger step sizes of 1 micrometer or more By specifying step sizes of 1 micrometer and higher verify that these steps are indeed within 20 of the 1 micrometer within 0 8 and 1 2 micrometers However make sure that the deviation from this step size is randomly dispersed about the 1 micrometers so that the average step size is still within 5 of the 1 micrometers A position measurement device such as a Mitutoyo Aurora Illinois USA 708 978 5385 MU Checker and
130. be called Track 2 and Track 4 will have no objects associated with it Undo The Undo button will undo the last action that was performed This button only goes back one action and becomes inactive once it is used until another action is per formed i e cannot continually step backward through multiple actions View Data The View Data frame contains two options for how to view the data in the 3D Viewer As Time Point This displays the data as individual time points with only one displaying at a time In this mode it is possible to scroll through the time points by using the controls at the bottom of the 3D Viewer As Time Series This displays all of the time points at once overlaid on one another In this mode there is no ability to scroll through each time point as they are all already displayed View Track Lines Checking the View Track Lines checkbox will display lines which follow the paths of each object The color of these lines will match the object s color which they are tracking Feature Selection In the Feature Selection frame the pertinent features those that differentiate an object class are selected and calculated Select All This button selects all of the available features Deselect All This button will deselect all selected features Calculate Statistics This button will calculate statistics for each of the chosen features and open the Track Statistics dialog Track Statistics Thi
131. bor selections will have values preset for you The default value for the Haze Removal factor is 0 97 and for the Z Kernel Width it is 3 The No Neighbor algorithm and Phase Content Expected are both disabled by default Note The Phase Content Expected should only be checked if the specimen is a Transmitted Light Brightfield dataset and exhibits significant phase characteristics e g areas of the specimen appear brighter than the background For this dataset leave the Phase Content Expected box unchecked 3 Click the Apply button The Nearest Neighbor deconvolution will be applied to the current slice and will be displayed in the current Slice Viewer To return to the original view of the slice click the Restore button To use the No Neighbor algorithm instead of the Nearest Neighbor algorithm check the No Neigh bor box on the Nearest No Neighbor Slice Operation box 4 Adjust the results by trying different values for the Haze Removal Factor and the Z Kernel Width also try the No Neighbor deconvolution by clicking in the flag box to activate and deacti vate it 5 Set the Haze Removal Factor to 0 98 the Z Kernel Width to 2 and leave the No Neighbor check box unchecked These values produce satisfactory results for the Nearest Neighbor or No Neighbor algorithm Click the Restore button to restore the optical section to its original view 6 Click the Close button to exit from the Nearest No Neighbor Slice Operation box The algori
132. by the above equation Doing so lessons the amount of deblurring that is possible along the axial dimen sion but there should still be substantial deblurring that is noticed For example it is common to process datasets that have Ad equal to 2 or 4 times that indicated by the above equation and sub stantial deblurring and noise reduction is still usually obtained albeit generally to a lesser degree Making Ad smaller than as indicated by the above Equation is the most critical deviation from the rule Generally it is best to avoid bending this rule even though it has mixed advantages and disadvantages Making the in plane sampling finer than the Rayleigh resolution limit will improve the in plane deblurring For confocal datasets however this desired improvement may come at the cost of degrading the axial resolution depending upon how well the other rules of thumb are followed as explained below In principle so long as the confocal pinhole aperture is stopped down to a point that the microscope is considered to be fully confocal then there should not be any problem with making this in plane sampling finer In fact doing so will ordinarily improve the in plane deblurring as explained On the other hand it is very often the case arguably most of the time that the confocalist does not have his her pinhole aperture stopped down to the fully confocal position because he she usually wants to detect more light to improve sensitivity Therefore
133. can change between datapoints If an object changes more than the set threshold it will be considered a new object and will not be included within that track Left click on the objects to be tracked Once all objects are selected click Track Manual Modify Clicking the Manual Modify button opens the Manual Track dialog wherein tracks can be added deleted and modified Manual Track The Manual Track dialog contains the controls to create delete and modify tracks If an object is erroneously included within a track it can be deleted or switched with a different object here Additionally in some instances multiple tracks can be merged into one track Track Menu On the right side of the Manual Track dialog is the Track Menu It contains a listing of all of the tracks in the dataset As tracks are added and deleted this menu will be updated Clicking on a track within the Track Menu will make the tracking line for that track become thicker in the 3D Viewer Additionally to rename a track click on it once to highlight it then click again and the name will become editable do not double click quickly After changing the track name click lt Enter gt on the keyboard Add Track This button will add a track to the Track Menu No objects will be assigned to that track until objects are added to that track manually This is done by highlight ing the track to add objects to in the Track Menu then clicking on the object to add to the track
134. cifically it will contain the following infor mation Slices per Time Point the number of optical slices in each volume that makes up the time series Total Time Points the number of time points that comprise the time series Channels Loaded which channels of R G B have been imported If a single channel set was loaded the channel selection will not be displayed Movie Parameters Choose Frame Range You may choose a range of time points to include in the time lapse movie using the Choose Frame Range frame in the Movie Parameters section Here you can choose the first time point Start Frame to include the last time point End Frame to include and the time step interval Include Every to use e g include every 3 frame in the time lapse movie Images that fall outside of the range will be ignored during the movie preview and generation The time step interval will be hidden until you select the Frame Range option Additionally when Frame Range is selected you can also set the save interval in the Include every x Frame s section Selecting 2 will save every second frame selecting 3 will save every third frame and so on Rotating Morph You may select an option for your movie to simply rotate through the chosen time point select option None to move through each time point while rotating select option Full Rotation Movie or to rotate through one time point at a time completing the prescribed rotation for each
135. click Open Select the dataset FitcDapi_crop tif and click Open 3 A dialog box will appear with the question The program has detected properties that indicate this dataset MAY contain an old AutoQuant Proprietary Multi channel TIFF Is this true sin Select Yes 4 From the File menu select Data Properties The Data Properties box will appear 5 In the Dimension frame verify the Width Height and Depth of the image in pixels Width 90 Height 120 Depth 49 6 In the Spacing X Y Z frame verify The X Spacing value is 0 07 The Y Spacing value is 0 07 The Z Spacing value is 0 15 If the spacings do not match those above select Deconvolution Settings gt Standard Settings from the Deconvolution menu to edit the spacings 7 Click OK to close the Data Properties box Note Do not close FitcDapi_crop tif Please continue on to the next section Numerical Aperture This lists the numerical aperture which was used to capture the dataset Scope Mode This lists the type of microscope used to capture the dataset March 2004 Page 64 AutoQuant Imaging Inc Manual and Tutorials Channel Wavelength Depending on the dataset there can be from one to three Channel Wavelengths listed This lists the wavelength of the channel present in the dataset Page Setup This function allows you to control the appearance of an image on the printed page You may change the placement of the image on the printed page by
136. cope having a fine depth of field DOF of about 0 4 micrometers onto a plane in a microscope sample which may be anywhere from a few microme ters e g chromosomes or epithelial cells to on the order of 50 to 200 micrometers e g neurons or brain slices The acquired image will contain both sharp in focus features originating from the plane of focus as well as hazy blurry features originating from out of focus planes that are above and below the plane of focus After capturing this image electronically we can refocus the micro scope to an adjacent plane that is on the order of a depth of field DOF away and acquire a sec ond image This process can be repeated until the entire specimen has been scanned The resulting dataset will be a 3D dataset or in other words a 3D image However it will be a rather poor image because it will contain all of the out of focus haze and blur The purpose of deconvolution is to remove the haze and blur and to restore sharpness and clarity to the image as illustrated for a neuron image below Figure 1 Illustrating the removal of background haze by deconvolution The image on the left is a 2 D projection of a 3D transmitted light brightfield image of a neuron sample The picture is shown with the gray levels inverted for clarity The image on the right is the result of deconvolving the image on the left March 2004 Page 27 AutoQuant Imaging Inc Manual and Tutorials The effects
137. criteria set forth in Steps 5 8 14 In the Object Counting Results dialog box click the Export Features button A Save As dialog will open Ensure that it is set to the Object Counting folder then type objectscounted into the File Name text box then click Save Click Close in the Object Counting Results dialog to close it Leave all datasets and dialog boxes open Tutorial for Object Tracking Note This tutorial assumes that the Tutorial for Object Counting has already been completed 1 In the Object Counting dialog box click on the To Tracking gt gt button This will open the Tracking dialog box 2 Click on the Auto All button This will track all of the counted objects through all of the time points Note the colored dots and lines which match but are darker than the color that was assigned in the Counting feature The dot denotes the center of the object at each time point and the line connects each time point for each object 3 Select all of the Tracking Features Displacement Velocity and March 2004 Page 211 AutoQuant Imaging Inc Manual and Tutorials Acceleration Click the Calculate Statistics button The Track Statistics dialog will open con taining all of the statistics about each of the counted objects In the Tracking dialog there is a text box that lists all of the objects tracked as Track 1 Track 2 etc Click on Track 1 notice that only one object in the 3D Viewer is colored and only one
138. crosshair Click and drag the crosshair to the location you wish use as the axis about which to rotate the image Once the crosshairs have been placed either click the arrows on either side of the rotation control the left arrow will rotate the image counter clockwise the right arrow will rotate the image clockwise or click on the slider and move it back and forth Rotate the object image until it is lined up Reposition The Reposition controls allow you to reposition the dataset to correct for movement or vibrations during acquisition Depress the Combine icon to overlay the two image Reposition the object image using the Reposition controls and observe the overlaid image on the left as the object image moves over the base image Slice Control The Slice Control adjust the slice which you are adjusting Clicking the right arrow key on the slider control will scroll to the next slice if you were working on slice 4 it will move to slice 5 Slice Status Box The Slice Status Box will indicate whether the displayed slice has been modified If the slice has been modified the word Unmodified will appear whereas if the slice has been modified the word Modified will appear Apply The Apply button when depressed will apply the changes to the selected slice then move to the next slice automatically The Apply button will be disabled until changes have been made to the slice Once the Apply button has been depressed the changes made to that slic
139. ct Object Classes frame will be disabled Perform Training Checking this button will activate the Add Define Redefine Classes and the Load Classes buttons so that object classes can be created or loaded Current Classes This box displays the classes which have already been defined As classes are added they will appear in this box March 2004 Page 204 AutoQuant Imaging Inc Manual and Tutorials Add Classes Define Classes Redefine Classes Clicking this button will open the Object Classes for Counting dialog in which the classes to be counted can be created and defined The name of this button will change based on whether the Perform Training option is selected and whether there are already classes defined Object Classes for Counting This dialog contains the controls to select objects and create a training set for use in counting all objects in a dataset Object Information In this frame enter the name of each object class to be created into the textbox Once the name is entered into the textbox click Define Object Up to eight separate objects can be defined Once the Object Selection process has begun described below it is not possible to move back to this step without undoing all steps in the Object Selection step so make sure that all desired object classes have been defined before moving on Object Selection In this frame as object classes are defined they appear in the menu within this frame To select objects t
140. cted dataset loaded If the 3D Viewer is open then it will simply be updated to display the cur rent volume selection The number of the frame being displayed in the 3D Viewer will be dis played in the box labeled Selected Frame Normalize Frame Intensities When selected this will normalize the intensities across all frames of the time lapse movie with respect to the global maximum and minimum values across all frames When unchecked the intensities will remain as captured from the 3D Viewer Generating the Time Lapse Movie When you are satisfied with the selections made for the Movie Parameters you can either click the Preview button or the Generate button If you click the Preview button then each frame that is to be included in the movie will be displayed in the 3D Viewer one after the other appearing the same as it would in the finalized movie However the frames that are shown will not be stored to disk When the preview finishes running the movie the view will be returned to the first frame of the movie which is not necessarily the view you had loaded at the time you clicked the Preview Movie button Clicking the Generate Movie button has the same effect as clicking the Preview button but will also store the movie to disk as it is generated When all frames have been generated and stored a new Untitled movie view that displays the time lapse movie that has been generated will be displayed As with existing movie views
141. ction select Iterative 4 In the DIC Image Settings select All Slices the current image is only slice 5 Still in the DIC Image Settings section select 135 from the Shear Angle drop down menu Once the angle is selected a sample will appear to the right of the drop down menu the light pat tern in this image should match that in the dataset 6 Select Low for Image Noise 7 Click Advanced to view the advanced features Verify that Total Iterations is set to 15 Verify that the Regularization Iterations and Regularization Contribution are both set to 10 Make sure all of the Processing Options are checked 8 Click OK Batch Process Under the Deconvolution menu click on Batch Process The Batch Processing box will appear Start This function starts the deconvolution process for the batch files in the queue Once the Batch pro cess has started you may cancel the processing of one or more of the files by clicking the Cancel button on the progress box This cancellation will only apply to the file currently being processed and will not effect the previously processed files or the files yet to be processed Note This menu item will NOT become available unless there are image stacks in the queue March 2004 Page 160 AutoQuant Imaging Inc Manual and Tutorials File Menu Open This option in the Batch Process dialog allows you to navigate to a folder and select the files you would like to add to the Batch Sel
142. ctuations caused by the statistical nature of quantum noise In the case of confocal fluorescence microscopy it is the purpose of deblurring algorithms to reduce these three undesirable effects and to thereby improve the utility of the microscope Deblurring is best carried out when the dataset is seemingly oversampled In other words best results are achieved when the pixel or optical section spacing is finer than what would be used normally without deblurring In plane deblurring is most successful when the sample spacing is finer than 1 2 the Rayleigh width If in plane deblurring is desired we will sample at 0 1 um or finer when the 1 2 Rayleigh width is 0 25 um Axial deblurring is substantial with widefield optics regardless of the z sampling The rule of thumb for confocal datasets without deblurring is to have the optical sections spaced according to the confocal spot size along z which is the reso lution element along the z direction calculated by the following equation Webb et al 1995 dE 1 44n NA where A is the wavelength in microns um y is the refractive index of the immersion medium and NA is the numerical aperture of the objective lens If Az is 0 5 um we will sample at 0 5 um or finer per slice The noise suppression mentioned above requires this type of fine sampling March 2004 Page 31 AutoQuant Imaging Inc Manual and Tutorials What is Blind Deconvolution Blind deconvolution is a term that is
143. d not have an XZ slice viewer open it would be automatically opened March 2004 Page 170 AutoQuant Imaging Inc Manual and Tutorials However if a dataset that appeared in the previous view has a slice viewer open but minimized in the new view then it will not be included in the new view s synchronization table the assumption being that it was minimized for a reason Continuing from the last example if Image_T001 did have an XZ slice viewer open but it was minimized then it will be left in its minimized state and will not be included in the updated synchronization table Scrolling through Slices The large scrollbar at the bottom of the Slice Synchronizer dialog box allows you to move through the slices in a manner similar to that of a single optical slice viewer i e the arrows at either end move one slice at a time and multiple slices can be jumped at a time by moving the slider about Additionally the text box to the left of the scroll bar indicates the global position of the slider effectively this is the position within the slice viewer that has the greatest number of slices If the Auto button is clicked the views automatically scroll through their slices Adding Datasets Datasets will be added to the synchronization table as they are opened in the workspace When a slice viewer oriented to the selected view is opened e g if the XZ view is selected and a new XZ slice viewer is opened th
144. d Deconvolution Chapter 24 in The Handbook of Biological Confocal Microscopy 2nd Edition James Pawley Editor Plenum Press New York 1995 Holmes T J 1989 Expectation Maximization Restoration of Band Limited Truncated Point Process Intensities with Application in Microscopy Journal of the Optical Society of America A 6 7 1006 1014 Holmes T J 1992 Blind Deconvolution of Quantum Limited Incoherent Imagery Journal of the Optical Society of America A 9 7 1052 1061 Holmes T J and Y H Liu 1989 Richardson Lucy Maximum Likelihood Image Restoration for Fluorescence Microscopy Further Testing Applied Optics 28 22 4930 4938 Holmes T J and Y H Liu 1991 Acceleration of Maximum Likelihood Image Restoration for Fluorescence Microscopy and Other Noncoherent Imagery Journal of the Optical Society of America A 8 6 893 907 March 2004 Page 225 AutoQuant Imaging Inc Manual and Tutorials Holmes T J Y H Liu D Khosla and D A Agard 1991 Increased Depth of Field and Ste reo Pairs of Fluorescence Micrographs Via Inverse Filtering and Maximum Likelihood Estima tion Journal of Microscopy 164 3 217 237 Janesick J R T Elliott and S Collins 1987 Scientific Charge Coupled Devices Optical Engineering 26 8 692 714 Joshi S and M I Miller 1993 Maximum a Posteriori Estimation with Good s Roughness for Three Dimensional Optical Sectioning Microsc
145. d select Programs 2 In the AutoQuant Folder select the name of the application either AutoDeblur or Auto Visualize AutoUpdate AutoQuant s products incorporate an AutoUpdate feature which will once a month connect to the internet to search for product updates automatically If you wish to search for an update without waiting for the default time to pass you can select Search for Update from the Help menu See the Help chapter for more information on the AutoUpdate feature March 2004 Page 19 AutoQuant Imaging Inc Manual and Tutorials Uninstalling the Software Go to Add Remove Programs under the Control Panel of your Operating System The Add Remove Programs box will list all currently installed programs Find and click on the name of the program you wish to remove Once the name of the program you want to remove is highlighted click on the Change Remove button The InstallShield Wizard will remove the program from your computer March 2004 Page 20 AutoQuant Imaging Inc Manual and Tutorials Limitation of Liability Limitation of Liability Notice To the maximum extent permitted by applicable law AutoQuant Imaging Inc disclaims all war ranties either expressed or implied including but not limited to implied warranties of merchant ability and fitness for a particular purpose with respect to the AutoQuant software package the accompanying written materials and any accompanying hardware Specific
146. datasets to correct for cross talk Typically 1f both donor and acceptor calibration images are available 1t is best to select Donor and Acceptor However 1f only one or the other set of calibration images are available for instance only the donor calibra tion images then select that set to correct in this instance select Donor Only Intensity Range Factor This divides the intensities into whatever number of sets is selected e g 1f the number 10 is cho sen then the intensities will be processed in 10 evenly distributed sets The recommended range is between 5 and 20 This number can either be typed into the text box or scrolled to using the up and down arrows next to the text box Note This is only available when using the Elangovan and Periasamy algorithm FRET Conversion Factor G This is the relationship between the loss of donor signal due to FRET with the Donor filter set DD and the increase in acceptable signal due to FRET with the FRET filter DA set Note This is only available when using the Elangovan and Periasamy or Gordon and Herman algorithm Acceptor Quantum Yield Qa The AutoQuant Imaging Inc algorithm requires the Acceptor Quantum Yield Qa which is the ratio between the number of fluorescence photons emitted and the number of photons absorbed This feature is only active when the AutoQuant Imaging Inc algorithm is selected in the Algo rithms section Result Name Prefix This text box allows the user to enter
147. detected Do you wish to load the entire sequence Select Yes 3 Generate a Sum Projection by clicking on the Sum Projection icon on the Toolbar 4 From the View menu select Single View and click on XZ to generate an XZ View of the Sum Projection Notice how the dataset appears darker as you go from the top to the bottom of the view March 2004 Page 116 AutoQuant Imaging Inc Manual and Tutorials 5 From the PreProcessing menu select Attenuation Correction The Attenuation Correction will be performed automatically on the dataset and the result will be displayed in the viewing window 6 Compare the raw XZ Sum Projection dataset to the newly corrected XZ Sum Projection dataset The bright to dark effect has been diminished in appearance Attenuation Correction is most often applied to Confocal datasets where there is frequently a decrease in image intensity due to attenuation light scatter and spherical aberration as the depth into the sample is increased Apply Attenuation Correction to the raw dataset before processing 7 Close all views before going on to the next section Background Equalization Note This section applies to users of Auto Visualize only This function allows you to remove unnecessary background from an image 1 From the File menu select Open or from the Toolbar click on the Open File icon Navigate to the SmallHip folder and select SmallHip avz The SmallHip avz dataset will be loaded
148. detected by noting that the highest abscissa value having a non zero histogram value is at 75 of the range of the abscissa An example of this is shown in Figure D below March 2004 Page 39 AutoQuant Imaging Inc Manual and Tutorials n o Q S o 5 o Q O Ya E 2 E 5 Z Gray values Minimum Maximum gray Maximum possible value in this frame possible gray gray value value Figure D Histogram of gray values in an image frame with no saturation Adjust the camera frame grabber gain and or illumination level such that the maximum gray value in the image frame is between 75 to 85 of the maximum possible gray value For instance for an 8 bit camera most RS 170 camera frame grabbers the maximum gray value is 255 For a 12 bit cooled CCD camera the maximum possible gray value is 4095 If your cooled CCD camera provides gain adjustments or frame averaging follow the manufac turer s directions to achieve the above described intensity histogram profile RS 170 Cameras Many RS 170 cameras provide some control over the video gain Some cameras provide a gain adjustment device on the camera itself while others provide this adjustment as part of a camera electronic control box Often frame grabber cards provide a software selectable gain factor Finally the lamp brightness control and the neutral density excitation filter control provide yet two more mechanisms for selecting the gain Please look through t
149. dimensional only Please close all views before going on to the next section Surface Area and Volume This feature allows you to measure the Surface Area and Volume of the selected image The view of the image must be in the XY projection in a Slice Viewer and be Segmented 1 Open the Pollen deb dataset from the Widefield folder Create a Slice Viewer Projection 2 Automatically segment the dataset using a Min Threshold value of 75 3 From the Analysis menu select Measurement and click on Surface Area and Volume The Measurements box will appear showing the Volume and Surface Area calculations Measurements x Volume cubic microns Surface Area square microns 826 6171 627 7574 1 869195 6 436887 828 4863 634 1943 Total Volume 828 4863 cubic microns Total Surface Area 634 1943 square microns Close 4 If you would like to keep your measurements for future work you may save them as a text file Click on the Save as Text File button and the Export Measurement box will appear March 2004 Page 201 AutoQuant Imaging Inc Manual and Tutorials Object Counting Tracking Note This feature is only available as an added plug in Contact your dealer for purchase infor mation Note It is imperative that the image information is correct in order to get accurate statistics mea surements The Object Counting Tracking mode allows the user to segment count and track up to eight classes of obj
150. displayed in the dropdown menu it can be entered into the textbox manually ROI Add This button calculates the efficiencies for a selected region of interest for the active image set March 2004 Page 180 AutoQuant Imaging Inc Manual and Tutorials Delete This button removes an already processed region of interest removes it from the table and erases all of the efficiencies calculated for it Highlight the desired ROI s to be removed then click Delete Save Statistics This button will save the data into a text file which can then be copied and pasted into an Excel spreadsheet Help This button will open the Online Help file opened to the section regarding FRET Close This button will close the FRET Analysis dialogue Correct FRET Cross Talk Tutorial 1 Navigate to the FRET folder in the tutorial images Open Image AAa Image AAf Image DAa Image DAd Image DAf Image DDd and Image DDf 2 From the Analysis menu select FRET 3 Select the desired algorithm from the Algorithms section 4 Associate each excitation emission specimen with an opened file by clicking on the arrows in the FRET Images section selecting the file with the Donor excitation and Donor emis sion applied to the FRET specimen for the DDf heading the file with the Donor excitation and Acceptor emission applied to the FRET specimen for the DAf heading and the file with the Acceptor excitation and Acceptor emission applied to the FRET s
151. ds on memory resources The sample 3D images that are supplied with the software require approximately 350MB of disk storage It is recommended that due to the inherently large size of 3D images the images be stored on large volume expandable media such as CD ROMs rewritable CDs or DVDs For rou tine use a disk drive of capacity 2GB or larger is recommended The Volume Projection Hardware selection in the 3D viewer requires a video card which sup ports OpenGL extensions It is recommended that an ATI Radeon 9600 series or higher be used for the 3D Visualization Following is an non inclusive list of consumer level video cards which support OpenGL coding ATI Radeon 8500 9000 9700 Diamond FireGL 1 nVidia GeForce 256 GeForce 2 GeForce 2 MX GeForce 3 GeForce 4 all Quadro lines March 2004 Page 16 AutoQuant Imaging Inc Manual and Tutorials 3D Labs Oxygen GVX1 and Wildcat family Matrox Parhelia Software Installation To install complete the following procedure 1 Insert the software CD ROM into your CD ROM drive 2 Choose the Start button on the taskbar usually located at the lower left hand corner of your screen and select Run 3 Type the letter of your CD ROM drive followed by a colon and type Setup E g E Setup Then choose OK Note E represents the letter of your CD ROM drive If the letter of your CD ROM drive is not E type the correct letter in its place The Setup wizard will th
152. e Channels dialog box will appear 3 Click on the Add button then click on the Select Data button An Available Datasets win dow will appear click on Open File Browse to the Multi Channel folder in the Tutorial data and select Malaria_Red 0 tif from the Multi Channel folder and click Open Your Tool s Option in Window s Explorer for hide file extension for known file types must be unchecked in order for you to view the tif extension Also in the Files of type section of the Open menu select All Files 4 A dialog box will appear with the question A sequence of files associated with this selected file is detected Do you wish to load the entire sequence Select Yes This question will March 2004 Page 61 AutoQuant Imaging Inc Manual and Tutorials come up again when you select for the Green and Blue Channels below Select Yes each time If you receive an error message stating that a file cannot be found open a windows explorer win dow browse to the Multi Channel folder sort by file type then delete the hdr files Warning Doing this to your own dataset will delete any image information you may have previously saved with it image spacing numerical aperture etc 5 Repeat steps 3 and 4 for the files Malaria_Green 0 tif and Malaria_Blue 0 tif 6 In the area labeled Select datasets from workspace click on the Malaria_Red 0 tif then click OK In the Import Channel window in box 1 b click on th
153. e Interval This field allows you to select the number of iterations that will occur between storage of decon volution results on the disk The possible values are integers from 1 to the number of Total Itera tions such that the Total Number of Iterations is evenly divided by the number of Save Intervals For example if the number of Total Iterations is 20 and the Save Interval is 5 the deconvolution will be saved at 5 10 15 and 20 iterations The deconvolution application automatically checks for available disk space before beginning the deconvolution If there is no entry or if zero is entered or the entry is greater than 5 000 then this field will be highlighted red indicating that it is outside of the acceptable range and the algorithm will not run until the field is corrected Performance This field allows you to speed up the Deconvolution process while sacrificing slightly on the results The default for this is unchecked Noise Level This feature allows you to set the noise level present in your image The options are Low Medium High and Other This option will already be selected for you if you have Automatic Deconvolution Settings selected If you choose to set your own noise level you can select the noise level that best matches your dataset The Noise Value to the right of the selection represents the amount of noise smoothing that will accompany that setting Selecting Other will allow you to enter a more specific number int
154. e Loop Mode button on the Movie player bar and then click the Play button Auto Visualize will display the frames of the movies in a continuous Loop pattern 8 Click Stop Change Frame Delay This feature allows you to set the time length each Frame or Slice is displayed This feature becomes selectable when a Movie file avm or avi or a Slice Viewer is the active view 1 Make active the Colrpollenrs avm movie 2 From the Visualization menu select Change Frame Delay The Change Frame Delay box will appear Type in the desired length of time you would like each frame displayed The time is measured in milliseconds ms 3 Click Continue This applies the Frame Delay change to the Colrpollenrs avm movie Please close all movies before continuing on to the next tutorial March 2004 Page 177 AutoQuant Imaging Inc Manual and Tutorials Tutorials for Analysis Menu Options FRET Note This feature is only available as an added plug in Contact your dealer for purchase infor mation This function allows for the elimination of Cross Talk so FRET can be accurately calculated This feature is predominantly useful for studying protein protein interactions with sensitized emission FRET techniques Correct FRET Cross Talk Algorithms In the Algorithm section there are three algorithms from which to choose There is Elangovan and Periasamy Gordon and Herman and AutoQuant Imaging Inc which is our own proprietary
155. e cannot be undone except by re manipulating the slice to its original format there is no undo once the Apply button has been depressed other than to close the Manual Alignment dialog box Undo If you have made changes to the slice but have not yet depressed the Apply button pressing the Undo button will return the slice to its original state if a prior change had been made to the slice after which the Apply button was depressed then additional changes were made to the same slice pressing the Undo button will return the slice to its state after the last time the Apply button was depressed This is not a step back feature all changes since the last time the Apply button was depressed for that slice will be undone Close Depressing the Close button will close the Manual Alignment dialog and will open a new untitled slice viewer incorporating any changes made to the dataset This new window can be saved March 2004 Page 123 AutoQuant Imaging Inc Manual and Tutorials Channel to Channel The Channel to Channel alignment feature allows the user to align multi channel datasets that have shift between the channels due to a filter cube or some other image acquisition problem To open the Channel to Channel Alignment feature go to the PreProcessing menu option select Image Alignment then Channel to Channel then select Automatic Alignment Direction This section allows the user to select the direction in which the image need
156. e filter When should I use each algorithm A The Blind Deconvolution is an iterative constrained algorithm that in most cases not all pro vides the best resolving power including the resolving power along the z axis The Inverse Filter is a fast linear method that gives remarkable deconvolutions with widefield dataset It does not work with confocal dataset With some types of samples especially those that have fine low con trast staining it gives even better results than the blind deconvolution Use the Inverse Filter if speed is the most important consideration or if the sample has fine low contrast detail that is important to see The Nearest Neighbor is the fastest deconvolution Its main advantage is speed It provides images typically within seconds It is a linear method Use the Nearest Neighbor algo rithm for quick feedback or for enhancing pictures for presentations but be careful about interpre tation of the images because it is not as robust against artifacts and noise as are the Blind Deconvolution and Inverse Filter Q Why do all the features in my deconvolved dataset look darker than before A The deconvolution will increase the dynamic range of the dataset as a result of removing the blur When an image is displayed on screen it is auto scaled between the maximum and mini mum intensities Therefore the background features will appear darker on screen Use the bright ness and gamma controls to better observe the features
157. e of the scroll bar displays the slice number being displayed in the Coloc Viewer A user can also enter a desired slice number into the textbox and that slice will then be displayed The scroll bar can be used to move through the slices sequentially by clicking on the pointer and dragging it from side to side The slice number in the text box will update when the scroll bar stops moving 2D Histogram The 2D Histogram displays the intensity distribution for the two channels The brighter an area is the higher the occurrence of that intensity The left side of the graph represents Image 1 and the bottom represents Jmage 2 Within the histogram are two lines forming an angle with boxes at the ends of them which act as sliders These lines create an intensity mask Only the intensities that fall between the two lines will be displayed The top line controls the displayed intensities for Image 2 in the Coloc Viewer The bottom line controls the displayed intensities for Image 1 in the Coloc Viewer To move the sliders place the cursor over the slider box and the cursor will turn into a hand Left click on the slider then move either up and down or left to right Color Map The Color Map selection changes the color map of the 2D Histogram The options available are Hot and Cool Intensity Range The Intensity Range frame displays the intensity ranges of each image This frame is not editable ROI The ROI frame contains the controls and stat
158. e radio button next to RGB From the drop down menu next to RGB select Red then click OK 7 Click Add then click Select Data and then repeat step 6 for Malaria_Green 0 tif and Malaria_Blue 0 tif assigning Green to Malaria_Green 0 tif and assigning Blue to Malaria_Blue 0 tif As you add datasets the bottom of section 2 of the Combine Channels dialog will update with each added channel shown individually and section 3 will show each channel com bined together 8 In section 2 click on the red circle underneath channel 2 and drag it upward without releasing the mouse When the circle is about halfway between the blue line and the top of the chart or the percentage below the circle notes around 150 release the mouse Note that the green in the 2D View updates with a much brighter green channel 9 In the Load Channels section highlight Malaria_Blue 0 tif then click Change In the Channel Specification section click the radio button for Fluorescent Probes then click on the drop down arrow Select Rhodamine 576nm from the drop down menu Click OK Note that areas that were previously blue are now yellow Highlight Malaria_Blue 0 tif again click Change then click the RGB radio button and select Blue from the dropdown menu 10 Click Create 3D Dataset A progress indicator will begin showing the writing of the red green and blue channels An XY Max Projection of the three channels is the output displayed This may open behind the
159. ec tion From the Deconvolution menu select Deconvolution Settings and click on Expert Settings The Expert Settings box will appear Expert Settings Subyolume Jw sr Montage Sub yolume overlap Pixels le al FT Z Montage gt Guardband Pixels le Le Dynamic Subsolumes Guardband Pixels le E Pre procezzing TF Intensity Correction J Accelerated 54 Detection _ Minimum Intensity Removal e Pre Condition Imported PSF Object First Guess Smoothed Aaw Image Super Resolution Activate Sub pixel Processing ee Factor fi Adaptive PSF Deconvolution Fired PSF Deconvolution PSF Processing Axial Stretch Factor E PSF Waist Airy Discs E F Disable PSF Constraints Subvolume section 1 This section of Expert Settings affects the handling of the object data The default values are as follows XY Montage is selected enabled March 2004 Page 137 AutoQuant Imaging Inc Manual and Tutorials 2 Z Montage is deselected disabled Dynamic Subvolumes is selected enabled Leave these fields at their default values Verify that the following fields are set to their default values The Subvolume overlap Pixels is 10 The XY Guardband Pixels is 10 The Z Guardband Pixels is 6 Pre processing section 3 Verify that the Intensity Correction box is unchecked 4 Verify that the Minimum Image Intensity Removal box is checked 5 Verify that the Accelerated SA D
160. ect a charge integration time for the first bias frame The accuracy of the bias frame dataset will increase with increasing charge integration time As a rule of thumb use 60 seconds for the first bias frame The charge integration time for the second bias frame needs to be twice that of the first bias frame so as a rule of thumb use 120 seconds for the second bias frame March 2004 Page 45 AutoQuant Imaging Inc Manual and Tutorials Collect a flatfield frame This will be used to calculate the flat field non uniformity of your cam era A flat field sample can be provided in a number of ways When using transmitted light brightfield the simplest way is to remove the sample from the microscope and grab an image of a blank stage A rather simple method with transmitted light brightfield is to take the biological sample extremely out of focus to a point where the image shows a completely flat field When using fluorescence the most reliable way is to prepare a flatfield slide by placing a drop of flu orescent dye onto a microscope slide and sealing it with a coverslip In case any debris particles may be contained in this droplet it is best to grab this image with the droplet well out of focus Some care must be taken to ensure that no out of focus remnants of debris appear in the image and that no other particles on the slide happen to come into focus See Table 1 Figure a and Fig ure b for an illustration of these co
161. ect the Multi Channel folder Select TobaMC tif and click Open 3 A dialog box will appear with the question The program has detected properties that indicate this dataset MAY contain an old AutoQuant Proprietary Multi channel TIFF Is this True e Select Yes 4 From the View Menu select Channels then select Red Channel An image consisting of only the red channel will be generated Green Channel This Channel feature represents either the intermediate length wavelength collected for a 3 channel dataset or the shortest wavelength collected for a 2 channel dataset 5 From the View Menu select Channels then select Green Channel An image consisting of only the green channel will be generated March 2004 Page 106 AutoQuant Imaging Inc Manual and Tutorials Blue Channel This Channel feature represents the shortest wavelength collected for a dataset 6 From the View Menu select Channels then select Blue Channel An image consisting of only the blue channel will be generated 7 Close all views before going on to the next tutorial Viewing the Channels and Slices of a Multi channel Data Set 1 The Untitled dataset should be open from the previous section If it is not open please Import the Multi Channel Data again From the View Menu select Channels then select Red Channel to view the red channel 2 From the Visualization menu select Slice Viewer or click on the Slice Viewer icon You may navigate t
162. ected Acceptor images by clicking the dropdown arrows and selecting the correct image from the list 3 Select a region of interest to process by clicking and dragging on one of the images around the area to be analyzed creating a rectangle around it then releasing the mouse button 4 Select a percentage range of intensities to analyze by adjusting the Fixed Threshold sliders to the desired percentages The minimum must be lower than the maximum March 2004 Page 182 AutoQuant Imaging Inc Manual and Tutorials 5 Select the dye pair used in the images in the Dye Pair s Foster Distance Rg section by clicking on the dropdown arrow and selecting the appropriate pair from the list Note If the dye pair that was used in the images is not listed but you know the Forster s distance you can enter that distance in the text box as well 6 Click Add The efficiencies will be calculated and displayed in the table in the dialogue box 7 Close all images before moving on to the next section Ratiometrics Note This feature is only available as an added plug in Contact your dealer for purchase infor mation This function allows you to observe changes in the sample environment such as changes in Cal cium concentration or changes in pH Images In this section the images to be analyzed are assigned to either the numerator or the denominator The feature will perform an image division and as such the image assigned
163. ecting Open from the File menu launches Windows Explorer Once the file of interest is located drag and drop it into the Batch queue Edit Menu This menu allows you to copy and change the settings for the files in the Batch The Tool menu items are Copy Optics Settings Paste Optics Settings Copy Deconvolution Settings Paste Deconvolution Settings Copy PSF File Settings Paste PSF File Settings Change Settings Change PSF File and Change Operation These features may also be accessed by moving your cursor over the file you wish to edit in the Batch and right click the mouse Copy Optics Settings This feature allows you to copy the Optics Settings from one of the files in the Batch 1 To use this feature select your source dataset 2 From the Edit menu select Copy Optics Settings This copies the Optics Settings parame ters from your source Paste Optics Settings This feature allows you to paste the Optics Settings into selected files in the Batch 1 To use this feature select the datasets you want to paste the Optics Settings into 2 From the Edit menu select Paste Optics Settings This pastes the Optics Settings parame ters from your source dataset into the selected files Copy Deconvolution Settings This feature allows you to copy the setup and deconvolution parameters from one of the files in the Batch 1 To use this feature select the file of interest from the Batch by clicking on it 2 From the Edit menu selec
164. ects and countless individual objects within each class as well as calculate statistics on their physical properties and movements The Object Counting Tracking feature can be accessed from the Analysis menu option on the main menu A dataset need not be opened to launch the Object Counting Tracking as it can be opened from the Object Counting Tracking module If a dataset is already opened and Object Counting Tracking is selected from the Analysis menu there will be an option to Use all images open on desktop Use active image open on desktop and Load new volume time series When selecting Use all images open on desktop make sure that all of the images have the same dimensions otherwise it will compromise the application s ability to properly count and track the objects Automatic Segmentation and Counting The Automatic Segmentation and Counting button will perform automatic segmentation and counting on the opened dataset on all opened volumes Using this option will not allow for multi ple object class definitions Segment The Segment Frame contains the controls used to segment the dataset The dataset must be seg mented before it can be counted Current Volume The Current Volume button will segment the current volume based on the most recently applied segmentation settings If no segmentation parameters have been applied then the default settings will be used All Volumes The All Volumes button will segment all opened volum
165. efault Fixed Threshold as active 4 Adjust the Minimum and Maximum Threshold Values by either sliding the scroll bars or by typing in a value in the corresponding text box 5 Click on the Apply to All Slices button Observe the segmented image Note The Apply to All Slices option is active with 2D Projections of the image The Apply to All Slices and the Apply to Current Slice options are both active when the dataset is in a Slice Viewer projection March 2004 Page 191 AutoQuant Imaging Inc Manual and Tutorials 6 You may change the Threshold value again The check next to the Change Threshold Parameters indicates that you may do this Clearing the check next to Change Threshold Parame ters locks the threshold values 7 To view the original image at any time click the Press and Hold to view the Original Image button This allows you to view the original image momentarily while you are clicking the button Releasing 1t brings back the segmented image 8 To return to the original image click the Restore button Note When your dataset has uneven illumination or an irregular background the Fixed Thresh old method is not suitable Use the Adaptive method in these cases Note Do not close TobaMC tif Max Projection Please continue on to the next section Manual Segmentation In order to segment a dataset manually the image must be in an XY orientation and in a Slice Viewer projection The labelling features for
166. election 147 Colors 83 Combine Manual Alignment 122 Combine Label with Automatic Segmentation 197 Combine Labels 194 Concatenate Movies 176 Movie List 177 Confocal image 28 Confocal Microscopes Molecular Dynamics 53 Sarastro 53 Constrain Rotation Group 90 Constrained Maximum Likelihood 33 Contents 214 Control Movie Points frame 93 End 93 Mid 93 Opposite Path 94 Start 93 Control Panel 81 Controls Manual Alignment 123 Cooled CCD Cameras 38 Cooled CCD Correction Setting Exposure Times tab 127 Cooled CCD Data Starfish Data 126 127 128 Cooled CCD Data Correction Browse 128 Cooled CCD Settings Dimensions 127 Exposure Times 127 Copy Deconvolution Settings 161 COPYING RESTRICTIONS 22 Copying Restrictions 22 Copyright 1995 2001 22 COPYRIGHT NOTICE 25 Copyright Notice 22 Correct Aspect 81 87 Correct XZ ZY Aspect Ratio 71 Correcting Cooled CCD Data 128 Data Correction 128 Launch 128 Correcting Data Cooled CCD 125 Correcting for Depth Attenuation 116 Count Objects Object Counting Tracking 206 Create 2D dataset Import multi channel data 61 Create 3D dataset March 2004 Page 231 AutoQuant Imaging Inc Manual and Tutorials Import multi channel data 61 Create Movie 79 95 Creating a Movie 171 Creating Three Orthogonal Views 68 Critical Measurement 64 Crop Cropping a Sub field 109 Crop Dimensions 111 Crop Icon Cropping a Sub field 109 Crop Rectangular Prism 130 Crop The Stack 108 Cube Surface 74 85
167. en proceed with the installation of the software to your hard disk drive usually the C drive You can find the installed folder by looking in the following directories C Program Files AutoQuant AutoQuant Combination Suite 9 3 Dongle Installation and Requesting a Permanent License All AutoQuant products will run for one month following their initial installation on your com puter During this time you will need to contact AutoQuant for a permanent license so that you can continue using your software beyond the initial trial period Once you have purchased a per manent license you will receive a CD and dongle at which point you can follow the instructions below Installing Version 9 0 and Subsequent Releases 1 Make sure that the dongle is not plugged into the USB port It will be plugged in after installation has completed 2 Run the setup exe program from the installation CD 3 Follow the instructions that come up during installation If the new version is being installed on a machine that already has the demo version installed a Maintenance Setup screen will appear providing the following options Modify Repair and Remove Select Repair this will re install the application using the dongle version of the program 4 When Setup has finished the dongle may then be plugged into an open USB port on your machine to allow the software to run March 2004 Page 17 AutoQuant Imaging Inc Manual and Tutorials Note Y
168. en that dataset will automatically be added to the synchronization table Also minimized slice viewers will not appear in the synchronization table If a slice viewer in the current view is restored from a minimized state it will be added back to the table Removing Datasets Datasets will be removed from the synchronization table as they are closed in the workspace When a slice viewer oriented to the selected view is closed e g if the XZ view is selected and one of the XZ slice viewers is closed then that dataset will automatically be removed from the synchronization table In addition minimizing a slice viewer in the current view will remove it from the synchronization table Movie Maker Note This section applies to users of the Auto Visualize software only Auto Visualize allows you to create a movie sequence To create a movie a series of frames are generated and then displayed in a contiguous manner A movie sequence consists of a series of rotations through a specified range and about a specified axis The movie generated can be played later in Auto Visualize or exported as an AVI file 1 Open the Colorpollenc tif dataset from the Multi Channel folder 2 From the Visualization menu select Movie Maker The 3D Viewer box will appear March 2004 Page 171 AutoQuant Imaging Inc Manual and Tutorials Note Images in the 3D Viewer will open in the XY view only The Movie menu of the 3D Viewer provides the tools
169. enerate the pair of images Auto Visualize will generate the stereo pair and display the two projections offset from each other by the Stereo Angle 1 Navigate to the SmallHip folder and open the SmallHip avz dataset 2 To make this dataset more recognizable from the Toolbar click on the XZ icon to create an XZ Max Projection view of the dataset The dataset will appear showing the bone structure from pelvis to the top of the rib cage Note A Stereo View may be created from any Single view 3 From the View menu select Stereo View The Stereo View dialog box will appear 4 Enter 10 Degrees for the Stereo Angle Click OK Auto Visualize will rotate the entire three dimensional volume 10 Degrees to create the appropriate stereo pair 5 To generate another Stereo View make the SmallHip avz XZ Max Projection the active view and click on the View menu then select Stereo View You may experiment with different Stereo Angles to show varying levels of depth 6 Close the Stereo Viewer s by clicking the Close X in the upper right hand corner Note Do not close SmallHip avz XZ Max Projection Please continue on to the next section Image Aspect Retain Aspect Ratio Stretch Aspect Ratio This feature allows you to change the size of the image by simply moving the cursor to the frame corner or side of a view and dragging the frame edge inwards or outwards There are two options under this menu item The default option is Re
170. entation menu select Apply Label and choose Apply to all slices Click the Apply button 3 From the Segmentation menu select Automatic Segmentation The Segmentation Parame ters dialog box will appear Using the Fixed Threshold set the Minimum Threshold Value to 35 and click the Apply to Volume button The labeled area will appear with a colored outline You may add another label by drawing another region out and clicking Apply in the Label Object box The new label will be outlined in a color different from the previous one 4 Close both the Segmentation Parameters box and the Label Object box 5 From the Segmentation menu select Combine Label with Automatic Segmentation Auto Visualize will combine the manually applied Label s with the Automatic Segmentation Fixed Threshold dataset and display the resulting Combined Label dataset The resulting dataset is com promised of only those manually applied labels that fall within the automatically segmented dataset March 2004 Page 197 AutoQuant Imaging Inc Manual and Tutorials iix This view shows the areas of the selected region that are above the Automatic Minimum Thresh old Value and contained within the manually Labeled area Load Labels This function allows you to load a previously saved Label file You must have previously saved the Labels as a seg file 1 To Load labels from backup you must have the dataset that the Labels file will be loaded onto al
171. entered into the AutoDeblur program are the x and y pixel sizes distance between pixel centers This may be obtained by using a microscope stage micrometer e g Edmund Scientific product number J30 593 or J30 088 Collect two images of the stage micrometer using the same lens with which you collected the optical sections You will need to perform two calibrations one for the x dimension and one for the y dimension Be sure that the graduations of the stage micrometer are properly aligned with the x and y axis This is done by repetitively adjusting the angle of the camera position on the optical tube and March 2004 Page 48 AutoQuant Imaging Inc Manual and Tutorials grabbing an image until it is clear that the graduation lines are perfectly vertical or horizontal see Table 2 for the x and y axis calibration respectively The stage micrometer will have a specifica tion of micrometers per graduation Denote this specification as Ax micrometers graduation Table 2 N 25 graduations N 512pixels 10x 25 x 12 7_ Ax 115x512 gt 0 54um pixels Table 2 Stage micrometer image used for calibrating the pixel size The distance between the first and the last of the six lines on the upper half of this image was measured to be 11 5cm W on a video screen The stage micrometer dimension was known from the man ufacturer to be 10um graduation The width of the entire screen W was measured to be 12
172. es based on the most recently applied seg mentation settings If no segmentation parameters have been applied then the default settings will be used Manual The Manual button allows the user to manually modify the segmentation of the dataset 3D datasets must first be segmented by clicking either the Current Volume or All Volumes button before entering the Manual mode Manual Segmentation In the Manual Segmentation dialog the user can use the mouse to add or subtract segmented area from a segmented dataset To do this hold down the left mouse button while moving the mouse March 2004 Page 202 AutoQuant Imaging Inc Manual and Tutorials over the desired area to be added or subtracted from the segmented dataset Essentially use the mouse to draw on or erase the segmented data Release the mouse button when done adding subtracting area Manual Segmentation When Manual Segmentation is selected use the mouse to draw an outline around an object to be added to the segmented data Do this by holding down the mouse button while tracing an outline around the object Once the outline is complete release the mouse button and the entire area within the outline will become colored indicating that it has been segmented The Manual Segmentation option is not available when segmenting a 3D dataset Mode The Mode frame contains the control buttons to select whether the segmented area is increased or decreased Add Clicking this b
173. es of having unsatisfactory experimental results It is best to follow each and every guideline to the letter wherever possible Do not violate a guideline for the sake of convenience Break only those guidelines for which you have no choice but to break because of restrictions caused by your experimental conditions Remember that with each guideline broken the risk of having unsatisfactory results increases Optics Alignment Before beginning any procedure be sure that the entire optical train is well aligned Follow the instructions provided by your microscope manufacturer on proper adjustment and alignment of the optical elements This should be performed daily prior to each experiment and should be re checked throughout the day if the microscope is receiving heavy usage Make certain of the fol lowing The fluorescence excitation lamp is properly centered and focused if fluorescence is being used The trans illumination lamp is properly centered and focused if transmitted light brightfield is being used The condenser is properly centered and focused if transmitted light brightfield is being used For transmitted light brightfield Adjust the condenser iris diaphragm numerical aperture NA according to the following March 2004 Page 35 AutoQuant Imaging Inc Manual and Tutorials For 3D digital imaging as performed here it is best to have the transmitted light be as incoherent as possible This implies
174. estore Settings will restore the settings to the last set tings entered during the current session Restore Settings Current Dataset will restore the set tings associated with the dataset prior to opening the Standard Settings dialog 5 On the Image Dimensions tab notice the following The Width value is 90 pixels 27 microns The Height value is 120 pixels 36 microns The Depth value is 49 pixels 19 6 microns Verify the X Spacing is 0 3 Verify the Y Spacing is 0 3 Verify the Z Spacing is 0 4 6 On the Microscope Settings and Image PSF Dimensions frame In the Modality box verify that Widefield Fluorescence is selected In the Lens NA field verify the value is 1 4 In the Refractive Index field verify the selection is Oil 1 515 7 In the Emissive Wavelength frame the wavelength for each channel is displayed To change the wavelength select the radio button for the channel of interest and either type in the wavelength nm or select the probe type from the Probe drop down menu For the first channel Channel 1 leave it blank For the second channel Channel 2 set the Probe to FITC and type 520 for the wavelength For the third channel Channel 3 set the Probe to DAPI and type 456 for the wavelength AutoDeblur checks each channel for dataset If the Channel is void of data it will automatically be skipped Calculate Spacings Clicking the Calculate Spacings button will produce tabs for Theoretical XY Spacings and
175. etection box is checked 6 Verify that the Pre Condition Imported PSF box is unchecked 7 Verify that the Object First Guess is set to Smoothed Raw Image PSF Section 8 In the Adaptive PSF Tab 9 Verify that the Axial Stretch Factor is 1 10 Verify that the PSF Waist is 1 11 Verify that the Disable PSF Constraints box is unchecked 12 Click OK to close the Expert Settings box This sets the Expert Settings for the FitcDapi_crop tif dataset 13 Close all views before going on to the next tutorial Expert Settings Explanations Z Montage The Z Montage setting allows the deconvolution application to break the dataset into sections along the optical axis and to deconvolve these subsections separately The valid settings are On checked or Off unchecked March 2004 Page 138 AutoQuant Imaging Inc Manual and Tutorials Guidelines The default for this option is Off It should only be turned On for image stacks with a large Depth setting e g gt 100 slices This option reduces the amount of RAM required by the deconvolution process It may also be useful in rare cases where the sample thickness is so large that the PSF changes dramatically along Z In such cases Z Montage allows the blind deconvolution to find different PSF solutions for different depths XY Montage The XY Montage option allows the deconvolution application to break the dataset into sub vol umes along the XY dimensio
176. f interest you would like to crop When Crop Dimensions is selected the Crop Dimensions box appears prompting you to specify the Top Left Front Corner and the Bottom Right Back Corner 1 Select a region of interest on the dataset by placing the mouse in the upper left hand corner of the dataset then clicking and dragging downward and to the right Release the mouse and a box will appear around the region selected 2 From the PreProcessing menu select Crop Dimensions A Crop Dimensions box will appear Specify the region of interest s Top Left Front Corner position and its Lower Right Back Corner by typing in the coordinate values 3 Click Continue and the region of interest selected will be cropped and displayed as a new volume March 2004 Page 111 AutoQuant Imaging Inc Manual and Tutorials Extend Slices An image can be extended have additional optical slices added to it along its Z axis This function allows you to generate additional image slices based on linear extrapolation and appends them to the top and bottom of an image It is not the same as adding blank slices to the top and bottom of a stack as mentioned in 1 below An extended dataset will have false slices attached to its top and bottom These false slices serve the purpose of a buffer to keep the guardband region from overlying meaningful features thereby keeping the meaningful data from being obscured in the deconvolution process This tutorial c
177. files are collected using the same microscope setup 1 To use this feature select the Pollen_psf deb file from the Batch1 queue Select Copy PSF File Settings from the Tool menu 2 Click on the TOSPollen_psf deb file to make it active 3 From the Edit menu select Paste PSF File Settings This automatically Pastes the Pollen_psf deb PSF parameters into the TOSPollen_psf deb file Copy All This feature will copy all of the setting Optics Deconvolution and PSF from the selected file in the batch 1 To use this feature select your source dataset 2 From the Edit menu select Copy All This copies the Optics Settings Deconvolution Set tings and PSF Settings parameters from your source Paste All This feature allows you to apply the Optics Deconvolution and PSF settings that have been cop 1ed from a selected file 1 To use this feature select your source dataset 2 From the Edit menu select Copy All 2 Select the file to which to paste the settings From the Edit menu select Paste All This pastes the Optics Settings Deconvolution Settings and PSF Settings parameters to your selected file March 2004 Page 162 AutoQuant Imaging Inc Manual and Tutorials Change Settings This feature allows you to change the setup and deconvolution parameters for a file in the Batch 1 To use this feature select the file from the Batch that you would like to change For exam ple the PollenPA deb file 2
178. first select the file to be deleted by clicking on it then click the Delete button March 2004 Page 163 AutoQuant Imaging Inc Manual and Tutorials Paste This function allows you to Paste a file that has been copied into the Batch queue Adding Files to the Batch 1 Select Batch Process under the Deconvolution menu Select Open from the File menu and navigate to the Tutorial Data directory Open the Batch folder and select the Pollen deb file 2 With the Pollen deb selected click and drag the file over to the Batch box and drop it into the Batch queue 3 From the Batch folder select the following files PollenPA deb PollenPA_psf deb TOSpollen deb and the TOSpollen_psf deb file While the cursor is over one of the selected files depress the left mouse button and drag the selected files into the Batch queue Note Do not close the Batch Processing box Please continue on to the next section Save Max Projection This feature allows you to save maximum intensity projections of batched operation results Click the checkbox in the Batch Processing dialog labeled Save Max Projection When you have checked this box a maximum intensity projection of the processed dataset from the XY perspective will be saved into the same directory as the processed dataset The file format of the max projection output will match the file format of the processed dataset Scheduling a Batch To schedule AutoDeblur to run a batch process
179. ge There are five options Use the current threshold If thresholding has already been performed this option will say Use the current threshold of x This option leaves the thresholding as it is Auto threshold This option automatically determines what is the best minimum threshold for that dataset Adjust threshold to This option allows the user to enter a minimum threshold value Binning Factor This option combines two or more intensities into one to allow for quicker rotations and renderings This decreases the amount of data while also slightly decreasing the detail See page 99 for more information on binning Use Smooth Data This option smooths the data out removing noise from it Height Map This feature displays a 3D image intensity height map of the rendered dataset March 2004 Page 74 AutoQuant Imaging Inc Manual and Tutorials Projection Menu This menu allows you to specify which 2D projection will be shown in the 3D Viewer You may choose from the following choices Maximum projection Sum projection Minimum projection Voxel Gradient Alpha Blending Best Focus Surface Slice All projection options are active when your image view is Volume Projection or Cube Surface If your view is Orthogonal Slices Surface Slice view will be the only active projection Surface Slice This feature allows you to view the first slice from each side of your dataset For exa
180. gs A Standard Settings box will appear 3 There are four sections in the Standard Settings the Deconvolution Methods the Optics Settings Deconvolution Settings and the Output Settings These are explained below Deconvolution Methods The Deconvolution Methods section allows you to select between Adaptive PSF Blind and Fixed PSF Non blind for the deconvolution 4 For Deconvolution Methods verify the setting is on Adaptive PSF Blind March 2004 Page 133 AutoQuant Imaging Inc Manual and Tutorials Optics Settings The Optics Settings section prompts you to set the microscope and dataset parameters obtained during the dataset collection Such parameters are the Numerical Aperture the Refractive Index the Microscope Modality the Emissive Wavelength the Image Dimensions and the PSF Dimen sions Experiment specific settings can be saved and loaded to save time on entering the settings each time an image is processed This can be done at the top of the Optics Settings frame in the text box to the right of where it says Select To create a saved setting once all of the optics settings are created type a name for the settings into the text box then click Add To load a previously saved setting select the desired setting from the text box drop down menu In addition to using previ ously saved settings there is an option to use the previous settings which will load the settings that were last used during deconvolution R
181. h 2004 Page 41 AutoQuant Imaging Inc Manual and Tutorials Saturated spike at zero Unsaturated spike near zero n o Q S o 5 5 o Q O Y E 2 E 5 Z Gray values Minimum possible Maximum possible gray gray value value Figure E Histogram condition for setting the offset black level of the camera frame grabber Look for an unsaturated spike at or near zero There may also be a saturated spike at zero March 2004 Page 42 AutoQuant Imaging Inc Manual and Tutorials Saturated spike at zero n o Q S o 5 5 o Q O Y E 2 E 5 Z Gray values Minimum Maximum possible possible value value Figure F A zero saturated histogram This condition is to be avoided when setting the offset black level Performing the Axial Scan Begin grabbing images at each optical section Start by focusing onto one end of the sample Grab a frame Do this with frame averaging if this feature is available on your setup This is especially important if the datasets are noisy due to low light levels It is recommended that you average 255 frames if possible Refocus the microscope to the next adjacent frame and grab the next image and so on Refer to file formats that are compatible with AutoDeblur If a TIFF format is used for instance as with some standard frame grabber cards store each frame in a file in sequentially numbered files with a numeric suffix i e files with names of t
182. he Time Lapse Movie Generator dialog The dialog dis played is an abbreviated version of the Time Series Movie Maker dialog described on page 173 For more information on the functionality of this feature refer to that section Cancel Clicking the Cancel button will close the Time Lapse Movie Generator dialog without creating or saving a movie Help Clicking the Help button will open the Online Help file for the Time Lapse Movie Generator Tutorial for Object Counting 1 From the Analysis menu select Object Counting Tracking 2 A Windows Browser will open Navigate to the Object Counting folder in the Tutorial images Open Nitellal tif A message will appear asking if you would like to load the entire sequence click on Yes Another message will appear asking if you want each file to be a time frame click on Yes Once the dataset has finished loading in the Import Cell Counting Time Series dialog click on Ok 3 In the Segment frame click on the All Volumes button This will segment all of the vol umes 4 In the Select Object Classes frame select Perform Training then click the Define Classes button This will open the Object Classes for Counting dialog 5 In the text box in the Object Information frame type in Class A Click the Define Object button then the Done button 6 In the Object Selection frame highlight Class A in the textbox by clicking on it March 2004 Page 210 AutoQuant Imaging Inc Manual a
183. he form IMAGE 1 IMAGE 2 and so forth If a raw 16 bit integer format is used for instance as with some of the cooled CCD cam eras then store all of the frames in sequential order and contiguously in the same file having an extension deb Repeat this image grabbing and storing sequence until you have scanned the entire sample Scan the entire depth that was chosen according to Figure 1 After collecting the datasets check the computer s directory for the file names that were chosen as a diagnostic check to be certain that all of the datasets were saved Check for their expected file sizes in numbers of bytes For instance if a raw 16 bit binary format is used as with a cooled CCD camera and if the image size is 1024 by 1024 with 85 slices then the number of bytes shown in the directory for this file ought to be 1024 x 1024 x 85 x 2 178257920 March 2004 Page 43 AutoQuant Imaging Inc Manual and Tutorials Be careful to move the plane of focus the exact same distance for each intermediate frame Check the specifications of your microscope and identify the amount of movement effected by a revolu tion of the fine focus knob 1 e the micrometers per revolution For example for the Olympus BH 2 the movement is 200 micrometers per full revolution of the fine focus knob These are well indicated by graduations on the knob with the knob having 100 graduations Thus the stage will move 2 micrometers for every graduation that the kno
184. he manuals provided by your microscope camera and frame grabber manufacturers to determine which if these options are available to you and methods to access them Similarly cameras and or frame grabber cards provide an offset black level adjustment mecha nism Again please refer the manufacturers manuals on methods to access the offset adjustment March 2004 Page 40 AutoQuant Imaging Inc Manual and Tutorials Once you have determined the above information the following procedure is recommended for setting these adjustments 1 Set the illumination light intensity for transmitted light brightfield or the neutral density filter for fluorescence first Do this by direct eye viewing and selecting the intensity so you can comfortably view the sample through the eyepiece If you are trying to maintain a very low light level to minimize photobleaching with a fluorescence sample this might not be possible Instead you may have to simply set the light level to a suitably low level which cannot be seen by eye 2 Set the offset value on the video camera and or frame grabber Ideally you want the offset to be adjusted so that a zero light level corresponds to a zero value on a histogram of gray values Block all light to the camera This may be accomplished either by turning the lamp power switch off for transmitted light brightfield or by closing the excitation shutter for fluorescence It may also be accomplished by sliding
185. he optics 45 46 Optical train 45 Optics Alignment 35 Options 81 Options menu 81 Origin 92 Original View 78 Ortho Slices 89 92 Orthogonal Slices 74 84 Orthogonal Slices frame 88 Out of band noise 33 Overview Collecting Images 35 OWNERSHIP OF SOFTWARE AND MEDIA 23 Page Setup 65 Parallel Slice 92 Performing Image Enhancements 103 Perspective View 82 87 PGM pgm 59 Phase content expected 217 Photobleaching 32 41 Photomultiplier 30 Piecewise Line 199 Pixel Sizes 48 Planeoffocus 27 Play Movie 77 Playing a Movie 57 Point Spread Function 27 32 point spread function 215 Poisson Modeling 33 poor signal to noise 216 Pre amplifier Bias 45 PreProcessing Menu Attenuation Correction 114 Crop 109 Depth Attenuation 114 Optical Density Correction 114 Preset Views frame 90 91 Probe 79 Probe Concentration 34 Processing an Image 147 Processing Current Slice 156 Processing Many Data Sets 34 Processing Stack 157 Projection 85 Image Alignment 122 Projection menu 75 Properties Group 87 PSF Experimental Measurement 32 Missing Cone 33 PSF Compensation 147 Q QuantumPhoton Noise 31 Quick Movies 77 R RO 180 Ratiometrics 183 March 2004 Page 235 AutoQuant Imaging Inc Manual and Tutorials B SI2 Sh2 184 Gaussian Smoothing 185 Grynkiewicz Equation for lon Concentration 183 Kd 184 Rmax 183 Rmin 184 Viscosity 185 Rayleigh Depth Of Field 37 Rayleigh Width 31 37 Red Channel 106 Refractive Index 37 13
186. he other direct rotational path i e the longer route Low res faster This feature allows the 3D Viewer to generate a movie quicker with only a slight decrease in res olution Play Movie frame This group controls the movie playback options and movie playback itself Stop This feature halts a movie in playback mode Play This feature begins a movie playing or puts the movie in playback mode The slider bar allows you to scroll through the movie frames On clicking Play playback will begin from the selected frame Step Angle This feature determines the size of the rotation angle between successive frames of the movie A smaller step size will cause more movie frames to be generated to move between the pathway points Frames sec This feature determines the frame rate of the final movie This does not affect the speed of the movie preview but is stored in the movie header so that a final movie file generated from the 3D Viewer replays at the desired rate March 2004 Page 94 AutoQuant Imaging Inc Manual and Tutorials Rock Mode This feature when checked sets the movie preview to rock from start to end then end to start and repeats When unchecked the movie preview will loop going from start to end then start to end and continues repeating Opposite Path The Opposite Path feature will run the movie in the opposite direction In other words if you select to rotate the object 30 degrees the
187. he subvolumes Transparency This feature allows you to toggle between whether the background areas of the object are trans parent or not Display Floor This feature allows you to toggle between whether the checkered floor pattern is On or Off in the 3D Viewer Perspective View This feature allows you to toggle between whether the perspective view tapered geometry toward horizon is On or Off Auto Rotate This feature allows you to toggle between whether the inertia based rotation 1 e toggles between whether or not the object can continue spinning after the mouse is released following a rotation is On or Off Stereo View This feature allows you to toggle between whether the 3D stereo split view is On or Off Subvolume Tab Subvolume frame This feature allows you to specify exactly a subvolume to be rendered In the edit boxes X from Y From and to you may specify the first and last slices that should be included in the subvol ume s region along each of the X Y and Z axes March 2004 Page 87 AutoQuant Imaging Inc Manual and Tutorials Once the edit boxes have been filled click Apply to make the changes take effect To quickly restore the full volume click the Whole Volume button Orthogonal Slices frame This feature allows you to specify exactly which slice along each of the X Y and Z axes should be displayed in the Orthogonal Slices view The values may range from 1 to the number of slices
188. hin the ROI Histogram Peak Selecting this algorithm will remove the most commonly occurring intensity level using the assumption that the majority of a dataset is background and contains no relevant data Minimum Value Selecting this algorithm will remove the lowest intensity value from the image Constant Value This involves entering an intensity value below which all intensities will be removed Background Image This involves taking an image of a blank specimen which will create a dataset that is entirely background and selecting this dataset from the drop down menu the dataset needs to already be opened for this to happen The algorithm will then analyze the intensity in that image and subtract that intensity from the desired dataset 1 Click on File from the main menu and select Open Navigate to the Ratiometrics folder and open HighCal 001 2 Select Background Subtraction from the Data Correction option under the PreProcessing menu The Background Removal dialog box will open 3 There are five different methods of background removal Constant Region of Interest ROD Histogram Peak Minimum Value and Background Image For the ROI option select a region of interest in the image that is empty background by click ing the mouse dragging it to create a box and then releasing Make sure that nothing is inside this box The Histogram Peak option requires no input it looks at a histogram of the intensi
189. his feature allows you to choose from 30 45 60 90 180 degrees for which the object will rotate about the X Axis Original View This feature allows you to reset the position of the dataset back to its original orientation Set Start Point This feature allows you to set the start point of the movie Set Mid Point This feature allows you to indicate that a view has been set as an intermediate point through which the rotating object should pass at the middle of the movie Mid Point Active This feature allows you to indicate that the rotating object will pass through the defined midpoint If the Mid Point Active is not set the movie will take the shortest path from the start to the end point Set End Point This feature allows you to set the end point of the movie Set Step Angle This feature allows you to select the size of the rotation angle between successive frames of the movie You may choose from 5 10 and 15 degrees A smaller step size will cause more movie frames to be generated between points Go to Point This feature allows you to go to the Start Mid or End point of the movie Loop Mode This feature plays the movie from start to end then start to end and continues repeating Rock Mode This feature plays the movie from start to end then end to start and continues repeating March 2004 Page 78 AutoQuant Imaging Inc Manual and Tutorials Opposite Path This feature is for any pa
190. his function allows you to import up to eight channels of data into one image set for viewing Selecting Import Multi Channel Data from the File menu will launch the Combine Channels dia log Combine Channels Dialog Load Channels This section contains the controls with which to add change and remove channels from the Com bine Channels dialog Add Click this button to add channels to the Combine Channels dialog Clicking this button will launch the Import Channel dialog Change This button allows you to change the attributes of a selected channel already added to the Com bine Channels dialog Select the channel to change by highlighting it then click the Change but ton Only one channel can be selected when you click the Change button Clicking this button will launch the Import Channel dialog Import Channel Dialog Clicking either the Add or Change button will open the Import Channel dialog In this dialog channels can be opened selected and assigned a channel specification Select Data Clicking this button will open an Available Datasets window If datasets are currently open those datasets will appear in the workspace with their complete file path As datasets are added they will appear in the workspace Available Datasets When the Select Data button in the Import Channel dialog is clicked this window opens From here you can add channels to the dataset Open File Click this button to add a file to the workspace A wi
191. hoton count datasets it also properly constrains the probe concentration and PSF to be nonnega tive This is something that is not done by methods based on Gaussian image models e g Least squares Jansson Van Cittert The model in AutoDeblur also takes into account the optical char acteristic of the dataset collection system including the sample and ensures that the estimated probe concentration and PSF agree with the physical system Advantage of an Iterative Method Because the method is iterative and nonlinear it is capable of properly constraining the solution i e constraining the probe concentration and PSF solu tions to be nonnegative This is especially important for both quantitation and for resolution improvement especially z resolution Our algorithm is capable of optimally restoring the miss ing cone of frequencies that are inherent in the PSF for widefield microscopes For a detailed description of the problem of missing cone of frequencies the reader is referred to the paper by Streibl see Bibliography Linear methods such as the well known Nearest Neighbor method and the Inverse Filtering methods see references are not capable of implementing such constraints and are thereby not nearly as suitable for quantitation or resolution improvement However imple mentations of both of these simple and fast deconvolution methods are included in AutoDeblur March 2004 Page 33 AutoQuant Imaging Inc Manual and Tutorials
192. hresholding 20 0 0 ceeceeeeseeeseceneceneeeeeeeeeesaecaeceeeeeeeeeesenaees 183 Use Grynkiewicz Equation for Ion Concentration cocoococnoncccnonnnononcnonanccnnnncnnnnanos 183 AAA TRONO 183 RDM tarada daa a ricos pibes 184 B S A A 8 eae al aae et 184 Calibrate scinne ewe el a 184 Numerator Wavelength oo ceecceeeseeeseceseceseeseeeceeescecsaeceecseeeeseeeaeeeaaenaeeees 184 Denominator Wavelength cee eeeeeecssceseeeseesseceseceseeeneeeaeesaecsaeceeeseeeeseeenaeed 184 Oia AA ea a a a cena een vied eae ae a 184 March 2004 Page 11 AutoQuant Imaging Inc Manual and Tutorials BOWS ece a ha scevd dida 184 O OOOO 184 Help a e a A leo ad ah eat ee lee ee ee ee ate es 184 O OE OOO 184 NISCOS aiii ia ie athe ee lies 185 PO O a o Coa 185 Remove Spot Noise miii eee doe aed condi 185 Gaussian Smoothing ses nia ai enna e ien ieii aad tices 185 Calculates scsi hit SA tetanus aisle AGE 185 SEAS Ei a AAA Bean Dede A Peat N bates cats 185 CLOS A Dead Vets Ferndown a a el 185 Helpoin iii leticia ladito leal ella 185 EAN 185 A A O 186 Mago l Image Lita A EA AA A A 186 MEO GEV TW A A E E A A EE 186 TOOIS ii NN T TE N NSN 186 2D Histogram heie eae a E A ON 187 Color Map neoaeae ea Ea EE Oa anion deni anes ab 187 Intensity Range A II E ia a aes ade ees ie ao eT 187 ROU E E E E A E E 187 A a a a a a Ud Ea 188 DEl n e a ere ES E a EAE rr eT cl catas Dd dea Bd ed eee 188 DAVE OLALISCI S x aii li ecavin S ENSS ONR 188 Generate Coloc m
193. hrough the slices composing the red channel Close this Slice Viewer projection and the Red Channel XY Max Projection before going on to step 3 Note When a color is selected from the View Menu the view projection formed depends upon the currently active view projection 3 From the View Menu select Channels then select Green Channel to view the green chan nel 4 From the Visualization menu select Slice Viewer or click on the Slice Viewer icon You may navigate through the slices composing the green channel 5 From the View Menu select Channels then select All Channels to view the Slice Viewer projection of the multi channel data 6 From the Windows menu select Close All A dialog box will appear with the question Untitled is not saved Do you wish to save it Select No This will close all open views March 2004 Page 107 AutoQuant Imaging Inc Manual and Tutorials Tutorials for PreProcessing Menu Items For this tutorial you may use your own data type Fluorescence Widefield Laser Scanning Confocal Brightfield Transmitted Light Spinning Disk Scanning Confocal Two Photon Fluorescence or you may follow along with the Tutorial recommended dataset Select Region The Select Region feature allows you to select a region within an image You have the choice of Rectangle Ellipse Polygon and Freehand selection tools 1 Open the Neuron deb dataset from the Confocal folder 2 To use this feature fr
194. i cal Imaging 6 3 228 238 March 2004 Page 227 AutoQuant Imaging Inc Manual and Tutorials Streibl N 1984 Depth Transfer by an Imaging System Optica Acta 31 1233 1241 Turner J N K L Szarowski S M A Marko A Leith and J W Swann 1991 Confocal Microscopy and Three Dimensional Reconstruction of Electrophysiologically Identified Neurons in Thick Brain Slices Journal of Electron Microscopy Technique 18 11 23 Van Trees H L 1968 Detection Estimation and Modulation Theory Wiley New York Veklerov E and J Llacer 1987 Stopping Rule for the MLE Algorithm Based on Statistical Hypothesis Testing IEEE Transactions on Biomedical Imaging 6 4 313 319 Visser T D J L Oud and G J Brakenhoff 1992 Refractive Index and Axial Distance Mea surements in 3 D Microscopy Optik 90 1 17 19 R H Webb and C K Dorey The Pixelated Image Chapter 4 in The Handbook of Biological Confocal Microscopy 2nd Edition James Pawley Editor Plenum Press New York 1995 Willis B J N Turner D N Collins B Roysam and T J Holmes 1993 Developments in Three Dimensional Stereo Brightfield Microscopy Microscopy Research and Technique 24 437 451 Wilson T 1987 The Size of Detector in Confocal Imaging Systems Optics Letters 12 227 229 March 2004 Page 228 AutoQuant Imaging Inc Manual and Tutorials Customer Feedback AutoQuant greatly appreci
195. i eee eee rile ea aes 179 Specified Cross Talk ci daa E E Ea ES 179 Donor Only Acceptor Only Donor and Acceptor seeseceeseeeereereererrrrrreeen 179 Intensity Range Factor seniicorinieaaa ia a aiia 179 PRET Conversion Factor Grisales 179 Acceptor Quantum Yield Qa oon eeccecesccesseceececeececeeeeeseeeeaeeeaaeceeaeceeeeecneeeeaas 179 Result Name Pr hs a ia A aie es 179 Use Cross Talk Coefficients ini iia 179 Calculate aena aE EE area 180 Clear A RR 180 Close ohea e l a nai oea E ar eet tay cedar derbies dud edt eet 180 Hliva A AAA AAA 180 Calculate FRET Efficiency cnica is 180 Data for FRET Efficiency Estimation 0 0 eeeecessecseceseceseeeseeeseesaeceaeceeeeeeeeeeeenaees 180 Image ROL uan do a dada 180 Fixed Threshold imei ad laicos 180 Dye Pair s Foster Distance RO oooococcnnncnionconoconannnnonnnonnconnocononnnnnnn itii espias 180 ROL una da A A aa 180 Add ci 180 Delete tia Ai Bae ee Pie 181 Save Statis S iii A A AAA A AE 181 Help Veo Adidas 181 CLOS A A A OE EEE EG 181 Correct FRET Cross Talk Tutorial ooonicnncnnnnninninnncocnocononoconannnnnnn conc con nonnnonnccnnncnnnoon 181 Use Cross Talk Coefficients Tutorial asis 182 Calculate FRET Efficiency Tutorial a 182 RAUOMICUICS dust A ai a ai a dese les geaes Goel 183 TASES EE E E dh Re eeecese ER Se 183 PTEPTOCESSIME iii aE E A a EEEa ERTE 183 Use Automatic Alignment eee eeseceseeeeceeeeeseesseecaeceseceseeeaeeeseesaeceaeceeeseneeeeeeaaees 183 Maximum Minimum T
196. ials 1 Navigate to the SmallHip folder and open the SmallHip avz dataset or you may follow along with your own dataset 2 From the View menu select 3D Viewer The 3D Viewer window opens with your dataset in the Volume Projection view Hardware if your system has the proper video card Software if it does not of its XY Max projection In the upper left hand corner of the 3D Viewer window the red line represents the X axis the green line represents the Y axis and the blue line represents the Z axis of the dataset 3 From the Rotation menu select Go to View and choose a view e g XZ The dataset will be rotated to the XZ view 4 To rotate the dataset click anywhere in the 3D Viewer window hold down the left mouse button and move your mouse The dataset will rotate in the same direction your mouse moves If you stop your mouse movement with the left button still depressed the rotation will stop If your mouse is moving while you release the left mouse button the dataset will continue rotating Clicking in the 3D Viewer window will stop the auto rotation 5 From the Color Map menu select Lookup Table then select Jet if you are using your own dataset and it is not grayscale then the options in all but the Background selection will be disabled The dataset will be pseudo colored with the color map displayed on the right side of the display 6 From the View menu in the 3D Viewer select Orthogonal Slices To change the dis
197. iding Backlash and Hysteresis 45 Bias and Flatfield Frames 45 Bottom of Sample 36 Confocal Data 51 RS 170 Cameras 40 46 Setting Exposure Gain Offset 38 Setting Number of Slices 37 Signal to Noise Ratio 54 Spatial Calibration 48 Top of Sample 36 Vibration Control 50 3 D Data Set 27 A About 214 About AutoDeblur 214 Acceptor Quantum Yield Qa FRET 179 Accurate deconvolution 215 Add Import multi channel data 60 Adjust All box 150 Algebra 189 Alignment 35 Channel to Channel 124 Manual 121 All Channels 147 All Slices 110 Alpha 86 Anaglyph 83 Anchor 89 Appendices 219 Application of AutoDeblur Deconvolution Package 30 Apply 93 Manual Alignment 123 Apply Seed Fill Label 193 Apply to all slices 110 Apply to current slice only 110 Apply to given slice range 111 Around 91 Freely 75 Object 75 Attenuation Correction 116 Depth Attenuation 117 Excitation Light Absorption 116 Fluorescent Emission Light Absorption 116 Photobleaching 116 Auto Rotate 81 87 AutoDeblur Installation 16 Software and Hardware Requirements 16 Starting 19 AutoDeblur File deb 58 AutoDeblur Files deb 58 Automatic Alignment Noise Thresholding 120 Automatic Segmentation and Counting Object Counting Tracking 202 AutoQuant Federal I D 25 Incidental Damages 21 Warranties 21 AutoVisualize File avz 58 Available data sets Import multi channel data 60 Axial Sampling Distance 37 Axial Smearing 31 Axis Best Axis 76 X Y Z axes 76 Axis menu 75
198. ificant phase characteristics e g areas of the specimen appear brighter than the background For this dataset leave the Phase Content Expected box unchecked 10 Click Start AutoDeblur will now execute the Inverse Filter deconvolution The status bar will indicate the progression of the Inverse Filter March 2004 Page 153 AutoQuant Imaging Inc Manual and Tutorials 11 When the Inverse Filter method has finished a new XY Max Projection view will appear This is the result of the Inverse Filter deconvolution Do not close the Inverse Filter result Unti tled dataset You will compare it to the results of the Nearest Neighbor in the Nearest Neighbor s section on pages 144 146 Inverse Filter Batch Processing This feature allows you to batch Inverse Filter operations Within the Inverse Filter Parameters dialog box enter all the necessary parameters for the inverse filter operation In order to add an inverse filter operation to a batch verify that the parameters in the dialog are valid and then click the Batch button 3D Inverse Filter FitcDapi_crop tif 1 3 Optics Settings Change Microscope Settings and Image PSF Dimensions Modality Widefield Fluorescence Lens NA 0 8 Refractive Index 1 PSF Pixels Spacing Probe FITC Wavelength Inm Image Pixels Spacing Microns E E Width 30 0 3 Channel 1 Height Channel 2 520 Depth Channel 3 520 PSF Genera
199. ight of the minimum intensity indicator and the minimum intensity indicator can only be placed to the left of the maxi mum intensity indicator The intensity values are used in a histogram stretching intensity filter For 8 bit data this filter sets all the pixel values that are greater than the maximum parameter to 255 and all the pixel values less than the minimum parameter to 0 Pixel values which fall in between the brightest and dark est parameter values are linearly scaled between O and 255 Image Enhancement may be done on a single color channel Red Green Blue or on All Chan nels by activating the appropriate choice under Channel 6 Click Show CDF line This displays the Cumulative Distribution Function line which maps the distribution of the intensities across the histogram 7 Click the drop down arrow next to Auto Levels A list will appear containing 4 differ ent percentage ranges Select 5 95 and note that the Brightness and Darkness indicators move to those percentages on the histogram 8 Close the Image Enhancement box by clicking the OK button Note When viewing in a Slice Viewer the maximum and minimum values for Brightness and Darkness Level are the true maximum and minimum values of the entire dataset For other projec tions the Brightness and Darkness Level values represent the maximum and minimum intensity values of the view Note Do not close SmallHip avz XZ Max Projection Please continue on
200. ill appear similar to the view shown below Note In order to observe the combined labels scroll to slice number 16 17 or 18 MS oxi 16 Auto JE Remove Label This feature allows you to choose which labels you would like to remove from the dataset To uti lize this function select Remove Label from the Segmentation menu When the Remove Label dialog box appears you can select the Label number s of the Label s you would like removed Clicking the Remove button will remove the selected Label s You may close all views before going on to the next section March 2004 Page 196 AutoQuant Imaging Inc Manual and Tutorials Combine Label with Automatic Segmentation To utilize the Automatic Segmentation function and the Manual Segmentation function together you must first either manually label the dataset and then choose Automatic Segmentation or vice versa Once both functions have been applied to the dataset select the Combine Label with Auto matic Segment feature and Auto Visualize will create the new dataset This dataset will consist of voxels with values that are above the Automatic Segmentation Minimum Threshold Value and are also contained within the manually labeled regions 1 Open the greenTobaMC tif dataset from the Multi Channel folder Create a Slice Viewer of the dataset Select the Freehand Select Region tool and trace out a shape similar to the one shown below 2 From the Segm
201. image Use the slider bar to spin the object about the anchor axis Angle This feature allows you to specify an exact angle of rotation about the X Y and Z axes Using the three field boxes you may set the Angle of rotation for the X Y and Z axes March 2004 Page 89 AutoQuant Imaging Inc Manual and Tutorials Note The order in which these rotations are executed depends on the anchor axis chosen After entering a set of rotation angles in the edit boxes use the Apply button to rotate the object to the specified point Alternatively you may use the Reset button to return the object to its original orientation 1 e its orientation upon being loaded into the 3D Viewer Round This button when clicked will round the object s rotation angles to the nearest whole degree interval as specified in the accompanying drop down menu The Apply button applies the current Anchor Axis and Angle to the object and the Reset button will set the object back to its original view Preset Views frame The XY XZ and ZY buttons allow you to automatically orient the object to the standard perspec tives of XY XZ or ZY Additionally the Flip button allows you to view the current orientation from the rear of the sample effectively rotating the object 180 degrees about the screen s Y axis Rotate Object frame The feature allows you to control the incremental rotations about a particular axis Axis This feature allows you to selec
202. iminate these problems Q How close together should my optical slices be A The optimal setting has them spaced equal to the depth of field for both widefield and confo cal This rule of thumb is flexible if other experimental constraints warrant a compromise Q What should I do after I load my dataset A Next go to Deconvolution Deconvolution Settings Standard Settings and fill in any entries that have not been previously entered including the type of deconvolution you want to perform Then go to Deconvolution Start 3D Deconvolution to begin your deconvolution Q How are saturated pixels handled A Saturated pixels are handled by a proprietary algorithm which heuristically treats them as unknown data points A special algorithm uses the blur information surrounding the saturated pix els to infer the information that should have otherwise been provided by the saturated pixel Q When should I use optical density or attenuation correction A Use optical density correction if you notice a flicker in your optical sections You will notice this flicker by creating a XZ View View menu gt Single View gt XZ and a Sum Projection Visualization menu gt Sum Projection If it is present the flicker will be obvious so when in doubt you may assume there is no flicker Flicker looks like dark horizontal lines through your dataset Use attenuation correction if you notice that the overall brightness of your picture bec
203. imized windows will not be tiled displayed To end the tiling click on each dataset and select the desired size from the zoom box Tile Vertically This function stacks vertically the open datasets in the window Closed or minimized windows will not be tiled displayed To end the tiling click on each dataset and select the desired size from the zoom box Close All This function allows the closing of all open views whether active or not and all docked datasets are minimized with only part of their title bar showing views all at once Window List This feature lists all current views on the screen March 2004 Page 213 AutoQuant Imaging Inc Manual and Tutorials Help AutoQuant Help This feature provides a help window opened to the table of contents Contents This feature provides a help window in the form of a table of contents unlike Search for Help On where it is more like an index Search for Help On This feature allows you to obtain help by searching for Key words or by selecting descriptive techniques Check for Update AutoQuant s products incorporate an AutoUpdate feature It is configured to check for updates once a month if your machine is connected to the internet If you would like to check for an update before that select Check for Update from the Help menu You will be prompted to click OK to search the internet for updates If you do not have a LAN connection but are using some other in
204. in the 3D Viewer Once an object is selected scroll to the next time frame by clicking the right scroll arrow on the bottom of the Manual Track dialog Click on the next object to add to that track in the 3D Viewer and repeat this for each frame Delete Track This button will delete the highlighted track in the Track Menu as well as the 3D Viewer The objects will remain but they will no longer be associated March 2004 Page 208 AutoQuant Imaging Inc Manual and Tutorials within a track To delete a track highlight it in the Track Menu by clicking on it then click the Delete button Only one track can be deleted at a time Delete Rest of Track Clicking this button when a track is selected will delete the rest of the track including the selected time frame Delete Current Object Clicking this button will delete an object from a track To do this click on the object to be removed in the 3D Viewer then click the Delete Cur rent Object button in the Manual Track dialog Merge The Merge button allows two separate tracks to be merged together This would be done if for instance Track 1 tracks an object in frames 1 3 while track 2 tracks the same object in frames 4 6 Highlight the two tracks to be merged in the Track Menu then click Merge The two tracks will become one continuous track and will be classified as the track that is listed highest on the Track Menu if Track 2 and Track 4 are merged they will now
205. in the diagnosis of disease or other conditions or in the cure mitiga tion treatment or prevention of disease in man or other animals or iii intended to affect the structure or any function of the body of man or other animals and which does not achieve any of its primary intended purposes through chemical action within or on the body of man or other ani mals and which is not dependent upon being metabolized for the achievement of any of its pri mary intended purposes c Usage with Confocal Microscopes This software may be used for visualizing or analyzing datasets from a confocal optical microscope so long as that confocal microscope is licensed in conjunction with U S Patent No Re 34 214 Most confocal microscope manufacturers have such a license It is the responsibility of the customer to determine if their confocal microscope is licensed in conjunction with U S Patent No Re 34 214 which may be done by enquiring with their confocal microscope manufacturer This software may not be used with confocal microscopes that do not have such a license AutoQuant will not be responsible for any infringement nor appearance of such infringement This restriction does not in any way imply any acknowledgment by AQI of the validity of U S Patent No Re 34 214 4 OWNERSHIP OF SOFTWARE AND MEDIA You agree and acknowledge that AQI transfers no ownership interest in the SOFTWARE or in any SOFTWARE copy to you under this Agreement or otherwi
206. ir of object positions where there are two possible rotational paths between the waypoints By default the movie generator will take the shortest rotational path When Opposite Path is selected the movie generator will take the opposite or longer rotational path Create Movie This feature allows the 3D Viewer to generate a finalized version of the specified movie and open it in the main viewing window Color Map Menu This feature allows you to select a color map in which to display your dataset You may choose from different colors look up tables wavelengths or probe types You can also select a color for the background as well as reverse the colors of the dataset Color This features allows you to select a color based on a single color The choices are Gray Red Green Blue Cyan Yellow and Magenta Look Up Table This feature allows you to select a color based on a look up table The choices are Red Fire Green Fire Blue Fire Black Body Copper Cool Jet and Spectrum Wavelength This features allows you to select a single wavelength to use as a color map for your image You may choose from 400nm 450nm 500nm 550nm 600nm 650nm 700nm and 750nm Probe This feature allows you to choose a color map based on the associated dye from a drop down list You may choose from some of the most popular fluorescent dyes like Dapi 456nm Cy2 506 Fluorescein 519nm Fite 520nm Lucifer Yellow 528nm GFP 540nm
207. is lists all of the available 3D features For this section it is imperative that the image information is correct in order to accurately calculate statistics Feature Display This section displays the statistics calculated on the selected features once the Calculate Statistics button is clicked The statistics will appear in spreadsheet form Save Training Sets Clicking this button will allow the user to save the data for the object definitions This can save time for future experiments if defining the same type of objects Each object will be saved as a separate csv file Ok Clicking Ok will load the training data back into the Object Counting dialog If the training data has not been saved a message suggesting to save the training sets will appear Clicking Yes will March 2004 Page 205 AutoQuant Imaging Inc Manual and Tutorials open the Directory to Save Training Set dialog Clicking No will close the Object Classes for Counting dialog and reopen the Object Counting dialog Cancel Clicking the Cancel button will close the Object Classes for Counting dialog without saving or loading any training data Counting will not be able to be performed until the training data is loaded Help Clicking the Help button will launch the Online Help system opened to the topic for Object Classes for Counting Load Classes This button allows the user to load a previously saved set of classes Clicking this button will open a browser
208. istics for the Region of Interest in the Coloc Viewer To add an ROI use one of the crop tools mentioned in the Crop Tools section March 2004 Page 187 AutoQuant Imaging Inc Manual and Tutorials Along the top of the ROI frame are the statistics that are gathered on the ROIs The first ROI listed is always the Active ROL If no ROI has been drawn then the Active ROI will be the entire Coloc Viewer Add Clicking this button will add an ROI that has been drawn using the crop tools to the statistics table Delete The Delete button will delete the selected ROI s Multiple ROIs can be selected by either click ing on the first and last ROIs to be deleted while holding the shift button or clicking and dragging from the first to the last ROI to be deleted Save Statistics Clicking the Save Statistics button will save the statistics as a txt file A Save As browser will appear in which the user can navigate to the desired directory and name the file Clicking Save will complete the save Generate Coloc Image Clicking this button will generate the image showing the colocalized areas based on the intensity ranges set up in the 2D Histogram This new image can be saved as an independent file Close Clicking this button will close the Colocalization dialog box Help Clicking this button will bring up the Online Help menu opened to the section on Colocalization 1 From the File menu option select open and navigate to the Mu
209. ith flipped data the projection is first created from the original data and then the new projection is flipped and displayed For Slice Viewer projections selecting Flip will flip all the slices in the dataset 1 From the View menu choose the Flip View option and select Horizontal The XZ Max projection will then be flipped along the horizontal axis of the view 180 degrees about the Y Axis Note Flipping the Image when the Slice Viewer or Montage View projection is active will result in flipping the entire 3 dimensional dataset not just the one slice being displayed 2 If you have added the Horizontal Flip View icon to your toolbar notice it is now depressed This indicates that the current view has been flipped horizontally from its original position By clicking this icon you can toggle between the flipped image and the original image Click on the Horizontal Flip View icon to restore the original view You may also wish to flip the view vertically by selecting Vertical from the Flip View options under the View menu Note Do not close SmallHip avz XZ Max Projection Please continue on to the next section Color Map This feature allows you to change the active view to one of the four Color Map options Gray Scale Hot Cool and Copper These various color maps can be used to obtain a different represen tation of the projections generated The default Color Map is Gray Scale 1 From the View menu choose Color Map and select Cop
210. k ccccccsecceeccesccescceeceeeceseeeeeeaseceseneeeees 229 March 2004 Page 14 AutoQuant Imaging Inc Manual and Tutorials How to Use This Manual The Tutorials of the manual go through every menu item in the Menu Bar Depending on the product you have purchased AutoDeblur Gold AutoDeblur Silver AutoDeblur 2D or AutoVisualize some sections may not apply to you this will be clearly indi cated at the beginning of the corresponding tutorial March 2004 Page 15 AutoQuant Imaging Inc Manual and Tutorials Installation Transferring a License Uninstalling and Hardware Requirements Sales Information AutoQuant Imaging Inc 877 25th Street Watervliet NY 12189 E mail salesO aqi com Website www aqi com Ph 518 276 2138 Fax 518 276 3069 Technical Support Internet support aqi com Ph 518 276 2138 Fax 518 276 3069 Software and Hardware Requirements Windows Systems Windows 98 ME 2000 XP 256MB RAM minimum 1GB RDRAM PC800 or faster recom mended 1GB free disk space 10GB recommended for image storage A minimum of an Intel Pentium 3 processor recommended Pentium 4 1 5GHz processor or better A minimum screen resolution of 1024x768 1280x1024 recommended A 24 bit SVGA display CD ROM drive Larger memory sizes are recommended for improved performance keeping in mind that the oper ating system the window interface and potential tasks of other concurrent tasks make strong deman
211. lay a logo in the lower left corner of the 3D Viewer The logo requirements are that it be a 24 bit bitmap file no larger than 256x256 If the file is larger than 256x256 the logo will automatically be cropped Perspective View This feature displays the view of the image in a depth perspective so that closer objects appear larger than distant objects Stereo Mode Off This feature is the default setting and keeps the stereo view turned off Anaglyph This feature shows contrasting colors that appear 3 dimensional when superimposed Only gray scale images can be viewed in anaglyph stereo mode LCD Glasses Note This option will only be available if you have the hardware necessary to support it This feature is for systems with OpenGL enable Stereo Viewing capability e g StereoGraphics CrystalEyes LCD glasses This capability splits the image into two alternating components to March 2004 Page 82 AutoQuant Imaging Inc Manual and Tutorials produce a 3D effect that is visible through special stereo goggles since this method does not require special coloring color images as well as grayscale images may be viewed in this mode Anaglyph Colors The feature splits the image into two differently colored components to produce a 3 dimensional effect This is the familiar Red Blue 3D effect though other color pairs may be selected like Red Cyan Red Blue Red Green Cyan Red Blue Red Green Red Left Only and Right Only
212. lect and load all three Field image stacks 1 Slow Scan Cooled CCD Data Correction x Files Parameters Raw Data AATLB star1 IV Flat Field Flat Field Load Unload JV Bias Field 1 ias Field 1 Load Unload JV Bias Field 2 ias Field 2 Load Unload Cancel Help 3 To load the Flat Field file 1t0 click the top Load button and navigate to the TLB folder 4 Select the star It0 file and click Open The File Open box appears with the message Dataset only contains one optical slice the orthogonal projection along XZ and YZ will be inac tive Click OK AutoDeblur will automatically load the Flat Field file March 2004 Page 126 AutoQuant Imaging Inc Manual and Tutorials 5 To Load the Bias Field 1 file click on its corresponding Load button and select the star bsO file from the TLB folder Click Open The File Open box appears with the message Dataset only contains one optical slice the orthogonal projection along XZ and YZ will be inac tive Click OK AutoDeblur will automatically load the Bias Field 1 file 6 To Load the Bias Field 2 file click on its corresponding Load button and select the star bs1 file from the TLB folder Click Open The File Open box appears with the message Dataset only contains one optical slice the orthogonal projection along XZ and YZ will be inac tive Click OK AutoDeblur will automatically load the Bias Field 2 file 7 Select the P
213. lgorithm Settings Total Iterations fio Save Interval fio a Noise Level Low f gt Noise Value 2 Performance Faster Processing Reduced Resolution Continue From Previous Result Browse V Use Recommended Expert Settings 4 Output Settings File Format TIFF 16 bit 7 Base Name for Deconvolved Data FitcD api_crop Save As I Save the PSF Files Go to Expert Settings Batch Carcel Hep March 2004 Page 144 AutoQuant Imaging Inc Manual and Tutorials Terminate Cancel and Preview 3D Deconvolution Once the Deconvolution has begun a progress dialog box will appear displaying what iteration is currently running on which channel along with an approximate remaining time There are also options to Terminate Cancel and Show Deconvolution Preview Terminate This feature will stop the deconvolution process but will save and open a new dataset based on the most recently completed iteration When this button is clicked you will receive a message saying Terminating Deconvolution Do you wish to finish the iteration and save the result Click ing Yes will finish the current iteration and open the result in a new dataset Clicking No will stop the deconvolution immediately and save and open a new dataset based on whichever was the last iteration completed for instance if you clicked Terminate during the 10th iteration it would save and open a dataset based on the 9th iterati
214. lti Channel folder Open colorpollenc tif 2 From the Analysis menu at the top of the screen select Colocalization 3 For Image 1 select colorpollenc tif Channel 1 with Red selected as the color next to it and for Image 2 select colorpollenc tif Channel 2 with Green selected as the color next to it 4 Move the maximum and minimum intensities for both images Notice how the colocalized areas change in the Coloc Viewer 5 Now move the Slice scroll bar back and forth Notice how the Coloc Viewer changes as the slices change Now click the All checkbox All slices will now be displayed as well as the colocalization in all of the slices March 2004 Page 188 AutoQuant Imaging Inc Manual and Tutorials 6 Click on the Coloc Viewer inside the image and drag out a rectangle creating an ROI Click Add The statistics for the selected ROI will be loaded into the statistics window Create another ROI and click Add to load it into the statistics window 7 Click Save Statistics Name the file statistics 1 txt and click Save 8 Highlight the first ROI by clicking on it then click the Delete button 9 Click Generate Coloc Image A new image will open displaying the colocalized areas Open the Slice Viewer from the new image Scroll through the slices noticing how the colocal ized areas change 10 Close all images before moving on to the next section Image Algebra Note This section applies to users of Aut
215. ltichannel dataset For this tutorial this feature should be disabled Alignment Log File The Alignment Log File allows you to name and save a log file which will detail the changes made to each slice Write Log Selecting this feature will prompt the Image Alignment feature to save a log of the changes made to each slice In the text box next to the right enter the file name you want the log file to be called Set folder path Click the set folder path button to open a windows browser to select the folder to which the log file will be saved March 2004 Page 120 AutoQuant Imaging Inc Manual and Tutorials 7 Click the OK button The application will launch the alignment process and will display the results upon completion The following images are the original dataset and the resulting aligned dataset Original Aligned 9 Create Slice Viewers of the original and the aligned images then open the Slice Synchro nizer from the Visualization menu Scroll through the datasets Notice the motion between the slices in the Unaligned avz dataset as compared to the Aligned avz dataset Please close all views before going on to the next section Manual Alignment Note This feature is only available as an added plug in Contact your dealer for purchase infor mation This feature allows you to correct for X amp Y translation and warp nonlinear distortion between frames as well as rotation Currently only Grayscale d
216. lue 1 378195E 08 6 Change the Order of Polynomial to 4 Notice how the dotted line more closely follows the intensity profile of the image Click Continue 7 Place the Untitled deb XZ Sum Projection view below the star 1 XZ Sum Projection view and compare the differences between the views Notice how the Optical Density Correction has reduced the amount of horizontal lines in the cor rected dataset Note The intensity profile displayed is that for a Grayscale dataset For a multi channel dataset the intensity profile displayed is the Red Channel This is the default You can view the intensity profile for each channel separately by making the appropriate selection under Select Channel For each channel you may chose an order of the polynomial which best fits the image intensity March 2004 Page 115 AutoQuant Imaging Inc Manual and Tutorials profile Other types of cameras often have a similar problem Ordinary Video Rate CCD cameras intensi fied CCD cameras digital cameras and other types of cameras will show this flicker effect There are several factors that contribute to this effect and it is unknown to what degree each factor con tributes Some camera manufacturers may claim that their cameras do not have these imperfec tions Even so the effect is still likely to arise and is due at least in part to other factors in the optical train that the camera manufacturer cannot control Such factors include
217. lving power increase or double the numbers Repeat this increasing until you are satisfied with the resolving power You will know you went too far if you notice obvious noise and arti facts which will be clear because structures will be shown which clearly cannot be present and which do not have remnants seen in the raw images If you notice such noise and artifacts cut the number of iterations in half With some experience 3 trials should be sufficient to determine the number of iterations necessary for your experimental setup and then all similar samples having identical conditions sample type lens magnification z spacing should use the same number of iterations These 3 trials can be done in a few minutes by cropping a small 64x64 section and experimenting with that section before deconvolving the whole volume March 2004 Page 216 AutoQuant Imaging Inc Manual and Tutorials Q How do I know the restored features are real A By looking at the raw and deconvolved images side by side compare to see if details are visi ble in both A structure if it is real will appear in the raw image although it will be more difficult to see due to having low contrast noise and blur Q When do artifacts appear A Artifacts such as ringing and mottling appear if the wrong parameters have been entered or if there is excessive noise Dead or hot pixels can also cause artifacts Using the Data Correc tion utilities can el
218. ly and the screen Z axis always runs along your line of sight regardless of how the dataset is cur rently oriented Best Axis When a rotation is being performed by dragging the mouse across the object the Best Axis setting determines the single axis that is closest to the mouse s direction of movement This allows you to rotate around one axis at a time without having to continually specify the explicit axis of rotation X Y Z axes This feature allows you to confine the rotation only about the enabled axis All mouse movements made to rotate the object will only rotate the object to the extent that the mouse moves along the selected axis Go to View This feature allows you to change the dataset to the XY XZ or ZY view Rotate 90 This feature allows you to rotate the view by 90 degrees around the X Y or Z Axis Oblique Menu This feature is used to manipulate the display of the Oblique Slice view Display Slice This feature toggles on and off the display of an oblique slice through the object When Display Slice is selected all other options under the Oblique menu are made active Go to Origin This feature allows you to reset the slice if you loose the slice off the edge of the object It sets the oblique origin to the last selected orthogonal slice intersection point March 2004 Page 76 AutoQuant Imaging Inc Manual and Tutorials Fix Plane This feature confines the position and orientation of the oblique
219. m display If the Slice Viewer for other views for example XZ ZY is displayed Auto Visualize will auto matically draw the current line in the other optical views This allows you to get a better feel for where the line you are drawing actually is located When drawing the line and cycling through slices the line in the other views will also move Note To measure angles Piecewise Line must be selected in the Line Mode frame Statistics This function provides you with specific statistical information about a dataset The Statistical Information displayed will be the following Min Intensity Max Intensity Total Number of Vox els Sum of Voxel Intensities Average Intensity Standard Deviation Variance ROI Region Of Interest area ROI perimeter and a Histogram of ROI of Current View You can obtain statistical information about the dataset in any view except for Montage view and Movie view 1 Open the TobaMC tif dataset from the Multi Channel folder 2 Under the Analysis menu choose Measurement and select Statistics or click on the Statis tics icon A Statistics dialog box will appear The Statistics box displays various Statistics which have been calculated from the active view The Statistics displayed will depend on what Projection type was chosen whether the image is in a Slice Viewer or 1f the image was Segmented If the image is in a Slice Viewer and or Segmented in addition to all the statistical information
220. may double click within the image to make the Standard Settings box appear 3 Verify the following dimensions In the Microscope Settings and PSF Dimensions frame In the Modality box verify that Fluorescence is selected In the Lens NA field verify the value is 0 7 In the Refractive Index field verify the selection is Oil 1 515 In the Image frame The Width value is 90 pixels and 32 724 microns The Height value is 80 pixels and 28 56 microns The Depth value is 110 pixels and 44 microns The X Spacing is 0 3636 The Y Spacing is 0 357 The Z Spacing is 0 4 In the Probe frame the Probe may be left blank Verify for Channel 1 the wavelength is 540nm Channel 2 and Channel 3 will remain blank 4 Click OK on the Standard Settings box 5 From the Deconvolution menu select Inverse Filter The Inverse Filter Parameters box will appear 6 The Optics Settings frame contains the dataset s Width Height Depth and Spacings Ver ify that they are the same as listed above 7 Leave the Spherical Aberrations checkbox unchecked If your dataset has spherical aber rations select this box then click the Detect button AutoDeblur will then detect the spherical aberrations so that they can be corrected when running the Inverse Filter algorithm 8 Select Medium from the Noise Level drop down menu 9 The Phase Content Expected should be used if the specimen is a Transmitted Light Bright field dataset and exhibits sign
221. me extent by averaging over several microspheres and or frames This is time consuming and effort intensive Second photobleaching of the microspheres limits the strength of the obtainable signal Third the actual PSF changes when the microsphere sample is removed and the biological speci men is inserted Fourth the PSF measurement must be done for each sample collection session and for every optical configuration used This is because the PSF is a function of the refractive index of the sample or medium and its nonhomogeneity Gibson and Lanni 1991 Finally the PSF may vary spatially especially along the optic axis All of the above complications are compounded with confocal microscopy where light levels are especially low and where the dependency of the PSF to the sample refractive index is especially strong Visser Oud et al 1992 March 2004 Page 32 AutoQuant Imaging Inc Manual and Tutorials By using blind deconvolution methods AutoDeblur eliminates the need for Point Spread Func tion PSF measurement improving both the accuracy as well as convenience of deconvolution This is a major advantage of using this package instead of those utilizing non blind deconvolution methods Another clear practical advantage of AutoDeblur is that it inherently reduces noise in the image especially in severely noisy cases This is partly because its mathematical foundations have quan tum photon noise as a fundamental assumption
222. mple with a darkfield dataset that was scanned well above and well below the sample its Surface Slice projection will have a dark slice projected for the Top view and Bottom view of the Surface Slice projection Note See Tutorials for Visualization Menu Items for an explanation of these projections Rotation Menu Free This feature allows you to view unconstrained rotations of the object An unconstrained rotation is produced when the object is rotated about multiple axes simultaneously your object will rotate in any direction your mouse moves Object This feature sets the object up to rotate around one of its three axes You may specify the rotation for the object axis by selecting Object and choosing the Axis of rotation X Y Z axis from the Rotation menu Once the axis is selected the object will only rotate along that axis The object axes are in relation to the dataset object itself that is the object X Y and Z axes always run along the width height and optical sections of the dataset respectively regardless of how the dataset is currently oriented March 2004 Page 75 AutoQuant Imaging Inc Manual and Tutorials Screen This feature constrains the rotations to the screen axis selected Under the Rotation menu you would select Screen and choose the desired axis The screen axes are in relation to your display that 1s the screen X and Y axes always run along the width and height of the screen respective
223. mplied 21 Wavelength 37 79 Wavelet PreProcessing Object Counting Tracking 204 Well capacity 39 What is Blind Deconvolution 32 When do artifacts appear 217 When should I use optical density or attenuation correc tion 217 Where to Save box 151 Whole Volume 81 88 89 92 Why won t my images load into Photoshop 218 Wiener Inverse Filtering 33 Wiener Regularization 128 Window 213 Window List 213 Wire frame Isosurface 99 Writeable CD ROMs 16 X X Spacing 63 XY Brush Size Object Counting Tracking 203 XY Guardband 139 XY Guardband Size 139 XZ YZ Slice Deconvolution 159 Y Y Spacing 63 Zero Pad 112 Z Montaging 138 Zoom 68 86 Manual Alignment 122 Zoom Step Manual Alignment 122 Z Spacing 63 March 2004 Page 238
224. n a processing sequence When you choose Selected as your option March 2004 Page 150 AutoQuant Imaging Inc Manual and Tutorials for Iteration s to Save you may specify the first or the Start iteration the End iteration and the Step of iterations the number of iterations to be skipped when saving the processed image 2D Deconvolution Results x Results Channels Save Where to Save HA Tutorial Data Widefield File Name Prefix colenese 1 m Data to Save Image C PSF Both m lteration s to Save Curent All C Selected Start End Step La aa a Save Close Cancel Help 2 For the Where to Save frame you may use the default location or click on the Browse but ton and navigate to the desired location 3 You may use the name already in the File Name Prefix box or change the name as desired For this example use colencsc 1 as the File Name 4 In the Data to Save frame the Image selection should be enabled 5 In the Iteration s to Save frame enable Selected For the Start value enter 15 for the End value enter 30 and for the Step value enter 15 This will Save the 15th and the 30th iteration when the Save button is clicked 6 Press the Save button A dialog box will appear with the Prefix File Name chosen above and the iterations to be saved as the suffix 1 e colencsc_15 30 tif 7 Press the Close button Close all datasets before co
225. n and editing the settings so that they do 3 In the Deconvolution Methods frame verify that Adaptive PSF Blind is checked In the Optics Settings frame verify the following On the Microscope frame In the Numerical Aperture field verify the value is 1 4 In the Refractive Index field verify the selection is Oil 1 515 In the Modality box verify that Fluorescence is selected In the Emissive Wavelength frame the wavelength of the dye used for each channel is displayed The dye name next to Probe indicates which dye name was entered last Verify the following Wavelength red nm None Wavelength green nm 520 FITC Wavelength blue nm 456 DAPI The Width value is 90 pixels and 6 3 microns The Height value is 120 pixels and 8 4 microns The Depth value is 49 pixels and 7 35 microns The Spacings microns The X Spacing is 0 07 The Y Spacing is 0 07 The Z Spacing is 0 15 On the Deconvolution Settings frame In the Total Iterations field enter a value of 10 In the Save Interval field enter a value of 10 This will perform 10 iterations of deblurring saving the 10 iteration March 2004 Page 142 AutoQuant Imaging Inc Manual and Tutorials 4 Verify that the Noise Level field is set to Low Verify that the Faster Processing Reduced Resolution box is unchecked Verify that the Continue from Previous Result box is unchecked if Deconvolution has not already been run this option will be dis
226. n in which the filename and type can be entered to save the dataset View Current Statistics Clicking the View Current Statistics button will open a new dialog Object Counting Results with the statistics for the current volume The statistics displayed are based on the defining characteris tics set up in the Select Object Classes procedure March 2004 Page 206 AutoQuant Imaging Inc Manual and Tutorials View All Statistics Clicking the View All Statistics button will open a new dialog Object Counting Results with the statistics for all volumes The statistics displayed are based on the defining characteristics set up in the Select Object Classes procedure Object Counting Results The Object Counting Results dialog contains the statistics on each object found including all of the defined object classes as well objects which do not fit into any of the defined classes These objects will be classified as Invalid Change Type Clicking this button allows the user to change an object from being classified as one type to another For instance if there are three classes of objects defined for the purpose of this example the classes are named A B and C and an object is assigned to class A but should be assigned to class B click on the desired object then click on Class B in the Change Type menu Click on the Change Type button That object will now be classified as Class A and will take on the color of that class Export Fea
227. n is drawn out you may then specify the desired rotation angle in the Rotation Off set edit box The ROI rectangle drawn in the image window will also be rotated to continue to show the region that is going to be cropped If after specifying the rotation angle you adjust the cropping coordinates by updating the text box entries then the ROI will be moved accordingly in the image window and the rotation offset will be maintained If however you click inside the image window and re specify the cropping coordinates by dragging a new region then the rota tion offset will be reset to 0 March 2004 Page 131 AutoQuant Imaging Inc Manual and Tutorials Adding Files Files may be added to the Apply To list in two ways First you may click Select Data which opens a dialog box that lists available datasets Second since it may be necessary to operate on more files than can reasonably be opened you may drag and drop files from the Windows Explorer directly into the Apply To list Removing Files To removing files select the file you wish to remove from the Apply To list and click the red X button to the right of the list box Please close all views before going to the next tutorial March 2004 Page 132 AutoQuant Imaging Inc Manual and Tutorials Tutorials for Deconvolution Menu Items Deconvolution Settings Note This chapter applies to users of AutoDeblur only Before you are able to decon
228. nd Tutorials 7 Select an object that is completely detached from the rest of the image and is round then click on that object with the mouse Once selected the object will change color Repeat this step for as many objects as can be located in the 3D Viewer To deselect an object that was mistakenly selected click on it again 8 In the Feature Selection frame click Deselect All then under 2D Features click on Area and Circularity 9 Click on the Calculate Statistics button The Feature Display frame will be populated with each selected object s location and statistics on each figure that was selected in the Features Selection frame in this case the Area and Circularity of each object 10 Click on Save Training Sets A Directory to Save Training Set dialog will appear Ensure that it is set to the Object Counting Folder then type trainingsetl into the file name textbox Click Save Click on Finish this will bring you back to the Object Classes for Counting dialog 11 In the Count Objects dialog ensure that the Separate Touching Objects box is unchecked 12 Click on the All Volumes button in the Count Objects frame This will count all of the objects in all opened volumes coloring each found object with a different color or shade of color 13 Click on the View All Statistics button This will display the location and pertinent statis tics in this case the Area and Circularity of each object found to match the
229. nditions In setting the exposure time when using transmit ted light brightfield first try the same exposure time that was used in collecting the optical sections during the axial scan Adjust the exposure time to ensure the conditions described in Fig ure C and Figure D When using fluorescence since the drop of dye will likely fluoresce at a dif ferent intensity than the biological sample it is especially important to carefully adjust the exposure time to ensure the conditions described in Figure C and Figure D To emphasize it is important that the maximum gray value in the image is at about 75 85 of the well capacity of the camera and that there are no saturated pixels in the image Carefully record all exposure times and file names for the bias and flatfield images since you will need them later RS 170 Cameras The bias and flatfield frame collection procedure needs to be repeated every day and for every dataset collection session This process is used to compensate for potential misalignments in the optics which will drift from day to day and to compensate for dust that may settle in the optical train which will change from day to day Collect one bias frame and one flat field frame The bias frame is taken with light to the camera blocked This may be accomplished by shifting the dichroic mirror filter set by turning off the trans illumination lamp if transmitted light bright field is used or by shuttering the excitation lam
230. ndows browser will open Browse to the desired file and click Open The selected file will be added to the workspace Select All This button is not active in this window OK Cancel To add a channel to the composite select that file by highlighting it then click the OK button Only one channel can be selected This will bring you back to the Import Channel dialog with the selected channel loaded and ready for channel specification To close the Available Data Sets window without adding a channel to the Import Channel dialog click the Cancel button March 2004 Page 60 AutoQuant Imaging Inc Manual and Tutorials Channel Specification In the Channel Specification section of the Import Channel dialog you can assign a color probe wavelength to each channel Click the radio button next to a selection this will activate a drop down menu that contains the color probe wavelength projections available for that option Click on the arrow next to the selected method Colormap RGB Color Composite depending on the dataset loaded one or the other will be available Fluorescent Probes Emissive Wavelength and select the desired color probe wavelength Clicking Cancel anytime before clicking OK will close the Import Channel window without affecting any changes Clicking OK will close the Import Channel window and the rendering in the Generate Datasets will update with the chosen channel displayed in the selected color probe wavelength Rem
231. ness of the scan selected as described earlier divided by the chosen axial distance between slices If you have limited disk storage space you may not be able to follow the above rule of thumb In that case you may set your axial distance between slices equal to a number larger than the depth of field AutoDeblur will still work in that case However as a trade off you can expect to experience some degrada tion in image quality The extent of the degradation will depend upon the axial sampling distance chosen relative to the DOF Deblurring is best carried out when the datasets are seemingly oversampled In other words best results are achieved when the pixel or optical section spacing is finer than what would be used normally without deblurring In plane deblurring is most successful when the sample spacing is finer than 1 2 the Rayleigh width If in plane deblurring is desired sample at 0 1 um or finer when the 1 2 Rayleigh width is 0 25 um Axial deblurring is substantial with widefield optics regardless of the z sampling The rule of thumb for confocal datasets without deblurring is to have the optical sections spaced according to the confocal spot size along z which is the resolu tion element along the z direction calculated by the following equation Webb et al 1995 _ 1440 AZ s where A is the wavelength in microns um y is the refractive index of the immersion medium and NA is the numerical aperture of the objective len
232. ng an image and Sizing zooming an image are different functions 1 Navigate to the Widefield folder and open FitcDapi_crop tif March 2004 Page 112 AutoQuant Imaging Inc Manual and Tutorials 2 From the PreProcessing menu select Resize The Resize Image dialog box appears This dialog box allows you to either specify a new Voxel Size a new Image Size or a new Resize Fac tor You also have the option of choosing Linear or Ideal as the method employed in the Resize function As one parameter is changed the others are automatically updated to preserve the true Image size Image Size For the Image Size Width enter a value of 250 For the Image Size Height enter a value of 250 For the Image Size Depth enter a value of 100 Voxel Size Verify the Voxel Size Width is 0 108 Verify the Voxel Size Height is 0 144 Verify the Voxel Size Depth is 0 196 Resize Factor Verify the X Resize Factor is 2 77777 Verify the Y Resize Factor is 2 083333 Verify the Z Resize Factor is 2 040816 Resize Methods Select Linear 3 Select OK A progress bar for the resizing of the image will be displayed When com pleted an XY Max projection of the newly resized dataset will be created Conversely if a dataset is too large to view easily the volume can be resized to a smaller three dimensional volume Please close all views before continuing on to the next section Optical Density Correction This feature is used to
233. not close the Pollen deb dataset Please continue on to the next section Nearest Neighbor No Neighbor Use with Widefield Fluorescence and Transmitted Light Brightfield datasets only The Nearest Neighbor algorithm is the fastest algorithm available It works by deconvolving one image slice at a time As a trade off in order to achieve this speed it is less accurate than either the Blind Deconvolution or the Inverse Filter It should be used in cases where speed is most impor tant Typical processing times are less than 1 second for a 256x256 single image slice and less than 1 minute for a 256x256x32 3D dataset Pentium III 450 Mhz AutoDeblur contains two methods for running the Nearest Neighbor deconvolution on a dataset One method is called Processing Stack and will run the specified deconvolution on the entire image stack The other method is called Processing Current Slice and is run while viewing one slice of an XY Slice Viewer of the image stack Processing Current Slice 1 Click on the Pollen deb dataset to make it the active view From the Visualization Menu select Slice Viewer The slice currently displayed will be the slice processed March 2004 Page 156 AutoQuant Imaging Inc Manual and Tutorials 2 From the Deconvolution menu select Nearest Neighbors and click on Processing Current Slice The Nearest No Neighbor Slice Operation dialog box will appear The Haze Removal Fac tor the Z Kernel Width and the No Neigh
234. not using spaces or special characters The dataset file can be saved as any of the following types AutoDeblur File deb AutoQuant s AutoDeblur format that uses an accompanying hdr file which contains the settings and other information about the AutoDeblur file AutoVisualize File avz AutoQuant s Auto Visualize format that uses an accompany ing hdr file which contains the settings and other information about the Auto Visualize file 8 bit character data This is a raw binary file type with 8 bits per pixel March 2004 Page 58 AutoQuant Imaging Inc Manual and Tutorials 12 bit data This is a raw binary file type with 12 bits per pixel Many cooled CCD cameras produce datasets stored in this form 12 bit swapped for UNIX This is a byte swapped format originating from a UNIX computer 16 bit data This is a raw binary file type with 16 bits per pixel Many cooled CCD cameras produce datasets stored in this form 16 bit swapped This is a byte swapped format originating from a UNIX computer 32 bit float This is a raw binary file type with 32 bits per pixel This allows you to store intensity values as real numbers with a precision of 6 or 7 significant digits TIFF tif Tagged Image File Format This is a common file type for standard image file format STK stk This is the Metamorph file format It is a proprietary format of Universal Imaging
235. ns Guidelines The default for this setting 1s On This option reduces the amount of RAM required by the deconvolution application It should be turned Off only if the deconvolution application is producing rigid box like artifacts in your dataset Z Montage The Z Montage option allows the deconvolution application to break the dataset into sub volumes along the Z dimensions The default for this option is unchecked Dynamic Subvolumes The Dynamic Subvolumes selection allows the deconvolution application to subdivide the dataset into the largest size the processing computer s RAM can handle The advantage of larger subdivi sions of data being processed is the increase in processing speed and therefore a decrease in the amount of time it takes to deblur a dataset Subvolume overlap Pixels The Subvolume overlap setting determines the number of pixels that the montaged subvolumes will overlap The possible values are integers from 0 to N 2 where N is the width or height of the XY field in pixels whichever is smaller Guidelines An overlap of 10 or 25 pixels usually works best If the result of the deconvolution contains artifacts having rigid lines edges or an obvious grid structure start with a value of 10 If doing so reduces the problem but does not eliminate it then increase this number again Overlap ping regions are deconvolved twice so making this number too large e g 100 will increase the deconvolution time XY
236. nt of the bad pixel s In this case the recommended Bad Pixel Treatment setting is Median Filter This is the most accurate means of treating bad pixels It replaces the bad pixel s intensity with the median value of its nearest neighboring pixels The Wiener Regularization setting is available because it executes much faster than the Median Filter setting but it is not as accurate as the Median Filter setting 10 Click OK to begin the data correction of the Slow Scan Cooled CCD dataset Data correc tion will take approximately 1 2 minutes depending upon your machine 11 Once the image has been corrected AutoDeblur will display the result in the viewing win dow Close all views before continuing on to the next section High Speed CCD The High Speed CCD selection under Data Correction is for datasets that were collected from a standard Video camera an intensified CCD camera a standard digital camera or any other camera besides the Slow Scan Cooled CCD camera There are two tabs the Files tab and the Parameters tab 1 Navigate to the Data Correction folder under the Tutorial Data directory Set the Files of type to All Files Open the Strfish tif dataset Create the ZY view of the dataset 2 From the PreProcessing menu select Data Correction and click on High Speed CCD The High Speed CCD Data Correction box will appear 3 Within the Files tab load the Flat Field file tif by clicking the top Load button and nav iga
237. ntinuing on to the next section Time Lapse Images You also have the option of selecting a region of interest and applying the region of interest to only part of a time lapse dataset March 2004 Page 151 AutoQuant Imaging Inc Manual and Tutorials 1 Click on the Open File icon browse to the Time Lapse folder and open Image_T001_S001_7001_C01 A message will appear saying A sequence of files associated with the selected file is detected Do you wish to load the entire sequence Select Yes 2 The Select Pattern dialog box will appear Select the pattern where the follows the letter T This instructs AutoDeblur where the time series indicator is in the file name 3 From the Deconvolution menu select 2D Blind Deconvolution The 2D Deconvolution dialog box will appear with the Standard tab displayed 4 In the Data to deblur frame select the Multiple Frames option 5 To set the range of images you would like to process select the Frames tab and in the Mul tiple Frames frame select Frame range Enter the desired starting Frame image number in the Start Slice box and enter the desired ending Frame number image number in the End Slice box 6 Click OK A Save Multi Frame Deblur box will appear You may use the default name for the resulting dataset or you may type in a name for your resulting dataset 7 Click the Save button A 2D Deconvolution will run on your dataset and upon completion you will be prompted
238. o Visualize only This function allows various mathematical operations to be calculated using two volumes The volumes must be in the AutoVisualize file type and must have the same dimensions The avail able Image Operations are Addition Subtraction Multiplication Division AND OR and XOR If Subtraction is selected the Second Image will be subtracted from the First Image If Division is selected the First Image will be divided by the Second Image The two original files will remain intact and a new data file will be created with the processed results To use the AND OR and XOR one of the datasets must be optically sliced in a Slice Viewer projection in the XY view and segmented 1 Navigate to the Widefield directory and open the Pollen deb dataset Generate an XZ Max projection 2 From the Widefield directory open the PollenPlus5 deb dataset 3 From the Analysis menu select Image Algebra When the Image Algebra box appears make PollenPlus5 deb the First Image and Pollen deb the Second Image The Scale for both the First and Second Image will be left at 1 This is the default value The Constant Value field will be inactive Note To add a Constant Value to an Image select None for the Second Image and enter a number in the Constant Value field The Constant Value is for intensity offset Every pixel s intensity will be either decreased by entering a negative number or increased by entering a positive number by the Const
239. o associate with an object class highlight that object class in the menu then click on the objects within the 3D Viewer that best represent the physical attributes of that class As objects are selected in the 3D Viewer they will become colored To deselect a selected object click on that object again To select objects for a new object class click on the new object class in the menu and click on the objects within the 3D Viewer that best represent the physical attributes of that class Repeat this for each object class that has been defined Delete Entry This button will delete an object class from the menu The Deselect All button must be pressed before clicking the Delete Entry button Deselect All This button will deselect all objects for all object classes Feature Selection In the Feature Selection frame the pertinent features those that differentiate an object class are selected and calculated Select All This button selects all of the available features By default all features are selected so this button is disabled until one of the features becomes deselected Deselect All This button will deselect all selected features Calculate Statistics This button will calculate statistics for each of the chosen features 2D Features This lists all of the available 2D features For this section it is imperative that the image information is correct in order to accurately calculate statistics 3D Features Th
240. o restore your dataset The Iterative process will yield better results whereas the Inverse Filter will yield satis factory results in less time DIC Image Settings Slice Selection In this section select the slice s to deconvolve Choose from All Slices Current Slice or Slice Range If Slice Range is chosen you must enter a range into the From and To text boxes Image Characteristics In this section input the data about your dataset to attain the best restoration results possible Shear Angle This is the angle at which the light in your acquisition equipment meets your object Select the proper angle for your equipment and the shadow box to the right of the drop down menu will mimic the lighting of your dataset Image Noise Select the image noise level that best matches your dataset Low Medium or High March 2004 Page 159 AutoQuant Imaging Inc Manual and Tutorials Channel to Deconvolve Select the channel to restore If you are restoring a grayscale image the only available option is Intensity if you are restoring a multi channel image select the channel to restore Advanced Settings Clicking the Advanced button will open the Expert Settings dialog Only change these settings if you are experienced with the DIC Restoration feature Otherwise use the default settings 1 Navigate to the DIC folder and open test tif 2 From the Deconvolution menu select DIC Restoration 3 In the Restoration Method se
241. o the Noise Value box Noise Value This box displays the noise value that is the default for the selected Noise Level Or if Other is chosen for the Noise Level then the Noise Value must be entered in manually Use Recommended Expert Settings It is recommended that you use the Use Recommended Expert Settings option As a general rule DO NOT MODIFY THE EXPERT SETTINGS until you have attained expertise in using the deconvolution application Expert settings should not be adjusted from their original default settings except in rare cases Do not change them casually All adjustments should be tested on a small sub field of the dataset e g 64 x 64 x 32 If you disable the Use Recommended Expert Settings uncheck the check box the Go to Expert Settings button becomes active Output Settings This field allows you to select the format in which the deconvolution application will save the deconvolution results The options are AutoDeblur File deb 8 bit character data TIFF 16 bit STK File SEQ File and TIFF Sequence 16 bit Guidelines The TIFF format allows the deconvolution results to be easily imported into other software The TIFF format uses no compression and is an 8 bit format March 2004 Page 136 AutoQuant Imaging Inc Manual and Tutorials Expert Settings Note This section applies to users of AutoDeblur only This tutorial continues with the FitcDapi_crop tif dataset from the previous Standard Settings s
242. ocal dataset It is used for applications where improving the resolving power is desired only along the optical axis This is a typical scenario for confocal dataset collection where empty lateral magnification is often avoided the X and Y dimensions show no distortion or haze Typical processing times are in a range from 2 to 5 minutes with a 256x256x32 dataset Pentium II 450 Mhz 1 Open the original Neuron_crop1 deb dataset from the Confocal folder 2 From the Deconvolution menu select Axial Blind Deconvolution The Axial Blind Decon volution box will appear The Use Power Acceleration is automatically selected This accelerates the algorithm and requires less iterations The number of Iterations is set to 10 Note It is recommended that you do not select the Use Power Acceleration feature if your dataset has a high noise to signal ratio 3 Click Start AutoDeblur will execute a one dimensional deconvolution of the Neuron_crop1 deb dataset A status box will appear indicating the progression 4 The deconvolution results will appear on the screen upon completion as a new untitled dataset 5 Generate an XZ View by clicking on the XZ view icon on the Toolbar 6 Generate a Slice Viewer of the Untitled1 deb XZ Max projection by clicking on the Slice Viewer icon on the Toolbar The median slice number approximately 32 will be displayed Com pare the axial deblurred result to the raw dataset XZ ZY Slice Deconvolution
243. of the control panel Channel 1 Channel 2 Channel 3 These controls will threshold the dataset If it is a single channel black and white dataset only channel 1 will be active If it is a multi channel dataset then the channels can be thresholded indi vidually or simultaneously by selecting the Link checkbox If the Link checkbox is selected then the threshold values will be linked with the threshold values in the Display tab Alpha This feature allows you to specify the Alpha value to be applied to Alpha projections displayed in the 3D Viewer Height Map Tab Display Options Frame Bin Factor Binning an image combines two or more intensities into one effectively decreasing the data which slightly decreases the detail of the image while speeding up the rotation and recalibration of the data thresholding adjusting the gamma etc A bin factor of 1 uses all of the available data whereas a bin factor of 10 uses the least amount of available data Select the bin factor either by entering in the desired bin factor 1 10 then clicking the Apply button or using the up and down arrows to scroll to the desired bin factor and then clicking the Apply button Height Factor The height factor controls the degree to which the height factor is displayed The higher the factor the more dramatically the difference between low and high intensities will be shown Either use the up and down arrows or enter a number between 0 1 and 1 0 in the tex
244. oints Control Panel The Control Panel allows you to access all features of the 3D Viewer It is recommended that the Control Panel not be used to make adjustments until expertise has been acquired in using the 3D Viewer Display Tab The Display tab allows you to specify from a choice of several parameters that will affect the viewing mode of the image Rendering View frame Rendering This feature allows you to select how your dataset is displayed Volume Projection Software 2D Textures and 3D Textures This feature displays a single 2D projection of the 3D volume as seen from your perspective at the object s selected orientation The Volume Projection Software will work slower than the other two options but will offer a higher resolution of the dataset The 2D Textures and 3D Textures projec tions require the proper video cards see page 16 for the recommended video cards and will ren der and rotate considerably quicker than the Volume Projection Software option The 3D Textures projection provides a more seamless rotation of your dataset than the 2D Textures removing some of the artefacts Orthogonal Slices This feature displays three single orthogonal planes oriented to the object s XY XZ ZY perspec tives These planes may be adjusted by holding down the CTRL key left clicking on a plane and dragging while still holding down the CTRL key March 2004 Page 84 AutoQuant Imaging Inc Manual and Tutorials
245. om the PreProcessing menu choose Select Region and click on Rect angle This is the default Select Region tool 3 Move your cursor over the dataset s region of interest Starting in the upper left corner click and hold the left mouse button while dragging the cursor This will create a rectangle shaped dotted line box around your region of interest If you need to further adjust the size of the rectan gle left click on one of the sides of the dotted rectangle region and drag the side inwards or out wards to resize as desired 4 To erase the dotted shape left click anywhere on the image outside of the dotted shape region Note Do not close Neuron deb Please continue on to the next section Crop This function takes a selected region of an image see Select Region above and creates a new document based on whatever pixels fall inside that selected region Crop the Stack 1 If the Neuron deb file is not open from the previous tutorial select the Confocal folder from the Tutorial data directory Select the Neuron deb dataset and open it March 2004 Page 108 AutoQuant Imaging Inc Manual and Tutorials Neuron deb 1009 2 Start in upper left corner of the view illustrated above to select a region 3 Press the left mouse button down and while still holding the left mouse button down drag the mouse to form a square similar to the one demonstrated in the above illustration Once the square is formed relea
246. omes progressively dimmer as you penetrate deeper into the sample Your dataset should be in a XZ View and the need for correction will be indicated by having a bright top and dark bottom Q When do I check Phase content expected A This is mainly used for brightfield samples which have not been stained and are viewed by closing down the condenser aperture on the microscope This can create for certain types of trans mitted light brightfield samples a phase character These types of samples cause a refraction of light passing through them rather than a simple absorption of light This type of sample is recog March 2004 Page 217 AutoQuant Imaging Inc Manual and Tutorials nized when 1 it is a transmitted light brightfield dataset and 2 you see regions in the image which are brighter than the background surrounding the sample By checking Phase Content Expected fine detail in the specimen will be preserved Q How do I deconvolve my dataset faster A First try the Inverse Filter under the Deconvolution menu This usually provides results in a few minutes It only works with widefield and not with confocal Secondly experiment with a cropped 64x64 section before deconvolving the whole volume Also set the Deconvolution Per formance to Best Medium Fast Finally make sure that you have maximized the amount of free memory on your computer Q How is blind deconvolution different from nearest neighbors and invers
247. on Cancel This feature will stop the deconvolution process and will not open a new dataset When you click on Cancel you will receive a message saying Do you wish to stop this process Clicking Yes will stop the process and return to the original dataset Clicking No will resume the deconvolution Show Deconvolution Preview Checking this feature will show a small preview of the dataset as it is being deconvolved and you will be able to view the improvement in the image with each iteration The default for this feature is unchecked Close all views before continuing on to the next section 2D Blind Deconvolution AutoDeblur s 2D Blind Deconvolution allows you to apply the Blind Adaptive Deconvolution to a single two dimensional image the image can be Fluorescence widefield Laser Scanning Confocal Brightfield Transmitted Light Spinning Disk Scanning Confocal and Two Photon Fluorescence without requiring previous knowledge of the microscope parameters or the image parameters The 2D Deconvolution algorithm is also capable of improving the resolution of an image for the restoration of features at a sub pixel resolution level You may process multi frame time series image sets individual color channels or intensity images The 2D algorithm is able to suppress noise while retaining quantitative accuracy total number of photons in the image thus allowing you the ability to make valid quantitative measurements Open an Image 1
248. oncentration and PSF that provide this maximum value solution This solution then in effect becomes our recon structed 3D probe concentration and PSF This optimization model is based on an assumption that the noisy camera data follow a Poisson random signal model This assumption is physically justi fied assuming a quantum photon limited photo detector which is certainly the case in many situ ations especially for very noisy low light level situations Thus AutoDeblur has a sound mathematical basis which makes it statistically optimal Partly due to this the algorithm inher ently reduces noise in the dataset Additionally it incorporates numerous improvements and valu able features March 2004 Page 34 AutoQuant Imaging Inc Manual and Tutorials Guidelines for Collecting 3D Image Datasets Overview For transmitted light brightfield and widefield fluorescence microscopy the main considerations are the following Collection of the optical sections Collection of bias frames Collection of the flat field frame Vibration control It is important to follow the guidelines outlined in the following sections The adage of garbage in garbage out is true for following these guidelines Even though AutoDeblur is relatively robust against non adherence to any of these guidelines with each guideline that is not followed and depending upon how severely the guideline is broken the microscopist increases his her chanc
249. ontinues with the Neuron deb dataset from the Crop tutorial above 1 From the PreProcessing menu select Extend Slices The Extend Image box will appear You have the option of extending the data by using false slices or by using the Zero Pad option which when selected gives you the ability to add blank slices to the Top and Bottom of your sam ple devoid of any information The Extend Depth indicates how many slices you want to add to the top and bottom of your sam ple In the Extend Depth box enter 4 for the Top and Bottom field values The new Depth value will be displayed in the New Dimension frame Leave the Zero Pad box unchecked 2 Click OK The extended dataset will be displayed Click on the XZ view icon and notice the difference between the real data slices and the false slices just generated 3 Close all views before going on to the next tutorial Resize This function allows you to change the overall size of an image There are two methods for doing so the Linear and the Ideal method The Ideal method is recommended however the Linear method is somewhat faster than the Ideal method Resizing a dataset resamples the data to produce a new stack of optical sections with dimensions different than those of the original stack unlike changing the size of the image on the screen This can be useful for reducing data storage requirements reducing pixelation blockiness in small datasets and improving rendering Note Resizi
250. opening the NA as far as possible For direct eye viewing on the other hand it is best to eliminate scattered and stray light in order to improve contrast and this implies closing the NA to a point just below the NA of the objective lens DO NOT DO THIS As a rule of thumb it is recommended to simply open your condenser NA as far as 1t will go If doing so makes viewing the sample overly difficult then lower the con denser NA until it is at about 1 2 20 higher of the objective lens NA If you are using an oil objective lens having an NA that is greater than 1 0 and using a condenser that allows usage of oil then you will need to do so i e you will need to place a drop of oil on the condenser in order to ensure this condition If you are using an oil objective lens for which the NA is higher than the highest available NA of the condenser for example a 1 4 NA objective lens and a 1 25 NA con denser then use the highest available NA that your condenser will allow and use a drop of oil on the condenser assuming your condenser allows you to use oil with it Collecting Optical Sections Setting the Top and Bottom of the Sample and of the Scan You need to select the top and bottom slice of the sample By performing a through focus opera tion with either direct eye or video camera viewing you should judge where the top and bottom of the sample are Note The top and bottom of the scan are not necessarily the same as the top and bo
251. opy Journal of the Optical Society of America A 10 5 1078 1085 Kasten F H 1993 Introduction to Fluorescent Probes Properties History and Applications Fluorescent Probes for Biological Function of Living Cells A Practical Guide Academic Press London in press Kimura S and C Munakata 1990 Dependence of 3 D Optical Transfer Functions on the Pin hole Radius in a Fluorescent Confocal Optical Microscope Applied Optics 29 20 3007 3011 Krishnamurthi V Y Liu T J Holmes B Roysam and J N Turner 1992 Blind Deconvolu tion of 2D and 3D Fluorescent Micrographs Biomedical Image Processing II and Three Dimen sional Microscopy San Jose SPIE 1660 95 102 Krishnamurthi V J N Turner Y Liu and T J Holmes 1994 Blind Deconvolution for Fluo rescence Microscopy by Maximum Likelihood Estimation Applied Optics in review Lalush D S and M W Tsui 1992 Simulation Evaluation of Gibbs Prior Distributions for Use in Maximum A Posteriori SPECT Reconstructions IEEE Transactions on Medical Imaging 11 2 267 275 Lange K 1990 Convergence of EM Image Reconstruction Algorithms with Gibbs Smooth ing IEEE Transactions on Medical Imaging 9 4 439 446 Llacer J and E Veklerov 1989 Feasible Images and Practical Stopping Rules for Iterative Algorithms in Emission Tomography IEEE Transactions on Medical Imaging 8 2 186 193 errata 9 1 112 1990 Lucy L B 1974
252. osition and ending position When the X Y or Z axis is selected for rotation you may rotate your dataset from and to 0 15 30 45 60 90 180 270 360 degrees Note The offsets used to generate the movie are relative to the view that is displayed at the time Apply is clicked Control Movie Points frame The controls in this group allow the creation of movies that rotate about more than one angle Start This field when checked indicates that a movie starting point is activated Mid This field when checked indicates that a view has been set as a pathway point through which the rotating object should pass at the middle of the movie End This field when checked indicates that a movie ending point is activated March 2004 Page 93 AutoQuant Imaging Inc Manual and Tutorials Each of these pathway points has an associated Set and Go to button The Set button in each case sets the current orientation of the object to be the associated pathway point The Go to button in each case reorients the object to display the currently selected position of the associated pathway point Opposite Path This feature is for any pair of pathway points There are two direct rotational paths between the pathway points By default the movie generator chooses the shortest rotational path between the two pathway points When this box is checked the movie generator will select t
253. ou can install the software onto as many machine as you would like and activate the soft ware by plugging the dongle into the machine you wish to use Transferring Moving a License For versions 9 0 and later refer to Installing Version 9 0 and Subsequent Releases in the previous section For all versions prior to 9 0 Floppy Disk License Transfers Floppy disk license transfers require a floppy disk an authorized copy of your product installed on the source PC and an unlicensed copy of your product on the target PC The transfer process does not jeopardize your license in any way and is completely secure because the floppy disk is registered to a specific PC in a specific location This ensures the license can only be transferred to the target PC you specify Example You want to transfer a license from PC 1 to PC 2 1 Ensure you meet the stipulations outlined above under Floppy Disk License Transfers 2 Start the program on PC 2 and select Kill License then select Transfer Into Computer Supply the floppy disk path The program imprints its registration on the disk 3 Remove the floppy disk from PC 2 and insert it into PC 1 4 Start the program on PC 1 and select Transfer Out of Computer Supply the floppy disk path The program reads the registration imprint file and writes a matching file to the floppy dec rementing the license at the source or discontinuing it if a single user license 5 Remove the floppy disk
254. ove Click this button to remove a channel from the Combine Channels dialog Select the channel s to be removed by highlighting 1t multiple channels can be selected by holding the shift key while clicking on the desired channels then click the Remove button The channel will be removed from the dialog as well as the renderings in the Generate Datasets section of the dialog Change Intensities This section contains the controls with which to change the intensities of the channels in your composite image Above the graph are numbers which correspond to the channel number Below each number is a red circle Use the mouse to click and drag the circle up or down to change the intensity of that channel moving the circle up will increase the intensity moving it down will decrease the intensity Beneath the graph are percentages which will update as the red circle is moved indicating the intensity of that channel Generate Datasets This section contains renderings of the combined channels as well as controls to create datasets from the combined channels Create 2D Dataset Click this button to create a 2D max projection of the composite The new image will be created in a new window which may appear beneath the Combine Channels dialog you may need to move the dialog to view the new image Create 3D Dataset Click this button to create a 3D max projection of the composite 1 Under the File menu select Import Multi Channel Data 2 The Combin
255. p if fluorescence is used If you are using frame averaging average as many frames as possible As a rule of thumb average 255 frames Collect a flat field frame This will be used to calculate the flat field non uniformity of your cam era A flat field can be provided in a number of ways When using transmitted light brightfield the simplest and most reliable way is to remove the sample from the microscope and grab an image of the blank stage Another way which will take more care is to take the sample extremely out of focus to the point where in effect the camera image shows a flat field see Table 1 We recommend doing this only if it is not possible to remove the sample without disrupting your experiment setup Otherwise one should avoid this method because it is more prone to errors and misjudgment For example Table 1 Figure a shows a common mistake which is quite often made This occurs when the sample is brought well out of focus but an obscure out of focus rem nant of the sample is still present Avoid this situation It is extremely important to have a flatfield image that represents a totally flat field as shown in Table 1 b When using fluorescence the most reliable way to prepare a flat field sample is to place a drop of dye onto a slide and to seal it March 2004 Page 46 AutoQuant Imaging Inc Manual and Tutorials with a coverslip Although in principle it may be possible to take the lens out of focus with
256. pecimen with the AAf heading Once a set of files has been assigned and processed the file associations will be saved in the header file and only one file association will need to be made in the FRET Image section the rest will automatically fill in upon clicking the second arrow 5 In the Use Calibration Images section associate the files with the appropriate headings as outlined in the preceding list It is advised that a region of interest be drawn in each one of the calibration sets This will speed up the processing Create the region of interest around the area in which FRET is suspected of occurring by clicking and dragging on the image creating a rectangle around the desired area If no calibration images are available proceed to the Use Cross Talk Coefficients section 6 Check the Use Automatic Alignment box 7 From the Subtract Background dropdown menu select Minimum Value 8 Select Donor and Acceptor in the Specified Cross Talk box 9 Enter tutorialsetl for the Result Name Prefix 10 Click the Calculate button Three new images will open all with the prefix tutorialset March 2004 Page 181 AutoQuant Imaging Inc Manual and Tutorials Use Cross Talk Coefficients Tutorial With the Gordon and Herman as well as the AutoQuant Imaging Inc algorithms there is an option to enter the Cross Talk Coefficients rather than loading calibration images It is still highly recommended that calibration images
257. per This will change the Gray Scale Color Map to the Copper Scale Color Map March 2004 Page 105 AutoQuant Imaging Inc Manual and Tutorials 2 Try changing the view to another Color Map selection to observe the different characteris tics Select either Hot or Cool under the Color Map selection in the View menu 3 To return to Gray Scale from the View menu select Color Map and choose Gray Scale Channels For this tutorial you may use your own data type Fluorescence Widefield Laser Scanning Confocal Brightfield Transmitted Light Spinning Disk Scanning Confocal Two Photon Fluorescence or 2 Dimensional images or you may follow along with the recommended data to use All multi channel data regardless of what microscope it came from or data type it is will be separable into its own channels Note The term Channel is used for delineating data that was collected at different wavelengths This menu item will be used for data that is imported as Multi channel data You may arbitrarily set any of the color channels to any of the emissive wavelengths collected The channel descrip tion set up below are guidelines for you with respect to which channel will correspond to what wavelength Red Channel This Channel feature normally represents the longest emissive wavelength collected for the dataset 1 From the File menu select Open or from the Toolbar click on the Open File icon 2 From the Tutorial Data directory sel
258. per Resolution Activate Sub pixel Processing This option will perform deconvolution on a sub pixel grid to achieve super resolved results It will also however result in longer processing times XY Factor If the Activate Sub pixel Processing feature has been checked the XY Factor feature will become enabled This feature allows you to select the number by which the pixels will be divided The options are 1 which is for when the Activate Sub pixel Processing feature is not activated 2 and 3 with three being the highest resolution and longest processing time PSF Section In the PSF section there are two tabs the Adaptive PSF Blind and the Non Blind The Adaptive PSF Blind tab Axial Stretch Factor This section allows you to set how much axial stretch to allow from the theoretical first guess PSF The default for Widefield datasets is 1 and for Confocal datasets the default is 3 March 2004 Page 140 AutoQuant Imaging Inc Manual and Tutorials PSF Waist The PSF Waist is the size of the narrowest part of the PSF usually measured in Airy Disc diame ters The default setting for both Widefield and Confocal is 1 Disable PSF Restraints This removes the limitations placed on the Point Spread Function PSF The Fixed PSF tab This tab contains the parameters to use with Gold s method Gaussian Width FWHM in pixels This field is based on the Noise Level found directly below If the Noise Level is set to Other then
259. performing a manual segmentation are Apply Label Apply Seed Fill Label Undo Label Highlight Labels Combine Labels Remove Label Combine Label with Automatic Segmentation Load Labels and Save Labels Apply Label This function will Label a selected region within your dataset 1 If the TobaMC tif Max Projection is not open from the previous section navigate to the Multi Channel folder and open the TobaMC tif dataset A dialog box will appear with the question The program has detected properties that indicate this dataset MAY contain an old AutoQuant Proprietary Multi channel TIFF Is this true Select Yes 2 Create a Slice Viewer of the dataset Scroll to slice 22 and select a region of interest simi lar to the one shown below using the Select Region Ellipse tool ioi xi act x Close Category New 1 y Help Apply to current slice only Apply to all slices Apply to given slice range From slice 117 to slice 32 March 2004 Page 192 AutoQuant Imaging Inc Manual and Tutorials Note The Select Region tools are as follows Rectangle Ellipse Polygon and Freehand You may select from any of these when you want to label regions of interest in a dataset 3 Under the Analysis menu select Segmentation and click on Apply Label The Label Object dialog box will appear Select Apply to given slice range and set the From slice to 17 and set the to slice to 3
260. pper and Lower Threshold limits to the defaults based on the type of object selected in the Look For drop down menu option With the Use Automatic Threshold checked the Upper and Lower Threshold controls will be disabled Upper Threshold This sets the upper threshold for the dataset Lower Threshold This sets the lower threshold for the dataset Set Threshold for All Volumes Clicking this button will set the selected thresholds for all volumes If the Upper and Lower Thresholds are changed from the default val ues this button must be clicked otherwise the default threshold settings will be used Segment Current Clicking this button will segment only the current volume Segment All Clicking this button will segment the entire dataset all volumes Close Clicking this button will close the Segmentation and Counting Expert Settings dialog and return to the Object Counting dialog Select Object Classes This frame contains the controls to create and define object classes to be counted Up to 8 differ ent classes of objects can be defined Alternatively the No Training Count All checkbox can be selected which will direct the application to count all objects with no class definitions or deter mining characteristics No Training Count All Checking this button will direct the application to count all objects with no class definitions or determining characteristics When this box is checked all other buttons in the Sele
261. pts itself to the real PSF of the microscope system which can be significantly different from the theoretical PSF and from the previously measured PSFs due to specimen and instrument variations The AutoDeblur Blind Deconvolution system is able to adapt to PSF changes within a specimen itself Thus the decon volved results are superior to those methods which utilize theoretical or previously measured PSFs For this example you may use your own data type Fluorescence Widefield Laser Scanning Confocal Brightfield Transmitted Light Spinning Disk Scanning Confocal or Two Photon Fluorescence images or you may follow along with the recommended dataset to use March 2004 Page 141 AutoQuant Imaging Inc Manual and Tutorials 1 Navigate to the Widefield folder under the Tutorial Data directory and open the dataset FitcDapi_decon tif This dataset has had the Standard Settings and the Expert Settings set up in the previous tutorial The information is contained in its header file and the header file is auto matically read when the dataset is loaded 2 From the Deconvolution menu select Start 3D Deconvolution The 3D Blind Non Blind Deconvolution box will appear Before starting a deconvolution it is a good practice to verify the Optics and Deconvolution Set tings If any of the following settings do not match what is in the Standard Settings box make the appropriate changes in the Optics Settings by clicking the Change butto
262. quential or incidental damages the above limitation may not apply to you In the event that AutoQuant is liable under implied warranty or incidental damages the period is limited to one year from date of purchase This notice will be governed by and interpreted in accordance with the laws of the State of New York in the United States of America March 2004 Page 21 AutoQuant Imaging Inc Manual and Tutorials Copyright Notice Copyright 1995 2003 by AutoQuant Imaging Inc MANUAL All rights reserved This manual may not be reproduced stored in a retrieval system or transmitted in any form by any means electronic mechanical by photocopying recording or otherwise in whole or in part without the written permission of AutoQuant Imaging Inc SOFTWARE United States copyright laws and international treaty provisions protect the AutoDeblur software package It is illegal to copy the software except as specifically allowed in the License Agreement stated below Any person found storing or using an unlicensed copy of this software is liable to be sued by AutoQuant Imaging Inc to the maximum extent permissible under applicable US and International laws and International Treaty Provisions Software License Agreement This is a legal agreement between you either an individual or an entity the end user and Lic ensee and AutoQuant Imaging Inc AQI or we the Licensor BY OPENING THE SEALED MEDIA PACKAGE or by
263. r any such termination deliver to AQI all software provided to you hereunder and copies thereof embodied in any medium 7 GOVERNING LAWS This Agreement will be governed by and interpreted in accor dance to the laws of the State of New York in the United States of America 8 LIMITATION OF LIABILITY NOTICE THE SOFTWARE IS PROVIDED ON AN AS IS BASIS WITHOUT ANY OTHER WARRANTIES OR CONDITIONS EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO WARRANTIES OF MERCHANTABLE QUALITY SATISFACTORY QUALITY MERCHANTABILITY OR FITNESS FOR A PAR TICULAR PURPOSE OR THOSE ARISING BY LAW STATUTE USAGE OF TRADE COURSE OF DEALING OR OTHERWISE SPECIFICALLY THE SOFTWARE IS NOT GUARANTEED TO WORK TO SATISFACTION ON EVERY 3D MICROSCOPY IMAGE EVEN IF THE IMAGE IS OF A TYPE THAT THE SOFTWARE IS STATED TO BE INTENDED FOR THE ENTIRE RISK AS TO THE RESULTS AND PERFORMANCE OF THE SOFTWARE IS ASSUMED BY YOU NEITHER AQI OUR DEALERS REPRESENTA TIVES LICENSORS OR SUPPLIERS SHALL HAVE ANY LIABILITY TO YOU OR ANY OTHER PERSON OR ENTITY FOR ANY INDIRECT INCIDENTAL SPECIAL OR ECO NOMIC LOSS EVEN IF WE HAVE BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES OR THEY ARE FORESEEABLE WE ARE ALSO NOT RESPONSIBLE FOR CLAIMS BY A THIRD PARTY OUR MAXIMUM AGGREGATE LIABILITY TO YOU AND THAT OF OUR DEALERS SUPPLIERS AND LICENSORS SHALL NOT EXCEED THE AMOUNT PAID BY YOU FOR THE PRODUCT THE LIMITATIONS IN THIS SECTION SHALL APPLY WHETHER OR NOT THE ALLEGED BREACH OR D
264. r instance entering 10 into the textbox will display gridlines and spacings of 10 20 30 40 and so on However when zooming in and out these will adjust for optimal display Scale Bar Checking this feature will display a scale bar which will assist in giving a perspective to the actual size of the object March 2004 Page 102 AutoQuant Imaging Inc Manual and Tutorials Help Tab This tab displays written instructions for manipulating the dataset in the 3D Viewer To rotate the object Left click on the image and drag in the desired direction To crop the volume or move an orthogonal oblique slice Hold down the Ctrl key left click on the plane slice and drag To rotate the oblique slice Hold down the Shift key left click on the oblique slice and drag To zoom the displayed image Right click on the image and drag from bottom right to top left or vice versa Image Enhancement Depending on the display of the machine running the software images may appear too dark or in other cases too bright To compensate for these differences it is possible to perform a Gamma correction on a particular view 1 Navigate to the SmallHip folder open the SmallHip avz dataset and generate an XZ view 2 From the View menu select Image Enhancement The Image Enhancement dialog box will appear 3 Check the Ignore Background box This will put the focus on the object and give a more detailed histogram of the data 4
265. r of slices along the corresponding axis March 2004 Page 88 AutoQuant Imaging Inc Manual and Tutorials In addition several predetermined rotational centers of interest may be selected and automatically calculated These centers are Manual Input This feature allows you to set the object s rotational center manually Whole Volume This feature allows you to set the object s rotational center to be the geometric center of the dataset s full volume This is the default center Subvolume This feature allows you to set the object s rotational center to be the geometric center of the cur rently selected subvolume Center of Mass This feature allows you to determine the coordinates of the object s center of mass and then sets the object s rotational center to these coordinates Ortho Slices This feature allows you to set the object s rotational center to the coordinates specified by the intersection of the three orthogonal planes as specified in the Orthogonal Slices frame Rotation Tab Rotate This feature allows you to determine whether the remaining controls on this tab will adjust the positioning of the Object or the Oblique Slice The dataset must be in slices for Oblique Slices to be active if not then only the Object may be selected Current Rotation frame Anchor This feature allows you to specify the axis about which the object rotates when the slider bar is used to rotate the
266. re allows the user to set the size of the display window The entire 3D Viewer will adjust its size to accommodate the size entered Enter the height and width of the desired display size in pixels in the appropriate text boxes Apply Clicking this button applies the changes of the Window Size March 2004 Page 98 AutoQuant Imaging Inc Manual and Tutorials Isosurface Tab It is best to threshold your image when using the Isosurface view such that the edges of your dataset have an intensity of O along all of the edges Any intensities detected along the edges will be displayed as triangles in the Isosurface viewer Appearance Frame Bin factor Binning an image combines two or more intensities into one effectively decreasing the data which slightly decreases the detail of the image while speeding up the rotation and recalibration of the data thresholding adjusting the gamma etc A bin factor of 1 uses all of the available data whereas a bin factor of 10 uses the least amount of available data Select the bin factor either by entering in the desired bin factor 1 10 then clicking the Apply button or using the up and down arrows to scroll to the desired bin factor and then clicking the Apply button Wire frame Selecting this button will display a wire frame of the triangles that create your image Deselecting this button will hide the wire frame Display Sides Selecting this button will connect the sides of the object
267. re is only available as an added plug in Contact your dealer for purchase infor mation This function displays and analyzes the overlay of two separate dyes in a multi channel dataset To use Colocalization a multi channel dataset must first be opened Image 1 Image 2 In the Image 1 and Image 2 sections the channels to be analyzed are selected and assigned a color in which to be displayed All available datasets will appear on the dropdown menus so make sure that the channels selected are the same dimensions If 2 channels with different dimensions are loaded into these dropdown menus an error message will appear stating The dimensions of this image are different from the other image Do you want to change to this new image for colocaliza tion analysis Selecting Yes will load the new image and clear the other image at which point a second channel will have to be selected Selecting No will not load the new channel and the chan nel loaded for the other image will remain loaded Next to each image dropdown menu is a dropdown menu with a color selector The channel selected in this dropdown menu will determine how the channel is displayed in the Coloc Viewer The options are Red Green and Blue Once a color is selected for one channel that color will not be available on the dropdown menu for the other channel Coloc Viewer Tools This section contains tools to manipulate the Colocalization image in the dialog el z E The zoom con
268. ready open 2 From the Segmentation menu select Load Labels navigate to the directory where you have saved your seg file Select the seg file and click on the Open button Auto Visualize will display only the Labels from the seg file on the dataset Note This operation will remove any previously unsaved labels in the current image Save Labels This feature allows you to save the Labels for a dataset in a seg file This file can then be reloaded onto a dataset at a later time 1 From the Segmentation menu select Save Labels A dialog box will appear prompting you for the file name and file location this is the location where the Label s file will be saved After entering a file name i e Triple Label segment and clicking the Save button the dataset will be saved as a seg file This file will be of the same dimensions as the raw image March 2004 Page 198 AutoQuant Imaging Inc Manual and Tutorials Save Segmentation Selected Labels This feature saves the dataset with the Labels as an image in a TIFF file format If your dataset is in a Slice Viewer Projection and you have changed either the gamma brightness or flip settings a box will appear with the question Save data as shown Selecting No will discard the gamma brightness and flip adjustments Selecting Yes will keep the gamma brightness and flip adjust ments A second box will appear with the question Do you wish to export as multiple files each of a
269. rease the threshold of the dataset click and hold on the desired Threshold button the button on the left increases the threshold the button on the right decreases the threshold until the dataset reaches the desired appearance Brightness es A To increase or decrease the brightness of the dataset click and hold on the desired Brightness button the button on the left increases the brightness the button on the right decreases the brightness until the dataset reaches the desired appearance Movie Controls The movie controls on the bottom of the 3D Viewer can be used for several uses The default use of the controls is to scroll through a 360 degree rotation March 2004 Page 83 AutoQuant Imaging Inc Manual and Tutorials along the X axis The Play button d ld h ward Step Backward buttons will move the dataset through one step of the 360 degree Ge will scroll through the entire 360 rotation The Step For rotation in either direction depending on the button clicked The Rock button will allow the dataset to rotate back and forth in the Play mode until the Stop button m is clicked once the Play button is clicked it turns into the Stop button The Scroll Bar can also be used to rotate the dataset along the X axis either by clicking along the Scroll Bar or by clicking and dragging on the pointer in the Scroll Bar If the dataset loaded is a time series dataset then these controls will move the dataset through the timep
270. rojec tions require the proper video cards see page 16 for the recommended video cards and will ren der and rotate considerably quicker than the Volume Projection Software option The 3D Textures March 2004 Page 73 AutoQuant Imaging Inc Manual and Tutorials projection provides a more seamless rotation of your dataset than the 2D Textures removing some of the artefacts See Software and Hardware Requirements Windows Systems in Chapter 2 for a list of video cards that will support the Volume Projection Hardware option Orthogonal Slices This feature displays three single orthogonal planes oriented to the object s XY XZ ZY perspec tives These planes may be adjusted by holding down the CTRL key left clicking on a plane and dragging while still holding the CTRL key Cube Surface This feature displays a cube which has on each surface a painted projection of the dataset as would be seen from directly above that surface or view The projection shown depends on the pro jection selected Isosurface This feature displays your dataset as a solid body with distinct surface and edge recognition and light reflection with quick rendering and rotation An advantage to this feature is when using the oblique slice a much clearer image is rendered as you scroll the slice through the 3D image Adjust Minimum Threshold When selecting the Isosurface projection a dialog will appear giving you the option to threshold the ima
271. ropped volume will only contain the slices specified by you 6 For this tutorial select the Apply to given slice range option and enter 18 for the From slice and enter 50 for the to slice 7 Press the Crop button A new Untitled dataset of the cropped region will appear in a Slice Viewer projection 8 You may repeat the steps above and choose Remove Region in step 7 to remove a region of interest If you desire to Crop out a different shape on each slice you should select the Apply to current slice only choice The Apply Region button must be clicked to have the current selected region take part in the Crop or Remove Region When a region is selected for cropping and the check box is checked the first highlight will be light blue Select another slice using the scroll bar at the bottom of the Slice Viewer and repeat the steps of selecting a region clicking the Apply Region button and checking the box left to the number in the list box The Clear Selected Regions button will remove the crop overlay from the regions that are cur rently selected 9 When you have chosen all of the desired cropping regions you should click the Crop but ton A cropped volume will be displayed with only the foreground image data from the high lighted regions all else will be set to background Note Do not close Neuron deb Please continue on to the next section Crop Dimensions This feature allows you to set the position and size of region o
272. s If Az is 0 5 um sample at 0 5 um or finer per slice The noise suppression mentioned above requires this type of fine sampling March 2004 Page 37 AutoQuant Imaging Inc Manual and Tutorials Setting the Exposure Gain and Offset Cooled CCD Cameras With cooled CCD cameras the exposure time is usually adjustable Consult the manufacturer s manual to determine how to change the exposure time To set the exposure time for collecting optical sections use the following procedure Focus onto a point near the center of the sample see Figure 1 Adjust the illumination light to a setting that you desire If you are using a transmitted light brightfield microscope adjust the light so that you can view the sample comfortably through the eyepiece Next try an exposure time set ting Usually if you have done this before you can start with a setting that has worked well for you in the past If you are doing this for the first time or if an approximate setting is not known start with an exposure time around 0 05 seconds Grab and view a single frame at this setting If the picture on your screen appears too dark increase the exposure time and repeat the frame grab bing and viewing procedures Be careful not to saturate make too bright any portions of the image Saturated regions can usually be identified as bright flat white regions in the picture as seen in Figure B Figure 1 1 Figure A Figure B
273. s 140 Object First Guess 1d A eas 140 Super Resolution 22 322 set sentient Aa 140 PSF SOCOM A reser arinei ii ai 140 The Adaptive PSF Blind tab oo eee ecccceeseceececeeeeeeeeeeaeeesaeceeeeceeaeecneeeeaas 140 The Fixed PSE tap tic e bes ceesk cial e aE ias 141 Start 3D Deconvolution sti ida 141 Terminate Cancel and Preview 3D Deconvolution ooococncnnnnnnnnicnnicananacacacacananano 145 Terminales tt iia ios 145 Caneel Ynienen ea e a e ee a aat iapa 145 Show Deconvolution Preview sssssssssssssessessseeseessressesseessessssesssessresstessesstessreseeesees 145 2D Blind Deconvolution A e eS 145 A E A E E E E EER 145 Setting Up a 2D Blind Deconvolution ocooccnnocccooncnnnoncnononcnonnnncnnnnncnnnnncnnnnccnnnanos 146 Selecta Regi n of tertulia ah een 146 Process an MALE ororen aE E nai Wie E E A EA 147 2D Deconvolution Results suscita saisis 148 To Further Process a Data Seti aetna en teat Ei 148 Viewing the Channels of a Processed Dataset 0 eeeesecseceeeeeeeeeneeeneeeaeeees 149 Saving a Processed Datsun ds 150 Time Lapse Uma ges ii A A ees 151 AAA A E it din etter eee 152 Inverse Filter Batch Processing 0300505 dotaciones dera ias 154 Nearest Neighbor No Neighbor A lead doS 156 Processing Cuento 156 o E E E EAE E N E E 157 Axial Blind Deconvolution isc da 158 MLN Slice DeconvGl ulin ossi a o aaee aas a eaaa 158 DIC Restoration disiras esras e 159 Restoration Method lt a A A E Bae es de AA 159 DIC Fae S
274. s dialog is launched by clicking the Calculate Statistics button and it displays the statistics of the tracked objects Only those features selected in the 2D Features 3D Features and Tracking Features will be displayed Clicking on an object within the Track Statistics dialog in the spread sheet will highlight that object in the 3D Viewer The Dropdown menu above the spreadsheet contains each of the tracks as well as an option for all Only the track s selected in this dropdown menu will be highlighted in the 3D Viewer and displayed in the spreadsheet March 2004 Page 209 AutoQuant Imaging Inc Manual and Tutorials Export Features This button will open Save As dialog in which the statis tics can be saved to a csv file Close This button will close to Track Statistics dialog 2D Features This lists all of the available 2D features For this section it is imperative that the image informa tion is correct in order to accurately calculate statistics 3D Features This lists all of the available 3D features For this section it is imperative that the image informa tion is correct in order to accurately calculate statistics Tracking Features This lists all of the available features relative to the movement of the tracked objects For this sec tion it is imperative that the image information is correct in order to accurately calculate statis tics Movie Generate Movie The Generate Movie button will open t
275. s to be aligned along the X axis along the Y axis or along both axes by checking both the X and Y axes The default setting is to align to both axes Base Slice This section is where the Base Slice the slice to which all others will be aligned is chosen The default setting is to let the application select the base slice Deselecting Select Automatically will enable the slice scroll bar with which the user will then need to scroll to the desired base slice Alternatively the user can enter the slice number into the text box to the right of the scroll bar Advanced Settings Clicking this button will open the Advanced Settings Dialog It is recommended that the default settings be used Clicking this button again with the Advanced Settings dialog showing will close the Advanced Settings dialog Noise Background Set Threshold This option sets the noise threshold for the dataset The default is set to 20 clicking the Set Threshold button allows the user to set the noise threshold to a different level Show Noise This option shows the noise on the image when the Set Threshold option has been selected The default is On Void Region Fill This function will put a frame like cover over the areas that no longer contain data After the data are aligned gaps and spaces are created at the edges This function chooses how those areas are filled in Note The Frame may have straight scalloped or irregular edges to it Black This will
276. s to the lower resolution version Correct Aspect This feature allows the visible dimensions of the object displayed in the 3D Viewer to be adjusted to take into account the pixel spacings When enabled the displayed voxels will be cubic in dimension Auto Rotate This feature enables the object to continually rotate once the object has been set in motion Display Floor This feature displays the black and white checkered floor pattern The floor is not visible when the Perspective View under the Control Panel is Off or the Volume Projection view is On March 2004 Page 81 AutoQuant Imaging Inc Manual and Tutorials Display Axes This feature will display colored bars on the axes to make it easier to identify the orientation of the object The X axis is red the Y axis is green and the Z axis is blue The axes can be labeled in the Captions Axes tab of the Control Panel Display Grid This feature will display a grid around the object in order to give a perspective of the size of the features within the object The spacings of the grid can be adjusted in the Captions Axes tab of the Control Panel Display Scale Bar This feature will display a Scale Bar in the lower right corner of the 3D Viewer which will adjust its size as the object is zoomed This is useful for giving a perspective to the size of the object and to a lesser extent the size of features within the object Logo This feature allows you to load and disp
277. s to the whole image Click on the Results tab and click the Full Image button in the Use Current Iteration Settings to Deblur frame A confirmation box will appear allowing you to confirm or change the total number of iterations to perform The number of iter ations that appears is based on where the slider was left in step 5 Click OK AutoDeblur will deconvolve the entire image using the current number of iterations and settings for each channel Viewing the Channels of a Processed Dataset A processed image may appear brighter in the region that has been processed than in the unproc essed region The 2D Blind deconvolution typically increases the dynamic range the maximum value a pixel can have becomes increased of the processed image compared to the original unprocessed image 1 Click the Channels tab You may view 1 2 or 3 of the channels simultaneously by check ing or unchecking the check boxes next to each channel March 2004 Page 149 AutoQuant Imaging Inc Manual and Tutorials 2D Deconvolution Results b x Results Channels Save Mixing lant Ki RM 330 MA G M 30030 Mf B V 30030 MY I Adjust All T PSF Original Current N m Display Controls BC Red HC Green MC Blue Apply Reset Minimum Maximum Gamma l Apply to All fo 2 a 8891 io _ MV AutoScale F Grayscale ss 2 To adjust the appearance of the processed dataset you may check the Adjust All box and u
278. saturated areas it can often recover information from within these saturated sections This is possible in situations where there are non saturated areas nearby the saturated areas The blur in the nearby non saturated region contains information about the saturated region which AutoDeblur restores The degree to which this is effective depends on the size of the saturated area and the numerical aperture of the lens Of course according to good laboratory practice it is always best to avoid saturation but this property of the algorithm makes data collection and experiment design much easier since the avoidance of saturation is now a flexible requirement and no longer an absolute requirement Q Are noisy poor signal to noise images going to be problem for AutoDeblur A AutoDeblur is able to process extremely noisy images and deliver restored image sets with very little noise Noisy images are common with a confocal microscope an intensified camera or whenever low light is used to minimize bleaching Q How many iterations should I apply How do I know I have performed sufficient itera tions What happens if I perform too many iterations A Start with the rule of thumb With the Use Recommended Expert Settings box checked for widefield and brightfield use 10 iterations for confocal two photon and spinning disk use 10 iterations If by inspecting the images you are satisfied with the resolving power stop If you want still better reso
279. se and that AQI and its licensors reserve all rights not expressly granted to you hereunder After you pay the applicable license fee and or purchase price for the SOFT WARE you will own the media on which the SOFTWARE was originally provided to you here under and on which you subsequently copy the SOFTWARE duly exercising the restrictions noted in Section 2 above AQI and its licensors shall retain ownership of all SOFTWARE and copies of the SOFTWARE or portions thereof embodied in or on such media including ownership March 2004 Page 23 AutoQuant Imaging Inc Manual and Tutorials of all copyrights mask work rights patents trademarks trade secrets and all other intellectual property rights subsisting in the SOFTWARE documentation enhancements adaptations and any modifications thereto 5 TRANSFER RESTRICTIONS You may not distribute rent sell sublicense or lease or otherwise transfer the SOFTWARE without express written permission from an authorized AQI official 6 ENFORCEMENT OF TERMS TERMINATION If you fail to fulfill any of your material obligations under this Agreement AQI and or its licensors may pursue all available legal remedies to enforce this Agreement and AQI may at any time after your default of this Agree ment terminate this Agreement and all licenses and rights granted to you under this Agreement You agree that if AQI terminates this Agreement for your default you will within thirty 30 days afte
280. se one of the scroll bars in the Mixing box to adjust the iteration number and or you may adjust the Minimum Maximum or Gamma values in the Display Controls box You may adjust the channels and the iterations individually by selecting or deselecting the Channel s of interest 3 While viewing an individual channel if you feel the specific channel appears dim in the Display Controls frame you may check that channel s Radio button and adjust the Gamma value You may also perform further processing to change the appearance of the Channels To process the image further see To Further Process a Dataset above Saving a Processed Dataset 1 In the Save tab you are able to select the Directory in the Where to Save frame This will be where the file you are about to save will be saved AutoDeblur will display the default name for the File name Prefix You can either accept this default name or create your own AutoDeblur will automatically append the File name Prefix with the slice range and the number of iterations performed in the resultant processed dataset If the Full Image was chosen then the slice range will consist of all the slices in the dataset In the Data to Save frame you can choose to save just the Image the PSF or Both Saving the PSF with the image will create a larger file by approximately 2 fold The Iteration s to Save features allows you to save the Current iteration All the iterations or only a Selected few iterations i
281. se the left mouse button The size of the above cropped area is approxi mately 100 by 100 pixels Note The cropped region s dimensions are concurrently displayed in the lower left hand corner on the main window status bar 4 From the PreProcessing menu select Crop and click on the Crop the Stack option or from the Toolbar click on the Crop the Stack icon The image will be cropped and displayed in the viewing window March 2004 Page 109 AutoQuant Imaging Inc Manual and Tutorials 5 From the File menu select Save As 6 When prompted for a file name to save the cropped image type Neuron_crop1 in the File name field and click the Save button You may close the Neuron_crop1 deb view Note Do not close Neuron deb Please continue on to the next section Slice by Slice Cropping Note This section applies only to users of Auto Visualize and this function is only available when the dataset is in a Slice Viewer projection If the Neuron deb file is not open select the Confocal folder from the Tutorial Data directory Select the Neuron deb dataset and open it 1 Create a Slice Viewer projection of the Neuron deb dataset by choosing Slice Viewer from the Visualization Menu 2 Click within the Slice Viewer projection and place your cursor in the upper left hand cor ner of the area of interest Click and hold down the left mouse button while dragging your cursor to the right and downwards A dotted line rectangle will
282. selec tion of the anaglyph color pair used in generating an anaglyph stereo image In addition you may choose to display only the left or right image of the stereo pair Angle This feature allows you to specify the angular offset between the two images of the stereo pair This number may be adjusted to optimize the 3 dimensional effect for a particular user the opti mum value for this is affected by your distance from the monitor and the distance between your eyes Stereo On This feature when checked displays the stereo view in the 3D Viewer window Apply Clicking this button applies the adjustments made within the Stereo View frame Display frame Perspective Angle This is the angular size of the 3D Viewer s field of view Reducing the angular size will produce many affects It will decrease the size of the field of view of the 3D Viewer it will reduce the 3D Viewer s visible area around the dataset and it will reduce the perspective effect on what is visi ble Conversely increasing the number increases the field size and increases the perspective effect Hardware Volume Projection Quality Provided your system has the required video card see page 16 for a list of acceptable video cards this section allows you to select the quality of the hardware projection The choices are Highest Medium and Fastest Apply Clicking this button applies the changes of the Perspective Angle of the display Window Size This featu
283. single slice You may save the Labels as a single TIFF file or as multiple TIFF slices Please close all datasets before going on to the next section Measurement Line Length This function allows you to obtain line length measurements from the dataset by drawing lines on any view Open a view you would like to draw lines on From the Analysis menu select Measure ment and click on Line Length An Intensity Profile Along Line box will appear Start by select ing from the Line Mode options Straight Line Piecewise Line or Freehand Line Left click the mouse within the view and move the cursor When using the Piecewise Line mode left clicking again will terminate the current line segment and initiate a new one The Intensity Profile Along Line box will show the data properties for the line s being drawn The distance of the line seg ment or the entire string of segments Piecewise Line are displayed at the bottom of the Intensity Profile Along Line box To stop drawing a line double click the left mouse button After you have stopped drawing lines you may erase all previously drawn lines by left clicking the mouse button within the view In the Intensity Profile Along Line box the Maximum intensity and the Minimum intensity will be displayed along with the Line Distance and the Angle The Intensity Profile is a graphical dis play of the intensity values of the voxels in the image that the current line spans Note For multi channel datasets
284. ss values 104 The Output File Format 136 The Region Selection Tools Shape tools 108 The Save Interval Rules of Thumb 136 Three Dimensional Line Lengths 200 Thresholds ee Isosurface 100 TIFF tif 59 Tile Horizontally 213 Tile Vertically 213 time lapse 215 Time Series Movie 173 Total Iterations 135 147 Tracking Object Counting Tracking 207 Training Object Counting Tracking 204 TRANSFER RESTRICTION 24 Trans Ilumination Lamp 46 Transparency 87 Triple View 68 Tutorials for File Menu Items 56 Tutorials for PreProcessing Menu Items 108 Tutorials for the View Menu Items 67 Tutorials for Visualization Menu Items 167 Two Views Image Alignment 122 U ndo Manual Alignment 123 ndo Label 194 ninstalling 20 nited States copyright laws 22 nsaturated Image 39 pdate AutoUpdate 214 S and International laws 22 JS GOVERNMENT RESTRICTED RIGHTS 25 accaca Vibration Control 35 50 Vibration Sources 51 Vibrations Air table 50 Anti Vibration Table 50 From Cooling Fans 51 Relative Motion 50 Shock 51 Video Gain 40 Video Rate CCD 128 View 67 73 XY 67 XZ 67 ZY 67 View menu 73 Viewing Channels 107 Viewing the Channels of a Multi Channel Data Set Red Channel 107 Viewing the Channels of a Processed Data Set 149 Viscosity March 2004 Page 237 AutoQuant Imaging Inc Manual and Tutorials Ratiometrics 185 Volume Projection 73 84 Voxel 171 Voxel Size 113 W Warranties 21 WARRANTY 25 Warranty I
285. ssee 53 Signal To Noise Considerations eseseseeseseeeeeseeresreerrsesrestrrissetststesstsresresserrtsressesees 54 2 Photon Dataset Coleco ias 55 Guide for Obtaining the Correct Collection Parameters ooooooccnnoccccnoncnononcninnnccnnnos 55 Sampling Criterion for X Y oei siruri a eA A E E aa Tie 55 Sampling Criterion fot Z o aean Er AEA S a ni eiae aaa 55 Tutorials for File Menu Items eters 56 DP e a 56 Openin YOUR TAOS A O O gees 56 Open Mov iii 57 ClOSE E EE E anal arene eae 57 Eaneh MArso s e a E a Ea es EESE AS E Teno ell 58 ed EE A e e RTS 58 Guidelines for naming your file s iii A A A a 58 Save Current View 2 2 con Coed dra i aaa E a as sends EA e AEEA Ra NS 59 Import Multi Channel Data sisci ascscsaspaccased seadcassaceevasedesaccusadcaceasncpeana badunedesnacesnisesesanadouses 60 Combine Channels Dialog soii oo ie 60 Load Ch nn lS iria d 60 DO OO EOS OOOO 60 CaN ur a td 60 Import Channel Dialoe vuitton 60 Channel Specification cespe iae reta ena Eee Eis Ri ant 61 IRGMON G a oes ola E A TOER 61 Change Intensities iiciin A A A PAL eee es 61 Generate Datasets rieien a a anita saa A CAN Ret cee rea 61 Create 2D Dataset iii cele ie iA ana 61 March 2004 Page 3 AutoQuant Imaging Inc Manual and Tutorials Create 3D DataSet neniatn ea stan pnascesecseentad avabancecuhged lune pesis 61 Data Properties tias 62 EAN E ae EEE E E EEEE PE E EE ET T E EE 63 A S S EEE A A sed cee eee 63 o ALEA E
286. ssion wavelength March 2004 Page 55 AutoQuant Imaging Inc Manual and Tutorials Tutorials for File Menu Items For this tutorial you may use your own data type Fluorescence Widefield Laser Scanning Confocal Brightfield Transmitted Light Spinning Disk Scanning Confocal Two Photon Fluorescence or 2 Dimensional images or you may follow along with the recommended dataset to use Open This function opens to a list of directory of files from which a chosen file can be loaded into AutoDeblur and AutoVisualize for processing For a list of file types that can be opened see Guidelines for naming your file s below Opening Your Image Note When opening a folder in any of the following tutorials if the folder appears empty select All Files in the Files of type field 1 From the File menu select Open or from the Toolbar click on the Open File Icon 2 From the Tutorial Data directory select the Widefield folder Select the dataset FitcDapi_crop tif and click on Open Note Various file formats may be handled such as AutoDeblur File Auto Visualize File 12 bit data 12 bit swapped data 16 bit data 16 bit swapped data IPLab File PGM File Bio Rad PIC TIFF File 8 bit character data 32 bit float AVI File Bitmap BMP File and STK File If your dataset is stored in a series of slice files the software will recognize the series and ask if you wish to load the entire series or simply the file that you selec
287. summed or voxel gradient projec tion or as individual optical slices No other package currently on the market combines these powerful processing tools with an environment for visualizing the results Q Can I process images that were acquired with a confocal microscope A Yes Confocal microscopes conventional two photon and spinning disk are effective at reducing the scatter from deep tissues In practice the z resolution is not good compared to the x y resolution Resolution along the z axis is typically 0 5 to 3 microns This means that structures March 2004 Page 215 AutoQuant Imaging Inc Manual and Tutorials are elongated along the z axis Applying AutoDeblur to a confocal image stack improves the z resolution dramatically often achieving beyond the theoretical 0 5 micron limit The signal to noise ratio of the deconvolved images is greatly improved and poor signal to noise is otherwise common with confocal microscopes especially in live cell imaging and where photobleaching is a problem Q What types of image files are supported by AutoDeblur A AutoDeblur supports most common image formats including 8 12 16 bit TIFF s Universal Imaging s STK BioRad s PIC Leica s native files BMP s AVI s Olympus Fluoview raw 8 12 16 integer 32 bit floating point data and others Q Can AutoDeblur handle the saturated portions of my image volumes A Yes Not only can AutoDeblur handle images that contain
288. t preferably heavy material Many laboratories have mounted tennis balls on such pallets as vibration isolators We recom mend first deflating the tennis balls by drilling a small hole on one end Otherwise we have expe rienced that inflated tennis balls are too rigid and do not sufficiently dampen building vibrations Collecting Confocal Datasets When collecting confocal datasets that will be used in conjunction with deblurring the following rules of thumb apply We emphasize that these are recommended rules of thumb for optimal operation Owing to the difficult trade offs among experimental conditions of signal to noise photobleaching photo damage field size and many other considerations you may find it neces sary to bend these rules These are recommended conditions and not hard rules You should go ahead and bend them if necessary Because of the robustness of AutoDeblur your deblurred results may still turn out fine but this will depend on many factors including your sample type and is less predictable than if you follow the rules To emphasize keep in mind the following adage garbage in garbage out AutoDeblur is designed to be robust against breakage of these guidelines Breakage of say one or two of them may cause no problem However with each addi tional guideline that is broken your chances of achieving satisfactory experimental results decreases As a general rule do not break any of the guidelines for sake of convenience
289. t Copy Deconvolution Settings This automatically copies the file s parameters If you paste the copied settings file into a text editor e g Windows Notepad you will be able to view the actual settings for the deconvolution Paste Deconvolution Settings This feature allows you to apply the setup and deconvolution parameters from one file into one or more files in the Batch This feature is generally used when similar files are collected using the same microscope setup 1 To use this feature select the file s from the Batch that you would like to copy the decon volution parameters to In this case the PollenPA deb file March 2004 Page 161 AutoQuant Imaging Inc Manual and Tutorials 2 From the Edit menu select Paste Deconvolution Settings This automatically Pastes the Pollen deb deconvolution parameters into the PollenPA deb file Copy PSF File Settings This feature allows you to copy the PSF File parameters from one of the files in the Batch 1 To use this feature select the TOSPollen_psf deb in the Batch by clicking on it 2 From the Edit menu select Copy PSF File Settings This feature copies the file s parame ters If you were to paste this file into a text editor you would be able to view the actual settings for the deconvolution Paste PSF File Settings This feature allows you to apply the PSF parameters from one file into one or more files in the Batch This feature is generally used when similar
290. t Labels The High light Labels box will appear Click in the box to the left of the number 1 Label The Label 1 area will show a blue pastel overlay see section Apply Label if you have closed the dataset from the previous section 2 Close the TobaMC tif XY SLICE VIEWER GREEN The Exit box will appear with the question Do you want to save the segment results Select No Combine Labels This feature allows you to join two or more overlapping Labels in a dataset You may choose either of the two Label Categories to place the new combination Label in or you may make a com pletely new one In order to combine labels the label edges must be touching March 2004 Page 194 AutoQuant Imaging Inc Manual and Tutorials Object Labeling MES oxi IESO Toba oxi View 1 View 2 View 3 To Combine Labels do the following Note Make sure the edges of the separate Labels are touching when you draw the labels out For this tutorial we are using the TobaMC tif XY SLICE VIEWER MULTI CHANNEL dataset 1 Scroll to slice 2 Using the Ellipse Select Region tool draw an Ellipse similar to the one shown above in the upper left hand corner of View 1 From the Segmentation menu select Apply Label Select the Apply to given slice range and set the slice range from 1 through 18 Click Apply 2 Scroll to slice 12 Using the Polygon or Freehand Select Region tool draw a polygon simil
291. t Mid Point 78 Set Start Point 78 Set Step Angle 78 Setting the Exposure Gain and Offset 38 Setting the Top and Bottom of the Sample and of the Scan 36 Setting the Top and Bottom of the Scan 37 Settings Stereo Tab 97 Setup 17 Shadow Isosurface 100 Shock Control 51 Sources 51 Single View 67 Skip Slices 69 Slice by Slice Cropping 110 All Slices 110 Slice by Slice 110 Slice Range 110 Slice Control Manual Alignment 123 March 2004 Page 236 AutoQuant Imaging Inc Manual and Tutorials Slice Status Box Manual Alignment 123 Slice Viewer 110 169 Smoothed raw image 140 Smoothing Factor 147 Software Copying Restrictions 22 Duplication 25 Software License Agreement 22 SOFTWARE USE RESTRICTIONS 23 Spacing Microns 63 Spatial Calibration 48 Specified Cross Talk FRET 179 Standard Settings 133 Save Interval 135 142 Total Iterations 135 142 X Spacing 64 Y Spacing 64 Z Spacing 64 Start 160 Statistical Information displayed 200 Statistical Optimality 33 Statistics 200 Step Angle 94 Stereo Mode 82 Stereo View 70 87 STK stk 59 Stop Movie 77 Sub resolution Microspheres 32 Subtract Background FRET 179 Subvolume 88 89 92 Subvolume frame 87 Sub Volume Overlap 139 Subvolume Tab 87 Sum Projection 167 Super Res Factor 147 Surface Area and Volume 201 Segmented 201 Slice Viewer 201 T Table of Parameters 220 Table of Z Spacings Z Step Sizes 219 TERM 25 TERMINATION 24 The Brightness and the Darkne
292. t box Project below This feature will display the XY view of the dataset on the bottom of the height map image Display sides This feature will display sides connecting the bottom of the image with the projection of the image if the Project below option is chosen to the height map March 2004 Page 100 AutoQuant Imaging Inc Manual and Tutorials Project on surface s This feature will overlay the image onto the height map Flip This feature will switch the height map to a depth map of the image The map will reverse the height map such that the map protrudes from the image towards you rather than away from you when looking at the image in its default orientation This feature allows you to analyze the inten sities side by side with the image in the XY perspective Wire frame This feature will display the height mapped dataset as a wire frame Display Method Frame Use projection Use this feature to use a projection mode when rendering the height map image From the drop down menu select one of the projections Maximum Projection Minimum Projection Sum Pro jection Voxel Gradient Alpha Blending Best Focus Surface Slice If Surface Slice is selected the text box to the right of the drop down will become active allowing you to select the slice to view either by using the up and down arrows or by entering the slice number into the text box Plane Select the plane to be mapped XY XZ ZY Use layered Dat
293. t from From the Movie menu Select Start Point 2 Now using your mouse rotate the view to a position that you want as the end point for the movie From the Movie menu select End Point 3 Select a Step Angle from the available options under the Set Step Angle option from the Movie menu 4 Click on Play Movie The movie will play in the direction in which you selected your Start Point and then the End Point 5 Again you may play the movie in Loop mode Rock mode and in the Opposite direction Note You may also select a Mid Point when generating a movie To do so rotate the view to a position that you want as the Mid Point From the Movie menu select Set Mid Point March 2004 Page 172 AutoQuant Imaging Inc Manual and Tutorials The last option under the Movie menu is Create Movie This feature opens an untitled movie file which may be saved It provides all the options to Play the movie Stop the movie Rock mode Play and Loop mode Play Close all views before continuing on to the next section Time Series Movie Maker This feature generates a movie from fixed angle projections of a volume over time You need to select the datasets that comprise the time series the angle at which to produce the projections and the projection to produce From the Visualization menu select Time Series Movie Maker The Time Lapse Movie Generator dialog box will appear ia Time Lapse Movie Generator 4 xi 1 Choose Data
294. t have obvious structures at the edges of the frame White This will put a white frame on the edge of the image where the aligned data has left a void This is recommended for Transmitted Light Brightfield datasets 5 Select Random from the drop down menu Noise Threshold This function allows you to set the noise threshold for which the Automatic Alignment feature will compensate By default Noise Threshold is disabled To enable check the Set Threshold box Noise Threshold is automatically set to 20 If this figure does not match your dataset move the pointer to the appropriate spot on the slider Base Slice This is the frame to which all other frames are aligned Find Automatically This function finds the base slice which has the highest similarity in features and structures to all other slices If the dataset is not opened in a Slice Viewer this is the only available option Current slice This is available only in a Slice Viewer This function uses the current slice being viewed as the base slice for alignment It allows you to select the base slice It is recom mended that Current slice be used if dataset has 50 or more slices 6 You will only be able to select the Find Automatically setting because the dataset is not in a Slice Viewer Base Channel In a multi channel dataset this feature allows you to select the channel upon which the dataset is aligned This feature will be disabled if the dataset is not a mu
295. t the axis about which to rotate the object The object will rotate about its own X Y or Z axis depending on the selection rather than the X Y and Z axes per ceived by you when you re looking at the screen Angle This feature allows you to select the angle by which to rotate the object about the specified axis Each time the Apply button is clicked the object will rotate the selected number of degrees about the selected object axis Constrain Rotation frame This feature allows you to select whether the object may rotate freely when manipulated i e rotate about multiple axes simultaneously or if the rotation of the object should be constrained to one axis at a time This can be useful to prevent unwanted rotations from occurring due to slightly imprecise mouse movements March 2004 Page 90 AutoQuant Imaging Inc Manual and Tutorials Around This feature allows you to choose the anchor of rotation your object will be constrained to Axis This feature allows you to choose which axis you would like the object rotated about It only is active if Object or Screen is selected for the Around type Oblique Tab Oblique Slice frame There are two settings Display Oblique Slice and Fix to Screen Display Oblique Slice This feature toggles between whether the display of an oblique slice is visible through the object or not Fix to Screen This feature when selected locks the position and orientation of the oblique
296. t the imaging micro scope system be isolated from vibrational disturbances to the extent possible The best approach to vibration control is to use a professional anti vibration table air table e g see the Ernest Fullam Inc catalog Latham NY or the Newport Catalog Irvine CA Often sufficient vibration control can be achieved for video microscopy systems without these air tables The following section offers some insight into the vibration isolation Most vibration related dataset collection problems arise from relative motion between microscope components This type of relative motion can often be caused by external energy sources Mounting the instrument on a maximally rigid platform can minimize relative motion between microscope components Remember that two types of rigidity are relevant dynamic and static Ideally the platform should be as rigid as possible dynamically as well as statically and yet be as light as possible and respond minimally to effects such as temperature changes For example Newport manufactures a special honeycomb sandwich structure that has these desirable proper ties In most quiet climate controlled laboratories an aluminum platform with a minimum thick ness of 0 5 1 inch may suffice Thicker platforms have higher static rigidity Try to minimize the length and width of the structure to the minimum necessary amount March 2004 Page 50 AutoQuant Imaging Inc Manual and Tutorials
297. t to appear in the table which can be arbitrary This means that the synchronizer is permitted to manipulate the currently viewed slice number of datasets that appear in this table If there are datasets that have different numbers of slices e g one dataset has 50 slices one has 30 slices and one has 90 slices all datasets will scroll only through the number of slices contained in the smallest dataset in this case all datasets would scroll through slices 1 30 If there are no XY slice viewers open then a message will appear in the bottom left region of the dialog informing you that you must open an XY slice viewer to be able to do anything Datasets are added and removed as they are added or removed from the workspace and also as they are minimized or restored in the workspace minimizing a dataset will remove it from the synchronizer restoring it will re add it Selecting a View You may choose to synchronize slice viewers of any of the three orthogonal views XY XZ or ZY When you change the selected view the slice synchronizer will attempt to make accessible each of the datasets that were already being synchronized This means that a slice viewer that is not open for the newly selected perspective but was open in the previous perspective will be opened automatically For example consider the synchronization table above _Pollen deb is being synchronized in the XY view If you were to change to the XZ view and _pollen deb di
298. tain Aspect Ratio which retains the origi nal aspect ratio of the view while changing its size The second option Stretch Aspect Ratio will not retain the original aspect ratio of the view Note Retain Aspect Ratio is the default for Image Aspect 1 Move the cursor over the edge of the image until it changes to a doubled pointed head arrow Hold the left mouse button down and drag the cursor outwards enlarging the image Notice how the image enlarges proportionally when Retain Aspect Ratio is active March 2004 Page 70 AutoQuant Imaging Inc Manual and Tutorials 2 Under View select Image Aspect and then select Stretch Aspect Ratio Move the cursor over the edge of the view until it becomes a doubled headed arrow Click and drag the edge and notice that the image enlarges disproportionately vertically or horizontally 3 Click the Retain Aspect Ratio option from Image Aspect again to reset the image back to its original proportions Note Do not close SmallHip avz XZ Max Projection Please continue on to the next section Correct XZ ZY Aspect Ratio This feature allows you to display the correct aspect ratio for the XZ or ZY views with respect to the physical dimensions of the voxels When viewing an XZ or ZY image without Correct XZ ZY Aspect Ratio on the view shown assumes that the voxels are perfect cubes If the Correct XZ ZY Aspect Ratio option is selected the view is shown such that the voxels have their correct
299. ted If your dataset was stored as 12 bit data on a UNIX machine and you are running the software on a PC you must select 12 bit swapped for UNIX as the file type This is also true for 16 bit data and when transferring 12 bit and 16 bit data from a PC to a UNIX machine 3 The dialog box shown below will appear Auto isualize AutoDeblur xj ep The program has detected properties that indicate m this data set MAY contain an old A4utoGuant Proprietary Multi channel TIFF Is this true TF answer is unknown select Mo IF your data appears incorrect open it again and answer Yes March 2004 Page 56 AutoQuant Imaging Inc Manual and Tutorials Select Yes to answer the question for the FitcDapi_crop tif dataset 4 The FitcDapi_crop tif dataset will be loaded and displayed in the XY Max Projection view Note If it is necessary to enter the dimensions of a particular dataset the first time it is loaded you will be prompted to do so The next time that dataset is selected the dimensions will automat ically be recalled for you Once a file is opened subsequent files with the same name but from a different directory will be numbered chronologically in the order they are opened For example if you have pollen deb opened from the widefield folder then you open another file name pollen deb from a different directory that file would be named pollen deb 2 and subsequent pollen deb files would be named pollen deb
300. ternet connection you will be prompted to click Configure to configure your internet set tings Any internet connections available on your machine will be listed in the Use Dial Up Con nection drop down menu Select the service you wish to use then click OK This will take you back to the original dialog box at which point you can click OK again AutoUpdate will now search for any available updates About This feature shows the version and copyright information about the product March 2004 Page 214 AutoQuant Imaging Inc Manual and Tutorials Frequently Asked Questions Q How does AutoDeblur perform an accurate deconvolution without a measured point spread function PSF A AutoDeblur s adaptive blind deconvolution automatically extracts point spread functions from the raw image dataset It does this by analyzing the spatial frequencies that make up the raw image volume and creating one or more PSF s that are based on this information Because the performance of a microscope varies across the field of view and especially along the optical axis AutoDeblur will often automatically derive several PSFs to process a single image volume This assures that results are accurate even though the optical system scope cover slip immersion medium mounting medium specimen is not linear Blindly restored PSFs are also more accurate than empirically measured PSFs because they are free from noise contamina tion and are based on
301. tes the slice number select the pat tern in which the pound sign falls at the end of the filename 2 From the Analysis menu select Ratiometrics 3 Click on the Use Grynkiewicz Equation for Ion Concentration box 4 Click on the Calibrate button 5 For the Numerator Wavelength section assign the LowCa1 001 and HighCal 001 images to the Low Ion and High Jon images respectively For the Denominator Wavelength section assign the LowCal1 002 and HighCa2 001 images to the Low Ion and High Jon images respec tively Click the OK button This will populate the Calibration Parameters frame in the Ratiomet rics dialog 6 Go to File then Open and navigate to the Ratiometrics folder then the Calibration folder and open BILT1 001 and B1LT2 001 For each file it will ask which pattern denotes the slice number select the pattern in which the pound sign falls at the end of the filename March 2004 Page 185 AutoQuant Imaging Inc Manual and Tutorials 7 Assign B1LT1 001 to the Numerator and assign B1LT2 001 to the Denominator in the Images frame of the Ratiometrics dialog 8 Verify that the Remove Spot Noise feature has been checked Click Calculate A new image will appear This is the result of the ratio 9 Click the Analysis button in the Ratiometrics dialog to display the statistical information on the new image 10 Close all images before moving on to the next section Colocalization Note This featu
302. the Skip Slice number to skip every other slice of the montage display Click OK to create the Montage viewer The slices will be displayed in the Montage frame The line separator designat ing a frame s boundary will be a maroon color for both grayscale and multi channel images 6 The Montage viewer created is of 24 subsequent slices To change the slices displayed click the Left arrow key or the Right arrow key on the keyboard and the previous slice or the next slice will be displayed respectively To change the entire Montage of slices click the Page Down key for the next set of slices or the Page Up for the previous set of slices Note When clicking on the Left arrow key the Right arrow key the Page Up key or the Page Down key the number of the slices that are being displayed in the Montage viewer are displayed on the Status bar at the bottom of the main window 7 To Close the Montage Viewer click on the X in the upper right hand corner of the Mon tage Viewer box 8 Close the brain dataset by selecting Close from the File menu March 2004 Page 69 AutoQuant Imaging Inc Manual and Tutorials Stereo View Note This section is applicable only to users of the Auto Visualize software This feature displays a Stereo View of the image file Stereo viewing is generally used for seeing depth in an image The Stereo View Parameters dialog box will prompt you for the Stereo Angle Degrees This angle will be used to g
303. the Statistics dialog box will also allow you to select the Slice Range from which you would like to collect your statistics Your range can be a Single Slice Multiple Slices or All Slices In both cases a Histogram of ROI of Current View is displayed to the left of the Statistical Infor mation box The grayscale values the intensity values are displayed as a percentage along the X axis Above the histogram is the Select Channel box for colored datasets This feature is not visi March 2004 Page 200 AutoQuant Imaging Inc Manual and Tutorials ble for grayscale datasets For a color dataset you may view the statistics for any of its Channels or All Channels by selecting the appropriate option from the Select Channel list box Three Dimensional Statistics Three dimensional statistics may be calculated when an image is in a Slice Viewer projection To calculate three dimensional statistics click on the Multiple Slices button or the All Slices but ton When the Multiple Slices button is clicked a Calculate 3D Statistics dialog box will appear prompting you to specify the Starting Slice number and the End Slice number from which the sta tistics are to be calculated Clicking on the All Slices button will preempt entering the range of slices and all the Optical Slices will be used Auto Visualize will then perform the calculations on the selected volume Selecting Single Slice will cause Auto Visualize to analyze the displayed slice two
304. the condition described earlier in Figure D When using fluorescence since the drop of dye will likely fluoresce at a much different intensity than the biological sample it is especially important to carefully adjust the gain to ensure the condition described in Figure D To emphasize it is important that the maximum gray value in the image is at about 75 85 of the well capacity of the camera and that there are no saturated pixels in the image as shown in Figure C Make a careful record of all gains and file names for the bias and flatfield images in your labora tory notebook Record all exposure times and numbers of frames averaged for all datasets col lected including the flatfield frames bias frames and the optical sections of the sample March 2004 Page 47 AutoQuant Imaging Inc Manual and Tutorials Table 1 Dust Spots Figure a Figure b Table 1 Figure a An incorrect attempt at a flatfield image by taking the sample well out of focus This image is not far enough out of focus An out of focus remnant of the sample is still visible Table 1 Figure b A correct attempt at a flatfield image by taking the sample well out of focus Note that no out of focus remnant of the sample is apparent This image will be used to correct the optically sectioned dataset for non uniform pixel sensitivity and for the dust spots that are highlighted Spatial Calibration Important parameters that are
305. the heading above the dropdown menu In other words for the first top dropdown menu select the image that is the corrected FRET image DAf If you have just run the Correct FRET Cross Talk algorithm the proper images will already be assigned All three images must be assigned in order to run statistical anal ysis on them Image ROI The Image ROI section displays the coordinates of the selected region of interest Select the region of interest by clicking and dragging on the desired image to create a rectangle around the desired region Top Left refers to the top left corner of the rectangle whereas Bottom Right refers to the bottom right corner of the rectangle For each the X Y and Z refers to the axial coordinates of the respective corners Fixed Threshold This section has two sliders which allows you to set the upper and lower percentages of the inten sities to be analyzed Set the intensities by either clicking on the pointer and dragging it to the desired location or by clicking on the desired location to incrementally move the pointer to that spot or by entering the desired intensity in the text box Dye Pair s Foster Distance Ro This section is where you can enter the dye pair used in the images This will allow for a calcula tion of the actual Forster s distance when calculating the FRET efficiency Select the proper dye pair from the dropdown list by clicking on the dropdown arrow or if the distance is known yet not
306. them through the image The maximum voxel value encountered along each ray is taken for the projection pixel value and the resulting image is made up of each maximum voxel value This projection high lights edges and prominent bright features 1 From the File menu select Open Navigate to the Widefield folder and select Pollen deb Click Open The dataset will be loaded in the XY Maximum projection view The XY Max Pro jection view is the standard default view except for transmitted light brightfield datasets which open in the XY Minimum projection view 2 Select XZ from the Single View option under the View menu 3 Set the XY Max Projection as the active view by left clicking in the view Note The status bar on the lower left hand corner of the main window will display the active view and projection Note Do not close Pollen deb XY Max Projection Please continue on to the next section Minimum Projection This function takes parallel rays perpendicular to the viewing surface and casts them through the image The minimum voxel value encountered along each ray is taken for the projection pixel value and the resulting image is made up of each minimum voxel value The Minimum projection provides volumetric representations in which foreground intensities tend to be suppressed This projection highlights edges and prominent dim features 1 Select Minimum Projection from the Visualization menu The Minimum Projection will be autom
307. ther button in the 3D Viewer toolbar is depressed use the right click button on the mouse and the click and drag to rotate 10 From the Projection menu select Surface Slice Click the Subvolume button left click on any of the cube faces and move your mouse to select a sub volume for display Useful Key amp Mouse Combinations for the 3D Viewer To crop the volume or move an Orthogonal Oblique Slice Select Display Slice from the Oblique menu click on the Subvolume button left click on the plane slice and drag To rotate the Oblique Slice Click on the Oblique button then left click on the Oblique Slice and drag To zoom the displayed image While the Rotation button is clicked right click on the image and drag from bottom right to top left or vice versa Alternatively click on the Zoom button then left click on the image and drag from bottom right to top left or vice versa Explanation of 3D Viewer Menus View Menu This menu allows you to specify which viewing mode to use You may choose from the following choices Volume Projection Orthogonal Slices Cube Surface Isosurface or Height Map Volume Projection Software and Hardware This feature displays a single 2D projection of the 3D volume as seen from your perspective at the object s selected orientation The Volume Projection Software will work slower than the other two options but will offer a higher resolution of the dataset The 2D Textures and 3D Textures p
308. thm chosen Nearest Neighbor and the values selected for the Haze Removal Factor and the Z Kernel Width are now saved for this specific dataset Note Do not close the Pollen deb dataset Please continue on to the section Processing Stack 1 Click on the Pollen deb XY MAX Projection to make it the active view From the Decon volution menu select Nearest Neighbor and select Processing Stack 2 The Nearest No Neighbor Parameters box appears The settings for the Haze Removal Factor 0 98 the Z Kernel Width 3 and the Nearest Neighbor algorithm were determined above in the Processing Current Slice section 3 Click Start The deconvolution will be performed on each slice of the entire stack A status bar will indicate the progression 4 Compare the results of the Nearest or No Neighbor deconvolution to the original dataset and note how the deconvolution removes haze and sharpens features Examine the Maximum pro March 2004 Page 157 AutoQuant Imaging Inc Manual and Tutorials jections and the Slice Viewers of the original Pollen deb dataset the Inverse Filter and the Near est Neighbor results in both the XY and XZ views Close all datasets before continuing on to the next section Axial Blind Deconvolution Use only with Confocal datasets Also known as 1D deconvolution this is a form of blind deconvolution that is especially custom ized for speed It is designed to be the fastest method for deblurring a conf
309. thms If it is not possible to obtain the calibration images DDd DAd AAd DDa DAa and AAa the Cross Talk Coefficients tab can be used to estimate the Cross Talk See the Use Cross Talk Coeffi cients Tutorial for more information on this March 2004 Page 178 AutoQuant Imaging Inc Manual and Tutorials PreProcessing Use Automatic Alignment Checking this box will align the channels using the Channel to Channel Alignment feature using 1ts default settings For more information on the Channel to Channel Alignment see page 124 Subtract Background This section allows the user to choose from three options regarding the Subtract Background algorithm None Minimum Value and Histogram Peak Selecting None will perform no back ground subtraction on the datasets Selecting Minimum Value will subtract the background by eliminating the minimum value present in the dataset Selecting Histogram Peak will subtract the background based on the most commonly occurring intensity level There are other options which can be performed on images individually available from the PreProcessing menu under the Data Correction option For more information on these see page 129 The Subtract Background feature should be used to clean up images that have background noise Doing so will allow for more accu rate results with the FRET algorithms Specified Cross Talk Donor Only Acceptor Only Donor and Acceptor This section allows you to select which
310. ties then removes the highest occurring intensity this is typically the background in an image in which the object takes up less than half of the image The Minimum Value option also requires no input and it removes the minimum intensity from the image The Constant method allows an intensity to be entered into the text box and anything at or below that intensity level will be removed from the image The Background Image option requires an image to be acquired with no object in it a blank image Select this file from the drop down menu If the background image file has not been opened open it using the Open button on the main toolbar Click on the radio button for the desired method of background removal March 2004 Page 129 AutoQuant Imaging Inc Manual and Tutorials 4 Click OK A new Untitled file will be created Image Macro This feature gives you the option to perform the same functions on several datasets at one time 1 Open the Neuron deb dataset from the Confocal folder 2 Select Image Macro from the PreProcessing Menu The Image Macro Functions box will appear 3 Click the Select Data button and the Available Datasets box will appear The Available Datasets box will list all datasets currently opened You may add datasets to this workspace by using the Open File button and opening as many datasets as you would like You may select the dataset you want to work with or you may use the Select All button
311. time point in succession select option Full Rotation Time Point When the Full Rotation Time Point option is selected the Rock option becomes available This option rocks the movie back and forth from the beginning to the end and back to the beginning before moving on to the next time point Time Stamp Format This section allows you to set up how the time stamp will be displayed on the movie or to select to not have it displayed at all Next to each radio button is shown the template for how that option will be displayed on the movie Caption This section allows you to enter a caption to be displayed on each frame To enter a caption you must first select the frame for the caption to be displayed on do this in the Selected Frame text box in the Preview Data section of the dialog Enter the desired text into the Caption textbox Do this for each frame you wish to enter a caption for March 2004 Page 175 AutoQuant Imaging Inc Manual and Tutorials Setting up a Movie The Setup Movie group includes the controls and options needed to interact with the 3D Viewer to prepare a time lapse movie View Selected Frame Click the View Selected Frame button the dataset selected in the table in the Choose Data group will be loaded into the 3D Viewer If multiple datasets are selected the lowest frame number will be displayed If when this button is clicked the 3D Viewer window is not open it will be opened with the sele
312. ting of High should be selected This will cause AutoDeblur to be extremely robust against the noise For SNR levels between 10 and 100 the Noise Smoothing setting of Medium should be selected For SNR levels of 100 or higher the Noise Smoothing setting of Low should work well See Table 3 to identify noise levels approximately by visual comparison Table 3 SNR 160 1 SNR 40 1 SNR 10 1 Table 3 Left to Right Simulated SNR of 160 1 40 1 and 10 1 respectively March 2004 Page 54 AutoQuant Imaging Inc Manual and Tutorials 2 Photon Dataset Collection Guide for Obtaining the Correct Collection Parameters Sampling Criterion for X Y Sample at 1 2 the Airy disk width with a wavelength that is the excitation wavelength The excita tion optics determine the width of the PSF This will be approximately 0 61A NA x 2 Where A is the emissive wavelength and NA is the numerical aperture Generally the result is 163 nanome ters or finer assuming a 1 4 NA lens If you can sample finer say 80 nanometers or 40 nanome ters try this also as an experiment It is expected that you may improve the x y resolution by at least 2 and possibly by 4 80 nanometers To improve the optical resolution by 4 to 80 nanom eters you need to increase the spatial sampling by 8 40 nanometers Sampling Criterion for Z Sample at 1 2 of the PSF axial width which approximately follows the formula a AJMANA
313. ting to the Data Correction folder Set the Files of type to All Files 4 Select the StrfishFfv tif file and click Open AutoDeblur will automatically load this Flat Field file 5 To Load the Bias Field file click on its corresponding Load button and navigate to the Data Correction folder Select the StrfishBfv tif file and click Open The File Open box appears with the message Dataset only contains one optical slice the orthogonal projection along XZ and YZ will be inactive Click OK AutoDeblur will automatically load the Bias Field file There is only one Bias Field for High Speed CCD Data Correction because Dark Current does not effect the image in this situation 6 Select the Parameters tab You may enter the value for the frame s per optical slice Leave the default setting of 1 for this example For Bad Pixel Treatment select Median Filter Click OK and AutoDeblur will correct the High Speed CCD dataset March 2004 Page 128 AutoQuant Imaging Inc Manual and Tutorials Note To slow down the viewing time per each frame increase the frame s per optical slice value Background Subtraction This feature should be used to clean up images that have background noise There are five differ ent ways to subtract the background from a dataset ROI This involves drawing an ROI around an area that is known to be the background The algorithm will remove all data with the same or lower intensity as the intensity wit
314. tion Load PSF from File t Theoretical PSF Algorithm Settings Noise Level _ gt Image Properties t4edium Phase Content Expected Output Settings MT Save the PSF Files Batch Cancel Help From the Deconvolution menu select Batch Process Within the Batch Processing dialog box the column labeled Operation allows you to know the type of operation that a specific task will per form There are two types of operations 3D Iterative Deconvolution and 3D Inverse Filter March 2004 Page 154 AutoQuant Imaging Inc Manual and Tutorials File Edit Help MT Save Max Projection Batch Job Name Bachi SCS The log file for this collection of 3D deconvolution tasks is a text file with the Batch Job Name and tst extension It will be located in the same directory of this AutoQuant product Batch Log File Directory C Program Files Auto0 uanttAutoO uant Imaging Suite 9 1 Browse Start Time 0414 2003 09 28 Operation Image File Name 3D Inverse Filter tantr utoO uant Imaging Suite 9 14 Tutorial Data idefield Pollen deb None Start lear List Delete Paste The batch will start in Cancel Timer You may switch between the two operations by selecting Change Operation from the Edit menu as shown below This menu is also accessible by right clicking on any of the tasks within the batch table File Edit Help Copy Optics
315. tion linkages between the focus control and the stage and thereby are prone to slippage under normal conditions There are a number of manufacturers that sell turnkey 3D dataset collection systems which includes both the needed hardware and software for widefield microscopy These products carry out the above instructions automatically Some of the manufacturers to check are listed here Uni March 2004 Page 44 AutoQuant Imaging Inc Manual and Tutorials versal Imaging Corporation Downington PA Compix Cranberry Township PA MediaCyber netics Silver Spring MD Nikon Melville NY Leica Bannockburn IL Avoiding Backlash and Hysteresis While stepping the focus always scan against the load If you have a stage focusing micro scope where the stage moves up and down as the focus knob is moved then always step the focus in the direction where the stage is moving upward This will cause you to scan the sample in the direction from top to bottom If you have a nosepiece focusing microscope then always step the focus in the direction where the nosepiece is moving upward This will cause you to scan the sample in the direction from bottom to top When adjusting the focus from one sequential slice to the next move the knob very slowly and cautiously Also be careful to move the knob only in one direction during the scan Once you have started grabbing frames never reverse the direction of the fine focus knob not even
316. to save the newly created file 8 Click the Save button A dialog box will appear asking you Do you wish to export as mul tiple files each of a single slice Select Yes AutoDeblur will show you the naming extension it will append to the dataset The new dataset will be saved under a name indicating the Start and End slices along with the number of iterations in its title Close all datasets before moving on to the next section Inverse Filter Use with Widefield Fluorescence and Transmitted Light Brightfield datasets only The Inverse Filter is a one step non iterative deconvolution method based on inverse filtering the ory It utilizes optimal linear filtering Inverse Filter is one of the simple deconvolution methods offered in AutoDeblur It is very useful for obtaining quick results but is not as accurate as Blind Deconvolution Inverse Filtering is typ ically more robust than the Nearest Neighbor or No Neighbor deconvolution methods The execu tion speed of the Inverse Filter is between that of Nearest Neighbor and Blind Deconvolution The Inverse Filter should be used in cases where speed is important A typical processing time is under 2 minutes with a 256x256x32 dataset Pentium III 450 Mhz 1 Navigate to the Widefield folder and open Pollen deb March 2004 Page 152 AutoQuant Imaging Inc Manual and Tutorials 2 From the Deconvolution menu select Deconvolution Settings then select Standard Set tings or you
317. to the next section Invert View This feature allows you to take a bright grayscale or color image on a darkfield or brightfield background and invert its pixels intensities For instance a bright grayscale image on a darkfield background after inversion will appear as a dark image on a bright background 1 From the View menu select Invert View Note Inverting the Image when the Slice Viewer or Multiple Slices Montage View projection is active will result in inverting the entire 3D dataset not just the one slice being displayed March 2004 Page 104 AutoQuant Imaging Inc Manual and Tutorials 2 To the right of the Image Enhancement icon on the toolbar click on drop down arrow select Add or Remove Buttons and select Invert View then click on the drop down arrow again The Invert View icon is displayed on the Toolbar The icon is displayed depressed to indicate that the current view is inverted from its normal state By clicking this icon you can toggle between the inverted image and the normal image Click on the Invert View icon to restore the original view When a newly inverted data projection is created the projection is created first using the original data and then inverted Note Do not close SmallHip avz XZ Max Projection Please continue on to the next section Flip Horizontal Vertical This feature will flip the current image over either horizontally or vertically When a new projec tion is created w
318. to the numerator will be divided by the image assigned to the denominator PreProcessing Use Automatic Alignment Checking this box will align the channels using the Channel to Channel Alignment feature using its default settings For more information on the Channel to Channel Alignment see page 124 Maximum Minimum Thresholding This section has two sliders which allow you to set the upper and lower percentages of the inten sities to be analyzed Set the intensities by either clicking on the pointer and dragging it to the desired location or by clicking on the desired location to incrementally move the pointer to that spot or by entering the desired intensity in the text box Use Grynkiewicz Equation for lon Concentration Select this option if you are measuring the effects of changes in calcium concentrations in your sample To get the best results out of this equation a calibration set of images should be taken This set should consist of four images two each at two separate wavelengths one with no cal cium and the other saturated with calcium Once a calibration has been performed the values can be entered into the textboxes manually in later analyses Rmax This is the ratio of the two wavelengths for the calcium saturated specimen March 2004 Page 183 AutoQuant Imaging Inc Manual and Tutorials Rmin This is the ratio of the two wavelengths for the calcium free specimen B SI2 Sh2 This is the ratio between the
319. trols will zoom the image in and out The button on the left enlarges the image and the button on the right decreases its size Holding either of these but tons down will continue to either increase or decrease the image s size depending on which button is being pressed The dropdown menu displays the image size relative to its original March 2004 Page 186 AutoQuant Imaging Inc Manual and Tutorials size as a percentage Alternatively it is possible to either select a predetermined percentage from the dropdown menu or manually enter a percentage into the textbox booed AAA The crop buttons allow the user to draw a region of interest in the colocalization image in the dialog For more information on how to use crop tools see page 108 The image on the left side of the dialog displays the two selected channels in the colors selected and shows the colocalized areas in white If the image has been enlarged such that it no longer fits in the allotted space scroll bars will appear on the bottom and right to allow the user to scroll to all areas of the image Directly below the Coloc Viewer are the controls to display and move the Coloc Viewer through the slices or display all of the slices Checking the All Slices checkbox above the scroll bar will display all slices and the scroll bar and textbox will become inactive To reactivate the scroll bar and textbox uncheck the Al Slices checkbox The textbox on the left sid
320. ts 210 Tracking Featur s must lili bl tds 210 MOVIE adie net alia iter te as ee ee da Ii 210 Generate MOVIE seid eich Aine at Aah om een er eee See ee ate 210 Cancino 210 Help iii A A aia 210 March 2004 Page 13 AutoQuant Imaging Inc Manual and Tutorials Tutorial for Object Counting coooonoccnonnnnnnonnconaconacnnnonnnonaconnoconannn nana non nc ona con ncnncannncnnnnos 210 Tutorial tor Object Tracking rain nooo osas 211 WINCOWS cada na 213 Casciari eones cda 213 TeHorizonta lat lts 213 Tle Vertical A A Ad 213 Clos AU ies E I oak a da a dd edad leed a Deh Fas Ad do 213 WindoWwJLastiaatia a ai 213 O 214 E A A A A A nO UT ee 214 LEOTE a REE E AAS A Dace cob EER E A ANE EEE ene sauteed ee 214 Search for Help ON encreire e dle 214 Check tor Update tn ai tai 214 ANO a ELS 214 Frequently Asked Questions ccoooooooncoccnnnncncnonnnnonannananannns 215 Appendices aiii idad 219 Table of Z Spacings Z Step Sizes A A A A ee 219 Table of Parametros a td sides ees 220 Toolbar cons er rra 221 Ele Toolbar Icons ita 221 Visualization Toolbar Icons ccccnnnnnonnconononononuneninonononcnnononanecnacocnnonononanecacicnnonona 222 View Toolbar Tons uni td 222 Analysis Toolbar Icons 23 0203 aa 223 Statistics Toolbar ICONS ooooonncncccnnononininicocncnnncnononananacocnnnnononnnnnnrncnccnononananicinoss 223 Zoom Toolbar LON iia cased cove cdedlne acecetcectuts a a Sae 223 Bibliography isis ii oia 224 Customer Feedbac
321. ts slices It can be thought of as a horizon tal slice through the XY view ZY This is a side view of the dataset or each slice It can be thought of as a vertical slice through the XY view 1 Navigate to the TLB folder and open the TLBview tif dataset Select XZ from the Single View option under the View menu Note Transmitted Light Brightfield datasets will open in the Min Projection view 2 Set the XY Min Projection as the active view by clicking within the XY Min Projection view Note The status bar on the bottom of the main window will display the active view and projec tion March 2004 Page 67 AutoQuant Imaging Inc Manual and Tutorials Triple View Note This section is applicable only to users of the Auto Visualize software Selecting Triple View allows you to create three orthogonal views of your dataset On the top left of the Triple View display is the XY view on the top right is the ZY view and on the bottom left is the XZ view of the dataset The TLBview tif XY Min Projection from the previous section should be active 1 From the View menu select Triple View The XY ZY and XZ views of the TLBview tif dataset are displayed in the Triple View box 2 You can also generate a Triple View for datasets displayed in a Slice Viewer projection Make active the TLBview tif XY Min Projection dataset From the Visualization menu select Slice Viewer The dataset will be displayed in a Slice Viewer
322. ttom of the sample The rule of thumb for selecting the top and bottom of the scan is described below using the terms illustrated in Figure 1 below Top of Sample Center of Sample 7 Bottora of Sample Bottora of Scan Figure 1 Shows the geometry and illustrates the terms used for 3D widefield dataset collection Note The actual axial distance that is scanned is about twice that of the sample The top and bot tom are over scanned by approximately half the thickness of the sample The above rule of thumb provides the best results If you have limited disk or memory space then you may not be able to March 2004 Page 36 AutoQuant Imaging Inc Manual and Tutorials follow this rule of thumb In that case scan the depth of the sample plus any additional amount allowed by your free hard disk space Setting the Top and Bottom of the Scan For best results the rule of thumb in setting the axial distance between slices is to have this dis tance equal to the Rayleigh depth of field DOF as given by the following equation A AZ tice DOF ____ Zolic 4n sin 0 5 sin NAMN where A is the wavelength if unknown use a value of 0 5 micrometers n is the refractive index of the immersion medium 1 0 for air 1 33 for water 1 515 for oil and NA is the numerical aper ture of the objective lens The total number of slices required then will be equal to the thick
323. tures The Export Features button allows the user to save the statistics into a csv file for further analy sis To Tracking gt gt Clicking this button in the Object Counting dialog will open the Object Tracking dialog Once this button is clicked there is no way to return to the Object Counting dialog It is advised that the sta tistics be saved from the Object Counting phase before moving on to the Object Tracking phase Close The Close button closes the Object Counting Results window Tracking This dialog contains the controls used to track the objects counted in the Object Counting dialog To perform Tracking the dataset must first be segmented and counted as detailed in the previous section Track The Track frame contains the controls to perform and modify the tracking feature Auto All The Auto All button will automatically track all counted objects The 3D Viewer will display the counted objects along with color matched bars indicating the paths that they take in the time series Before tracking the Tracking Tolerance dialog will appear Distance Tolerance This slider sets the threshold for the distance an object can move and remain in a track It is based on a percentage of the length of the object s radius Thus if the threshold is set to 300 then any thing that moves more than 300 the length of its radius will be overlooked as an object within that track March 2004 Page 207 AutoQuant Imaging Inc M
324. used to describe methods of deconvolution which do not require the point spread function PSF of the system to be explicitly known prior to the deconvo lution AutoDeblur is based on blind deconvolution Indeed a reconstructed estimate of the PSF is produced by AutoDeblur concurrently with the deconvolved image dataset There is no need to provide AutoDeblur with a PSF of any kind The important practical implication of blind deconvolution is that it adapts to the real PSF of the microscope system which can be significantly different from the theoretical PSF see Figure 6 and from previously measured PSFs due to specimen and instrument variations The AutoDeblur blind deconvolution system is able to adapt to PSF changes within a specimen itself Thus the deconvolved results are superior to those of methods that utilize theoretical or previously mea sured PSFs Figure 6 Shown on the left is a theoretical PSF for a confocal microscope On the right is the PSF as revealed by AutoDeblur Direct experimental measurement of the PSF is an arcane and difficult procedure that is not straightforward Hiraoka Sedat et al 1990 for routine usage and thereby impedes the wide usage of deblurring algorithms that require it The PSF measurement method typically involves imaging a sub resolution fluorescent microsphere This approach has several limitations First noise in the imaging system also shows up in the measured PSF This can be overcome to so
325. ution Through Error Energy Reduc tion Optica Acta 21 709 720 Gibson S F and F Lanni 1991 Experimental Test of an Analytical Model of Aberration in an Oil Immersion Objective Lens Used in Three Dimensional Light Microscopy Journal of the Optical Society of America A 8 10 1601 1613 Gordon G Berry G Liang X Levine B and Herman B 1998 Quantitative fluorescence reso nance energy transfer measurements using fluorescence microscopy Biophysical Journal 74 2702 2713 Grynkiewicz G Poenie M and Tsien RY 1985 A new generation of Ca2 indicators with greatly improved fluorescent properties Journal of Biological Chemistry 260 3440 3450 Hebert T R Leahy and M Singh 1988 Fast MLE for SPECT Using an Intermediate Polar Representation and a Stopping Criterion IEEE Transactions on Nuclear Science 35 1 615 619 Hiraoka Y J W Sedat and D A Agard 1987 The Use of Charge Coupled Device for Quan titative Optical Microscopy of Biological Structures Science 238 36 41 Hiraoka Y J W Sedat and D A Agard 1990 Determination of Three Dimensional Imaging Properties of a Light Microscope System Partial Confocal Behavior in Epifluorescence Micros copy Biophysics Journal 57 325 333 Holmes T J Bhattacharyya S Cooper J A Hanzel D Krishnamurthi V Lin W Roysam B Szarowski D H Turner J T Light Microscopic Images Reconstructed by Maximum Like lihoo
326. utorials The Sum Projection Rendering Range is from 0 to 100 The default Rendering Range of Voxel Gradient Best Focus and Alpha Blending is from 20 to 100 The default Alpha Value is 0 5 Load Settings This feature allows you to load settings previously saved using the Save Current Settings As fea ture see below Load Settings will include all fields found in the Standard and Expert Deconvo lution Settings For more information on Standard and Expert Settings see page 133 Save Current Settings As This feature allows you to save the Standard and Expert Settings as text files for later use with either the same or another dataset The file is saved with an set extension This section of the tutorial assumes you have the FitcDapi_crop tif dataset open 1 With FitcDapi_crop tif as the active view from the File menu select Save Current Settings As 2 In the File name field type the name you would like for these settings For example Test_parameters set 3 Click on Save You have now saved the Settings for this file This dataset will be used later in the tutorial section for the Deconvolution Menu items Please close all views before going on to the next tutorial Exit This function closes the software and all files that are open You do not need to save the images generated in this tutorial section Recent Files List This feature displays five of the most recently loaded and closed datasets This list is
327. utton will add more area to the segmented region by placing the cursor in the region where segmented area is to be added then holding down the left mouse button and scrolling over the desired area Erase Clicking this button will erase area from the segmented region by placing the cursor in the region where segmented area is to be erased then holding down the left mouse button and scrolling over the desired area XY Brush Size The XY Brush Size frame contains three different size options for manually modifying segmenta tions The larger the brush size the more area will be added or erased based on the Mode selected in the segmentation In the Z Brush Size frame the size of the brush will determine the depth of the segmentation how many z slices will be segmented Finish Accept Changes Clicking this button will accept and save the changes that have been made to the segmentation of the dataset Undo Changes Clicking this button will undo all changes that have been made since the last time Accept Changes was clicked Close Clicking this button will save the changes made to the segmenta tion and return to the Object Counting dialog Use Recommended Expert Settings Checking this button will use the recommended expert settings when performing segmentation on the dataset To change the expert settings deselect Use Recommended Expert Settings then click the Go To Expert Settings button Go To Expert Settings Clicking this
328. volve a dataset you must set the Standard Settings and the Expert Settings The objective of this tutorial is to give you a feel for setting both the Standard and the Expert Settings Note This tutorial contains many explanations and guidelines that are important to the under standing of Standard and Expert settings It is recommended that you read over the explanations and guidelines to assist you in specifying the Standard and Expert settings Standard Settings The Standard Settings allow you to specify parameters needed for deconvolution Inputting these parameters is the first step in the deconvolution process Pausing the cursor over a field requiring input will launch a tool tips description of that item Additionally any values that are outside the expected range will be shaded in a color coded fash ion Yellow indicates that the value is outside the expected range but the algo rithm will still run Red indicates that the value is outside the expected range and the algo rithm will not run until the value is corrected Cyan indicates that the dataset appears to be oversampled but the algo rithm will still run 1 From the Widefield folder open the FitcDapi_crop tif dataset and create an XZ Sum Pro jection A significant amount of haze can be observed in the FitcDapi_crop tif dataset especially in the XZ Sum Projection 2 From the Deconvolution menu select Deconvolution Settings and click on Standard Set tin
329. will not be displayed The threshold range is 0 to 100 Alpha This feature allows you to specify the Alpha value to be applied to Alpha projections displayed in the 3D Viewer Display Range 0 100 This feature allows you to adjust image brightness levels to improve visibility of features of inter est Brightness This feature allows you to specify a threshold percentage above which all intensities are increased to maximum Darkness This feature allows you to specify a threshold percentage below which all intensities are decreased to minimum Gamma This feature allows you to apply the specified gamma correction factor to the image Zoom This feature allows you to specify a zoom factor for the image Higher values enlarge the image and lower values reduce the image Apply This button applies new values to your dataset Default This button resets the Image Appearance values to their defaults March 2004 Page 86 AutoQuant Imaging Inc Manual and Tutorials Properties frame Correct Aspect This feature allows you to rescale the image along Z so as to more accurately reflect the actual proportions of the object as defined through its X Y and Z spacings Plane Outline This feature allows you to outline the boundaries of the dataset around orthogonal planes and around subvolume boundaries This aids in judging the orientation of the dotted line and in judg ing the relative positions of the planes and t
330. working with the selections under Orientation and Margins Print This function prints the active screen You may print the active view by selecting Print from the File menu Operation Settings This function allows you to specify information regarding the File directory and the Rendering Range for various projections of a dataset Files You have the choice of changing the location of the temporary files or the startup directory by either typing in the new location or browsing to the new location The startup directory is the loca tion in which the software application will look for image sets Note You are advised to place the temporary files directory on a drive with at least 1 gigabyte of free space Rendering The Rendering tab allows you to set the range of values a pixel may have depending on the pro jection chosen or the threshold values for a projection The projection rendering ranges can be set for Max Min and Sum Projections The threshold values can be set for Voxel Gradient Best Focus and Alpha Blending projections Note You are advised to leave the Rendering settings at their default values You may want to change the Rendering settings in the future for noisy or photobleached datasets The default Rendering Range values are as follows The Max Projection Rendering Range is from 0 to 100 The Min Projection Rendering Range is from 0 to 100 March 2004 Page 65 AutoQuant Imaging Inc Manual and T
331. ysis menu select Segmentation and choose Automatic Segmentation The Segmentation Parameters box will appear Note When selecting Automatic Segmentation from the Analysis menu you will notice that all of the Labeling functions are inactive This is because as will be explained in the next section in order to use Manual Segmentation a dataset must be in an XY Slice Viewer projection March 2004 Page 190 AutoQuant Imaging Inc Manual and Tutorials Segmentation Parameters Fired Threshold Change Threshold M Parameters Minimum Threshold Value 5 E y Maximum Threshold Value 2 96 g pr Minimally Maximally Adaptive Adaptive 4 Ye a E Ls rA salt Close Help Press and Hold to view the Original Image The two methods for Automatically segmenting a dataset are the Fixed Threshold and the Adap tive Threshold methods The Fixed Threshold which is the default allows you to specify the lower and upper boundaries of the threshold that is the Minimum and Maximum Threshold val ues The pixels that fall within these boundaries retain their value and are turned white all other pixels are blackened out The Adaptive Threshold compares a pixel with its neighboring pixels and selects those which are above a specified value and are turned white all other pixels are blackened out In both cases the end result is a binary black and white image 3 Leave the d
332. ze T Optical Density Correction ll Attenuation Correction 2 Background Equalization a Invert Data x Image Algebra Statistics Toolbar Icons p Line Length Y Statistics Surface Area and Yolume Zoom Toolbar Icon 200 me March 2004 Page 223 AutoQuant Imaging Inc Manual and Tutorials Bibliography Agard D A 1984 Optical Sectioning Microscopy Cellular Architecture in Three Dimen sions Annual Review of Biophysics and Bioengineering 13 191 219 Aikens R S D A Agard and J W Sedat 1989 Solid State Imagers for Microscopy Meth ods in Cell Biology 29 291 313 Ayers G R and J C Dainty 1988 Iterative Blind Deconvolution Method and Its Applica tions Optics Letters 13 7 547 549 Bertero M P Boccacci G J Brakenhoff F Malfanti and H T M Van der Voort 1990 Three Dimensional Image Restoration and Super Resolution in Fluorescence Confocal Micros copy Journal of Microscopy 157 1 3 20 Carrington W A 1990 Image Restoration in 3D Microscopy with Limited Data Bioimaging and Two Dimensional Spectroscopy Los Angeles SPIE 1205 72 83 Cohen A R B Roysam and J N Turner 1994 Automated Tracing and Volume Measure ments of Neurons from 3 D Confocal Fluorescence Microscopy Data Journal of Microscopy 173 2 103 114 Conchello J and E Hansen 1990 Enhanced 3 D Reconstruction From Confocal Scanning Microscope Im

AutoQuant 8.0.2 Suite User's Manual

Contents

Download Pdf Manuals

Related Search

Related Contents