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Huygens Professional User Guide

1. Working with Movie Projects Using the Timeline Export to AVI or Tiff Series If the result is satisfactory press the record but i ton below the preview See Figure 13 4 to render the final movie and export it to AVI or a ae Tiff series Before the save dialog appears the 640 Huygens Movie Maker will show the animation settings dialog See Figure 13 5 where e g the AVI quality can be set Note that large movies Hone i will take several minutes to render High quality V Height Frame rate 12 Once the movie has been exported to AVI it can be opened in a movie player like Apple Quick Time or Windows Movie Player To quickly FIGURE 13 5 The animation open the last saved movie in the operating sys settings dialog tem s default movie player press the button labeled Open AVI in external movie player B AVI quality 90 Cancel Accept Saving Projects The collection of keyframes transitions and animation settings is called a project The image itself does not belong to the project To save the current project to disk press the save button or choose FILESSAVE PROJECT in the menu The Movie Maker project files have the extension hgsa Huygens animation template The Movie Maker will ask if the project should be saved when one attempts to close the Movie Maker while there are unsaved changes to the project Reloading and Appending Projects If a saved project
2. This little bar reports Pipe P ch 0 8 obj Pipe S ch 1 17 obj 2 selected anchors e left the number of objects currently segmented in each surface pipe e right the total number of objects selected as anchors Table and Analysis Shortcuts 19 These widgets give quick access to some table operations e Auto clean checkbox this option makes the table to be cleaned whenever the seg mentation or analysis conditions change to always have a fresh start Deselect this option to keep all data and keep accumulating rows in the table e y Clear statistics table manually delete the table contents e y Filter opens a dialog that allows you to filter objects out based on the reported parameters see below e c Analyze all Analyze sel This button runs the analysis procedure on all the cur rently segmented objects for objects on the pipes selected by the Pipe mode radio buttons See Figure 19 1 item 6 When a 2D area has been selected See Figure 19 1 item 9 this buttons analyzes only the objects under the selection Filtering Objects The Filter button opens a pop up dialog that allows you to discard objects Huygens Professional User Guide for version 4 1 125 e Based on any of the reported parameters e Using a certain arithmetic operator e Tocompare the parameter with either a fixed value or with another reported param eter The pipes that are filtered can be controlled with Based on
3. e Quality change threshold This number gives the minimum quality increase between subsequent iterations If the increase of quality drops below the Quality change threshold the deconvolution process will stop The lower the threshold value and the larger the number of itera tions the higher is the quality of deconvolution e Other parameters like Brick mode Iteration mode and Padding mode are best kept in their default modes More information on these modes can be found in the SVI wiki pages Pressing the RUN button will end up with a restored result in the destination image Figure 3 11 You may compare this result with the original image using two linked Slic ers see step 3 on page 12 After inspecting the result you may want to perform another deconvolution run Maybe you also wish to change one or more parameters In case the output image destination is not changed you will be asked if you like to do a completely new run or replace the pre vious result 5 http support svi nl wiki BrickSplitting 6 http support svi nl wiki IterationMode 7 http support svi nl wiki PaddingMode 20 Huygens Professional User Guide for version 4 1 Step 8 Saving your Image e Huygens Professional File Edit Deconvolution Visualization Analysis Options Help I 3 E xw A mW S Gb RBR New Open Save ss Del Del all Clear Copy Paste Repl Crop S Split XS A Comb Z Comb T Tools T lj aa
4. eee eee eee eee 101 Interaction with the ODbJectSs 25 9 a ua I e REIS Ido Tak e 104 Render Pee 105 Object SSN 106 StOLING VOLI Results acido o coe AS 110 Fore Reading a ania trices op oE Sry ON ANTI RUE d alee ROR a RC Md dion 110 CHAPTER 18 Object Analyzer Geometry Measurements 111 ISO SULIACE poaae ao ex MER Gap NO WERE RON an A UU nie RP d 111 Principali 3lsoeda sam eunti td aoi Woe app neis v deis e odd eue 111 Letieth and WAH san ac ssa carpet nep d Gre dec ere a 111 Meriva NS 112 AP Ribe Ve 113 More Pat meters and Filtering s iius ehe RAE RS see NERE ES Seed 113 CHAPTER 19 Object Analyzer Component Reference 115 Main window compohentsesdsstesebns avs eek iis RR Udo ER RUE 116 CHAPTER 20 The Colocalization Analyzer 131 How to use the Colocalization Analyzer cc ccc cece ec ee eee cence 131 Iso colocalization object analysis 0 cece cee cece ee cece eee eee eens 134 Backgrounds vs thresholds in colocalization ccc cee eee cece ee eee 135 RONCO atere eine Oe RE EEE PE euis 136 CHAPTER 21 Establishing Image Parameters 137 mage SIZE P PR 137 Signal to Noise Ration cods05 oe rx eee 137 Black Ka M mer 139 Sampling Density tesa seed Sd SG 139 Computing the Backprojected Pinhole Radius and Distance 140 CHAPTER 22 Improving Image Quality eee eee eee 145 Data Acquisition Pl
5. Once the proper parameters have been set and verified they can be saved to a Huygens template file hgst These templates can be applied at the start of the parameter wizard or in the parameter editor The LOAD A TEMPLATE button will allow the selection of a template from a list of saved template files which reside both in the common templates directory and in the user s personal template directory The Huygens common templates directory is named Templates and resides in the Huygens installation directory See Table 2 1 on page 8 The user s personal templates directory is called SVI Templates and be found in the user s home directory 26 Huygens Professional User Guide for version 4 1 Using a Measured PSF Using a Measured PSF The Intelligent Cropper Measured PSF s improve the deconvolution results and may also serve as a quality test for the microscope If the measured PSF contains less channels than the image a theoret ical PSF will be generated for the channels where there is no PSF available See Chapter 5 The PSF Distiller on page 33 and The Point Spread Function on page 149 for more information Once the deconvolution wizard is opened a previously created deconvolution template and or a measured Point Spread Function PSF can be loaded A measured PSF should only be used for deconvolution if the image and the bead s were recorded with the same microscope at the same parameter settin
6. gt xi t Xio i 1 Help Plot ofthe average distance ofthe objects from the origin of their tracks versus the frame number Help Plot of the MSD of the objects from the origin of their tracks versus the elapsed time Ifthe histogram above is considered a half normal distribution then the values in this plot are the variances TIP Click and drag in the plot to measure the slope FIGURE 16 8 The slope of the mean squared displacement plot indicates a diffusion coefficient of 9 32 um s 2 The Speed Tab EQ 2 There is also a close relation between the MSD and the histogram in the first plot Figure 16 7 In case of pure Brownian motion and a large number of objects the histo gram will show a half normal distribution In this special case the variance of that dis tribution is given by MSD t The rate of change of this variance is known as the dif fusion coefficient D which thus can be estimated by measuring the slope of the MSD plot Figure 16 8 Just click and drag in any of the plots to measure dis tances and slopes This tab displays information on the velocity of the tracked objects The first plot is a his togram that shows the distribution of the speed of the objects while the second plot shows the average speed over time 96 Huygens Professional User Guide for version 4 1 The Track Analyzer The third plot informs about the velocity distribution of the object
7. See Figure 5 3 Huygens PSF distiller rt Help File Input beads Help Reports PSFs amp Accus Searching aligning and averaging of beads Microscopic parameters loaded Beads 3 Accumulator chO Averaging beads There are currently 9 beads in the accumulator Distiller status Load an additional bead image or click Distill to a Startthe PSF distiller Total beads averaged 5 Per image averaged beads Beads 9 Loadanotherbead Distill Ready Dragging on FIGURE 5 3 The averaging stage in the PSF Distiller wizard Confocal and Two Photon Bead Images Images from 160 nm beads should look like smooth fuzzy blobs with hardly visible noise Use the default SNR settings If available it is a good idea to average 2 to 5 beads 36 Huygens Professional User Guide for version 4 1 Distillation Stage Distillation Stage Finalizing the result Two photon bead images may look slightly noisy If so set the SNR to 20 and average 4 to 10 beads To load more bead images press LOAD ANOTHER BEAD in the averaging stage and either select an image from the main window or open a new one Widefield Bead Images Images from 160 nm beads should look like smooth fuzzy blobs with no visible noise Use the default SNR settings Averaging beads is not necessary for widefield images The distillation stage usually requires no user intervention though in some cases a pop up w
8. The Huygens Orthogonal Slicer shown in Figure 9 1 is designed to show the same point in 3D space from three orthogonal directions e axial or xy top left e frontal or xz bottom left e transverse or yz bottom right Huygens OrthoSlicer File View Plot Help Value ch 0 522 S AJL D e sl ss BE s I 4 23 8 46 7 ch 1 122 6 46 12 Time frame Contrast FIGURE 9 1 The Huygens Orthogonal Slicer If you move one of the slices the others will follow to make sure that the center of each of the slices intersects in the same point in space This behaviour makes the Ortho Slicer a useful tool to study small objects in 3D Huygens Professional User Guide for version 4 1 55 The Crosshair Cursor Visualization parameters Measurements Auto Zoom Display Options The position of your mouse is projected as a cross hairs pointer on all slices The value besides the center of the cross hairs gives the distance of the mouse position to this pro jection If this number is positive it means that real pointer is more towards you in front of your screen Changing the visualization parameters in the Orthogonal Slicer is similar to the Huygens Twin Slicer on page 47 There are tools to e change time frames e zoom in out fit or zoom 1 1 e change display colors e tune the brightness and contrast Panning can be achieved by right clicking and dragging an image
9. e Colocalization Analyzer f PSF Distiller M Movie Maker N Enable new pre released features S Object Stabilizer E Time series v Surface Renderer visualization x Chromatic Shift Corrector a RBNCC option for colocalization T Object tracker Huygens Professional User Guide for version 4 1 159 160 Huygens Professional User Guide for version 4 1 A Address iv 157 Analyze object 104 Anchor 122 Animation frame count 70 frame rate 70 Movie Maker 73 SFP 64 Surface Renderer 70 AVI files 76 B Background 18 29 135 Backprojected pinhole radius 25 pinhole spacing 25 Batch Processor 39 Beads 33 36 Black level 15 29 Bleaching 20 147 Bleaching correction 30 Bounce 78 Brick layout 30 Bricks 20 Brightfield images 155 C Center scene 70 Channels 31 Clipping 15 29 146 CMLE 30 Colocalization 113 131 coefficients 133 map 133 Color mode 45 64 Colors 151 Concurrent tasks 43 Contact SVI 157 Contrast 46 Convert 24 Correlation 113 Coverslip position 25 152 Cropper 14 27 D Deconvolution templates 42 wizard 23 Discard object 104 Distiller 33 Distributors 157 Drift 32 E E mail iv 157 Emission transparency 63 wavelength 25 Excitation fill factor 25 154 Huygens Professional User Guide for version 4 1 photon count 25 transparency 63 wavelength 25 Experiment preset 107 123 F Fax number iv 157 File Formats 2 24 Open 24 Save 32 Series 150 Fil
10. e Distillation Stage in this stage the PSF is measured from the averaged beads for all available channels e Finalizing the result in case it is desired to combine results from earlier distillations with the current result to obtain a multi channel PSE an earlier result can be added here It is also possible to add single or multi channel previous results to a current multi channel result e Save the result The next sections of this chapter will explain the wizard stages in detail See Loading an Image on page 24 and Saving the Result on page 32 for more information on handling image files Starting the Distiller After launching Huygens Professional open the first bead image via FILE gt OPEN If the license includes the PSF Distiller option start the PSF Distiller via the menu DECONVO LUTION PSF DISTILLER When the window is opened one or more accumulator images will be created into which later on the averaged beads will be kept Now the start stage will be entered Verifying Microscopic Parameters See Verifying Microscopic Parameters on page 25 for more information on the micro scopic parameters Next to the optical parameters listed in Table 4 1 on page 25 it is in particular important to check the sampling densities Huygens Professional User Guide for version 4 1 35 Averaging Stage Do not use undersampled bead images If any of the entry fields for the sampling density turns orange or red the
11. tion of the mouse pointer Changing Display Colors Click an option in the Color panel to select a color scheme e Greyscale the image is displayed in gray tints For single channel images this gives a higher contrast than the emission or global colors e Emission colors if the the emission wavelengths are set correctly this gives the most intuitive view e Global colors the colors as defined in the global color scheme The global color scheme applies to all visualization tools and can be modified via the Huygens Profes sional main menu OPTIONS PREFERENCES EDIT GLOBAL COLORS e False colors a false color is given to each intensity value This view gives a high con trast and makes it easy to spot areas of homogeneous intensity Tuning the Brightness and Contrast The brightness can be adjusted in the most right Brightness panel using the buttons and dragging the slider or putting the mouse pointer over the slider and using the scroll wheel The Gamma panel provides a linear and some nonlinear ways of mapping data values to pixel intensities These are e Linear default pixel values are mapped to screen buffer color intensities in a linear fashion Note that the actual translation of the screen buffer values to the actual brightness of a screen pixel is usually quite nonlinear e Compress where an image contains a few very bright spots and some larger darker structures using linear mode will result in poor visibili
12. Advanced Usage Advanced Usage from the microscopic sampling sizes and number of pixels the image is composed of If the microscopic sampling sizes of the image are incorrect then the object size is set according to some default parameters and may not be related to the actual object size Saving Scenes Choose FILE gt SAVE SCENE to save the rendered scene as a Tiff file SFP Fundamentals The voxel values in the image are taken as the density of fluorescent material In case of a multi channel image each channel is handled as a different fluorescent dye Each dye has its specific excitation and emission wavelength with corresponding distinct absorp tion properties The absorption properties can be controlled by the user See the trans parencies in Table 11 1 on page 63 The different emission wavelengths give each dye its specific color To excite the fluorescent matter light must traverse other matter The resulting attenua tion of the excitation light will cause objects which are hidden from the light source by other objects to be weakly illuminated if at all The attenuation of the excitation light will be visible as shadows on other objects To optimally use the depth perception cues generated by these shadows a flat table below the data volume is placed on which the cast shadows become clearly visible In Figure 11 2 the table is rendered as a mirror After excitation the fluorescent matter will emit light at a lo
13. By using the right click on a channel checkbox all channels can be turned on or off except for the selected channel Cropper Custimizations and Options The cropper is extended with extra visualisation options which are available under Options LOCK RATIO fixes the aspect ratio of the cropping box for each projection sepa rately PIXEL PROPERTIES shows the pixel intensities and position of the mouse when you hover over the image The intensities however are only shown when one time frame and one channel are selected When you turn on PREVIEW a small renderer shows the result ing image after cropping The histogram is an important statistical tool for inspecting the image It is included in the deconvolution wizard to be able to spot problems that might have occurred during the recording The histogram shows the number of pixels as a function of the intensity gray value or groups of intensities If the image is an 8 bit image gray values vary between 0 and 255 The x axis is the gray value and the y axis is the number of pixels in the image with that gray value If the image is more than 8 bit then gray values are collected to form a bin For example gray values in the range 0 9 are collected in bin 0 values in the range 10 19 in bin 1 etc The histogram plot now shows the number of pixels in every bin 28 Huygens Professional User Guide for version 4 1 Estimating the Average Background Estimating the Average Back
14. Microscopic Parameters TABLE 4 1 Optical parameters explained Parameter Microscope type Numerical aperture Objective quality Coverslip position Imaging direction Backprojected pinhole radius Backprojected pinhole spac ing Lens refractive index Medium refractive index Excitation wavelength Emission wavelength Excitation photon count Excitation fill factor Explanation Select from widefield confocal spinning disk or fourPi For a multiphoton microscope select confocal and insert an exitation photon count higher than 1 The NA of the objective lens Select from perfect poor or something in between The position of the glass interface between the immersion and embedding medium in um relative to the first slice of the stack Select from upward or downward Upward means that the objective lens is closest to the bottom slice in the stack Downward stands for an inverted microscope The radius in nm of the pinholes in the spinning disk as it appears in the specimen plane This is the physical pin hole radius divided by the total magnification of the detection system The distance in um between the pinholes in the spin ning disk as it appears in the specimen plane This is the physical pinhole distance divided by the total magnifica tion of the detection system The RI of the immersion medium for the objective lens The RI of the specimen embedding medium The wavelength in nm of the exci
15. Save current ROI to file Load ROI from file Add ROI from file Intersect with ROI from file Subtract ROI from file Center ROI on the anchor CM align the Center Of Mass CM of the currently defined ROI with the CM of the selected anchors Clear ROI Keep only objects inside the ROI discarding anything else The relaxed selection mode in the options also affects how objects partially inside the ROI are handled Huygens Professional User Guide for version 4 1 117 e Analyze ROI volume computes and reports information on the table about the ROI itself e Analyze all objects inside the ROI reports in the table information about objects inside the ROI or partially outside it depending on the relaxed selection option e Help on Regions of Interest ANCHORS e Select all objects as anchors e Deselect all anchors e Invert current anchor set e Setanchors by filtering This opens a filter dialog as explained in Table and Analysis Shortcuts 19 on page 125 but allowing you to select or deselect anchors instead of discarding objects e Keep anchor objects discarding anything else e Discard all anchor objects e Analyze only objects select as anchors FILTER some useful predefined filters to remove objects based on their features and access to a full control filter tool and to reload the original data e Quickly remove objects that are touching the borders of the image as they are surely incomplete e Quickly r
16. See Tuning the Brightness and Contrast on page 49 for an overview If the Link channels box is checked this means that the way of mapping data values to pixel intensities is the same for all channels if not the range is automatically adjusted for to minimum and maximum in each channel 50 Huygens Professional User Guide for version 4 1 Using the Slicer in Advanced Mode Linking Controls The LINKING menu can be used to change the way in which both slicers communicate The options in this menu are listed in Table 8 2 Note that settings get synchronized once the controls are being used TABLE 8 2 The options in the Twin Slicer s LINKING menu accessible in advanced mode Option Description POINTER LOCATION Shows the position of the mouse pointer in the other slicer SLICE POSITION Makes sure that the cutting plane for the right slicer crosses the center of the left slice and vice versa TIME FRAME Synchronize the time ZOOM LEVEL Synchronize the level of magnification PANNING This does not affect position of the cutting plane but it shifts the right slice such that the projection of the center of the left slice is in the center of the right slice and vice versa ROTATION Makes sure that the rotation angles for both cutting planes are the same ACTIVE CHANNELS The left and right slicer will have the same channels enabled and disabled COLOR SCHEME Makes sure that the left and right slicer use the same color
17. To center the slice press the Center button the blue dot at the lower left of the image or press c To overlay a ruler on the image hold the left mouse button and drag The length of the line in microns is displayed beside it Left click and drag the end points of the ruler to make adjustments Note that the other orthogonal directions show a projection of this ruler Press and hold Ct x1 while dragging an end point to change length without chang ing direction Left click and drag the middle of the ruler to move it in its entirety with out changing length or direction Press and hold the Ct rl key while dragging the ruler to move it perpendicular to its direction To remove the ruler left click somewhere else on the image When a ruler is drawn the help pane will be replaced by a plot that shows the intensity profile along it See the SVI wiki for more information about the data plotter s capabili ties When you click the middle mouse button the Orthogonal Slicer will automatically cen ter and zoom in on the brightest spot in a 3D neighborhood around the mouse pointer The VIEW menu allows you to show or hide information and guides within the image overlay including pointer coordinates time intensity zoom rotation and the wire frame The Global value range option in the PLOT menu uses the maximum and minimum value of the image s to determine the visible range of the plot otherwise it uses the maximum and minimu
18. e Destination The destination image in which the result will be stored e Signal Noise per channel This is the value that was estimated in step 6 e Max iterations 4 http www svi nl RestorationMethod Huygens Professional User Guide for version 4 1 19 The deconvolution process will stop when the maximum number of iterations is reached or when the increase of quality drops below the Quality change threshold Depending on initial quality of your image a higher number of iterations will result in a better restoration result e Search for background The option Auto will choose the best estimation mode for you and calculates the background value In Manual mode the background value should be specified in the field below This value was calculated in step 5 e Backgr per ch absolute or 96 Multi channel images may have different background values per channel If the previ ous parameter was set to Manual this list of values holds the background for each of the channels Since the backgrounds established by the survey are conservative values unless In near object is selected they can be increased by a percentage For instance 1096 means all background estimates are increased by 1096 Negative per centages are also valid e Bleaching correction If this parameter is set to If possible bleaching correction will be performed for widefield images or confocal time series showing exponential behavior
19. seed 524 07 garb 1 objects 9 ROI vol 0 34587 um 3 CM 170 13 213 42 9 1209 frame 0 1 V Conditions pipe 1 chan 1 thresh 203 seed 254 garb 1 objects 17 ROI vol 0 34587 um 3 CM 170 13 213 42 9 1209 frame 0 1 3 1 ol P 355 48246 75232 61899 0 169549 56 9 16177 42609 13 3981 4 2 ol PL 4911 94659 38924 57894 Q 323823 32 1 34402 46413 23681 5 3 i F 1677 60 758 2659 6 7792 o 730828 35 7 268129 34 511 15 137 6 4 0 P 514 23813 78466 65037 0 220270 08 5 53358 76415 26321 7 5 i F 396 16171 53145 7 1591 0 164795 01 e 50684 10735 74654 4 m The contents of the table can be copied to your clipboard or stored to a file in disk by using the FILE menu The Table Columns and Their Headers 20 When you move your mouse over the column titles at the top of the table you get a description of each parameter at the very bottom of the window See Figure 19 1 item 20 There are many parameters that measure complex things so in the column title there is only room for a cryptic label You can always find out what each parameter is by looking at its tooltip The description of each parameter will be also stored in your file when you export the table later Distance to first neighbor same pipe um 3 http www svi nl ObjectAnalyzerNeighbors 126 Huygens Professional User Guide for version 4 1 Main window components This brief parameter description plus longer explanations are
20. 62 Advanced USE avvek 63 Spee AMOS nes es hides a deus sere 64 The Surface Renderera s 3 53 ERREUR OF basi USABOL da ucsoE Eo race NR ES 68 IAW AINE GUS ACG s ep EUR EP bbb eae tr brine tardan irt 69 Simple AniIManOns eese ee 70 The Movie Maker sorcerer eee eee eee E DR RTO ATL Je sr ee 73 Creating and Adjusting Kofranesvsaavvavsvvrrsrrvr PERDER RAM Leve ied 74 Usine the Storyboard eo y doe Vd RC o V vU RO Cp RE SOR eR pes 75 Working with MOVISLPEOJGCES 12 54 dep ocio xe ew aes a oe eee awe tees 76 Usine the Limieline 2 x vv aei Peces oc IR dpud obedece se Mido ropa do 76 Advance d TOD e See 77 The Chromatic Shift Corrector saaya essens Starting the Chromatic Shift Corrector ccc eee ce cece ee eee eee 79 Estimation of the chromatic SDifS 42 wea v sr 80 Visualization of the chromatic shifts 0 5 5o esee e EHE S Y eee 80 Editing the chromatic shift VeCtOrs sw ses vasse serer CI eens 81 Working withitemplates s sew Uo EE d eee 82 The Object Stabili ooa a deo Pre memo 8 Stabilization of 3D TimeSeries eee ccc eee cece m nnn 83 Alignment Or Slices 3D Stacks ustedes vert uero NRS 88 The Obec ITO NOE vaar ses teste oso 9T mrd TND 91 Huygens Professional User Guide for version 4 1 THe ODE racket Wizard ve 91 Trek erea aa ties a A EE 94 CHAPTER 17 Introduction to the Object Analyzer 99 Starting the Object Analy Zen Ne 99 Segmenting the Objects Setting the Threshold 0
21. Contact Information on page 157 3 http www svi nl 10 Huygens Professional User Guide for version 4 1 Step 1 Start Huygens Professional CHAPTER 3 Step 1 Start Huygens Professional Getting Started This chapter will help you to get through the basic procedures in deconvolving an image and to become familiar with the user interface components of Huygens Professional Note that the steps 4 and 5 described below are automated in the deconvolution run step 7 but they will give you some insight in the process of deconvolving an image On Linux start the software by clicking its icon or by typing huygenspro into a Unix shell On Mac OS X and Windows click its icon When Huygens Professional is started from a Unix shell it is a good idea to navigate to your image directory first The huygenspro command starts a graphical user interface GUI that comes up with four empty thumbnail images Figure 3 1 e Huygens Professional Je File Edit Deconvolution Visualization Analysis Options Help a 3 ial i 2 f d New Open Saves Del Del all Clea Cop Paste Rep Crop Thumbnail overvie dg E b c psf Tcl shell Task reports Ready Memory usage 0 MB FIGURE 3 1 The main window on Microsoft Windows Four empty thumbnails are shown by default Huygens Professional User Guide for version 4 1 11 Step 2 Load an Image Step 3 Inspect your Image Thumbnail
22. Cropping stage Select channel Inspecting the image histogram Background Estimation e Deconvolution Stage Setting the final deconvolution parameters Run the deconvolution Select final result e Postprocessing Stage correct for Z drift if any and accept the result e Save the result The next sections of this chapter will explain the wizard stages in detail Huygens Professional User Guide for version 4 1 23 Loading an Image Select FILE gt OPEN to open the file dialog browse to the directory where the images are stored and select the image e g faba128 h5 A demo image fabal28 h5 is placed in the Images subdirectory of the installation path see Table 2 1 on page 8 Most file formats from microscope vendors are supported but some of them require a special option in the license to be read See the SVI support Wiki for updated informa tion When the file is read successfully select the image to process Then either the DECONVO LUTION WIZARD button can be pressed right upper corner to begin deconvolving the image or to first process the data using the tools under the TOOLS button some tools are described in the next subsections If a bead image was loaded then one can also proceed by selecting PSF DISTILLER in the DECONVOLUTION menu to generate a point spread function from measured beads See Chapter 5 The PSF Distiller on page 33 A special license is needed in order to launch the PSF Distiller Co
23. Details on page 158 Notice that the Object Analyzer See page 99 also provides colocalization measure ments at the object level The Colocalization Analyzer works more at the level of the whole image despite local statistics of the colocalizing regions can be easily retrieved Both analyzers work in a sense in complementary ways The Object Analyzer allows to define objects segmentation and observe how much they overlap in volume or intensity Objects defined like this can overlap with other objects or not The Colocalization Analyzer explores the whole image to search for colocalizing regions based on the usual colocalization coefficients These regions are then segmented and treated as objects to analyze These objects are therefore always volumes of intersection To start the Colocalization Analyzer in Huygens Professional right click on an image s thumbnail to open the contextual menu then select COLOCALIZATION ANALYZER Alter natively an image s thumbnail can be selected then in the menu bar select VISUALIZATION gt COLOCALIZATION ANALYZER The image must be multi channel See Multi channel Images on page 16 as the colocalization is based on the overlapping of l http www svi nl ColocalizationTheory 2 http www svi nl ColocalizationCoefficients Huygens Professional User Guide for version 4 1 131 r A N Huygens Colocalization analyzer Coloc Analyze cimi E File Options He
24. Edit vector tool Click on the Correct image button below to correct the image for Ichromatic shift as defined by the shift vectors in the table A new corrected image will be created Active channels Select Color scheme Global colors Custom colors 0 v Q Zoom Colors Correct image FIGURE 14 2 The Chromatic Shift Corrector after estimating the shifts A plot shows the gap between two channels The estimated shift is drawn as a vector on the slicer The Chromatic Shift Corrector will return accurate and reliable estimations of the exist ing chromatic shifts Nevertheless the possibility to edit and customize the estimated shifts exists so that the user can reach more precission if necessary The user can also edit the shifts estimated automatically by the Chromatic Shift Correc tor To apply a different customized correction a channel has to be selected for edition The components of the selected shift vector can be modified by using the edit tool which allows to shorten and lengthen the estimated shift Huygens Professional User Guide for version 4 1 81 Working with templates While the shift of the edited channel is shortened or lengthened the plot updates itself to show how the gap between the reference channel and the edited channel is increased or decreased In this way the contents of the estimated shifts can be modified while checking in advance whether the image will be corrected appropria
25. Garbage Volume Level A quick way of removing disturbing objects is the GARBAGE VOLUME You can find this entry in the alternative slider view of the threshold to which you can switch by clicking the small button fi at the top right of the histogram The garbage entry will be shown right below the seed slider Objects with a number of voxels below the garbage level are 102 Huygens Professional User Guide for version 4 1 Segmenting the Objects Setting the Threshold discarded This means that when you set it to 1 no segmented object is discarded but if you set it to e g 100 any object with a volume smaller than 100 voxels will be removed So far for the segmentation You can apply some post segmentation filtering in complex ways the details are explained in the expert tutorial In the top menu you can also find some predefined Filters that you can use for quick access to discard objects based on some basic geometrical properties or on the way they relate to other objects By now these two tools threshold and garbage level are already powerful enough to continue our exploration of other Object Analyzer features Using the watershed segmentation An extension of the seed and threshold segmentation method is the watershed segmen tation In case of using the seed and threshold method it is possible that objects merge at a certain threshold level To solve this problem the watershed segmentation is imple meted If you do n
26. Guide for version 4 1 27 The Image Histogram Cropping in X Y and Z The borders of the proposed cropping region are indicated by a coloured contour The initial position is computed from the image content and the microscopic parameters at launch time of the cropper The three views shown are maximum intensity projections MIPs along the main axes For more information about these projections read Chapter 10 The MIP Renderer on page 57 By default the entire volume including all time frames is projected The red yellow and blue triangles can be dragged to restrict the projected volume The cropper allows manual adjustment of the proposed crop region To adjust the crop region drag the corners or sides of the cropbox to the desired position or use the entries in the Specifications panel To crop the original image press the CROP button To create a new cropped image press the EXTRACT button The EXTRACT button is however not vis ible when opened through the deconvolution wizard Do not crop the object too tightly because that would remove blur information relevant for deconvolution Cropping in Time and removing channels To crop in time select the from and t o frame under Specifications There are entries for the time selection if the image is a time series Only a continuous range can be selected to crop in time All available channels are listed under Channels in the operation window and can be removed individually
27. Guide for version 4 1 149 File Series There are many ways in which Tiff files or other file series are named These files can have multiple counters referring to slices time frames or channels and these counters can have arbitrary prefixes and ordering Numbered Tiff Series If a series is simply numbered like slice001 tif slice002 tif sliceOnn tif then Huygens Professional will read the series into a single 3D image Because Tiff files usually carry no additional microscopic information check the parameters carefully Leica Numbering Huygens Professional natively supports both reading and writing Tiff series with Leica style numbering if there is more than one channel slice or time frame A single channel 2D time series would be numbered according to the scheme im tNN tif Here NN is replaced by the time index for each frame A more complex multi channel 3D time series has this pattern im tNN zNNN cNN tif In this series the second channel of the fourth slice of the third time frame has the file name im t02 z003 cO1 tif The File Series Tool WB Huygens File Series Tool Although Huygens Professional uses Leica o style numbering for writing files the soft Huygens Professional scanned the directory ware attempts to detect any type of file CJUsers edwin Desktop ImagesY for matching file names i series for reading Whenever a file i
28. Iso colocalization object analysis So at startup the iso colocalization surface sliders are deactivated as we have to calculate a colocalization map first by pressing COMPUTE Together with a surface renderer a MIP renderer is available The viewpoint of the MIP and surface can be selected by moving the Tilt and Twist slide Also try changing the zoom The obtained colocalization map is represented in the renderer window by iso colocal ization surfaces These iso surfaces represents points which all have the same colocaliza tion value thus regions in which the degree of colocalization exceeds a certain value become objects This certain value can be controlled by the threshold slider in the iso colocalization surface parameters The transparency and the brightness of this surface pipe can be controlled with the correspondent sliders The color range in which the objects are displayed can be modified using a hue selector page 151 One can also switch the surface pipe off Some modes generate two channel colocalization maps colocalization of red with respect to green and vice versa e g in case of the Manders M1 and M2 coefficients In these cases the iso colocalization surface parameters will offer the possibility of render ing any of the two channels and thus the threshold is referred to the active one By clicking on the rendered objects local colocalization parameters are computed and reported which will be discussed in more
29. SLICER The Slicer enables the user to show a single 2D plane extracted from a 3D volume There are controls to select any plane orientation in space zoom and scroll through the available planes For time series a separate control is available to scroll through the available time frames Furthermore controls are available to set the contrast brightness and color scheme for the different channels Color schemes include spectral colors i e the dis played color of a channel matches the true color of the emission wavelength in so far this is possible You can open as many Slicers as you like on the same image or on different images Figure 3 2 Multiple Slicers can be linked and listen to other Slicer windows When linking is active right clicking on the main Slicer will change the other view s center to the clicked coordinate regardless of its orientation Note that the option other from the linking panel has to be selected in order listen to Slicer windows of other images More information on the Slicer and other Huygens visualization tools can be read start ing from The Slicer on page 45 onwards 12 Huygens Professional User Guide for version 4 1 Step 3 Inspect your Image oe is Linking Position V Orient V Center FIGURE 3 2 Two slicers opened from the same image V Listen I Master Contrast e Huygens Slicer fabal28 commi e Huygens Slic
30. Table 10 2 gives an overview of the different render options that are available through the TABLE 10 2 Render options for the MIP Renderer Option RENDER SIZE COLOR MODE SHOW SVI LOGO OPTIONS menu Description Adjust the size of the rendered image When the render size exceeds the display area then use the right mouse button to pick up and move the scene canvas Choose between GREY EMISSION COLORS GLOBAL PALETTE See Adjusting the Global Color Scheme on page 151 or FALSE COLOR Hide or show the SVI logo at the bottom right Huygens Professional User Guide for version 4 1 59 Simple Animations Templates All scene settings i e both the render options and all parameters can be exported to a template file via FILE gt SAVE SCENE TEMPLATE The template files have the extention hgsv and can be applied to any image that is loaded in the MIP Renderer The Huygens Movie Maker See The Movie Maker on page 73 allows to create easily sophisticated animations using the MIP SFP and Surface Renderer Without the Movie Maker the MIP Renderer has the option to make simple animations of the image changing the view point in different movie frames Set the render parame ters for the first frame and click MOVIE tab SET 2FIRST SCENE Now adjust the view point for the final frame and click SET gt LAST SCENE Set also the frame count frame rate and other general options such as colors Finally press th
31. Table onl v ist neighbor of the object s 1st neighbor same pipe Scene too Distance to first neighbor same pipe um Advanced modes All parameters listed above will be reported on the Correlation between channel table cem sg This parameter set will report for each objectthe distance and the ID of the nearest neighbor in its same pipe Some selected parameters will be also on the Calculate color shift interactive screen for the object pointed to You will also get the neighbor ofthe neighbor you can To enable even more parameters and configure your use itto find and filter couples of objects own presets go to Options Configure reported parameters Distance to reference objects Location inside a flat nucleus Location inside a round nucleus Colocalization intersection Intensity in the other channel OK amp Help FIGURE 17 8 The Experiment Presets dialog Collections of parameters can be selected to be reported on the table and interactively on the scene depending on the experimental needs On its left column a series of different experimental needs are listed When you click on each of them a new list of parameters is listed in the middle column and a description is shown on the right column Even more when you hover with your mouse over the listed parameters you get a tooltip text explaining each parameter with more detail Please read
32. The Batch Processor e 0 00 0 0 090 09 0 09 09 09 09 09 00000000 090 9 90 0 9 0 0 O6 39 The Batch Processor Window 4499 eB Gr 39 LER NE dao e E aetna ers ds 40 huie me P 43 CHAPTER 7 The Slicer 0 09 0 0 0 0 09 0 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 9 09 09 9 09 9 09 099 06 RE oO 45 err Ct CT 45 Contrast surre ee 46 IUe ec ae 46 bm ucc aes RON CIO e AIT ans ere 46 CHAPTER 8 The Twin Slicer 0 0 0 0 0 0 0o 0 0o 09 09 09 09 0e 09 09 09 09 09 09 09 09 09 09 09 9 0 ERR ERE ERE 47 Using theliceran Dasic Mode Lau ru ee de Ceu EE RUN eve toes 48 Usine the Shcer in Advanced Mode 203 eo ea Eee oe arie d adt 50 WeASULCINCN sa ers euch xa TK ub ido eke er ah d XR Euer ie i 52 CHAPTER 9 The Orthogonal Slicer 0 0 09 0 09 0 0 09 0e 0 0e 09 0o 09 09 09 0o 0e 0e 0o 000000 55 The Grosshait Cursor Hvasser vate DRS 56 Visualization parameters oda e Rs SKE CHR PURI E REND REN ERU 56 Measure MENES opes uid eR Seed 56 ii Huygens Professional User Guide for version 4 1 CHAPTER 10 CHAPTER 11 CHAPTER 12 CHAPTER 13 CHAPTER 14 CHAPTER 15 CHAPTER 16 NMS ZOOM see 56 Display Omar 56 The MIP Renderer uuu RC CERE Basic Ude ee 57 Advanced Valur eee Ver pP wage 59 SIMpIC ANNAN s Subs US OE o uA OS RIO AD EO bred A ERO paie 60 The ST P Rendere io vois emm edet bah SE sae meta o toas OL DASIC USAR ia deste ed e wee buPS DE S US e M End a eS Aud a v dris
33. V Listen Time Zoom Other Slicing Slice center at voxel coordinate 0240 0216 035 2 e b I View options v Link channels Stretching Global Gamma Compress Color Contrast gt Time Ready Canvas size 443 x 410 Interactive FIGURE 3 9 The PSF in x z view False coloring and a compressed contrast highlight the low intensity values showing the typical diabolo shape in lateral view Note that although this step gives you insight in the PSE it is actually better letting the deconvolution run generate the PSF on the fly Especially while using Huygens Professional User Guide for version 4 1 17 Step 5 Estimate the Average Background Value Step 6 Estimate the Signal to Noise Ratio SNR large images that are processed brick wise see Brick Wise Processing on page 137 the PSF is prevented from being unnecessary large it adapts its size to the brick size In case of a multi channel image the PSF is generated for one channel at the time instead for all at once All images can be cleared using the command EDIT gt ZERO IMAGE in the main window You can do this for the image psf as it will not be used in the next steps Huygens estimates the average background in a volume image The average background is thought to correspond with the noise free equivalent of the measured noisy image It is important for the search strategy that the microscopic parameters of the i
34. a too large SNR value might be risky when restoring noisy originals because the noise could just being enhanced A noise free widefield image usually has SNR values higher than 50 A noisy confocal image can have values lower than 20 A good starting SNR value can be estimated simply by visual inspection from the quality of the image Figure 21 1 shows some examples of recordings where different noise levels Original SNR 30 SNR 15 SNR 5 FIGURE 21 1 Examples of different SNR values Same image different noise levels were added to an original restored image Estimating the SNR in noisy images is fairly easy It is based on the idea of establishing the voxel intensity value s corresponding to a single photon hit by looking for such an event in a dark areas of the image Knowing the intensity value M of the brightest voxel in the image one can now calculate how many photons are involved in it The SNR is now defined as SNR M EQ 1 E If a significant blacklevel is present it should be subtracted from M and s See Black Level on page 139 and the SVI Wiki for more details In low noise images this is much more difficult as single photon events are no longer easily observed Fortunately in such cases the establishment of the precise SNR is not very important for the restoration method and a rough estimation based on the appear ance of the image is usually enough See Figure 21 1 2 http www svi nl SNR
35. also given in the selection and configuration dialogs that selects or configures the reported parameters For columns with numeric values basic descriptive statistics of all values in the column are also reported along with the parameter description You can select whether the sum runs for both pipes ALL or PS or only for one of the pipes P or S depending on the selected pipe mode See Figure 19 1 item 6 This provides a quick way of finding the total number of voxels in the object list or those that are colocalizing for example Many interesting questions can be answered by this summations and ratios between them You can right click on a column to pop up a contextual menu from which you can retrieve more detailed statistics STATS for that column The descriptive statistics will analyze pipes separately and together and also include ROI information if present The reported values are the maximum MAX the median MED the minimum MIN the number of items considered N the summation of the values SUM the average value AVG and the standard deviation for the N items SD N reported also as a percentage relative to the average value Clicking on a column title selects the whole column for you to copy Multiple columns can be selected by holding the Ct r1 key To plot a his togram of the distribution of values in a column select the column or a subset and select Histogram from the pop up menu IE 1N CMCM 0 1 N 53
36. are finished it should be quite fast in this example move your mouse over the table rows and see what happens The object corresponding to the current row will be highlighted on the canvas and the distance to its nearest neighbor will be shown 38 1 5 14 10279 92715 13 935 0 2636 93 2 17649 8 1495 6 2257 8 1499 37 1 s 7 14458 163 71 14 ol 1316 35 8 3 0245 27868 35824 2 0866 1 7127 9 4394 10 844 4 0386 ol 1983 05 g 1273 15 083 0 8238 29 2 2 2204 17 244 s 11 32 902 78 999 15 5 40 77 396 94902 15224 1 l 39 40 Ready rendered Canvas size 678 x 577 Dragging on 38 S 1NP CMCM 4 2358 um FIGURE 17 10 Exploring the table Hovering the cursor over the table rows also highlights the corresponding object on the scene A good way to find an object in a very long table is by clicking on it on the rendering canvas while the Analyze object mode is active the table will be shifted to show its corre sponding row and it will be highlighted By moving the mouse over the column titles you get an expanded description of the parameter plus some basic statistics of all the cells in that column You can also right click on a column and select STATS for more detailed statistics To plot a histogram of the distribution of values in a column select the column or a sub set click with the right mouse button and select HISTOGRAM from the pop up menu Huygens Professional User Gu
37. be anchors for example to act as references to measure distances from other objects when asking for local statistics When you set an object as a reference anchor it will light up and change color on the screen to indicate its new status It is possible to select a group of anchor objects and you can operate with them through the Anchors menu e Rotate scene interacts with the full image to rotate it in the space by dragging the mouse pointer on the rendering view That can also be achieved by moving the Tilt and Twist sliders along the rendering e ap Pan scene interacts with the full image to move it in space laterally This means that you can pan the scene in the 2D plane of your screen not along the third dimen sion along your line of sight e 8 Pan canvas is similar to pan scene but not exactly the same It allows you to explore the canvas by not re rendering the scene This only makes sense when you have a canvas larger than your rendering window of course See OPTIONS VIRTUAL RENDER SIZE in the top menu e Shift the ROI This is the only mouse mode that is not always enabled you need to have defined a region of interest ROI before moving it around For the advanced users some of these mouse modes have shortcuts in other mouse modes Most of the times you can pan scene independently of the selected mouse mode if you use your mouse right button instead of the left one Similarly you can pan canva
38. buttons column there is a colored reference cube that will help you in orienting in space when you rotate the dataset specially with large zoom factors that do not let you see the surrounding box frame in the ren dered image The initial view of this cube is the blue top face corresponding to z 1 Hovering the mouse over the cube faces brings a tooltip with the face label x y and z with values 0 or 1 The Interactive Rendering Canvas 8 11 The canvas shows the scene the result of the ray tracing algorithm rendering the seg mented objects The scene is determined by the objects orientation tilt and twist the zoom the bright ness of the pipes what point is centered on the view and so on All that is taken into account by the renderer that generates the scene and puts it in the canvas on the screen for you to see it Notice that depending on the render size OPTIONS VIRTUAL RENDER SIZE the canvas can be larger than your screen The On Screen Reported Parameters and Tooltips 8 The currently selected Experiment preset See Experiment Presets EEETIEEEE MR IN CHCM 5 0821 um 14 on page 123 selects the statistics to report many parameters to the table See Figure 19 1 item 20 21 and 22 A few of these parameters can be also reported on the screen for the current object for easy reading The magnitude of the dis tance that is plotted on the screen is followed by a triple dash The Selected Are
39. coverslip Because in many cases the coverslip position does not coincide with the first plane in the data this position can be set in the microscopic parameter editor To our knowledge none of the existing microscopic image files record the coverslip position in the meta data 152 Huygens Professional User Guide for version 4 1 Setting the Coverslip Position Next to direct numerical input the Cov E tait mia Coverslip Parameters erslip position and imaging direction can be set using a visual editor Imaging direction I Slide position amp Far away Close to object Z 12 83 um Figure 23 3 reachable from the Zoom 14 61 px um 3870x parameter editorby clicking the wrench button Inverted Microscope The editor shows the coverslip position and imaging direction relative to the data as read from the microscopic file BEDER In an inverted microscope with the Estimate position Cance o objective physically below the speci FIGURE 23 3 The coverslip position editor men it is likely that the first xy plane in showing an xy MIP of the data along they the data corresponding with the lowest y direction The coverslip position can be adjusted by dragging the blue line The imaging direction here upwards is indicated by the position of the objective location in the xz maximum intensity projection MIP on the screen corre sponds with the xy plane sca
40. cross the image borders and the contrast between object and background should be high Pushing the NEXT button on the pre processing screen immediately starts the alignment process because this method does not require any additional user input When the Object Stabilizer has finished measuring the displacements the stabilization screen is shown Continue reading Stabilization Settings on page 87 86 Huygens Professional User Guide for version 4 1 Stabilization of 3D Time Series IE Huygens Object Stabilizer Loc8800 File Edit View Help Position 239 7 l Edit tracks Time oa kede EIME so 0 0 Cpt Color scheme Custom Customcolors 0 NO Zoom X position vs time lt lt Previous 2 ste Contrast FIGURE 15 5 The Edit tracks screen in the Object Stabilizer Stabilization Settings The stabilization settings screen shows the mea sured displacements in x y and z direction and the rotation in four plots The three sliders above the plots can be used to filter outliers drift and noise from the displacement curves When the displacement curves shows sudden steps then the outlier filter can be used to remove those steps The drift and noise filters can be used to respec tively remove the drift and keep the random motion or keep the drift and remove the random motion The effect of those filters is shown in Figure 15 6 At the bottom of the stabili
41. displayed through the VIEW menu Zooming Use the scroll wheel to zoom in or out on the location of the mouse pointer or access the Zoom tab The four buttons in this tab respectively zoom out 3 zoom in 2 zoom 1 1 Q the x sample distance matches 1 pixel and view all 7 Rotation The three radio buttons in the Rotate tab can be used to switch between axial xy fron tal xz and transverse yz orientations The Twist slider rotates the cutting plane around a z axis while the Tilt button rotates the the cutting plane around an axis in the xy plane The tilt and twist angles can be displayed through the VIEW menu Note that the wireframe box in the bottom left of each view port gives visual feedback about the position and orientation of the slice Changing Display Colors Click the Colors tab key to view the color settings panel The Active channels drop down menu can be used to enable or disable channels In addition to the color schemes that are available in basic mode Changing Display Col ors on page 49 the advanced mode allows the use of custom colors Use the color picker to manually select a color for each channel Tuning the Brightness and Contrast The brightness and contrast controls are accessible in the Contrast panel The brightness can be changed per channel or for all channels at once The Gamma drop down menu provides a linear and some non linear ways of mapping data values to pixel intensities
42. for updates PECES m 9 standard RGB scheme V Check for updates when starting applic m The CBF scheme selects a colorpallet which is Channel 4 ms e unambiguous both to colorblinds and non colorblinds Threads restrictions E Limit threads Max threads 2 RGB scheme Accept Other settings Alternative dialogs Safe widgets V Flush Execution Log Cancel ox FIGURE 23 2 The global color scheme can be modified through the Preferences window There are two color scheme available which are the RGB and CBF schemes The RGB scheme Red Green Blue scheme is the colorscheme that starts with the red green and blue colors for the first 3 channels The CBF scheme Color Blind Friendly scheme are contrasting colors that are unambiguous both to colorblinds and non colorblinds The hue selector is a component that allows adjustment of the color range in which objects are displayed See The Surface Renderer on page 67 and The Colocalization Analyzer on page 131 Objects belonging to different channels can be represented in different hue ranges to make them clearly distinct The gradual differences inside the 2 Published by Okabe and Ito How to make figures and presentations that are friendly to Color blind people J FLY 2002 Huygens Professional User Guide for version 4 1 151 Image Statistics Setting the Coverslip Position selected range make
43. in these planes might be affected by hard to correct bleaching they might even reduce the quality of the deconvolution result In any case never crop the objects of interest As a rule of thumb leave about one extra um above and below the objects Brick Wise Processing Deconvolving images requires much computer memory than the image size because all computations are done in 32 bit floating point format and because several extra hid den images are needed to store intermediate results To reduce the memory require ments Huygens Professional will split the images into bricks deconvolve the bricks sequentially and fit the bricks together in a seamless fashion Brick wise processing is an automatic feature of Huygens Professional To find out the best number of bricks let the software run in automatic mode for splitting It will consider many options and go for the most optimal one More information can be found on the SVI Wikil The Signal to Noise Ratio referred to as SNR or S N is in the Huygens Software used as a regularization parameter i e as a parameter that controls the sharpness of the restora tion result The higher this value the sharper the restored image will be Therefore it l http www svi nl BrickSplitting Huygens Professional User Guide for version 4 1 137 should not be considered as a parameter describing the original image but more as a tunable parameter that controls the deconvolved result Using
44. independent objects distinguishable Also a range can be collapsed to have all objects in a channel displayed with exactly the same color In Huygens the hue selector does appear in two flavors Hue Range n This selector allows the adjustment of a hue range The objects on which this selector acts will get a color that lies within this range The assignment of colors is based on the position of an object or on another parameter Hue Range and Saturation a This selector allows the adjustment of a single hue value and a satura tion The upper triangle defines the color while the lower triangle sets the saturation for this color left is white right is fully saturated Right click on a thumbnail image and select SHOW PARAMETERS from the pop up menu This window shows besides the parameter settings statistical information of the partic ular image Amongst them are the mean sum standard deviation norm and position of the center of mass When there is a mismatch between the refractive index for which the microscope s objective is designed and the actual refractive index of the embedding medium the shape of the point spread function PSF will be distorted due to spherical aberration See Refractive Index Mismatch on page 145 As deeper layers in the specimen are imaged moving away from the coverslip this distortion will progressively worsen To compute the spherical aberration it is necessary to know the distance from the
45. interest in the image as shown in Figure 15 1 The NEXT button E Huygens Object Stabilizer H2B_apoptosis_Bunka Mao File Edit View Help Position 3 87 53 86 0 08 Correlation settings Time frame O valen Click and drag in one ofthe views to create a spherical region of interest Without a ROI the Stabilizer will use the whole image Detect rotation J Press Nextto start the alignment lt lt Previous Abort Next Color scheme Greyscale M Custom colors 0 MF m Zoom Colors Contrast FIGURE 15 1 The Correlation settings screen in the Object Stabilizer will start the alignment process When the Object Stabilizer has finished measuring the displacements the stabilization screen is shown Continue reading Stabilization Settings on page 87 The Model Based Correlation Method When the geometry of the imaged object did not change much during the acquisition then the time series can be stabilized using a model of the object The stabilizer creates the model automatically Pushing the NEXT button on the pre processing screen immediately starts the alignment process because this method does not require any additional user input When the Object Stabilizer has finished measuring the displacements the stabilization screen is shown Continue reading Stabilization Settings on page 87 The Multi Object Tracking Method When your image contains well defined objects i e
46. is reloaded in a Movie Maker that has the same image attached then the final movie will be exactly the same However the Movie Maker allows the user to apply saved projects to different images or append saved projects to the current story board To load or append a project from disk select FILE gt OPEN PROJECT or FILE gt APPEND PROJECT from the menu respectively Besides projects from disk the Movie Maker has some presets that can be appended to the movie These can be found in the PRESETS menu Visual Feedback The timeline is an interactive plot which shows the frame number on the horizontal axis and the value of a render parameter on the vertical axis See Figure 13 6 It gives a more detailed visual feedback on values of each of the animated parameters A mouse click somewhere in the timeline area will select the corresponding frame and display a pre view of the frame in the preview area The left and right arrow keys can be used to navi gate through the frames To zoom in on the timeline click near the frame of interest and use the scrollwheel or the magnifying glass buttons below the timeline to change the zoom level 76 Huygens Professional User Guide for version 4 1 Advanced Topics Advanced Topics Timeline FIGURE 13 6 The timeline area gives a detailed visual feedback on the animated parameters Changing Render Parameters The keyframe nodes are displayed in green and can be
47. more details on their utility Aim 11 Objects under your cursor are shown highlighted Objects that have been already analyzed are also shown enclosed by a box when the mouse moves over them or over the correspondent table row To ana lyze an object just click on it while in the Analyze object mouse mode or press the ANALYZE ALL button See Figure 19 1 item 19 Objects in the Primary pipe are framed with a red box and objects in the Secondary pipe with a green one A small label showing the object s number ID is also shown when pointing at it The background color also indicates if it belongs to the primary red or the secondary green pipe Yellow labels are shown whenever the pointer has two objects below it from different pipes If a distance is configured to be reported on screen See Figure 19 1 item 8 it is also plotted when pointing to an object If you hover over an objects row in the table and point to a cell containing a distance parameter it will also be plotted on the rendering canvas Like this you can interactively explore many reported distances Notice that depending on the active pipe mode See Active Pipe Mode 6 on page 120 the interaction with the scene may highlight and affect objects in one pipe only 122 Huygens Professional User Guide for version 4 1 Main window components Scene Control Sliders 12 Three sliders run along the canvas vertically on its right and ho
48. packages are natively used by Ubuntu and other Debian based Linux distribu tions Double click the package file e g huygens 4 1 0 p1 i386 deb and fol low the steps in the package manager To install the package through the command line dpkg i muygens 4 l 0 5l 1386 deb RPM RedHat Package Manager packages are natively used by RedHat Fedora SUSE and other RPM based Linux distributions Double click the package file e g huygens 4 1 0 p1 i386 rpm and follow the steps in the package manager or install the package through the command line rpm Lvl force huygens 4 1 0 p1 1596 x pm l http www svi nl Huygens Professional User Guide for version 4 1 After the Installation After a first time installation there is not yet a license available However still the soft ware can be start ed Without a license it will run in Freeware mode The System ID nec essary for generating a license pops up See Figure 2 1 when opening Huygens Professional and it can be found in the HELP gt LICENSE menu The next section explains how to obtain and install a license string File Edit Deconvolution Visualization Analysis Options SubTasks Help E SG amp q S k amp amp New Open Del all Thumbnail overview Task reports No Valid License Could not find a valid license 2 Continuing in freeware mode You can request a free test license to test all the different options in this program If this computer is co
49. result is selected you can restart again save the deconvolution template quit the wizard which discards the results or press DONE to export the result to the main window of Huygens Professional Multi channel Multi channel images can be deconvolved in a semi automatic fashion to give the oppor tunity to fine tune the results obtained with each individual channel After the prepro Images SMOD OMM cessing stage the multi channel image is split into single channel images named imagename Ch0 lt imagename gt Ch1 etc The first of these is automatically selected for deconvolution The procedure to deconvolve a channel in a multi channel data set is exactly the same as for a single channel image Therefore multiple reruns on the channel can be done manu ally just as with single channel data When everything is done press ACCEPT in the last stage This will cause the next channel to be selected for restoration Proceed as usual Huygens Professional User Guide for version 4 1 31 Z drift Correcting for Time Series Saving the Result with the remaining channels If it is not needed to process all the channels in an image one or more channels may be skipped When the last channel has been processed the wizard allows to select the results which should be combined into the final deconvolved multi channel image This means that up to this point it is still possible to decide which of the results to combine even in wh
50. running tasks the places should be defined were the results are saved Save location field and a file format in which the results should be stored OPTIONS gt OUTPUT FORMAT Selecting Input Files The Batch Processor has a wizard to guide in creating new tasks with only a few clicks By clicking ADD task button 9 below the task field a new field titled Selected images is expanded at the right See Figure 6 2 on page 41 Either a complete folder containing images can be selected or folders can be browsed to select single files If a file is selected containing multiple sub images e g a Leica LIF file a secondary menu will pop up to select which sub image to deconvolve Each selected sub image will be added as a new task in the queue After selecting the images to restore the NEXT button can be clicked to select or cre ate a microscopic template 40 Huygens Professional User Guide for version 4 1 Usage r f Huygens Batch Processor File Edit Options Help Save location C Users edwin Desktop Images Tasks Task Image file Microscopic template Days At 13 2 000 Processing overview Short introduction to the Batch Processor FEE EEC a ct Selected images 28 C Users edwin Desktop Images e959 2ch ics 4 C Users edwin Desktop Images WF original ics C Users edwin Desktop Images fabal28 comb time h5 Deconvolution template Status The Batch Processor ex
51. see three tabs See Figure 20 1 of which the first is visibile In this tab the colocalization coefficients can be calculated You can choose the timeframe select colocalization coefficients set the background settings and choose a colocalization map In default start up all these settings are set for you such that you can inmediatly press COMPUTE First we select the data to analyze For time series the Frame slider selects the time coor dinate We follow the usual naming convention in colocalization theory for the two compared channels Red R for the first channel Green G for the second channel We can select in the lower part of the window which data channels from our image are the Red and Green channels to be compared A two channel histogram is calculated by default and updated whenever we change the Red or Green channel selection This histogram is already an indication of the degree of overlapping between the selected channels for two channels with a high degree of over lapping the histogram pixels trend to concentrate along the diagonal y x line In con trast total absence of overlapping would produce a 2D histogram with values only on the coordinate axes Along the x and y line of the 2D histogram there are 1D histograms for each color chan nel separately The enlarged versions of these 1D histograms are shown when clicking on the SHOW HISTOGRAM PER COLOR CHANNEL button at the top right of the histogram By movi
52. sizes To appreciate the details better you can increase the zoom factor up to 1 30 to see what is shown in Figure 17 4 fem E Huygens Object Analyzer Advanced objectAnalyzer test image caste mm File Options History ROI Anchors Filter Help Mode Zoom m OD 1 30 m S S e Frame selection Q Frame 0 1 EA 0 Sl Object Segmentation EE Primary Scnd MIP EE seed threshold on histogram wl ROI Transparency 0 60 n Brightness 0 80 0 107 Chan 0 Data ROI preferences lac Transp ae Brigt Apply to P sj es An Twist 0 i Select statistics Experiment preset Pipe P chan 0 25 objects No anchor objects selected V auto clean ag wv EN Label Chan Pipe Voxels C Masx C MassY C MassZ Ready rendered Canvas size 678 x 577 Dragging on FIGURE 17 4 Different segmentation conditions Lowering the threshold increases the objects size and adds more objects to the scene In this new segmentation after lowering the threshold we have made the original objects larger but also included many new ones that may be in the way If you are not interested in all those new objects now you can try to remove them Here is where the seed plays an interesting role but we decided to forget it by now We can also filter the objects based on their measurable properties or just use a garbage voxel level to discard objects that are too small Setting a
53. slice of the 3D dataset in its correspond ing location Render Parameters Table 12 1 gives an overview of all render parameters in the Surface Renderer TABLE 12 1 Surface render parameters Parameter Description Frame Set the time frame in case of a time series Threshold surface Adjust the threshold level for the selected pipe i e the intensity for which the iso intensity surfaces are defined See Threshold on page 68 Threshold MIP Set the soft threshold level for the MIP pipe See Threshold on page 58 Garbage volume Volumes that contain less voxels than defined by the garbage volume parameter will not be rendered This is useful for rendering only sig nificant objects in noisy images Transparency Set the level of transparency to other pipes Brightness Adjust the brightness for the selected pipe Slice Z position Set the position of the slice in the Slicer pipe Huygens Professional User Guide for version 4 1 69 Simple Animations Render Options Table 12 2 gives an overview of the different render options that are available through TABLE 12 2 Render options for the Surface Renderer Option Description ANIMATION FRAME COUNT Set the number of frames that will be rendered in a movie 180 frames with a frame rate of 24 fps result in a movie with a duration of 7 5 seconds ANIMATION FRAME RATE Adjust the frame rate a rate of 24 frames per second is fine for smooth movies VIRTUAL REN
54. stabilization methods available which are explained in the next paragraphs e Cross correlation e Model based correlation e Multi object tracking e Center of mass alignment If a 2D time series was read in as a 3D stack then the Huygens Tcl command convertZ2T can be used to convert move z dimension to the time dimension In Huy gens Essential this function is available from the operations windows main menu EDIT gt CONVERSIONS gt XY Z TO XY T Pre processing The first step in the wizard is the same for all methods In this screen there is a button to launch the cropping tool See The Intelligent Cropper on page 14 and a selector for disabling the channels that are not relevant The channel selector is hidden for single channel images Which step will follow next in the wizard depends on the stabilization method chosen Huygens Professional User Guide for version 4 1 83 The Cross Correlation Method This can be considered an all round method It can correct for both x y z translation and axial rotation Adjacent time frames are compared and the program tries to find the best alignment by maximizing structural overlap A spherical region of interest can be defined to stabilize a particular part of the image After the pre processing step the correlation settings step is shown This screen allows to enable or disable rotation detection rotation detection takes more time and it is possi ble to select a region of
55. that we have the first pipe Primary and the second pipe Secondary abbreviated P and S in some places We could have called them also Red and Green pipes independently of their real colors like it is common in conventional colocalization analysis and the name may so be changed in future versions of the software We keep numbers to refer to image chan nels which is something different because in each of these pipes you can put any chan nel you want in any order You can even segment the same channel twice with different conditions in each of the pipes All the different objects in a pipe are colored differently to be able to distinguish them The range of colors assigned to each pipe can be controlled with a HUE SELECTOR See Hue Selector on page 151 You can collapse this range completely if you want that all objects in a pipe get the same color There is also a maximum intensity projection MIP pipe that doesn t interfere in the analysis but that can be used to set a spatial reference to our eyes Before continuing our exploration let s put channel 0 again in the primary pipe with a threshold of 4096 488 in absolute terms nothing Off in the secondary pipe and chan nel 0 again in the MIP so we see something like Figure 17 7 FIGURE 17 7 Objects and MIP Objects in a surface pipe rendered together with a MIP pipe for spatial reference The data channel is the same one in both pipes The Object Analyzer can measure
56. the Movie Maker user interface showing the storyboard 1 the preview area 2 and the timeline About Keyframes A keyframe defines a control point within a transition This can be either a start point end point or an intermediate point in time The appearance of the 3D rendered image is fixed in these frames In between keyframes the Huygens Movie Maker calculates a smooth or linear transition a technique called tweening Inserting New Keyframes To add the first keyframe to the storyboard one of the renderers should be launched from the Movie Maker window by pressing the corresponding button g g or e In the renderer a scene can be defined using the available controls see Chapter 10 Chapter 11 and Chapter 12 for more information on these windows Once the looks of the first frame are satisfactory press the add keyframe button Ei to capture this config uration and add the keyframe to the storyboard All render parameters are captured and most of them can be smoothly animated Because a movie needs at least two keyframes the start and end point of a transition the same renderer should be used to define a second configuration Once the looks of the second frame are satisfactory press the add keyframe button again The storyboard will now show two keyframes with an arrow in between This arrow indicates transition from one keyframe to another 74 Huygens Professional User Guide for version 4 1 Using
57. the SFP renderer excitation and subsequent emission of light of fluorescent materials is simulated Each subsequent voxel in the light beam is affected by shadowing from its predecessors The transparency of the object for the emission light controls to what extent the viewer can peer inside the object l http www svi nl SFP Huygens Professional User Guide for version 4 1 61 Basic Usage Orientation and Zoom Adjust the viewpoint by moving the Tilt and Twist sliders See Figure 11 2 or by clicking e Huygens SFP Volume Renderer e959 2ch FEE File Options Help Zoom Object Background Table Movie Extras Time series Time frame 0 0 m HF E Tilt I 45 deg Global Emission Custom Custom color p 0 100 Twist 234 deg ll D nad dd E B Ready FIGURE 11 2 The SFP Renderer window and dragging the mouse pointer across the scene The magnification can be adjusted by using the Zoom slider or the scroll wheel Notice that the scene is rendered with low quality while being edited which allows for fast scene edition The scene is automatically rendered with good quality when the edi tion is finished and the scene released This automatic rendering can be deactivated by pressing the SWITCH OFF rendering button 4 However when either the FAST MODE render button or the HIGH QUALITY render button 4 are pressed the automatic rendering occurs again
58. the descriptions briefly to get an impression of them Let s select now the preset called CORRELATION INSIDE CHANNELS and inspect it in detail Please read the descriptive text This parameter set will report for each object the distance and the ID of the nearest neigh bor in its same pipe For each object we are interested in we are going to find also the closest object Notice that from the listed parameters in this preset only one is selected by default to be reported on the scene All of them will be calculated and reported on the table but only the selected ones will be shown on the rendering canvas as well when you interact with the objects In this case it is smart to report only the distance there Once you have selected this CORRELATION INSIDE CHANNELS preset press OK to con tinue Huygens Professional User Guide for version 4 1 107 Measuring the Objects Make sure you have the Analyze object mouse mode selected click on a nice object on the screen and notice the new columns that appear on the statistics table Before showing the actual object details a row in the table informs you about the seg mentation conditions for this pipe Then comes the data itself When you move your mouse over the column titles at the top of the table you get a description of each param eter at the very bottom of the window istance to first neighbor same pipe um AS many parameters are available sometimes measuring things co
59. the principal moments of inertia of the object are defined For one of these axes the rotation inertia of the object is minimal around this axis the object would rotate with the least effort This axis usually lies along the length of the object The other two axes are orthogonal to it and orthogonal to each other One can easily define a box with dimensions L pBoxW0 pBoxW1 in the system of refer ence of the principal axes that encloses the object completely The sides of this box are in general not parallel to the main planes of the image because the principal axes do not coincide with the image x y or z axes in general It is as if the principal box is rotated with respect to the image in order to properly enclose the object which may not be aligned with any of the image axes Huygens Professional User Guide for version 4 1 111 Sphericity The length of the object is the largest distance measured along the three principal axes it coincides with the largest dimension of the principal box L One could use the other two dimensions of the principal box as width and thickness of the object but for some practical uses this may be too simplistic The width of the objects is actually computed with a search algorithm that acts as a vir tual caliper held perpendicular to the length axis To find the largest width of an object one would rotate the caliper around the object and repeat this procedure while sliding the caliper al
60. to check the alignment result carefully in using the x z and y z views of the Twin Slicer See Using the Slicer in Basic Mode on page 48 Huygens Professional User Guide for version 4 1 89 90 Huygens Professional User Guide for version 4 1 Introduction CHAPTER 16 Introduction The Object Tracker Wizard The Object Tracker The Huygens Object Tracker wizard and the Track Analyzer allow to study the 3D motion of cells and smaller particles By manually selecting a few objects and background regions the Object Tracker is trained to differentiate between object and background volumes and thus to detect new objects In this way not only can bright spot tracking be done but also tracking of more complex scenes Because the Object Tracker is designed to detect and track objects with a well defined center it works best with spherical disk like flat or slightly elongated objects The Track Analyzer is launched after the Object Tracker wizard has finished processing the time lapse data This tool helps to filter edit and analyze the results It presents the properties of the measured tracks in informative graphs and allows to export the track data to a file The Preprocessing Stage When the Object Tracker is launched through the main menu ANALYSIS gt OBJECT TRACKER or the thumbnail pop up menu it will start in the prepro cessing stage Figure 16 1 The wizard checks if the sampling distances and tim
61. too The signal to noise ratio SNR or S N is used as a parameter that controls the sharpness of the deconvolution result It is best estimating these values manually because auto matic SNR estimation is tricky The easiest way to obtain some SNR is to look at the tex tures of bright areas in your object image Figure 21 1 on page 138 shows examples of an image having different noise levels Inspect your image and decide if your image is noisy SNR wiki which is accessible in a fast way by typing the command help SNR in the Tcl shell 18 Huygens Professional User Guide for version 4 1 Step 7 Perform a Deconvolution Run Step 7 Perform a In the operations panel there are different deconvolution methods to choose from Devconvoluti n Bun These choices are Classic Maximum Likelihood Estimation CMLE Quick Maxi mum Likelihood Estimation QMLE and Quick Tikhonov Miller restoration Quick TM Quick TM is intended for very special circumstances only CMLE is the most gen eral restoration method available and should be used in most circumstances For more information on these restoration methods visit the SVI wiki After clicking the CMLE button the following parameter values can be adapted Figure 3 10 _ Huygens Operations faba128 c0 mcm File View Channels Linking Convert Arithmetic Filter Restoration Manipulation Analysis Visualization Help 5 DM sy ES m a SAA
62. users The recommendations go on the direction of obtaining the highest quality images from the microscope from the point of view of acquiring as much information as clean as possible This alone is worth the effort but it will also be very valuable for the deconvo lution afterwards Some basic guidelines to improve the deconvolution results are also listed More infor mation can be found in the SVI Wikil Refractive Index Mismatch A mismatch between the refractive index of the lens immersion medium and specimen embedding medium can cause several serious problems e Geometrical distortion Frequently referred to as the fishtank effect The axial sam pling distance that is recorded in the image file is the step size by which the objective lens moves along the z axis The focal point inside the sample however shifts due to the fishtank effect by a different step size Therefore objects will appear elongated or shortened in the image data Huygens Professional will automatically adapt the PSF to this situation but it will not modify the image geometry After deconvolution the geometric distortion can be cor rected by multiplying the z sampling distance by the medium refractive index divided by the immersion refractive index e Spherical aberration this phenomenon causes the oblique rays to be focused in a different location than the central rays The distance in this focal shift is dependent on the depth of the focus in the spe
63. values exceeding the dynamic range are all converted to 4095 This phenomenon is called clip ping information in the clipped samples is lost In practice be suspicious if the data contains intensity values at the extremes of the numerical range These ranges are 0 255 for 8 bit data 0 4095 for 12 bit data and 0 65535 for 16 bit data Read more on the SVI Wiki Undersampling One of the rules of measurement that is often overlooked is sampling according to the Nyquist rate See Sampling Density on page 139 Especially the sampling distance along the optical axis is frequently too large too few xy slices are imaged This leaves as result a 3D stack in which there is hardly any relation between the adjacent slices It is important to know how the sampling conditions should be established in order to recover an image from the sampled values How the objects should be sampled depends on the microscope type widefield confocal etc and on the microscopic parameters used like the numerical aperture and wavelength The SVI Wiki has an on line tool that computes the ideal Nyquist sampling distances for any widefield confocal spinning disc and 4 Pi microscope Do Not Undersample to Limit Photodamage Sometimes undersampling is done to limit photodamage to live cells However if photo damage plays a role it is actually better to limit the number of photons per sample than to http www svi nl TotallInternalReflect
64. view ports to move the cutting plane back and forth This can also be achieved using the buttons adjacent to the slider and the up down arrow keys on the keyboard or by placing the mouse pointer over the slider and using the scroll wheel The play button moves the cutting plane through the data volume The pointer coordinates can be displayed through the VIEw menu Note that it is possible to move the cutting plane out of the volume Pressing the center button or pressing the C key centers the plane again The button centered at the top of the window enables switching between basic and advanced mode In basic mode all controls are visible in the panels below the view ports See Figure 8 1 In contrast to the advanced mode which allows independent control of the left and right slicer See Using the Slicer in Advanced Mode on page 50 the basic mode shows a single set of controls that apply to both slicers 48 Huygens Professional User Guide for version 4 1 Using the Slicer in Basic Mode Changing Time Frames Drag the slider in the lower Time frame panel to change the time frame or press the play button to animate the time series The time frame can be displayed through the VIEW menu Orientation Make a selection in the most left Orientation panel to change the plane used to display the image Zooming Click the buttons in the Zoom panel or use the scroll wheel to zoom in or out on the loca
65. 0 60 Brightness 0 80 nul prer erences Transparency 1 00 Brightness 0 00 Twist 23 23 Select statistics _ Correlation inside channels statistics Correlation inside channels Pipe P ch 0 9 obj Pipe S ch 1 17 obj 2 selected anchors 18 BI MASS ASS SSG Frame IN MOM IN Mb TIN CCo TN Chi hiz 20 m 34 659 38 924 57884 46413 23 691 040052 3 o P 1677 60 758 2659 67792 0 730828 35 7 26128 3451 16137 35528 4 0 P 514 23813 79465 65037 0 220270 08 5 53358 76415 26321 0 6554 16171 53145 71581 0 164795 01 10735 74654 4 769 22 cn oo 00 to Ready rendered Canvas size 678 x 577 Dragging on Mouse buttons 1 2 3 draw selection pan canvas shift selection FIGURE 19 1 The Object Analyzer interface Different regions are enumerated to describe them in sections of this chapter Huygens Professional User Guide for version 4 1 115 Main window The OA main window is shown in Figure 19 1 with most of its components in an active state Different regions of the interface are enumerated to describe them conveniently components along this chapter When the OA is launched not all the buttons in the toolbox are enabled Most buttons are automatically enabled when they are usable depending on conditions set by the user Main Menu 1 FILE Entries to save the rendered image export or clear the object statistics and analyze all time fra
66. 138 Huygens Professional User Guide for version 4 1 Black Level Black Level Sampling Density The blacklevel also called base line or electronic shift is the output of the photo multi plier if no light is coming through A positive black level See Figure 21 2 will do no harm to the deconvolution since it is 5 83e405 7 09e405 3 89e405 4 73e405 2 36e405 0 72 7 145 218 FIGURE 21 2 Left The histogram of an image without black level Right The histogram of a similar image with a significant black level automatically accounted for in the background estimation stage A large black level value however will reduce the effective dynamic range of the microscope Besides that a large black level will prevent the bleaching decay analyzer to do its job correctly and the bleaching correction may turn out to be poor or even impossible To prevent this Huy gens Professional has a tool to adjust the base line of an image and the Batch Processor templates show the option to remove the black level as a preprocessing step It is also possible that the black level is negative In the image histogram this will show as a spike on the left This causes clipping See Clipping on page 146 in the lower inten sity limit and it is impossible to correct clipped images should be reacquired More information can be found on the SVI support Wiki The sampling density is the number of recorded sample
67. 84 46 35 1 40 6092 87 086 14 049 0 770594 9 15209 12936 1893 0 76123 47 1 14 10279 92715 13935 0 263693 2 17649 81435 62257 8 1499 48 37 1 7 14458 163 71 14 OF 131535 8 30245 27868 35824 20866 49 zwei 17284 14 0 113053 8 42358 45366 44955 20866 50 S 11 32302 78 999 18 of 1983305 9 1273 15083 10844 17127 51 s iD 77396 94902 15224 0 823829 2 22204 17244 40388 34394 Ready rendered Canvas size 678 x 577 Dragging on 38 S 1NP CMCM 4 2358 um 34 S 0 82804 36 S 1 7649 If you select columns instead of rows in the table the tooltip will display the correspond ing parameter values for the same object Do not highlight columns and rows at the same time by now it may be too confusing to interpret The Status Bar and Tooltip 23 The bottom part of the window is a status bar that also shows an contextual tooltip Ready rendered Canvas size 678 x 577 Draggingon Mouse buttons 1 2 3 draw selection pan canvas shift selection The left side reports the current status of the renderer and analyzer You can see whether a long computation is running or if the analyzer is ready for further interaction Then the current size of the canvas is reported When you first start the OA the canvas size is adapted to the exact room left for the scene rendering but it can be larger or smaller at wish OPTIONS VIRTUAL RENDER SIZE The Dragging status refers to the automatic rendering of the scene while the user
68. 9 86 px um 2612x Estimate positions fol Slide position Far away Close to object mounted on the slide select Close to object upper right corner Drag the coverslip to its proper location When this location is at some distance from the data it might be necessary to zoom out The image can be dragged by hold ing down the right mouse button In terms of imaging quality when there is a refractive index mismatch between embedding medium and immersion medium this is not an ideal situation LX 2 EJ 2s lam Cancel OK since the light from and to the objective FIGURE 23 4 The Coverslip editor with must travel hundreds of wavelengths the slide position set to Close to object through the embedding medium pos sibly resulting in strong spherical aber ration induced bloating of the PSF Excitation Beam In confocal microscopes the entry pupil of the microscope objective is illuminated by a Overfill Factor laser beam Usually laser beams have a Gaussian intensity profile As a result the illu mination intensity is not constant over the pupil but will decrease towards the edges Lower edge intensities will lower the effective NA and therefore negatively affect resolu tion In most confocal microscopes this is remedied by using a beam width which is sig nificantly larger than the entry pupil at the cost of loss of excitation power The ratio between the beam width a
69. DER SIZE Adjust the size of the rendered image When the render size exceeds the display area then use the middle mouse button to pick up and move the rendered image TRANSPARENCY DEPTH This option defines how different surfaces are seen through the others Simple see through one surface the surface closest to the viewer Quite often this is sufficient Normal see through two surfaces Deep consider many more screening levels making the final rendering computationally more complex BOUNDING BOX Enable or disable the bounding box or adjust the line color SCALE BAR Enable or disable the scale bar SHOW SVI LOGO Hide or show the SVI logo at the bottom right High quality MIP Render the MIP pipe in high quality mode CENTER SCENE Undo both the panning of the projection center right mouse button and the rendered image itself middle mouse button the OPTIONS menu The ANIMATION FRAME COUNT ANIMATION FRAME RATE and REN DER QUALITY apply to the rendering of simple movies as explained in the next section Templates All scene settings i e both the render options and all parameters can be exported to a template file via FILESSAVE SCENE TEMPLATE The template files have the extension hgsv and they can be applied to any image that is loaded in the Surface Renderer The Huygens Movie Maker See The Movie Maker on page 73 allows to create easily sophisticated animations using the MIP SFP and Surface Renderer Without t
70. ERER from the main menu Huygens Professional User Guide for version 4 1 67 Basic Usage Orientation and Zoom Adjust the viewpoint by moving the Tilt and Twist sliders See Figure 12 2 or by drag Huygens Surface Renderer 959 2ch Co ese File Options Help Zoom x 124 Position settings Set M Go Frame selection 0 Frame 0 0 LJ Object Segmentation Primary Send Tertiary Threshold 3 AL 8 Garbage vol 1 Transparency 0 00 Brightness 0 92 0 111 Chan0 Tilt 21 MP Slicer Threshold 2 m l 6 Transparency 0 60 Brightness 0 90 Actions High quality Animate Twist 123 l Ready rendered Canvas size 794 x 666 Dragging on Mouse buttons 1 2 3 rotate pan canvas pan scene FIGURE 12 2 The Surface Renderer window ging the mouse pointer on the large view The magnification can be adjusted using the Zoom slider or by using the scroll wheel Use right mouse button to pan the center of the projection Threshold Use the Threshold slider in the Render pipes panel to apply different thresholds to the data channels See Figure 12 3 Voxels that are spatially connected and have intensities above this threshold define closed vol umes These volumes are represented by the 3D iso intensity surfaces containing them each object having FIGURE 12 3 A contour line a different surface color for an interpolated value of 5 Because 5 is
71. For example batch scripts can be programmed with Huygens Scripting which makes it possible to run scripts written in Tcl using the extensive set of Tcl Huygens image pro cessing commands Batch processes can also be configured easily with the interactive Huygens Batch Proces sor The Batch Processor is the tool to do large scale deconvolution of multiple images within Huygens Professional The Batch Processor To launch the Batch Processor first open Huygens Professional then click on the menu DECONVOLUTION gt BATCH PROCESSOR Window The different elements that form the window initially with no tasks see Figure 6 1 on page 40 are e The Save Location This is the directory where the resulting images will be placed during the batch run With the two folder buttons a location can be respectively selected or a new location can be created in the currently selected folder e The Tasks area shows a list of tasks empty at start Tasks are jobs that will be pro cessed by the Batch Processor one by one Each task line consists of an image a microscopic template and a deconvolution template These templates can be updated after a task line is added to the list to tune the values in each particular case In the Usage section this is explained in more detail e Thebutton bar has at the left side a clock to delay the beginning of the processing and at the right side buttons to delete duplicate stop start and add tasks to the list on
72. Huygens Professional User Guide for version 4 1 Scientific Volume Imaging B V Huygens Professional User Guide for version 4 1 Scientific Volume Imaging B V Copyright O 1995 2012 by Scientific Volume Imaging B V All rights reserved Mailing Address Scientific Volume Imaging B V Laapersveld 63 1213 VB Hilversum The Netherlands Phone 31 35 6421626 Fax 31 35 6837971 E mail info svi nl URL http www svi nl Cover illustration Macrophage recorded by Dr James Evans White head Institute MIT Boston MA USA using widefield microscopy as deconvolved with Huygens Stained for tubulin yellow green actin red and the nucleus DAPI blue CHAPTER 1 CHAPTER 2 CHAPTER 3 CHAPTER 4 Contents MON TUistallattoAn saper baskere oS DELE b P Microsol VV INOW Si a abeo E DO ee 5 MAC OSX oe 5 Enes DD gasser 5 Le PP Lea 5 After the Installations 4a eyes RENE EA WC SRXqEESEK wes Olu awe A CHE EA a 6 The benene drug 6 IJpdatine the Sof WAEei eve ex ideo aero USO Ea eR Qe P diam ed ep N 8 Removing he Sol Wales vede saine ceu oq EE a Wears Re Sud aq qs 9 System Requirements for Huygens Professional eee eeeee 9 Support on Installation assistere 10 GNR Tf Step D Start Huygens Professionale siu dar e RIDGE REP ACCRUE QA ode 11 SED 2 Load dn rev n 12 Step 3 Inspect your Image vase sva aw IRSE LTS 12 Step 4 Generate a Poin
73. Images Huygens Professional initially loads four empty images named a b c and psf These images are tagged with default microscopy parameters but the image data consists of just zeroes The fourth image psf will be used in the following steps as a container for the point spread function First an image file has to be opened Select FILESOPEN from the menu in the main win dow and find the image that you would like to deconvolve visualize or analyze In the distribution you will find the demo image faba128 The image consists of the file pair faba128 ids and faba128 ics The IDS file contains the image data and the ICS file the meta data including the microscope parameters You may click on one of these files to open the image In a Unix shell steps 1 and 2 can be combined in a single command that loads the images automatically after starting Huygens Professional huygenspro fabal28 ics dama ids amp This command loads the files faba128 and dama and shows their thumbnail images in the main window The ampersand symbol amp makes sure that the command prompt will return If you would like to open a series of Tiff slices please read Numbered Tiff Series on page 150 for the naming convention in order to be able to read a multi dimensional image as a whole The slicer Select the faba128 image and open the Slicer via the menu VISUALIZATIONSSLICER in the main window or by right clicking the thumbnail and choosing VIEW
74. Imaris classic TIFF file series with arbitrary numbering Zeiss LSM Metamorph STK Biorad PIC Olympus TIFF Fluoview and SIS Delta Vision IMSubs r3d MRC files IPLab IPL Image Pro SEQ OME XML and plain text TXT and CSV Optionally reads Zeiss ZVI and LSM Leica LIE Olympus OIF and BioVision IPM and IPM Tiff Writes HDF5 ICS Imaris classic Biorad PIC TIFF Leica style and classic numbered TIFF OME XML Delta Vision IMSubs r3d plain text TXT and CSV and AVI MJpeg Huygens Professional User Guide for version 4 1 What is Huygens Professional Analysis Functions Threshold and label 3D image Analyze labelled objects compute center of mass volume and integrated intensity Estimate background Measure distance Plot line profiles Compute image ratio Compute colocalization coefficients Compute the co occurrence matrix of an image Visualization Capabilities Thumbnail images Multiple Slicers on one or more images Each Slicer is able to show x y x z or y z slices for selectable points in time while optimizing contrast on a global or per plane basis besides x y x z and y z slices show slices in any arbitrary orientation display multi channel images in spectral or false color report individual pixel voxel positions and values swing through planes or time dynamically link the position orientation time and zoom View your data from three sides simultaneously u
75. In some microscopes with pinholes that are not circular a geometrical correction will also be needed The Airy Disk as Unit for The Backprojected Pinhole Some confocal microscopes report their pinhole size with the diameter of the Airy disk as unit shows how to compute the backprojected pinhole radius 0 612 N A4 ry NA EQ 3 with NA the numerical aperture of the lens N the number of Airy disks and 3 the excitation wavelength In principle using 1 is not correct because the Airy diffraction pattern is formed by the emitted light However we suspect microscope manufacturers prefer to use the excitation wavelength because it is better defined and does not depend on settings of devices like adjustable band filters Note that this relation bypasses the need to know internal system and lens magnifica tions Square Pinholes and the Shape Factor The shape correction from a square to a circular pin hole is based on equalizing the area for both pinholes 7 or See Figure 21 4 the shape factor c for a square pinhole becomes l 4 n 0 564 For circular pinholes c 0 5 to just Because the area A d d r d Jn convert the diameter to a radius FIGURE 21 4 square and If the size of a square pinhole is given in Airy disk disk with an equal area A units then the backprojected radius is a combination of Equation 3 and the shape factor c for a square pinhole Taking into account that Equation 3 alr
76. OE gt Thumbnail overview EH Task reports LLEIGLIUI O Iteration 29 Iteration 30 Iteration 31 Iteration 32 Iteration 33 Iteration 34 Iteration 35 Iteration 36 Iteration 37 Iteration 38 Iteration 39 Iteration 40 quality i quality 1 quality 1 quality 1 quality 1 quality 1 quality 1 quality 1 quality 1 quality 1 quality 1 quality 1 quality 1 7323 928 933 937 941 945 948 951 954 957 960 962 962 Step 8 Saving your Image Maximum no iterations reached iterations stopped Final quality factor 1 962 Elapsed time 4 74 sec Scaling of output not necessary faba128 Tcl shell 1 img open C Program Files SVI Images fabal28 h5 query logEnable series tool gt fabal28 2 fabal28 show mode thumb HuProThumbnail processMethod 4 3 fabal28 cmle psf gt c 4 iA fl MV Y AA Ready Memory usage 7 MB FIGURE 3 11 The main window with a deconvolved image c Note that the Task reports panel shows information on the deconvolution process If you are done and wish to save the result select the thumbnail of the restored image and save the file by selecting FILESSAVE AS from the menu in the main window There are different formats to choose from Why is automatic deconvolution essential If the procedure is not automated what should you do to deconvolve an n channel image You will have to split your
77. RPM package Mac OS X Drag the installed version to the waste basket Sy stem Tables Table 2 3 Table 2 4 and Table 2 5 list the requirements for Windows Mac OS X and Linux Requirements for H uygens TABLE 2 3 System requirements for Microsoft Windows T rofess ional Operating system Huygens runs on Microsoft Windows 2003 Server XP 32 and 64 bit Vista 32 and 64 bit and Windows 7 32 and 64 bit Processor AMD Athlon 64 or Intel Pentium 4 and higher Memory 2 Gb or more Graphics card Any fairly modern card will do TABLE 2 4 System requirements for Mac OS X Operating system Processor Memory Graphics card Huygens runs on Mac OS X Tiger 32 bit only and Snow Leopard 32 and 64 bit G5 PowerPC or Intel 2 Gb or more Any fairly modern card will do a OSX 10 5 or higher with X11 is required for full 64 bit capabilities TABLE 2 5 System requirements for Linux Operating system Processor Most popular distributions like Ubuntu RedHat Fedora and SuSE are supported 32 and 64 bit AMD Athlon 64 or Intel Pentium 4 and higher Huygens Professional User Guide for version 4 1 TABLE 2 5 System requirements for Linux Memory 2 Gb or more Graphics card Any fairly modern card will do S upport on If any problem are encountered in installing the program or the licenses which could not Installation be solved with the guidelines here included please search the SVI Wiki or contact SVI See
78. Selected columns also act as special parameter selector The value of the parameter for selected columns will be reported on the status bar See Figure 19 1 item 23 when you explore cell values for given objects Like this you can quickly compare different parameter values for the same object by looking at the report in the status bar while you move your mouse over the table cells Ready Canvas size 400 x 255 1 47 14 8 A similar thing can be also done by selecting rows see below When multiple columns are selected the STATS popup dialog will report descriptive sta tistics for all them Conditions Report 21 When new segmentation or analysis conditions are set they are reported when you ana lyze one or all objects One row is added to describe the image and the time of the analy sis and another row per active pipe is added that reports the image channel in that pipe the segmentation conditions the number of objects in that pipe and the volume and geometrical center of mass of the ROI Conditions pipe 0 chan 0 thresh 504 8 seed 540 52 Garos la objects 12 BOIS vol 1105 um 3 OM 150 115 10 The parameters about the ROI volume and CM are the same for both pipes in case two are active This is because there is only one ROI for all pipes and channels and the Cen Huygens Professional User Guide for version 4 1 127 ter Of Mass CM here reported is calculated considering the ROI as a uniform solid o
79. a 9 The Select area mouse mode lets you define a region in the image so you can do different things with it analyze objects inside it anchor them as references or use the selected volume to define a region of interest ROI Therefore the selection is not yet a 3D ROI but simply a 2D area That is why inside is quoted in the previous paragraph objects are inside the selection only from the current point of view so it would more appropriate to say below it It allows quick and simple interaction with the objects specially on flat images here the objects remain more or less l http www svi nl RayTracing Huygens Professional User Guide for version 4 1 121 in a plane but only by defining a 3D ROI you have full control on what objects are removed when you want to discard some of them or when you want to crop the dataset In the Select area mouse mode you can use the right mouse button to shift the defined selection around and reuse it multiple times in different locations Anchor Objects 10 Objects that act as references anchors are shown in the rendering with magenta or violet colors depending on the pipe they belong to so that they are clearly distinct from other regular objects There are different ways to set objects as anchors interactively See Selection Interactive Operations 3 on page 119 or by using filtering operations top ANCHORS menu See the on line article about anchor objects for
80. a lot of different parameters on the segmented objects but only a few are reported by default Otherwise the statistic table would be too satu rated with information and you will not be able to find your way in it To obtain object information you can click on each object interactively while you are in the Analyze object mouse mode or press the ANALYZE ALL button 6 amp on the top right of the table to auto matically process all the segmented objects in all pipes When you have an area selected on the screen enclosing a few objects this button analyzes only these objects 106 Huygens Professional User Guide for version 4 1 Object Statistics There are many parameters you can measure for each object not only about its internal information but also about its relationship with other objects and regions in the image Let s see now how to retrieve all this information Configuring the Reported Parameters To simplify the usability of the OA there is a big button next to Select Statistics on the top left of the table that reads EXPERIMENT PRESET at start up Click on it and you will get a preset selection dialog like the one in Figure 17 8 Select experiment report preset mesm All parameters are reported on the table i i insi DE E Sel pupgupicupe dub frp Correlation inside channels Table onl w Sum of all voxel values ees nid Table onl Label of the first neighbor same pipe Correlation inside channels
81. ad the original dataset with this button e A Undo last operation Active Pipe Mode 6 a These radio buttons control for most of the interactive ROI and selection operations on which of the active data pipes Primary Sec P s s is ondary or both and channels Selected pipe or All they act Pipes are explained in Render Pipes 16 17 on page 124 Examples of operations controlled by these buttons are e Interactively clicking on objects When you click on the scene only objects in the active pipe are considered e Analyze all Analyze selection e Set ROI to the visible objects e Sum all column values when hovering over a column header in the table e Apply the ROI to crop the dataset 120 Huygens Professional User Guide for version 4 1 Main window components There are only a couple of practical differences between the PS and All modes When cropping data by applying a ROI you can crop the channel in the Primary pipe P the channel on the Secondary pipe S on both PS or all channels in the image All even if they are not shown in any active pipe When summing cell values in a column of the table you can include in the summation cells for the channel in the Primary pipe P the channel on the Secondary pipe S both PS or all cells All including those reporting about the ROI For all the other operations the PS and All modes are equivalent Reference Cube 7 At the end of the
82. ast option determines the deconvolution algorithm The default is Classical Maxi mum Likelihood Estimation CMLE but it can also be set to Quick Maximum Likelihood Estimation QMLE The wizard offers these two algorithms but Huygens Professional also has Quick MLE time Quick Tikhonov Miller and Iterative Constrained Tikhonov Miller algorithms available If done press ACCEPT to proceed to the deconvolution stage The following parameters for the CMLE algorithm can be set The options for QMLE are almost the same except that the Quality threshold and the iteration mode are not avail able 1 Number of iterations MLE is an iterative process that never stops if no stopping cri terion is given This stopping criterion can simply be the maximum number of itera tions This value depends on the desired final quality of the image For an initial run the value can be left at its default To achieve the best result this value can be increased to e g 100 Another stopping criterion is the Quality threshold of the pro cess See Item 3 2 Signal to noise ratio The SNR is a parameter than controls the sharpness of the res toration result Using a too large SNR value might be risky when restoring noisy orig inals because the noise could just being enhanced A noise free widefield image usually has SNR values higher than 50 A noisy confocal image can have values lower than 20 3 Quality threshold Beyond a certain amount of iterations ty
83. at order Once ACCEPT is pressed a multi channel image named decon final is created For 3D time series the wizard shows an additional stage to enable correction for move ment in the z direction axial that could have been occurred for instance by thermal drift of the microscope table In case of a multi channel image the corrector can survey All channels and determine the mean z position of the channels or it can take One chan nel as set by the Reference channel parameter After determining the z positions per frame the z positions not the image can be fil tered using a median Gaussian or Kuwahara filter of variable width If the drift is grad ual a Gaussian filter is probably best In case of a drift with sudden reversals or outliers a median filter is best In case the z positions show sudden jumps we recommend the Kuwahara filter After deconvolution when the final result is selected and exported to the main window the result can be saved Select the image to be saved and select FILESSAVE As in the menu bar The HDF5 file format preserves all microscopic parameters and applies a loss less compression To see which other fileformats the huygens software support see http www svi nl FileFormats Select FILE gt SAVE STAGE REPORT to store the information as displayed in the Report tab 32 Huygens Professional User Guide for version 4 1 CHAPTER 5 Beads suited for PSF Distillation The PSF Distiller Huyge
84. bject There is another way to calculate the CM of the ROI not considering it homogeneous but taking into account the real image intensities in envelops These intensities being different per pipe yield to different ROI s CM per pipe This is not reported in the condi tions rows but as object rows in the table when you select Analyze all In this case the ROI itself is treated as a new object in each pipe and more detailed information is reported in separate rows in the table See the wiki article about ROI The Table Rows How to Explore Them 22 Each row in the table is either a Conditions report See Conditions Report 21 on page 127 or information about one of the segmented objects In the second case the object the row refers to is identified by some mandatory parame ters parameters that are always reported e Label an integer index that is unique per object inside its pipe e Chan the image channel this object belongs to e Surf the surface pipe in the current analysis the object belongs to P is Primary S is Secondary e Voxels the number of voxels in that object e C Mass three columns x y z for the center of mass location of the object in the image coordinate system with voxels as units The second letter in the Surf column informs whether the object you clicked was in the front F or the back B position This is only relevant in the following situation it may happen that two objects in diffe
85. cene is automatically rendered with good quality when the edi tion is finished and the scene released This automatic rendering can be deactivated by pressing the SWITCH OFF rendering button 3 However when either the FAST MODE render button or the HIGH QUALITY render button 4 are pressed the automatic rendering occurs again upon releasing the MIP scene Huygens Professional User Guide for version 4 1 57 _ Huygens MIP Renderer Imaginal disc2 60 original DECON ChO o mn File Options Help Zoom Object Box Extras Movie 7 Time series Time frame 0 0 Channel parameters Soft threshold 0 01 Ei Color mode Emission Global Tilt Grey Twist 17 deg l d 9 aod B Ready FIGURE 10 2 The MIP Renderer window Threshold The Soft thresold slider in the Channel parameters pannel at the right affects the thresh old level The application of a threshold is a preprocessing step that reduces the back ground in the image i e voxels with intensity values below the threshold value become transparent Contrary to a standard threshold which is all or nothing values above the threshold are kept values below it are deleted the soft threshold function handles images in a different way It makes a smooth transition between the original an the deleted value Saving Scenes Choose FILESSAVE SCENE to save the rendered scene as a Tiff file 58 Huygens Professi
86. ched widefield image The point spread function PSF is the way in which an imaginary infinitesimal small light source would be sampled by your microscope Often this object is no longer a point but blurred and spread out The aim of the PSF generation is to estimate the amount of blurring along the x y and z axes In the final step of deconvolution the PSF is used to come to a measured deconvolution in contradiction to the so called blind deconvolu tion A PSF can be obtained either by recording small beads with known bead diameter 180 nm beads work fine and reconstructing a mea sured PSF from the bead image or by calculating a theoretical PSF from the information about your micro scope settings Huygens Professional has many tools to handle experi mental PSF s but these are beyond the scope of this user guide A theoretical PSF can be generated by clicking the button Theor PSF from the operations panel in the operations window This function computes a PSF from the micro scopic parameters By default the result will be displayed in the image named psf but any available image can be selected as destination Press ing the RUN button will execute the function The Slicer can be used to examine the generated PSF in more detail Figure 3 9 e Huygens Slicer psf File View Channels Linking Help Voxel data At Twist 000 deg I YZ Linking V Position V Orient V Center
87. cimen If the mismatch is large e g when going from oil immersion into a watery medium the PSF will become asymmetric at depths H http www svi nl 2 http www svi nl FishtankEffect 3 http www svi nl SphericalAbberation Huygens Professional User Guide for version 4 1 145 of already a few microns This is especially harmful for the deconvolution of wide field images A workaround for this problem is to keep the z range of the data the number of slices as small as possible The solution is to use a water immersion lens instead e Total internal reflection When the numerical aperture of the objective lens is larger than the medium refractive index total internal reflection will occur This is causing excitation light at high angles to be bounced back into the lens and therefore limiting the effective NA If spherical aberration is unavoidable the image can still be improved during restora tion using an adaptive point spread function See Refractive Index Mismatch on page 145 Clipping The intensity of the light emitted by the microscopic sample is converted to electrical signals that pass an adjustable amplifier These electrical signals must be converted to numbers processed by the computer This conversion is done by the CCD camera Most scientific CCD cameras have a 12 bit converter limiting the output numbers to the range 0 4095 Negative input signals are usually converted to 0 while positive input
88. cking the undo button 3 Changing Transitions Transitions can be changed by double clicking the arrow BLE that joins two keyframes This will pop up a window in Daon 2 tl which the duration of transition in frames or seconds Twist Smooth CW can be changed as well as the transition type of the TT parameters that are animated See Figure 13 3 For most a parameters the Huygens Movie Maker can calculate lin ear or smooth transitions An exception to this is the twist which also requires a direction of rotation clock FIGURE 13 3 The wise or counter clockwise and a number of rotations IransiHon Settings Cancel Accept dialog is shown by double clicking a i transition arrow in Playing a Preview Movie the storyboard To preview the movie just press the play button 6 in Preview the Preview area See Figure 13 4 The Movie Maker quickly creates a low resolution movie and displays it in the preview area If loop mode is on the movie will be repeated until the stop button is pressed To change the animation settings aspect ratio size frame prame rate fps 12 rate etc press the render settings button in the Pre I view area The High quality setting and the AVI quality are 3 M M t3 not reflected in the movie preview but only noticeable in i i the final result FIGURE 13 4 The preview area Huygens Professional User Guide for version 4 1 75
89. d setting to a physical size and from there convert to the backprojected radius An Example the Olympus FV1000 As reported by Olympus engineers the FV1000 confocal microscope has a square pin hole and an internal magnification of 3 82x The pinhole size reported in the Olympus software is the side length of this square pinhole Therefore c 1 n 0 564 and Mp 3 82 Using a 100x objective lens and a reported pinhole size d of 150 um the backprojected pinhole radius r in nm is 3 d 150 10 gp 56S ec EQ 6 eS 5711005082 eel J sys The on line calculator knows the system magnifications and pinhole shapes for most popular microscopes Checking the Bio Rad System Magnification The Bio Rad MRC 500 600 and 1024 microscopes have a very high magnification in the detection system The fixed system magnification is according to Pawley 8 http www svi nl YokogawaDisk 9 http www svi nl BackprojectedPinholeCalculator 10 Pawley J B Handbook of Biological Confocal Microscopy 27 edition 1995 Plenum Press New York and London ISBN 0 306 448262 Page 30 142 Huygens Professional User Guide for version 4 1 Computing the Backprojected Pinhole Radius and Distance 53 x Mape With m between 1 0 and 1 56 factor 1 25 for the fluorescence attachment multiplied by factor 1 25 for the DIC attachment The factor of 53 includes the 8x eyepiece just below the scan head but does not include that variabi
90. data is unusable for distilling PSF s If there are multiple bead images then the parameters of bead images loaded at a later stage should match the ones to establish in this stage a warning of any mismatch will be given After setting the bead diameter and estimating the Signal to Noise Ratio SNR the image is searched for beads that meet the following selection criteria e lt A bead should not be too close to another bead If a bead is too close to another bead their signal will interfere with each other In widefield bead images this is quite prob lematic due to the large size of the blur cone Fortunately widefield PSF s can be derived from single bead images e Abead should not be too close to an image edge After all another bead might be located just over the image edge e The intensity of a bead should not deviate too much from the median intensity of all beads If it is brighter then it may be a cluster of two or more beads If it is dimmer then it is not likely to be a bead In both cases the object geometry is unknown so they are unusable If for some reason no usable beads are found an explanation and some advice will be displayed in a pop up window The software will try to find beads first with ideal selec tion criteria If this does not yield a single bead then it will automatically retry with reduced inter bead distance criteria After having added the last bead recording press DISTILL to enter the distillation stage
91. der Pipes Render Pipes e 4 Pan canvas is similar to pan scene but not exactly the same It allows you to explore the canvas by not re rendering the scene This only makes sense when you have a canvas larger than your rendering window of course See OPTIONS gt VIRTUAL RENDER SIZE in the top menu e sy Shift the ROI This is the only mouse mode that is not always enabled you need to have defined a region of interest ROI before moving it around The buttons in the second third and fourth groups are not mouse modes they do not set new behaviors for your mouse when interacting with the objects view but execute opera tions on previously defined conditions for example deleting the objects under a selected area or discarding every object that has not been selected as anchor We will not con sider them in this basic tutorial as you don t need them to perform basic measurements Please see the Object Analyzer Component Reference on page 115 for a detailed description of these toolbox buttons At the end of the buttons column there s a colored reference cube that will help you in orienting in space when you rotate the dataset specially with large zoom factors that do not let you see the surrounding box frame in the rendered image The initial view of this cube is the blue top face corresponding to z 1 Hovering the mouse over the cube faces brings a tooltip with the face label x y and z with values 0 or 1 The Analyze obj
92. detail in Iso colocalization object analysis below There is one Maximum Intensity Projection MIP pipe available to redirect the data channels to The MIP rendering of one channel maybe one different from those used for colocalization or the two channels under analysis can be a good spatial reference for the objects from the colocalization map When an original channel is selected the threshold slider can be used to select what voxels are considered for the MIP rendering depending on their intensities Notice that here the threshold is simply used for representation If both R and G channels are selected to be rendered their correspondent backgrounds as selected in the histogram will be used as projection thresholds As with the surface pipes the transparency and the brightness of this MIP can also be controlled All the obtained information can be saved to external data text or image files through the FILE menu One of the features of the colocalization analyzer is iso colocalization object analysis Figure 20 3 on page 135 It allows to determine quickly the properties of the different colocalization regions in the data This is realized by visualizing the colocalization map as iso colocalization surfaces In this way regions in which the degree of colocalization exceeds a certain value become objects By clicking on the objects local colocalization parameters are computed and reported To relate the iso colocalization objects to the or
93. dragged vertically to adapt the value of the parameter The interpolated transition frames are displayed as smaller red dots See Figure 13 6 If a render parameter has been changed the Movie Maker will recalculate the transitions update the thumbnails in the storyboard and update the still of this frame in the preview area To select the render parameter which value should be shown in the graph open the drop down menu below the timeline and choose the preferred parameter Because the amount of parameters that can be animated is huge the menu only lists the ones that change during the movie If Other render parameter is selected a dialog window will pop up that shows a list of all available parameters In order to change one of the parameters in a frame which is not a keyframe the frame can be converted into a keyframe To do this select the frame in the timeline and choose EDIT gt CONVERT TO KEYFRAME from the menu Stretching Movie Length The number of frames in a transition can be changed by double clicking the transition arrow However if a movie gets complex it is easier to use the stretch tool to e g double the number of frames in all transitions This tool can be found in the menu TOOLS gt STRETCH MOVIE The stretch tool shows the frame rate the number of Stretch time Ex frames and the duration in seconds See Figure 13 7 ER i The two sliders can be used to change the frame rate Movie length and
94. e consider following the expert on line tutorial where you will be told on many other powerful things you can do with this versatile analyzer A reference description of the Object Analyzer components can be found in Chapter 17 on page 99 3 http www svi nl ObjectAnalyzerExpertTutorial 110 Huygens Professional User Guide for version 4 1 Iso surface CHAPTER 18 Iso surface Principal Axis Length and width Object Analyzer Geometry Measurements In the Object Analyzer the threshold set for the segmentation is also used to define an iso surface around the object An iso surface is a 3D surface representation of points with equal values in a 3D data dis tribution Is the 3D equivalent of a 2D contour line See Figure 12 3 on page 68 Based on this one can measure volume and surface in high resolution by fine polygonization at a sub pixel level Segmented objects are geometrically analyzed in terms of their principal moments of inertia In this sense the recorded light intensity registered in the image is used as den sity the pixel values are interpreted as local mass so brighter regions weight more The principal axes of an object establish a natural system of reference based on its mass distribution When you rotate an object around one of its principal axes the angular momentum is parallel to it This does not happen in general and is what makes these axes so special Around these axes
95. e nm 10 0 4 0 6 0 8 1 1 2 1 4 Numerical Aperture FIGURE 21 3 Critical sampling distance vs NA The curves above show the critical sampling distance in axial and lateral directions for wide field and confocal microscopes The emission wavelength in both cases is 500 nm aperture for a wavelength of 500 nm To apply this plot of to another wavelength simply scale the vertical axis by that wavelength For example if a widefield microscope with NA 1 3 is used there can be noticed from the plot that the critical lateral Nyquist sam pling distance at 500 nm emission is 95 nm For an emission wavelength of 570 nm this becomes 500 500 x 95 108 nm In the confocal case it is the excitation wavelength which determines the Nyquist sample distance In theory the pinhole plays no role but larger pinholes strongly attenuate fine structures at the resolution limit Therefore as a rule of thumb with a common pinhole diameter of 1 Airy disk the lateral critical sampling distance may be increased by 50 96 with negligible loss of information In cases were the pinhole is much larger the lateral imaging properties much resemble those of a widefield system and the sampling distance can be set accordingly We do not recommend to increase the axial sampling distance appreciably beyond the critical distance In a multi photon excitation microscope it is the excitation wavelength divided by the photon count which determines the sampling Mo
96. e squared values of the amplitudes Taking the absolute square destroys all phase informa tion one would need to effectively apply deconvolution Fortunately in the brightfield case the detected light is to a significant degree incoherent Because in that case there are few phase relations the image formation is largely governed by the addition of intensities especially if one is dealing with a high contrast image In practice one goes about deconvolving brightfield images by inverting them using DECONVOLUTION2 CONVERT BRIGHTFIELD IMAGE and processing them further as inco herent fluorescence widefield images The Tikhonov Miller algorithm was proven to work excellently for brightfield data This algorithm is available in the Huygens Profes sional only With the MLE algorithm one should watch out sharply for interference like patterns periodic rings and fringes around objects in the measured image As a rule these become pronounced in low contrast images After the deconvolution run a reverse to the original contrast setting is possible Huygens Professional User Guide for version 4 1 155 156 Huygens Professional User Guide for version 4 1 Contact Information CHAPTER 24 Support and Contact Information Contact Information Addresses and Phone Numbers Mailing Address Scientific Volume Imaging B V Laapersveld 63 1213 VB Hilversum The Netherlands Phone 31 35 6421626 Fax 31 35 6837971 E mail mmfodsvisnl URL
97. e Global Color Scheme Hue Selector Note that the selection has to be unique i e it is impossible to have ignored variables without having a Slice Time Frame or Channel counter In the second third and fourth row the range for each of the counters can be defined A range from 0 to 9 with step size 2 will load the files 0 2 4 6 and 8 Note that the time in seconds and z sampling intervals in nm are not adapted to the step sizes Press the LOAD SELECTION button to load all files in the series into a single image Before the dialog is closed the tool will check if all files in the selection are really present in the directory Huygens Professional uses a global scheme for coloring the different channels in multi channel images These colors can be adjusted through the Preferences window via OPTIONS2 PREFERENCES EDIT GLOBAL COLORS See Figure 23 2 Preferences mm General options Directories Shell color scheme Regular C Hacker G Image thumbnail colors C Global colors Exa Emission colors Global channel cola Global channel colors cameo M O About Edit global colors aie This dialog allows you to assign colors to each channel Channel 1 FE Q of an image These global colors are available in Export format EE thumbnails slicers and renderers to display any image Q Channel 2 pr spe EN mn EN m The RGB scheme button below sets the colors to the Check
98. e animate button H and select a directory to save the AVI movie or the TIFF frames to The exported AVI files use the MJPEG codec and can be loaded in most movie players including Windows Movie Player and Apple Quicktime The TIFF frames are useful to combine multiple animations or edit the movie in e g Windows Movie Maker l http en wikipedia org wiki Mjpeg 60 Huygens Professional User Guide for version 4 1 CHAPTER 11 The SFP Renderer The SFP Renderer generates realistic 3D scenes based on the 3D microscopy image which is taken as a distribution of fluorescent material l he computational work is done by the Simulated Fluorescence Process SEP algorithm simulating what happens if the material is excited and how the subsequently emitted light travels to the observer See Figure 11 1 The properties of this algorithm allow to render the object at different depths unveiling layers under the object s surface The SFP algorithm is not limited by boundaries or sharp gradients and is exclusively suited to render 3D microscopic data sets Since the SFP algorithm is based on ray tracing techniques it does not require a special graphic card To start the SFP Renderer right click on a thumbnail and select VIEW gt SFP RENDERER from the pop up menu or choose VISUALIZATION gt SFP RENDERER from the main menu AW view point excitation light projection plane z 4 Shadows on table FIGURE 11 1 In
99. e by one or many at the same time Huygens Professional User Guide for version 4 1 39 Usage E e ap Huygens Batch Processor File Edit Options Help Save location C Users edwin Desktop Images 53 Eg Tasks Task Image file Microscopic template Deconvolution template Status Daysb At13 H 00 29 P e Processing overview Shor introduction to the Batch Processor The Batch Processor executes tasks one by one or in parallel Tasks are represented by lines in the Tasks panel above Each task is described by an input image a microscopic template which will be applied to that image and a deconvolution template which specifies how the image will be deconvolved The results of all tasks will be saved in a common folder set in Save location Usage Progress Detailed ri Ready Scheduler ready FIGURE 6 1 The Batch Processor main window e The Usage Progress and Processing report tabs in the Processing overview area give information about the whole process in its different stages e The status tabs at the bottom of the batch processor window supply some state infor mation about Huygens Professional The most left tab shows the state of Huygens Professional and the tab to the right of it shows information of the batch scheduler The most right of these tabs gives information about the button the mouse is cur rently pointing at Before starting with creating and
100. e interval are reliable and will pop up a parameter editor Figure 3 3 on page 13 if those parame ters need attention Depending on the image dimensions 2D 3D single multi channel the preprocessing stage will give the option to create an axial maximum intensity projection MIP select relevant channels or crop the image See The Intelligent Cropper on page 14 These preprocessing operations help to speed up and enhance the tracking process Detection Settings In this stage the parameters that control the object detection can be adjusted The first screen of this stage shows the choice to use a wizard The option of loading detection set tings from a file is not yet available in this release Huygens Professional User Guide for version 4 1 91 Huygens Object Tracker Loc8800 a amp den File Edit View Help Preprocessing Continue with a 2D maximum intensity projection of the image to speed up the tracking process Create MIP Select only the relevant channels to speed up tracking and improve results Select The source image may be cropped in volume or in time to speed up the tracking process Press the button to launch the cropper Launch cropper Previous Color scheme Custom Custom colors 0 O Zoom Colors Contrast Overlay settings FIGURE 16 1 The preprocessing stage in the Huygens Object Tracker The next screen gives the optio
101. e objects colocal ize so that the intersection volume of the two pipes is not empty a ROI can defined with this operation and applied it to discard objects and parts of objects outside it so that only the colocalizing regions remain Envelop anchors use the currently selected anchors to define a 3D ROI so that also holes inside the objects are filled in and in the ROI Enclose anchor in a box define a prism that is an envelop to the selected anchors Make a spherical ROI of a given diameter centered in the image You can shift it later with the shift ROI mouse mode Set to all volume maybe a good starting point for further interactive modifica tions Modify Fill inner cavities a ROI defined by using a threshold may contain inner cavities visible when selecting the deep Transparency Depth This operation fills them in automatically Fill inner and cutoff cavities A cutoff cavity is a hole in the surface of a ROI that touches the image limits Grow shrink the currently defined ROI can be enlarged or reduced in a certain number of VoXels independently in the xy plane or in the z direction A 3D xyz reduction is also possible here the number of voxels in the xy plane will be partially adapted to the entered voxels along z to considering the voxel anisot ropy grow shrink the volume proportionally Outer shell re define the ROI considering only an outer shell of given thickness Invert the ROI volume Storage
102. e the new license i N License License overview Your current license is desktop system The license options are freeware License file Your license file contains the following licenses e Your license file C Program Files SVI bin huygensLicense N Add License Enter your new license here Cancel Add license Explain Copy to clipboard HuE 2 4 0 wc d2 Bf mp eom2012Jul01 51 7c825a336b979 edwinfsvi nl 7753e366dfbbaeaez oaq Delete Add new license Help Report FIGURE 2 3 The license window allows to add delete and troubleshoot licenses Huygens Professional User Guide for version 4 1 Updating the Software Location of the License File The license string is added to the file huygensLicense in the SVI directory Table 2 1 on page 8 TABLE 2 1 The default installation paths per platform Platform Installation path Windows C Program files SVI Windows 64 bit Edition C Program files x86 NSVIN Mac OS X Applications SVI Linux usr local svi a The path name on Mac OS X depends on where the software is installed This is a typical example On Irix and Linux and Mac OS X an alternative location is the user s home directory On OS X this is especially convenient when updating frequently Troubleshooting License Strings The license string as used by N Explain License Es a SVI has the same appearance License deta
103. e with them e die Deselect all anchors Huygens Professional User Guide for version 4 1 119 et Keep the selected anchors discarding anything else e Nu Discard the selected anchors More operations are available at the top ANCHORS menu The main purpose of an anchor is to serve as analysis reference in some experiment presets for example to measure dis tances relative to them But you can also use the selected anchors to delete some objects or to define a 3D ROI based on them ROI Interactive Operations 4 e Reduce the current ROI to the volume under the selected 2D area intersects the ROI with the selection e VG Add the volume under the selection to the ROI volume union of the ROI with the selection C Remove the volume under the selection from the defined ROI difference of the ROI with the selection e amp Set all objects inside the ROI as anchors This requires a ROI to be defined of course e SV Keep objects inside the ROI discarding anything else e VG Discard the defined ROI reverting to the whole image Data Cropping and Restore Operations 5 e Once a ROI is defined you can use these scissors to crop the dataset and delete set to zero all voxels outside the ROI a Export the current data as cropped by the ROI or after deleting objects interac tively to the original image in order to save it Bir deleted data was not yet exported to overwrite the original you can always relo
104. eady converts a diameter into a radius the relation becomes 0 690 Nag a A EQ 4 6 http www svi nl BackprojectedPinholeCalculator 7 http www svi nl AiryDisk Huygens Professional User Guide for version 4 1 141 Converting from an Integer Parameter Unfortunately quite a few microscopes do not report the physical pinhole size or the Airy disk size Instead often an integer size parameter p in the range 0 255 8 bit is specified If p maps to a physical size in linear fashion then Equation 5 can be used to translate that parameter into a backprojected pinhole radius u 103 M8 293 max Smin uk jg Ip MobjMsys EQ 5 Here c is the shape factor ands and s are the minimal and maximal pinhole size n in um The factor 10 converts from um to nm The backprojected Pinhole Spacing for Spinning Disks As is the case for the backprojected pinhole diameter the distances between the pinholes in spinning disks must be divided by the system magnification For the frequently used Yokogawa disks for example the physical spacing is about 253 um This can be checked by imaging a stopped disk Using a 100x lens for example the backprojected distance is about 2 53 um If an extra zoom lens is placed between the disk and the sample its mag nification must be also considered A Supplied Calibration Curve If a calibration curve was supplied with the microscope best use that curve to convert the displaye
105. ect mouse mode is the default one Click on an object and see what happens You probably already did by now and noticed that some parameters were reported on the table at the bottom of the window We will see how to report even more in a minute First we need to briefly explain what a pipe is just to know how to select what channels from the image you want to analyze When we opened the analyzer we got the first channel of the image directly shown on the screen but we can change that and select the other channel from the multi channel original image to explore it For that we simply need to select another channel to be shown in the PRIMARY PIPE where it reads Chan 0 you just select Chan 1 See Figure 17 6 Object Segmentation Primary Scnd MIP seed threshold on histogram B Transparency 0 60 Brightness 0 30 0 60 Chan 0 e FIGURE 17 6 Rendering pipes control panel But what if you want to explore both channels at the same time Huygens Professional User Guide for version 4 1 105 Object Statistics The term pipe suggests that data go from your original image to the final rendering in the Object Analyzer through a computational duct in which some processing occurs You have two of these pipes in the OA to redirect data through So we have a secondary pipe too You can activate its control pane by clicking on the tab that reads SCND Secondary does not mean here less important it is just
106. ecutes tasks one by one or in parallel Tasks are represented by lines in the Tasks panel above Each task is described by an input image a microscopic template which will be applied to that image and a deconvolution template which specifies how the image will be deconvolved The results of all tasks will be saved in a common folder set in Save location iz Usage i Progress Ready Scheduler ready Delete selected Filter series v Auto load series MEE Select files Select folder Detailed reports UTER FIGURE 6 2 The Batch Processor with the Selected images field expanded Microscopic Templates Describing the Images Overrule meta data The parameters in this template can be used to overrule the parameters found in the meta data of the image files Keep all meta data h Overrule all meta data Overrule unreliable meta data Keep all meta data FIGURE 6 3 The Batch Processor To guarantee the quality of the deconvolution it is very important that the image acquisition conditions are properly described Luckily the parameters that the Huygens algorithms need are just a few The microscopic parameters can be edited and saved to a template so that they can be reused metadata selector in the future In addition to editing the micro scopic parameters Huygens also offers the pos siblity to deconvolve the image with the microscopic parameters stored i
107. ed for instance by thermal drift of the micro scope table Basic Image Processing Capability to handle multiple images Time series support Multi parameter multi channel image elements stacked or packed Basic data types unsigned byte 16 bit signed integer 32 bit float 2x32 bit complex Per image undo redo capabilities An Image History that keeps track of all operations This history can be saved along with the files or as a Huygens script to automate tasks Scripting and batch processing environment based on Tcl Tool Command Lan guage Core Image Processing Functions Create delete copy copy block convert split join zoom rotate iso sample shift replicate image Add remove border shift to sub pixel accuracy mirror image swap image octants Arithmetic operations on two image operands one image operand and a scalar mathematical functions on one image operand soft clipping amp thresholding 4D Gaussian filter of arbitrary widths 4D Laplacian filter Generate solid and hollow bandlimited spheres generate Poisson and Gaussian noise Real and complex 4D Fast Fourier transforms Reporting amp Display Operations Image statistics Report sampling density with respect to Nyquist rate Image histograms of images with up to two channels Plots of energy flux as function of time and axial position Plots of intensity profiles along lines in any orientation Image File I O Reads HDF5 ICS
108. egular change of this parameter in the plot right click on the transition arrow and choose SYNCHRONIZE TIME FRAMES from the pop up menu The Movie Maker will set the transition type to linear and add or remove some frames from the transition to achieve a 1 1 2 1 1 2 etc synchronization Creating Loopable and Bouncing Movies In two simple steps a movie can be made loopable i e it can be played seamlessly in repeat mode 1 Copy the first keyframe and paste it to the end 2 Right click on the final keyframe that has just been pasted and click SKIP THIS FRAME in the pop up menu The result is the best when all transitions are set to smooth To create a bouncing animation i e an animation that is played in reverse when the last frame is reached mark the bounce option in the animation settings dialog Doing so will not insert extra keyframes on the storyboard but the frames are appended in reverse to the final AVI file or Tiff series About Movie Quality In the Huygens Movie Maker two types of quality can be set 1 The High quality check box determines if the quality of the renderers should be set to the highest possible setting when rendering the final movie 2 The AVI quality scale bar determines the compression level of the AVI file Set to 100 96 the quality is the best but then the file size will be large These quality settings are not reflected in the movie preview but only noticeable in the final re
109. elds indicate a 9 X nm detrimental to the deconvolution result Please Y nm fields indicate dense sampling beyond of 9 Z nm e T s All parameters were read from file metadata Wizard Parameter checking Sampling intervals FIGURE 4 1 Parameter wizard check sampling density Red coloring indicates a suspicious value and orange a non optimal value F ms f Edit Microscopic Parameters Templates Save Edit microscopic parameters of image fabal28 Sampling interval Backprojected pinhole r nm 50 692 Excitation wavelength nm 50 692 Emission wavelength nm 202 766 Excitation photon count 1 000000 Excitation fill factor Optical parameters Reports Microscope type Confocal Numerical aperture 1 30 Objective quality Good Coverslip pos um n 0 000 Imaging direction Upward Pinhole spacing um 2 500 Refractive indexes Lens Oil 1 515 Medium Oil 1 515 Meta data All verified FIGURE 4 2 The microscopic parameter editor This window can be opened by right clicking on the thumbnail and selecting PARAMETER EDITOR The parameter editor and parame ter wizard can be found by right clicking on the image thumbnail and selecting PARAMETER EDITOR or PARAMETER WIZARD The parameter wizard can also be found in the DECONVOLUTION menu Figure 4 1 shows the parameter wizard The parameter editor is shown in Figure 4 2 Microscopic Template Files
110. em and it should not be blocked at this moment the beginning of the queue processing can be delayed by using the timer Just adjust the delay in days zero for today and set the time of the day in which the processing should start The timer checkbox is selected automatically deselect it to dis able the timer Exiting the Batch Processor If the Batch Processor is closed while running tasks it will stop all running tasks The Batch Processor window can be scrolled down while running tasks Just exit the Batch Processor after all the jobs have been finished The FILE menu can be used to save the tasks list for future reference In this menu also the information reported during the batch processing can be saved The EDIT menu can be used to duplicate or delete tasks in the list The OPTIONS menu has three sub menus e OUTPUT FORMAT this sub menu shows several options for the file format to select for saving the restoration result e THREADS PER TASK this sub menu allows to set the number of processors per job Typically in a run where tasks are processed sequentially the computational work will still be distributed over the available processors depending on license limita tions The number of threads Huygens can use in parallel is by default set to AUTO but in cases where it is required to restrict the computing resources set the number of threads CONCURRENT TASKS if the system has multiple processors it can be cho
111. emove objects based on size or sphericity e Quickly remove non colocalizing objects e Quickly find pairs of objects inside the same pipe or by combining the two pipes e Advanced filtering shows the same filter dialog explained in Table and Analysis Shortcuts 19 on page 125 HELP shows on line help and tutorials Mouse Modes 2 These buttons control the Mouse mode what the mouse does when you left click on the image When you hover with the mouse over these buttons you get a tip on what they are for If they have a keyboard shortcut to activate them it is also shown here For example the first mouse mode is intended to analyze objects and you can always activate it from within the rendering view by pressing the keyboard key 1 From left to right and from top to bottom e Analyze object lets you click on different defined objects and obtain the local sta tistics e Select area lets you define a 2D region on the current view of the image so you can do different things with it analyze or discard objects below it anchor them as references or interactively define regions of interest ROI We will see what all this is useful for e J Discard object lets you discard irrelevant objects one by one Just select this mode and click on the disturbing objects 118 Huygens Professional User Guide for version 4 1 Main window components xi Select object as anchor lets you select and deselect objects to
112. er fabal28 File View Channels Linking Help File View Channels Linking Help m Zoom 2 83 0 Ej Tilt 90 0 XY xz Twist 050 deg I vz Twist 000 deg Voxel data Linking Voxel data At 0053 0047 033 T Listen V Position V Orient V Center At 0069 0050 032 1s 1 Value 026 Time Zoom Other Value 021 Slicing Slicing Slice center at voxel coordinate 0066 0062 032 Slice center at voxel coordinate 0064 0064 032 gt I l View options View options Chlo False v EH cn I Master chlo False v B ed ch Ready Canvas size 443 x 410 Interactive Ready Canvas size 443 x 410 Interactive Zoom V Other gt e Microscopic Parameters Verify the microscopic parameters needed for the generation of a PSF like NA microscope type sample size etc by selecting EDIT gt EDIT MICROSCOPIC PARAME TERS from the main menu Figure 3 3 Did you use the correct sampling size during the image acquisition To find out select ANALY SISSNYQUIST RATE from the main menu which displays an informa tion window as shown in Figure 3 4 Read more about this important topic at the SVI wikil It is also important to make sure that the sample sizes reported by the microscope during the acquisition are correct edit Microscopic Parameters re f gt Templates Load Save Help Edit microscopic parameters of image fabal28 Sampling interval Backprojected pi
113. etection algorithm can be configured to automatically adapt its settings per time frame to correct for bleaching for example The tracking algorithm can be configured to use the geometry and orienta tion of the detected objects besides the position brightness and filter values for con necting the tracks default The NEXT button will start the tracking process When this is finished the edit tracks screen is shown See Figure 15 5 on page 87 In this stage the user can select gt break 5 and delete 4f the detected tracks The Object Stabilizer averages the remaining tracks to correct for the average displacement of the objects When the NEXT button is pressed again the stabilization screen is shown Continue reading Stabilization Settings on page 87 Huygens Professional User Guide for version 4 1 85 IE Huygens Object Stabilizer Loc8800 eJ File Edit View Help 4 4 ds j Select bg Mark obj Mark bg Cn Delete Select Break Delete nvert Histogram Width vs number of objects Width N Lower threshold 0 um Upper threshold 1 410 um lt lt Previous Color scheme Custom Custom colors 0 v e Zoom Colors Contrast FIGURE 15 4 The Optimizing object detection screen in the Object Stabilizer The Center of Mass Alignment Method This method works best if the image contains a single large object No objects should
114. etting the Threshold Segmenting the Objects Setting the Threshold Huygens Object Analyzer Advanced objectAnalyzer test image cJ File Options History ROI Anchors Filter Help Mode i Oo 2 ev Frame selection e Frame 0 1 pw ET Object Segmentation Primary Send MIP Obj ects seed threshold on histogram ROI Data Transp Apply to I E s Ps All _ Select statistics Experiment preset Pipe P chan 0 11 objects No anchor objects selected V auto clean ET Ww FN Label Chan Pipe Voxels C MassX C MassY C MassZ Ready rendered Canvas size 678 x 577 Dragging on FIGURE 17 2 Start up screen of the Object Analyzer The objects in the images first channel are automatically segmented with some default parameters calculated based on the intensity distribution The segmentation method currently available is the Object Analyzer is more flexible than simply setting a threshold it uses a combined seed and threshold method see Figure 17 3 The seed acts as a secondary threshold level so that objects that do not reach it in intensity in at least one voxel are discarded In this introductory tutorial we will basi cally ignore the seed an leave it at the automatic linked value slightly larger than the threshold itself For more details on how it works you may refer to FIGURE 17 3 Visual repesentation of the seed and threshold segmentation met
115. evel and panning Dragthe sash to the left to make the left slicer a bit smaller Select the Zoom tab at the bottom and click the view all button ZZ Huygens Professional User Guide for version 4 1 57 Measurement Now the right slicer can be used to zoom in on the data while the left slicer shows the position in the image See Figure 8 2 f Huygens Twin Slicer advanced mode File Linking View Plot Help Imaginal disc2 60 Basic mode falue ch 0 172 5143 ch 1 225 6900 oom 10 83 px um 2866x Imaginal_disc2_60 EE d ea eo Select Active channels Select N Global colors v Color scheme Global colors v 0 gt A Custom colors 0 gt Time frame Zoom Rotate Fi Contrast Left mouse button draws a ruler Middle mouse button zooms in on bright spots Press lt c gt to center the slice FIGURE 8 2 The Twin Slicer in advanced mode with all controls but zoom and panning linked Markers Double click in one of the images to place a marker at the position of the mouse pointer As configured in the VIEW menu the marker shows the coordinates and intensity values besides it To remove the marker click it and press the Delete key Rulers To overlay a ruler on the image hold the left mouse button and drag The length of the line in um is displayed beside it Click and drag the end points of the ruler to make adjustments Press and hold the Ct x1 key while drag
116. fractive index Lens 25 Medium 25 Mismatch 145 Region of interest 116 Render pipes 68 105 124 SFP 61 size 64 70 Surface 67 Restoration methods 19 ROI 116 S Saturation 29 Segmentation 101 seed and threshold 101 watershed 103 Select area 104 Select object 104 SFP 61 Signal to noise ratio 30 Slicer 12 Slide position 153 SNR 18 30 Spherical aberration 145 148 152 Sphericity 112 Statistics 106 152 Storyboard 75 Support 157 Surface Renderer 67 System ID 6 159 System requirements 9 T Task 42 Templates Deconvolution 42 Microscopic 26 41 Visualization 64 70 Threads per task 43 Threshold 62 68 101 Time series 24 Timeline 76 Total internal reflection 146 Tracking 91 Transitions 75 Transparency 69 Transparency depth 70 U Undersampling 146 Uninstall 9 Updates 7 8 V Velocity 96 W Watershed segmentation 103 Wavelength Emission 25 Excitation 25 Windows 5 11 Wizard 23 35 Huygens Professional User Guide for version 4 1
117. ging an end point to change length without changing direction Click and drag the middle of the ruler to move it in its entirety without changing length or direction Press and hold the Ct r1 key while drag ging the ruler to move it perpendicular to its direction To remove the ruler click it and press the Delete key Intensity Profiles When a ruler in the left slicer is selected the right slicer will be replaced by a plot win dow and vice versa See the online SVI Wiki for more information on the data plotter Select PLOT gt PLOT BOTH SLICERS from the menu to show the intensity profiles for both the left and right image in the same plot The graphs for the left slicer will have solid lines while the graphs for the right one are dashed See Figure 8 3 l http www svi nl DataPlotter 52 Huygens Professional User Guide for version 4 1 Measurement r Huygens Twin Slicer advanced mode File Linking View Plot Help WF original n HBH elv FEHE zom HE Time frame Zoom Rotate Colors Contrast Hold the lt ctrl gt key to drag perpendicular Press Delete to delete the line FIGURE 8 3 Measuring the intensity profile along a line The plot can be configured such that it shows the profile of both images left solid right dashed Huygens Professional User Guide for version 4 1 53 54 Huygens Professional User Guide for version 4 1 CHAPTER 9 The Orthogonal Slicer
118. greyed out here you can only modify the microscopic parameters in the main microscopic parameter window or the PSF wizard e The Help pane 3 http www svi nl RecordingBeads 34 Huygens Professional User Guide for version 4 1 The Processing Stages in the Wizard The Processing Stages in the Wizard Explains the different steps and displays links to relevant wiki pages to explain every step in detail e The Distiller status pane Keeps track of all the steps during the distiller run and reports the progress e The Reports pane Displays the progress report of the distiller process and the status of the distilling process e The PSF and Accus pane Displays the intermediate results of the distilling process The thumbnails can be inspected with the Slices The slider on the right side can be used to scroll down if many files are displayed e The Wizard pane Shows the steps that will take you from checking image parameters to distilling and averaging the beads from your images The following steps and stages are to be followed Loading an image e Start Stage here the possibility exists to load a microscopic parameter template and check the microscopic parameters e Averaging Stage in this stage all channels of the image are searched for beads that meet the selection criteria After each successful or unsuccessful search there is the possibility to load in additional bead images or go to the next stage
119. ground The histogram in Figure 4 4 shows that the intensity distribution in the demo image is of reasonable quality The narrow peak shown at the left represents the background pixels all with similar values The height of the peak represents the amount of background pixels note that the vertical axes uses logarithmic scaling Histogram log scale In this case there is also a small black gap at the left of the histogram This indicates an FIGURE 4 4 The image electronic offset often referred to as black histogram The vertical i i mapping mode can be selected level in the signal recording chain of the fr SEE li ud ar logarithmic or microscope sigmoid If a peak is visible at the extreme right hand side of the histogram it indicates saturation or clipping Clipping is caused by intensities above the maximum digital value available in the microscope Usually all values above the maximum value are replaced by the maxi mum value On rare occasions they are replaced by zeros Clipping will have a negative effect on the results of deconvolution especially with widefield images The histogram stage is included in the deconvolution wizard for examining purpose only It does not affect the deconvolution process that follows In this stage the average background in a volume image is estimated The average back ground corresponds with the noise free equivalent of the background in the measured noisy image It is importa
120. gs The time needed to deconvolve an image increases more than proportionally with its volume Therefore the deconvolution can be accelerated considerably by cropping the image Huygens Professional is equipped with an intelligent cropper which automatically sur veys the image to find a reasonable proposal for the crop region See Figure 4 3 In computing this initial proposal the microscopical parameters are taken into account making sure that cropping will not have a negative impact on the deconvolution result Because the survey depends on accurate microscopical parameters it is recommended to use the cropper as final step in the preprocessing stage press YES when the wizard asks to launch the cropper but it can be launched from outside the wizard through the menu EDIT CROP r amp Huygens Crop WF deconvolved Specifications Origin Span X 124 3270 Y 29 3 291 zn i Channels v O Mi Options 7 Link Sliders E Lock Ratio V Auto Contrast Cropbox color fo 9 Pixel properties Value X 24 293 Y 151 Zz 71 Preview Hide options E Extract Crop FIGURE 4 3 The crop tool in Huygens Professional 2 The user home directories are usually located in C NUsers on Windows 7 and Vista and in C NDocuments and Settings on Windows XP and lower On Mac OS X they are usually in Users and on Linux in home Huygens Professional User
121. he Movie Maker the Surface Renderer has the option to make simple anima tions of the image changing the view point in different frames Set the render parame ters for the first frame and click SET gt HOME in the Position settings panel at the right Now adjust the viewpoint for the final frame and click SET gt END Also the frame count frame rate or other render options in the OPTIONS menu may be adjusted Finally press the Animate button in the Actions pane and select a directory to save the AVI movie or the TIFF frames to 70 Huygens Professional User Guide for version 4 1 Simple Animations The exported AVI files use the MJPEG codec and can be loaded in most movie players including Windows Movie Player and Apple Quicktime TIFF frames are useful to com bine multiple animations or edit the movie in e g Windows Movie Maker l http en wikipedia org wiki Mjpeg Huygens Professional User Guide for version 4 1 71 72 Huygens Professional User Guide for version 4 1 An Overview CHAPTER 13 An Overview The Movie Maker The Movie Maker is a tool that allows to easily create sophisticated animations of multi channel 3D images using the powerful Huygens visualization tools Animations from the MIP Renderer See Chapter 10 on page 57 the SFP Renderer See Chapter 11 on page 61 and the Surface Renderer See Chapter 12 on page 67 can be combined in a single movie The Movie Maker assists the use
122. he micro scopic parameters attached to the data Because a theoretical PSF can be generated without any user intervention Huygens Professional does the calculation in the back ground without any notice Images affected by spherical aberration See Refractive Index Mismatch on page 145 are better restored using a theoretical depth dependent PSF 2 Measuring a PSF By using the PSF Distiller a measured PSF can be derived from images of small fluorescent beads See The PSF Distiller on page 33 Measured PSF s improve deconvolution results and may also serve as a quality test for the microscope Deconvolution as it is done in Huygens Professional is based on the idea of finding the best estimate of the object that is imaged by the microscope To assess the quality of an estimate Huygens Professional simulates the microscopic imaging of each estimate the estimated is convolved with the PSF and compares the simulation with the measured image From the difference a quality factor is computed The difference is also used to compute a correction factor to modify the estimate in such a way that the corrected esti mate will yield a better quality factor The quality factor as reported by the software is a measure relative to the first estimate and therefore a number greater than or equal to 1 If the increase in quality drops below the quality threshold the iterations are stopped l http www svi nl ImageFormation Huygens Professional User
123. he objects Depending on what of the previously reported parameters we choose for W and T we have different practical definitions of axial sphericity Probably the most intuitive one is the axial sphericity of the principal box axSphPB the ratio of the volume of an ellipsoid with axes L pBoxW0 pBoxW1 to the volume of a sphere circumscribed around the principal box see above using the largest side the length of the object L as diameter Equation 1 i 1 3 AxSphPB Box We pboxW EQ 1 L 2 Another definition reported as AxSph involves the lateral and axial widths dis cussed above obtained with the virtual caliper algorithm Equation 2 WiAx Wi 1 3 AxSph 7 WiAx MAC L EQ 2 The roughness sphericity characterizes the roughness of the iso surface it measures how close the volume to surface ratio is to the one of an ideal sphere This is conven tionally defined as 73 i ev 1 where V is the iso volume and A is the iso surface of the segmented object Both sphericity values become 1 for an ideal sphere The SurfSph is the inverse of the sur face factor fs used in Goetze et al A spect Ratio Again the aspect ratio of an object can be defined in terms of different dimensions e The axial aspect ratio AxRatio is the ratio of the object length to its axial width WiAx e Similarly the lateral aspect ratio LatRatio is the length divided by WiLatC More Parameters Many more parameters geometr
124. he website can also be consulted for updates Twice a year April and October new releases will become available During and shortly after this period it is advisable to consult more frequently Download the new version from the SVI web site Proceed with the installation as explained above Do not uninstall the old version as this will delete the license string The newer version will by default automatically replace the older one However for the MAC OS X we 2 http www svi nl Huygens Professional User Guide for version 4 1 Removing the Software advise to first uninstall the software and then reinstall a newer version In that case please make a backup of the license string in a safe place before removing the previous installation Removin g the Removing the software will also cause the license string to be removed If it is preferred So ftware to uninstall the current version prior to installing a newer one take care to store the license string in a safe place Table 2 2 on page 9 shows the uninstallation procedure for each platform TABLE 2 2 The uninstallation procedure per platform Platform Procedure Windows Open the start menu and select PROGRAMS gt HUYGENS SUITE UNINSTALL 2 REMOVE THE HUYGENS SUITE Linux Open the package manager search for huygens and unin stall it This could also be handled with the command line type dpkg r huygens to install a Debian package or rpm e huygens to install an
125. histogram indicates saturation as is the case in the right histogram in Figure 3 6 Saturation is caused by overexposure e g by raising the laser intensity above the maximum pixel value available on your microscope Usually all values above the maximum value are replaced by the maximum value On rare occasions they are replaced by zeroes Clipping will have a negative effect on the results of deconvolution especially with widefield images See Clipping on page 146 for more information A histogram may also show an offset at the left hand side which indicates a positive blacklevel A large positive blacklevel value will reduce the effective dynamic range of your microscope but will do no harm to the deconvolution with Huygens Professional For more information see Black Level on page 139 or the SVI wiki page DE Cc 3 TT LL 75 100 125 Canvas size 321 x 255 45 31 13 75 ad Canvas size 321 x 255 26 87 6 722e004 FIGURE 3 6 Two image histogram windows In the right image the spike at the right hand side indicates clipping saturation 3 http support svi nl wiki BlackLevel Huygens Professional User Guide for version 4 1 15 F Huygens Operations faba128 o amp s File View Channels Linking Convert Arithmetic Filter Restoration Manipulation Analysis Visualization Help B A WM P i FR SAG Lin Lad CMLE QMLE Quick TM XYZto XYT XYT to XYZ Av Sphere
126. hod the expert on line tutorial Let s try different segmentation parameters and see what happens First we lower the threshold value from the automatically calculated value down to something around 360 the mentioned numerical values refer to this particular test image of course You can drag the blue line in the channel histogram and shift it to lower values or click on the blue font label showing the threshold value to enter any number The threshold line on the histogram can be found at the right of the window in a pane labeled 2 http www svi nl ObjectAnalyzerExpertTutorial Huygens Professional User Guide for version 4 1 101 Object Segmentation Next to it in magenta there is the seed level As it is linked to the threshold by default when you shift the blue line the other one goes with it You can also switch to a percentage representation of threshold and seed by clicking the small button f at the top right of the histogram In this alternative slider view the only one before Huygens 3 5 you can also control whether the seed is linked to the threshold or it remains independent We see many things happening when we shift the threshold to lower values First the objects we had defined grow in size because more voxels around them get attached to them Some objects that were separated before grow so much that the now get con nected and they define new single objects We also get more and more objects of all
127. http www svi nl We are directly reachable by phone during office hours CET or by e mail 24 7 Distributors An up to date list of distributors can be found on our web site Support SVI Support Wiki The SVI Wiki is a rapidly expanding public knowledge resource on 3D microscopy and deconvolution Based on the WikiWikiWeb principle it is open to contributions from every visitor In addition it serves as a support medium for SVI customers and relations to discuss different aspects of the Huygens software This is a list of useful starting points in the SVI Wiki to learn more about the Huygens software and microscopical imaging in general e Information on the parameters describing the imaging conditions sampling numer ical aperture pinholes etc http www svi nl MicroscopicParameters l http www svi nl distributors 2 http www svi nl Huygens Professional User Guide for version 4 1 157 License String Details Information on the restoration parameters signal to noise ratio background quality criteria etc used by the deconvolution algorithms http www svi nl RestorationParameters A step by step example on how to tune these parameters to achieve the desired resto ration results http www svi nl DeconvolutionProcedure Important issues regarding image acquisition and restoration sampling clipping etc http www svi nl ImportantFactors Typical acquisition pitfalls spherical aberrat
128. i photon and 4 4 Pi experimental microscopes Server flag Determines the number of cores that are enabled for multi thread ing A hyperthreaded core is counted as a single core It can be desktop 2 cores s small server 4 cores m medium server 8 cores 1 larger server 16 cores and x extreme server 512 cores Option flags This is a set of characters that list the enabled optional modules An overview of these modules is given in Table 24 2 158 Huygens Professional User Guide for version 4 1 License String Details TABLE 24 1 The building blocks of the Huygens license string Substring Description Locking policy A set of characters that indicate to which properties the license is locked These can be d expiry date e e mail address m sys tem ID n number of cores p processor type and s pro cessor details Expiry date The date on which the license or maintenance ends When this substring starts with eom then only the maintenance expires the license remains valid System ID A 16 character hexadecimal string containing hardware identifica tion numbers E mail address The customer e mail address Checksum A 20 character hexadecimal checksum on the previous substrings TABLE 24 2 Identifiers for the optional modules Character Module a Object Analyzer A Advanced Object Analyzer b Small file reader bundle B Complete file reader bundle
129. ical and of other kinds can be calculated reported and duel EM used to filter the data by the Object Analyzer Please refer to the on line tutorials to learn 5 how to use the different parameters and Experiment presets Parameters are available that report e Correlation inside and between channels by analyzing neighbor objects e Location of objects with respect to reference objects anchors e Location of objects inside regions of interest for example bodies inside a cell nucleus e Colocalization of objects by computing the volume and the intensity overlap of seg mented objects in different pipes Any of the calculated parameters can be used to filter out objects and further segment your image in elaborated ways 1 Goetze et al Molecular Cellular Biology 27 p 4475 4487 2007 Huygens Professional User Guide for version 4 1 113 114 Huygens Professional User Guide for version 4 1 CHAPTER 19 Object Analyzer Component Reference This section describes the components of the Object Analyzer OA interface This section is intended as a quick reference See Introduction to the Object Analyzer on page 99 to learn how to use the basic components in context LE IE Huygens Object Analyzer Advanced objectAnalyzer test image ee File Options History ROI Anchors Filter Help 1 Frame Bi Object Segmentation Primary Scnd MIP seed threshold on histogram dg Transparency
130. ide for version 4 1 109 Storing your Results Further Reading IE 1N CMCM 0 1 N 53 Ready Canvas size 400 x 255 1 47 14 8 FIGURE 17 11 Histogram representation of one column values After computing all objects sphericity parameters for example and having them reported on the table we can plot a histogram of the particle sphericity distribution In the top menu you can do FILESSAVE OBJECT STATISTICS to export the table you see on the screen to a file that you can import elsewhere The file can be read directly or imported in conventional spreadsheet programs or data plotters The table can be stored as a plain text file a csv file that can be imported in e g Microsoft Excel OpenOffice Calc or GNUplot and as an m file that can be imported in Matlab to do further analysis and or calculations You can also save the current scene as you see it on the rendering canvas to a TIFF file on disk with FILE gt SAVE SCENE In the OPTIONS menu you can find different options that affect how the scene is rendered You can set the MIP pipe to high quality for example or render each analyzed object together with its numeric ID label so that you can link them with the exported data visually In the HISTORY menu you can save your analysis history as a template to reproduce it using the same or other dataset This tutorial has covered the very basic features to the Huygens Object Analyzer If you want to learn much mor
131. ight side of the wizard shows a his Optimizing object detection togram of the number of objects versus 7 varde are markea with a plus their score The score is a qualitative Tek de WINE recede uie re Ee measure for the probability that the i detected object should indeed be classi fied as object By default all objects with a positive score are taken into account but this threshold can be changed in order to make the Object Tracker less or more sensitive To change the threshold score tick the lower threshold box and drag the m tower threshold 24 54 slider Figure 16 4 i Upper threshold Inf Press Next to recompute e e J A Oo A 5 2 1 2 0 I e ol Besides the score thresholds can be applied to the number of objects and the FIGURE 16 4 Increase the threshold on width and brightness of the objects The the score to reject more objects width and brightness can be limited by both a lower and upper threshold There is by default a threshold of 100 objects on the number of objects An important thing to note is that all thresholds are applied not only the currently selected one When one of the thresholds is modified the NEXT button will reload this screen instead of advancing to the next When the detection is satisfactory press NEXT again to advance to the next step Huygens Professional User Guide for version 4 1 93 The Track Analyzer Tracking Parameters Th
132. iginal data the surface objects can be blended with a MIP projection of the data See The MIP Renderer on page 57 The color range in which these objects are displayed can be modified using a hue selector page 151 134 Huygens Professional User Guide for version 4 1 Backgrounds vs thresholds in colocalization File Options Colocalization Surface MIP Renderer Object Analysis Object colocalization statistics Clear Help Analyze all Selected colocalization coefficient Pearson Conditions Threshold 1 04e 05 Seed level 1 09e 05 5 Garbage level 2 Total objects 69 Frame 0 Red chan 0 bg 40 48 Green chan 1 bg 47 62 4 Ready Dragging on FIGURE 20 3 Tab three of the Colocalization Analyzer if for object analysis for the colocalized regions of the colocalization map B ackground S Vs Backgrounds are for removing signal prior to the calculation In an ideally restored image thresholds in that would not be necessary because all the signal present in the image gives a valid rep resentation of it The background would have been removed during the image restora colocalization tion Thresholds are used to split the colocalization maps in two regions what are interesting objects and what are not Local colocalization values are calculated for every image voxel but the zero value would be very rarely achieved In most of the cases there are some non zero colocalization everywhere
133. ils explained on all supported platforms Here all details will be listed for the license which is shown in blue after For each product it ls this paragraph Each license element will be shown on a separate line If a license or license element is not valid for the current product then required to have a license this will be shown in red string installed Select a license string in the license HuEss 4 0O wenp4 d2 B tvACMsxTc dmp 2011Nov03 S51 7c825a33 bS7S daniel svi n1 7158S01icabaS EEd There was one possible reason found why this license may not be valid to run this particular product window HELP gt LICENSE ee ME and press the EXPLAIN This license product name matches the currently running product LICENSE button All details Version 40 The licensed verson is equal to the currently running product version for the current license will be Microscopes wcnp4 listed Figure 2 4 If run w enables Widefield microscope functionality enables Confocal microscope functionality ning into licensing problems n enables Nipkow disk microscope functionality p enables multi photon microscope functionality this information can be used 4 enables 4 Pi microscope functionality toanalyze the problem 4 FIGURE 2 4 The Explain License window lists all license details When the system is attached to the internet a pop up window will appear when a newer version is available T
134. indow will be displayed with a question or a warning All channels will be processed automatically In the final stage a previously obtained PSF image can be added as a channel before or after the current result Press ADD CHANNELS if this is desirable The Distiller will com pare the microscopic image parameters of the selected PSF image and check its content In case there are problems the software will ask to decide between ignoring the differ ences and discarding the selected file Press DONE if you are finished adding channels to the distilled PSF Finally press EXPORT amp CLOSE This will export the distilled PSF to the main window where the result can be saved or used immediately in a subsequent deconvolution run Read more about PSF s and the PSE Distiller on the SVI Wiki 4 http www svi nl PsfDistiller Huygens Professional User Guide for version 4 1 37 38 Huygens Professional User Guide for version 4 1 The Batch Processor Window CHAPTERG The Batch Processor Once you are familiar with a particular kind of dataset and the restoration parameters are determined a couple or more of similar datasets can be restored automatically This is called batch processing A batch process consists of a number of image restoration tasks one per image which are executed one by one until all are finished Depending on the multithreading capabil ities of the computer multiple tasks can be executed in parallel
135. ing it pressed while moving the image to the desired position For each channel one out of five color schemes can be chosen e Grey Pixel values are assigned different shades of grey ranging from black for the lowest values to white for the highest values e Emission The displayed color of a channel matches the true color of the emission wavelength in so far this is possible e False Pixels values are assigned different colors ranging from black dark purple for the lowest values to bright red for the highest value e Global In a multi channel image colors can be assigned to the different channels by suing the global color palette e Custom Huygens Professional User Guide for version 4 1 45 A custom color can be chosen for each of the channels As with RGB pixel values are assigned different shades of a particular color ranging from black for the lowest val ues to the brightest possible shade for the highest values Contrast For the whole view three basic gamma contrast settings can be chosen e Linear In this mode the pixel values are mapped to screen buffer color intensities in a linear fashion Note that the actual translation of the screen buffer values to the actual brightness of a screen pixel is usually quite non linear e Compress When an image contains a few very bright spots and some larger darker structures using the Linear mode will result in poor visibility of the darker structures Res
136. inter acts with it In very slow systems the dragging is turned off automatically and the ren dering only happens after the user released the mouse buttons or finished moving the segmentation sliders The tooltip the region with light yellow background shows contextual information e Alonger description of the reported parameters when you hover over the table head ers See Figure 19 1 item 20 and a sum of the cell below them e The value in the cell under the cursor when you point at table rows plus selected ref erence values Huygens Professional User Guide for version 4 1 129 e The different actions bound to the mouse buttons when the cursor is inside the ren dering canvas 130 Huygens Professional User Guide for version 4 1 How to use the Colocalization Analyzer CHAPTER 20 How to use the Colocalization Analyzer The Colocalization Analyzer The Colocalization Analyzer provides information about the amount of spatial overlap between structures in different data channels for 3D images and time series As this overlapping can be defined in many ways Huygens gives the colocalization coeffi cients most commonly used in literature Object Pearson Spearman Overlap Intersec tion and Manders M and K More information on these coefficients can be found in the SVI Wiki The Colocalization Analyzer is an extended optional tool and is enabled by a C flag in the license string See License String
137. into the license mail message with the EDIT gt PASTE menu item of the mail program This dialog box also contains a button to Check for updates on the SVI company server Installing the License String Select the license string in the e mail message and copy it to the clipboard using EDIT2 COPY in the mailing program Start Huygens Professional and go to HELPOLICENSE a dialog box pops up Then press the ADD NEW LICENSE button and paste the string into the text field Figure 2 3 Complete the procedure by pressing ADD LICENSE this will add the string to the huygen sLicense file Please try to avoid typing the license string by hand any typing error will N About x e About the software Huygens compute engine 4 0 0p4 64b Huygens Essential for Win amp 64 Built on Aug 4 2011 10 55 33 Copyright C 2001 2011 by Scientific Volume Imaging B V Laapersveld 63 1213 VB Hilversum The Netherlands Email info svi nl URL http www svi nl More about the software Software updates Check for updates Download file System ID This computer has the following system ID 51 7 c825 a336 b979 Copy FIGURE 2 2 The HELPS ABOUT window The system ID is shown at the bottom invalidate the license With an invalid license the software will remain in Freeware mode When the license is correct the message Added license successfully will appear Restart Huygens Professional to activat
138. ion http www svi nl DynamicRange http www svi nl ClippedImages I O U A http www svi nl NyquistCalculator 146 Huygens Professional User Guide for version 4 1 Data Acquisition Pitfalls limit the number of samples Having less photons per sample means that the overall pho ton dose can remain largely constant as a result bleaching does not need to get worse Although this results in an apparently noisier image there is actually more information in the data because sampling density is higher Deconvolution with Huygens Profes sional removes the noise and may enhance the image quality dramatically Of course there are limits but a fair trade off can be often found Concluding it is better to record 10 separate noisy slices 100 nm apart than two slices 1000 nm apart and averaged 5 times in order to reduce noise Bleaching Bleaching is a practically unavoidable phenomenon in fluorescence microscopy Because the image planes are acquired sequentially bleaching will vary along the z direction Assuming it is not strong it will not affect deconvolution results on confocal or two pho ton images But in widefield deconvolution bleaching is more of a problem Fortunately the bleaching in widefield images can usually be corrected quite easily Huygens Profes sional will do so automatically However if the bleaching is strong the correction might not be perfect resulting in lower quality deconvolution results Illumina
139. ion undersampling bleaching etc http www svi nl AcquisitionPitfalls Information on recording beads to measure a PSF http www svi nl RecordingBeads Tutorials and detailed information on using the different aspects of the Huygens soft ware restoration visualization analysis programming etc http www svi nl Tutorials Uploading images to SVI http www svi nl SendImagesToSvi Detailed information about the installed license strings can be displayed via HELP gt LICENSE Select the license string of interest and click EXPLAIN LICENSE A Huygens license string consists of a set of substrings separated by dashes These substrings describe e g the product version number options etc The checksum at the end of the string should match with all other substrings A complete string looks like this HuPro 4 l wcnp d tvAC emnps eom2013Dec31 e7b7c623393d708e user idomain comj 4fceO0dbe86e8ca4344dd Table 24 1 lists the building blocks from which this string is composed TABLE 24 1 The building blocks of the Huygens license string Substring Description Product The product to which the license string applies This can be HuEss HuPro HuScript and HuCore Version The version number of the product Microscope types This substring consists of one or more characters representing the microscope types for which the deconvolution is enabled These are w widefield confocal r Spinning disk mult
140. is La CMLE QMLE Quick TM XYZ to XYT XYT to XYZ Av Spheres Distill PSF Theor PSF Image Stats Estimate Bg Slicer Operations Parameters LJ Classic Maximum Likelihood Estimation Zoom a PSF if available psf Output T Destination c Signal Noise per channel 20 20 20 20 Max iterations 40 Search for background Auto hi Tilt Backgr per ch absolute or 0 0 0 0 0 0 0 0 00 deg Bleaching correction If possible bl Brick mode Auto Quality change threshold 3 01 Iteration mode Optimized hl 0 xy Padding mode Automatic hl XZ YZ Twist 000 dea u Voxel data Linking At El V Position V Orient V Center 4 Listen n Time Zoom Other Slicing Slice center at voxel coordinate 0064 0064 032 DR ae View options J Link channels Stretching Global Gamma Linear Tel Cmd faba128 cmle psf gt c Color Contrast Time Run Command Ready Canvas size 440 x 400 Interactive FIGURE 3 10 The operations window where CMLE is selected The right panel shows the parameters for the CMLE deconvolution run e PSF if available From the list of all opened images an image can be selected that is used as the PSF This can be either a measured PSF or an earlier generated PSF using the PSF button from the operations window If you do not use a measured PSE it is recommended to select an empty image In this case a PSF is not available and the software uses the microscopic parameters to generate PSF s on the fly
141. is is the final stage before the automated tracking starts The parameters that can be modified in this screen affect the linking of the detected objects between subsequent time frames The first parameter limits the distance over which an object is allowed to move between time frames This means that the tracker will not connect two objects in subsequent time frames if their mutual distance is larger than this value The second option enables or disables the use of object geometry and orientation for linking objects Enabling this option makes sense if the objects of interest have distinct sizes Quick Edit Tracks When all time frames are processed and the detected objects have been linked into tracks the wizard advances to the quick edit tracks stage In this stage broken tracks can be connected and wrong tracks can be deleted or broken down into smaller pieces that can be re connected The graph shows the position of the objects in the xy plane versus the time frame num ber Figure 16 5 The projection angle can be changed using the slider below the graph amp B 2192 Cf R alete Select Bresk Undo Redo Connect Delete Invert XY position vs frame number p a ec A XY projection angle 0 c NG 4 Previous Abort Next gt gt FIGURE 16 5 The quick edit tracks stage allows to connect break and delete tracks An angle of 0 projects on the x axis while 90 projects
142. isting estimated shift dashed line Therefore the plot serves as a comparison between the reference channel the edited channel and the corrected edited channel This comparison allows us to see to what extent the intensity of edited channel is shifted with respect to the reference channel At the same time it shows in advance whether the estimated shift vectors will correct for the existing chromatic shifts properly The result of a chromatic shift estimation is shown in Figure 14 2 The shift between the intensity profiles of the reference channel and the edited channel is visible in the embed ded plot Additionally the dashed line in the plot shows the intensity profile of the edited channel as if it were corrected with the estimated shift Ideally the intensities of the corrected channel dashed line and the reference channel would show no gap or shift having similar shapes and peaks roughly located at the same X positions Huygens Chromatic Shift Corrector File View Plot Estimation method amp A 49 82 SHE vs o Chromatic shift templates Correlation IcShift 2010 12 14 CM alignment Reference channel DES Shift vectors microns 0 24 0 05 0 03 4 Edit vector Channel 1 Help The plot shows a profile of a specific channel with the shift vectors from the table dashed line applied Prior to correcting the image the shift vectors can be modified for each channel with the
143. its linked mode the seed is set referred to the span between the actual threshold and the maximum and ranges from the threshold value itself 096 to the image maximum 10096 This is because the seed being an upper threshold can never be lower that the threshold It is also useful to be able to set the seed to 096 so that it is not used at all and you retrieve the classical threshold only segmentation Still you may find convenient to express the seed relative value in the same terms you use for the threshold relative to the image intensity rage or to be able to set the seed to a fixed value independent of the threshold For that deselect the seed checkbox so that 096 also represents the same value as the 096 threshold the image minimum or zero and the two sliders are unlinked The linking mode of the seed does not really affect the segmentation it is just a matter of convenient representation of relative values what is applied to the image as threshold and seed are always the absolute numeric values shown on the entry widgets and next to the histogram lines that you can edit directly by clicking on them All the different objects in a pipe are colored differently to be able to distinguish them The range of colors assigned to each pipe can be controlled with a Hue Selector See Hue Selector on page 151 You can collapse this range completely if you want so all objects in a pipe get the same color General Object Information 18
144. king on FIGURE 20 4 The RBNCC option is enabled and a the visible image name The reference image can be selected reference image should already be opened in the main window of Huygens Professional and should have the same dimensions as the original When the reference image is selected you only need to press COMPUTE and the coefficients will be corrected automatically The correction factors applied to the coefficient are shown in the RBNCC frame where the abbreviations stand for a specific coefficient e S Spearman coefficient e P Pearson coefficient e OS Object Spearman coefficient e OP Object Pearson coefficient pe http www svi nl ColocalizationAnalyzer http www svi nl RBNCC e 136 Huygens Professional User Guide for version 4 1 Image Size CHAPTER 21 Image Size Signal to Noise Ratio Establishing Image Parameters The deconvolution algorithm needs to know some of the parameters describing the image acquisition These are not too many but careful determination may significantly enhance the deconvolution results The amount of computing time involved in deconvolving images is more than propor tional to the image size It is therefore sensible to limit the data size as much as possible Regarding widefield images we recommend to not record planes below and above the object which only contain blur Huygens Professional does not need these planes to restore the object Since the blur
145. l factor 25 Filter Objects 125 Filter tracks 94 Flow 97 Freeware mode 6 G Garbage volume 69 102 Geometrical distortion 145 Global color scheme 151 H hgst file 26 hgsv file 64 70 Histogram 15 28 109 l Image Convert 16 Imaging direction 25 Installation 5 Linux 5 Mac OSX 5 path 8 Uninstall 9 Windows 5 Iso surface 67 Iteration mode 30 Iterations 19 30 K Keyframes 74 L Leica numbering 150 Lens refractive index 25 License string 6 158 Linking 46 Linux 5 11 M Mac OSX 11 Mac OSX 5 Mean squared displacement 96 Measure Aspect Ratio 113 Colocalization 113 131 Correlation 113 Length 111 Principal Axis 111 Sphericity 112 Medium refractive index 25 Memory 9 Microscope type 25 Microscopic parameters 25 Mouse mode 104 118 Movie Maker 73 MSD 96 Multi channel images 31 N Number of iterations 30 Numerical aperture 25 Nyquist rate 13 14 146 O Object Analyzer 79 99 Object size 62 Objective quality 25 Operations Window 16 Orthogonal Slicer 55 Overfill factor 154 P Parameter templates 26 Parameters 25 Edit 13 16 Microscopic 13 Restoration 19 Phone number iv 157 Photodamage 146 Pinhole radius 25 spacing 25 Pipe Mode 120 Render 68 105 124 Point spread function 17 33 149 Presets 76 107 123 Principal axis 111 Processor 9 PSF 149 Asymmetric 145 Distiller 33 Experimental 148 Theoretical 17 Q QMLE 30 Quality factor 20 149 Quality threshold 30 R RBNCC 136 Re
146. l of the Chromatic Shift Corrector The saved templates can be imported again to apply its shift vectors to other images The template vectors will be loaded and listed in the vector table showing the intensity pro files as if the ESTIMATE SHIFTS button had been pressed The image can then be corrected by clicking on the CORRECT IMAGE button which will create a new corrected image In order to be able to work with the Chromatic Shift templates properly it is recom mended to apply these templates to images that have the same emission and excitation wavelengths as the image of the template 82 Huygens Professional User Guide for version 4 1 Stabilization of 3D Time Series CHAPTER 15 Stabilization of 3D Time Series The Object Stabilizer The Huygens Object Stabilizer measures and corrects for cell motion thermal drift shaking and other types of movement i e translation in x y and z and axial rotation Both the measurement and subsequent stabilization are done in 3D and at sub pixel level The Stabilizer not only stabilizes 2D or 3D time series but it also allows the align ment of slices within a 3D stack To launch the stabilizer select an image and select DECONVOLUTION OBJECT STABILIZER from the main menu Stabilization over time works best if the 3D time series has been deconvolved first Deconvolution enhances resolution and reduces noise which helps to analyze motion For time series there are four
147. lection red 92 Huygens Professional User Guide for version 4 1 The Object Tracker Wizard It is important to make sure that the diameter of the object selections is about the same as the diameter of the real objects because the Object Tracker uses that information to tune the detection filters The size of the background selection does not affect the detec tion When at least one object selection and one background selection is created pressing the NEXT button will initialize the object detection and advance to the next step in the wiz ard Note that you can choose any slice for training but the objects and background selection is only possible in one slice During training Linear Discriminant Analysis is used to find the best features to distinguish between object and background In combination with the watershed segmentation method See Using the watershed segmentation on page 103 objects are defined This process is done for each slice Optimizing Object Detection After pressing the NEXT button in the object detection stage the wizard will highlight the detected objects with a light blue overlay A plus symbol marks the center of each detected object Figure 16 3 In this screen it is still possible to add delete or modify the selections In case the selections are altered the NEXT but ton will reload this screen instead of advancing to the next stage FIGURE 16 3 Detected objects The r
148. lity in magnifica tion due to the variations in tube length that are result from the aligning the system The high system magnification allows the viewing of the diffraction pattern Airy disk at the pinhole plane directly by eye To enable the correctness verification of the values for the system magnification used in the on line calculator the way the system magnifi cation was derived is now explained In a Bio Rad MRC600 with a NA 1 3 60x objective the Airy disk has a diameter of around 2 to 2 5 mm at the pinhole plane The diameter of the first Airy zero ring is 7 6 lateral optical units o u using Equation 7 to express a distance r in dimensionless o u WS r ENA EQ7 In the system described here an o u is 0 3 0 033 mm At the specimen plane backpro jected a lateral o u is in this case around 61 nm The total magnification is in that case 4918x the system magnification 4918 60 82 9 x This value corresponds well with the largest possible system magnification for the MRC600 53 x 1 25 x 1 25 83 ll http www svi nl OpticalUnits Huygens Professional User Guide for version 4 1 143 144 Huygens Professional User Guide for version 4 1 Data Acquisition Pitfalls CHAPTER 22 Data Acquisition Pitfalls Improving Image Quality In this chapter discusses basic suggestions on how to acquire better microscope images These are based on common problems that we find frequently in data provided by
149. lp Colocalization Surface MIP Renderer Object Analysis Two channel Histogram Frame selection show histograms per color channel uu Frame 0 1 ll V All RBNCC Reference image Coloc Reference Correlation Correction S 1 051 P 1 013 OS 1 042 OP 1 016 Colocalization coefficients Pearson Frame 0 Red chan 0 bg 0 0033 Green chan 1 bg 0 0302 7 ObjectPearson Pearson 0 715 RBNCC ObjectPearson 0 690 RBNCC Spearman 0 897 RBNCC v Spearman ObjectSpearman 0 869 RENCC Overlap 0 78 4 ObjectSpearman xi 0 233 K2 2 649 Mi 0 991 M2 0 989 v overlap Intersection 0 738 I1 0 808 I2 0 895 v k12 Frame 1 Red chan 0 bg 0 0014 Green chan 1 bg 0 0058 4 Manders Pearson 0 723 RBNCC E ObjectPearson 0 686 RBNCC V inters Spearman 0 909 RBNCC ObjectSpearman 0 873 RBNCC 4 i12 Overlap 0 787 K1 0 236 K2 2 628 M1 0 992 M2 0 990 Intersection 0 750 I1 0 816 I2 0 903 1 36 Clear Red channel Green channel Background 0 0033 Background 0 0302 Thresh range 0 Thresh range 0 Cho Chl Ch0 amp Chi Colocalization map Pearson si Compute Resdy Dragging on 285 369 1 FIGURE 20 1 The first tab of the Colocalization Analyzer showing the colocalization coefficients and histograms different channel intensities Wait for the analyzer to initialize and to compute the first 2D histogram with the default settings When the colocalization analyzer is opened you will
150. m together and assigning them a distinct name or label This is done interactively by the Object Analyzer To remove too small objects in an early stage from the analysis a garbage level can be set below which objects are discarded After that detected objects are automatically labeled and sent to a continuous iso surface renderer See Chapter 12 on page 67 The Object Analyzer is an extended optional tool and is enabled by a special flag in the Huygens license string This chapter is written in the form of a step by step introductory tutorial to the basic functions of the Object Analyzer A reference guide that describes all the components of this tool can be found on page 115 Launch Huygens Essential or Huygens Professional e Loadan image you want to analyze To explore all the OA possibilities better use a multi channel image e Select the image thumbnail and in the top menu go to ANALYSIS2 OBJECT ANALYZER ADVANCED You can find this introductory tutorial on line in the SVI Wiki from where you can also download the test image we will use in the following explanations Huygens Professional User Guide for version 4 1 99 Please explore the image with the Twin Slicer See Chapter 8 on page 47 to get an impression of it This is a deconvolved image to reduce noise and blur artifacts It is always a good idea to perform object analysis with deconvolved datasets FIGURE 17 1 Maximum intensity projection of the tes
151. m values of the plot data l http support svi nl DataPlotter 56 Huygens Professional User Guide for version 4 1 Basic Usage CHAPTER 10 Basic Usage The MIP Renderer The Maximum Intensity Projection MIP Renderer enables the possibility to obtain an orthogonal projection of 3D data from any given viewpoint XJ view point The MIP renderer projects the image voxels on the screen by tracing near parallel rays from a viewpoint far away through the data volume Along each ray the maximum inten sity encountered is taken for the rendered image See Figure 10 1 Notice that this implies that two MIP renderings from oppo site viewpoints show symmetrical images projection plane To start the MIP Renderer right click on a data volume thumbnail and select VIEW MIP RENDERER i i FIGURE 10 1 schematic overview from the pop up menu or choose of MIP rendering The maximum VISUALIZATION2MIP RENDERER from the intensities on rays parallel to the main menu screen are projected Orientation and Zoom Adjust the viewpoint by moving the Tilt and Twist sliders See Figure 10 2 or by click ing and dragging the mouse pointer across the scene The magnification can be adjusted by using the Zoom slider or the scroll wheel Use the center mouse button to pan the cen ter of the projection Notice that the scene is rendered with low quality while being edited which allows for fast scene edition The s
152. mage are cor rect especially the sampling distance and the microscope type are of importance The average background estimation is invoked by selecting ANALYSIS ESTIMATE BACKGROUND from the menu in the operations window The following choices are possible e Lowest value Default The image is searched for a 3D region with the lowest average value The axial size of the region is about 0 3 um the lateral size is controlled by the radius parameter which is set to 0 5 um by default e In near object The neighborhood around the voxel with the highest value is searched for a planar region with the lowest average value The size of the region is controlled by the radius parameter e Widefield First the image is searched for a 3D region with the lowest values to ensure that the region with the least amount of blur contributions is found Subsequently the back ground is determined by searching this region for a planar region with the lowest average value Again the size of the region is controlled by the radius parameter In case of the image faba128 the best choice is Lowest value because we are looking for the background outside the object in a confocal image After pressing the RUN button the calculated value is displayed in the Task reports panel of the main window It is one of the values that should be known in the next steps Note that like PSF generation this step is automated in the deconvolution run step 7
153. menu bar or from the quick access buttons Note that the left side of the operation window is similar to the Slicer Each function can be controlled by editing the parameters in the operations panel To execute the operation press the RUN button Note that the corresponding Tcl command is displayed at the bottom of the panel and after execution also at the Tcl shell in the main window Using the operations window it is also possible to change or convert the dimensions of an image By selecting a menu item from EDIT gt CONVERSIONS you can e g change xyz into xyt or convert a 3D stack into a 4D time series 16 Huygens Professional User Guide for version 4 1 Step 4 Generate a Point Spread Function PSF Step 4 Generate a Point Spread Function PSF Intermezzo Bleaching Correction This extra check is given here for the sake of completeness for those using this sche matic approach for widefield images or confocal time series From the menu in the operations win dow select ANALYSIS gt PLOT FLUX A typi cal example is shown in Figure 3 8 Note that the demo image fabal128 is a regu lar confocal image a bleaching correc tion test makes no sense here Q flux MV3Crop3 Z position um Ready Canvas size 400 x 255 0 6178 14438768 FIGURE 3 8 flux plot generated by the Plot flux tool The plot shows a typical example of the intensity distribution along the z axis of a blea
154. mes in a series OPTIONS Rendering options and advanced statistics configuration e Virtual render size sets the size of the canvas on which the Ray Tracing algorithm renders the image This canvas can be larger that the OA window or even the screen that s why you can pan the canvas to inspect other regions Like that you can render and save high resolution TIFF images e Transparency depth controls the number of surfaces considered by the renderer in order to show inner cavities and objects inside objects e Bounding box shows or hides the reference 3D bounding box e Scale bar shows a scale bar on the scene The distances are calculated based on the voxel sizes in the original image microscopic parameters e Show ID labels render the scene showing the numerical ID label of each of the ana lyzed objects e Show SVI logo e High quality MIP enables or disables the high quality rendering mode for the MIP pipe e Show reference cube See Reference Cube 7 on page 121 e Show on screen tooltips for interactive actions e Relaxed selection when active objects partially outside the selected 2D area are also considered e Center scene moves the point of view to show the center of the dataset e Configure statistics report shows all available parameters to let you select which ones are calculated and reported on the table This is intended for advanced users begin ners should better use the Experiment presets See Experiment Pre
155. mplex to describe there is only room for a cryptic label in the column title You can always find out what each parameter is by look ing at its tooltip The description of each parameter will be also stored in your file when you export the table later The tooltip of the column header also shows the sum and the average of all the cells below it that you can optionally restrict to include data of a given pipe only Click on another object to add its data to the table You will see that the conditions are not reported again because they have not changed A checkbox option at the top right of the table clears it automatically when the segmentation or report conditions change so you always have an organized table Deselect this option to simply accumulate rows on the table so you can save it later and process it with another program Another button next to that checkbox clears the table manually whenever you want IN CMCM 5 6053 um FIGURE 17 9 Interaction with the objects When the distance to the nearest neighbor is computed it is also displayed on the screen for the object aimed at You may also have noticed that something else happens when you interact with the ren dered objects In the example of Figure 17 9 the user clicked on object 11 A line joins the center of mass CM of this object with the center of mass of the nearest neighbor and this distance is reported at the top of the window This is the only parameter repor
156. much i i closer to 6 than to 12 the The three render pipes in the Object Segmentation distance of the contour to frame referred to as primary secondary and tertiary the voxel with value 12is allow us to define three threshold levels that can be larger than the distance to applied to the same or to different data channels The the bottom right voxel data channel can be selected using the menu button in A the Object Segmentation panel The color range in which the different objects inside a render pipe will be displayed can be adjusted with the hue selector next to it 12 6 6 Saving Scenes Press the HIGH QUALITY button in Actions panel to apply full scene anti aliasing to the rendering and choose FILE gt SAVE SCENE to save the rendered scene as a Tiff file 68 Huygens Professional User Guide for version 4 1 Advanced Usage Advanced Usage Adding a Maximum Intensity Projection Besides the surface pipes there are additional rendering pipes to redirect data to the scene The MIP pipe works projecting the voxels with maximum intensity laying in the path of the rays traced along the viewing direction See Chapter 10 on page 57 In com bination with the surface pipes very clear representations can be obtained of the differ ent objects in the image The MIP of a channel can be a good spatial reference for the objects in other channels Adding a Slice The Slicer pipe is available to represent a single
157. n Huygens Professional splits the image into bricks in two situations a The systems memory is not sufficiently large to allow an image to be deconvolved as a whole b Spherical aberration is present for which the point spread function needs to be adapted to the depth Press DECONVOLVE to start the restoration process See Figure 4 5 Pressing STOP DECONVOLUTION halts the iterations and retrieves the result from the previous iteration If the first iteration is not yet complete an empty image will show 30 Huygens Professional User Guide for version 4 1 Finishing or Restarting a Deconvolution Run Deconvolution wizard Beta File Options Help Images gt ag x Number of vo s 0 5000 10000 15000 Intensity Help Reports Deconvolution setup Deconvolution Deconvolving the image In this stage the actual deconvolution of the image takes In this stage channel 0 ofthe original image will be deconvolved on the place based on results from the previous stages basis ofthe PSF and background as computed in the previous stages The result will be stored in image deconV2 Ch0 The deconvolution is done in an iterative fashion The Ne PIT EE Below are the default values for deconvolving images If necessary you iterations is reached or some quality measure can change these values decon final Ch1 Maximum iterations 50 See also About deconvolution Signal to noise ratio 40 Q
158. n be close to 70 degrees When a ray has to cross the cover glass to medium interface at such an angle total reflec tion may occur To be precise total reflection occurs when the NA of the lens is higher than the refractive index of the embedding medium This will reduce the effective NA of the lens Huygens Professional User Guide for version 4 1 147 Deconvolution Improvements Acquire an Experimental PSF A point spread function or PSF is the image of a single point object The degree of spreading blurring in the image of this point object is a measure for the quality of an optical system The imaging in a fluorescent microscope is completely described by its PSF Although in many cases a theoretically calculated PSF very well matches the real one ideal theoretical calculations can not predict actual misalignments or other prob lems inside the optical path Therefore it is always recommendable to measure an exper imental PSF and if it is very different from the ideal one use it for deconvolution instead of the theoretical one The experimental PSF can be measured by acquiring the image of a small bead When the size of the bead is known then the PSF Distiller can distill the real shape of the PSF from the recording See Chapter 5 The PSF Distiller on page 33 Spherical Aberration Correction When there is a refractive index mismatch then the Huygens software automatically correct for spherical aberration by ada
159. n the image metadata Because different manufacturers save the microscopic data with different confidence levels the Batch Processor allows you to choose whether only reliable metadata should be taken into account for the deconvolution see Figure 6 3 It is therefore important to understand what the different microscopic parameters refer to and know how to establish them A typically conflictive one is the backprojected pin hole radius This parameter is not difficult to calculate especially with the assistance of the online backprojected pinhole calculator As mentioned above the entered parameters can be stored in templates for conveniences sake so the same template can be applied to a series of images acquired with the same physical conditions l http www svi nl ConfidenceLevels 2 http www svi nl BackProjectedPinholeRadius 3 http www svi nl BackprojectedPinholeCalculator Huygens Professional User Guide for version 4 1 41 Click the NEXT button again when finished with the image description possibly having reused a previous template Deconvolution Templates Configuring the Restoration Process Like the microscopic parameters the deconvolution parameters can also be gathered in a template Again it is important that it is understood what the role of these parameters is Please see The Deconvolution Stage on page 15 and the Restoration Parameters article in the SVI Wiki A typicall
160. n to filter on brightness only This disables other image fil ters and is faster and usually good enough for bright or dark spot tracking The ignore artifacts choices give the option to discard objects that are detected close to the image borders as image borders can be sensitive to false positives Object Detection Stage In the object detection stage the user is requested to select a few objects and background areas In this stage one can also create a region of interest ROI such that only objects within this ROI are analyzed By pressing the DEFINE A ROI a new window will be opened in which an ROI can be created When done press the CLOSE AND EXPORT ROI button The ROI will be visualized in the tracker as a new overlay image The search for objects is then limited to object within the ROI Position 60 26 44 12 3 50 un To start the training use the object selection Time frame 0 Value ch 0 45 tool 45 to draw green outlined spheres to select objects Use the background selection tool 4 to draw red outlined spheres to select regions containing mostly background voxels Figure 16 2 Like the Orthogonal Slicer page 55 the Object Tracker displays the same point in space the image center from three orthogonal directions xy xz and yz The object and background selections are visible and can be drawn and modified in either of these projec tions FIGURE 16 2 Two object selections green and a background se
161. nd the pupil diameter is the excitation beam overfill factor See Figure 23 5 and is typically in the range from 2 to 4 Relative Beam Intensity 1 00 exp 2 x 1 2 exp 2 x 2 2 exp 2 x 4 2 0 75 0 50 0 00 5 0 2 5 0 0 2 5 5 0 Relative Distance from Optical Axis FIGURE 23 5 Lens entry pupil red beam profile with overfill factor 1 blue 2 green and dark red At overfill factor 1 the beam intensity is 14 of the maximum at overfill factor 2 the edge intensity is 61 of the maximum 3 http en wikipedia org wiki Gaussian beam 154 Huygens Professional User Guide for version 4 1 Brightfield Images Brightfield Images The overfill factor can be set as a microscopic parameter in Huygens Professional and is taken into account when computing the point spread function Brightfield imaging is not a linear imaging process In a linear imaging process the image formation can be described as the linear convolution of the object distribution and the point spread function hence the name deconvolution for the reverse process So in prin ciple one cannot apply deconvolution based on linear imaging to non linear imaging modes like brightfield and reflection One could state that the image formation in these cases is linear because it is governed by linear superposition of amplitudes However microscopes do not measure light amplitudes but rather intensities i e the absolut
162. nformation window Values tool leaves you with values smaller then one smaller than one indicate the image appears to be undersampled Your undersampling future recordings may benefit from this infor mation Note that supersampling is not a prob lem although your dataset becomes unnecessarily large without providing extra information For now we continue with the z undersampled demo image The Intelligent Cropper The time needed to deconvolve an image increases more than proportional with its vol ume Therefore deconvolution can be accelerated considerably by cropping the image Huygens Professional is equipped with an intelligent cropper that automatically surveys the image to find a reasonable proposal for the crop region In computing this initial pro posal the microscopic parameters are taken into account making sure that cropping will not have a negative impact on the deconvolution result To start the cropper select EDIT2 CROP from the menu in the main window Initially the cropper suggests that the bottom planes from faba128 can be removed without signifi cant loss of information that will decrease the deconvolution quality Figure 3 5 Huygens Crop WF deconvolved REI Specifications Origin Span X 270 Y 29 20 gt Z2 35 mm Options V Link Sliders E Lock Ratio V Preview 7 Auto Contrast Cropbox color a Pixel properties Value X 24 293 Y 151 Z 71 Previe
163. ng the Slicer in Basic Mode The View Menu Use the VIEW menu to show or hide image properties and guides These are listed in Table 8 1 TABLE 8 1 The options in the Twin Slicer s VIEW menu Option Description POINTER COORDINATES Display the position of the mouse pointer in um or in voxel coordinates TIME Display the time for the current slice in seconds or frame num bers INTENSITY VALUES Display the intensity values for all channels on the current pointer location ZOOM Display the zoom value in screen pixels per micron magnifi cation factor is displayed as well using the pixel density for the monitor this value gives an estimation for the absolute magnifi cation ROTATION ANGLES Display the tilt and twist angles in degrees DROP SHADOWS Enhance the contrast for the overlayed lines and text by show ing drop shadows SLICE BOUNDARIES Draws the slice boundaries for the left image in the right one and vice versa This is helpful when both slicers are used WIREFRAME BOX Show or hide the wireframe box which gives visual feedback on the position and orientation of the cutting plane green and the displayed slice gray in the data volume red SVI LOGO Show or hide the SVI logo in the lower right of the view port Panning Click and hold the right mouse button on the slice to move it around Clicking the center button or pressing the c key centers the slice Slicing Drag the slider below the
164. ng the colored background lines in either the 2D histogram the enlarged 1D histograms or by changing the numeric values in the input fields you can specify the 132 Huygens Professional User Guide for version 4 1 How to use the Colocalization Analyzer File Options Help Colocalization Surface MIP Renderer Object Analysis Zoom iso colocalization surface 1 00 Threshold 1 04e 05 12 Seed thr 5 Garbage vol 2 Transparency 0 78 Brightness 0 79 Tilt 62 Off MapO MIP of original image Transparency 1 00 Brightness 0 64 Channel R 0 G 1 v Twist 35 Ready rendered Canvas size 693 x 604 Dragging on Mouse buttons 1 2 3 sel object pan pan scene FIGURE 20 2 Tab two of the Colocalization Analyzer shows surfaces for regions of overlapping channels backgrounds for the two selected channels These values are subtracted from the voxels intensities when calculating the coefficients if the result is negative it is understood as a zero Generally the colocalization coefficients depend much on correct estimation of the image background and resolution For these reasons we strongly recommend to compute colocalization coefficients only on deconvolved images One sets a background value prior to the calculation to remove signal that would other wise lead to spurious colocalization This is intended for minor tuning or just in case it is really needed to calculate colocalization in ra
165. nger wavelength Since this emitted light has changed wavelength it is not capable to re excite the same fluorescent matter multiple scattering does not occur Thus only the light emitted in the direction of the viewer either directly or by way of the semi reflecting table is of importance By sim ulating the propagation of the emitted light through the matter the algorithm computes the final intensities of all wavelengths the spectrum of the light reaching the viewpoint The properties of the interaction between object and light transparency both for exci tation and emission can be adapted interactively by the user to produce different scener ies Render Parameters Table 11 1 gives an overview of all render parameters in the SFP Renderer TABLE 11 1 SFP render parameters Parameter Description Time frame A selection of the time frame for time series Object size Adjust the total transparency of the rendered object See Object Size on page 62 Excitation Adjust the excitation transparency for the matter in the selected channel Emission Adjust the emission transparency for the matter in the selected channel Object brightness Set the intensity level for the excitation light source for the selected channel Soft threshold Adjust the threshold level for the selected channel See Threshold on page 62 Huygens Professional User Guide for version 4 1 63 Simple Animations TABLE 11 1 SFP render paramete
166. nhole r nm 250 9 X nm wo Excitation wavelength nm 488 Y nm 50 692 Emission wavelength nm 520 9 Z nm 80 Multi photon excitation 1 T s 1 000000 Excitation fill factor 2 00 Optical parameters Reports Microscope ype SEER Numerical aperture 1 30 Objective quality Good v Coverslip pos um Fa 0 000 Imaging direction Upward GO Pinhole spacing um 2 530 Refractive indexes Lens immersion Oil 1 515 Embedding med Oil 1 515 Meta data All verified Revert Cancel Accept FIGURE 3 3 The parameter editor The values in this window should be checked carefully before deconvolution l http support svi nl wiki NyquistCalculator 2 http support svi nl wikiCalibrateAxialDistances Huygens Professional User Guide for version 4 1 13 In case of the faba128 image we see that the Q fs image is fairly good sampled in the x and y F opina rate wart Nyquist criterion directions since the Nyquist rate is 0 93 which Up X dir 0 93 Y dir 0 93 Z dir 0 82 f a is near to the value of one However the z Sampling precisely according to the Nyquist criterion direction has a Nyquist rate of 0 82 which would involve sampling distances nm j gt pr indicates undersampling the sampling was too coarse It is advised to sample in the z direction using a sample distance of 166 nm or FIGURE 3 4 The Nyquist less It can be concluded that if the Nyquist i
167. nimize black borders lt lt Previous Abort Next gt gt JU BP Color scheme Greyscale M Custom colors 0 MF m Zoom E Colors E Contrast FIGURE 15 7 The stabilization result can be cropped manually to cut out the objects of interest The original image borders are shown as grey rectangles allows the user to manually cut off the black borders crop the image to the original size or keep the full size 3D alignment can be applied prior to deconvolution if the stack suffers from severe mis alignment between adjacent slices In those cases the Object Stabilizer may improve the deconvolution of 3D stacks and can be applied beforehand In all other cases applying deconvolution first is preferable The 3D alignment method always uses the cross correlation method for comparing adja cent frames and it can correct for x and y translation and rotation The steps in the wiz ard are the same as for the stabilization of time series as explained in The Cross Correlation Method on page 84 A comparison between a misaligned and aligned z stack is shown in Figure 15 8 FIGURE 15 8 An x z slice of a misaligned z stack left and the alignment result right The chromatic shift in stack like this one can be correcting using the Chromatic Shift Corrector 88 Huygens Professional User Guide for version 4 1 Alignment of Slices in 3D Stacks Note that 3D alignment deforms the volume It is advisable
168. nnected to the internet you can simply request a license at the SVI server Otherwise please send an email to license svi nl including the following system ID bc37 557b 77c1 af57 Ready Memory usage 0 MB FIGURE 2 1 The startup window of Huygens Professional If no license string is installed the software runs in freeware mode The License Strin g The license key used by all SVI software is a single string per licensed package It may look as follows HuPro 4 1 wcnp d tvAC emnps eom2013Dec31 e7b7c623393d708e user domain com 4fce0dbe86e8ca4344dd At startup Huygens Professional searches for a license file huygensLicense which contains a license string This license string is provided by SVI via e mail Installing the license string is the same for all platforms Obtaining a License String If upgrading is not handled from a previous installation it is likely that a license is not yet available To enable us to generate a license string we need the fingerprint of the com puter used the so called system ID number 6 Huygens Professional User Guide for version 4 1 The License String If Huygens Professional is not already running please start it The system ID pops up as long as no valid license is available and is displayed in the HELP gt ABOUT dialog Figure 2 2 Send it to sales8svi nl anda license string will be provided To prevent any typing error use the COPY button to save the ID to the clipboard It can be printed
169. nned clos relative to the data as shown The z est to the objective This situation is position shown top left in the image shown in REF TO FIG However since indicates the distance in um of the scan directions and data planes might coverslip to the first data plane have been reordered this match is not guaranteed Fortunately it is often easy to spot the flat side of the object where it adheres to the glass so the orientation can be verified Upright Microscope In an upright microscope and a z scan starting away from the coverslip the first plane is also likely to be physically the lowest plane In that case the imaging direction should be set to downwards and the coverslip position in the top part of the xz MIP projection However if the scan started close to the coverslip while storing these first planes first in the data set the MIP projection will show the data upside down Consequently the cov erslip position will be in the lower part of the MIP and the imaging direction is upward Slide Position When the specimen is mounted on the coverslip the distance from the object to the slide is probably in the range from 50 to100 um outside of the image In this case or in the case there is no slide select Far away in the top right selector Huygens Professional User Guide for version 4 1 153 r Edit Micr Coverslip Parameters When the specimen is close to or Imaging direction Z 0 20 um Zoom
170. ns Professional is optionally equipped with the PSF Distiller This wizard driven tool guides through the process of measuring a microscope s Point Spread Function PSF It guides you in selecting your bead images creating and saving the PSF for fur ther use in deconvolution runs The wizard is able to measure a PSF from one or more images of fluorescent beads each containing one or more beads It is also able to distill multi channel PSF s from information collected from multi wavelength beads or assem ble a multi channel PSF from single channel PSF s Measured PSF s improve deconvolution results and may also serve as a quality test for the microscopel The measured PSF acts as a calibration of the microscope in the sense of relating a physical known object with what the microscope actually measures Figure 5 1 shows an example of a theo retical PSF next to a measured one The PSF Distiller is able to measure a PSF from one or more 3D stacks each containing one or more fluorescent beads It can distill multi channel PSFS FIGURE 5 1 An xz cross section of i a theoretical PSF left versus a from information collected from multi wavelength measured PSF right for the beads or assemble a multi channel PSF from single same confocal setup channel PSF s The PSF distiller works by inspecting average images from small beads that are almost sub resolution in size so they contain much of the PSF information In order to meas
171. nt for the search strategy that the microscopic parameters of the image are correct in especially the sampling distance and the microscope type The following search strategies are available e Lowest value default The image is searched for a 3D region with the lowest average value The axial size of the region is about 0 3 um the lateral size is controlled by the radius parameter which is by default set to 0 5 um e In near object The neighborhood around the voxel with the highest value is searched for a planar region with the lowest average value The size of the region is controlled by the radius parameter e Widefield First the image is searched for a 3D region with the lowest values to ensure that the region with the least amount of blur contributions is found Subse quently the background is determined by searching this region for the planar region with radius r that has the lowest value Press the ESTIMATE button in the wizard to continue For checking the estimated value hover over a background area in the image the intensity values are displayed at the top The value can be adapted either by altering the value in the Absolute background field or in the Relative background field Setting the latter to 10 for example lowers the esti mated background by 1096 3 Learn more about histograms at http www svi nl ImageHistogram Huygens Professional User Guide for version 4 1 29 The Deconvolution Stage The l
172. nt to prepare series of tasks for the same image Just push the button and the copy of the selected line will be ready for mod ification For example it might be required to vary one deconvolution parameter to find the optimal value Running the Batch Job When the batch process is configured its configuration can be saved for future reference by selecting FILESSAVE in the menu 4 http www svi nl RestorationParameters 5 http www svi nl SetTheSignalToNoiseRatio 6 The user home directories are usually located in C NUsers on Windows 7 and Vista and in C NDocuments and Settings on Windows XP On Mac OS X they are usually in Users and on Linux in home 42 Huygens Professional User Guide for version 4 1 Menus Menus Add as many tasks as required single files of complete folders the Huygens Batch Pro cessor will run them all By pushing the start button 5 the Batch Processor will start and go over the task list The progress of the Batch Processor and the report for each individual task are shown on the tabs in the Processing overview area The status of each task in the task list changes accordingly to the progress of the process The restored images are saved in the selected destination directory as soon as they are ready along with the image history and an independent task description that can be loaded later in the Batch Processor to re execute it If the computations are very demanding for the syst
173. nuclei or small particles then the image may be stabilized using object tracking Objects are tracked over time and their average movement is used to stabilize the time series 84 Huygens Professional User Guide for version 4 1 Stabilization of 3D Time Series After the pre processing step the detection settings Detection settings step is shown Figure 15 2 In this screen the detec 7977 tion algorithm can be configured to filter on bright Filter on brightness only Ignore objects at ness only This is a little faster and usually sufficient F x andy boundaries of the sices i v top and bottom of the stack for tracking bright or dark spots in images with a high contrast between the spots and the background FIGURE 15 2 The Detection settings screen When the detection algorithm finds many false objects at the image borders this may happen for very small object sizes then the Object Stabilizer can be configured to ignore objects at x and y boundaries and at the top and bottom of the stack Next the user is asked to use the Select object tool and N Select background tool amp to mark a few objects and back ta i ground areas in the first time frame Figure 15 3 shows an example of selected objects green selection and back ground red selection It is important to make sure that the size of the object selections roughly matches the real size of the objects because this is a
174. nverting a Dataset Before deconvolving a 3D stack can be converted into a 3D time series using the convert3D24D command or vice versa or a 3D stack can be converted into a time series of 2D images using the convertZ2T command or vice versa Time Series time series is a sequence of images recorded along time at uniform time intervals Every recorded image is a time frame Huygens Professional is capable of deconvolving automatically of 2D time or 3D time data There are some tools that are intended only for time series as the widefield bleaching corrector or the z drift corrector Adapting the image In the DECONVOLUTION menu you can find a contrast inverter helpful for the processing of brightfield images See Brightfield Images on page 155 A Crop tool is also avail able but its use is recommended only after properly tuning the image parameters and will be explained in a later stage When the coverslip is on top it is recommended to flip the image using the mirror command This is specially important in case of a refractive index mismatch read more at Setting the Coverslip Position on page 152 l http www svi nl FileFormats 24 Huygens Professional User Guide for version 4 1 Verifying Microscopic Parameters In Huygens Professional you are not obligated to verify your microscopic parameters but it is recommended to check them either with the parameter wizard or the parameter editor Verifying
175. of voxels lower than the garbage level You can see the seed as a secondary threshold The first threshold segments the data and makes independent objects but then only objects with intensity that goes above the seed level remain while the rest are discarded At start up the threshold and seed levels are repre Hos ee ee sented by vertical blue and magenta lines respec Primary Send MIP _ tively on top of a histogram of the channel in the v5 ll pipe An alternative representation of these levels as sliders is available that also show their values as a I Use watershed segmentation Sigma i percentage of the channel maximum and allows you 20 tolink the seed with the threshold The garbage vol d ume control is also in the slider pane not in the his m 44 togram To switch between the histogram and the Garbage volume 1 sliders control panes you have to click on the small button 93 at the right of the pane title 2 http www svi nl SeedAndThreshold 124 Huygens Professional User Guide for version 4 1 Main window components The threshold ranges between the minimum and the maximum values in the channel intensities considering all the time frames Its percentage representation refers to the maximum value By default the seed is linked to the threshold value so when you vary the latter the seed absolute value also changes in such a way that its relative value remains constant In
176. ogo at the bottom right of the scene Adjust the size of the rendered image When the render size exceeds the display area then use the middle mouse button to drag and drop the rendered image Choose between EMISSION COLORS or GLOBAL PALETTE See Adjusting the Global Color Scheme on page 151 All scene settings i e both the render options and all parameters can be exported to a template file via FILESSAVE SCENE TEMPLATE The template files have the extension hgsv and can be applied to any image that is loaded in the SFP Renderer Keep in mind that the sampling sizes of the data affect the transparency The Huygens Movie Maker See The Movie Maker on page 73 allows to create easily sophisticated animations using the MIP SFP and Surface Renderer 64 Huygens Professional User Guide for version 4 1 Simple Animations Without the Movie Maker the SFP Renderer has the option to make simple animations of the image changing the view point in different frames Set the render parameters for the first frame and click the MOVIE tab SET gt FIRST SCENE Now adjust the viewpoint for the final frame and click SET gt LAST SCENE Set also the frame count frame rate and other general options such as colors and light properties Finally press the animate but ton H and select a directory to save the AVI movie to The exported AVI files use the MJPEG codec and can be loaded in most movie players including Windows Movie Pla
177. on the y axis To break tracks down into smaller pieces simply select the break tool 57 and click between the nodes that should be disconnected Multiple tracks can be selected using the CTRL key If the nodes of the selected tracks do not overlap in time then those tracks can be joined by pressing the connect button 4 Pressing NEXT will close the wizard and load the tracks in the Track Analyzer The Filter Tab The Track Analyzer can be used to analyze the tracks that have been generated by the Object Tracker The scene on the top left displays these tracks Tracks can still be edited 94 Huygens Professional User Guide for version 4 1 The Track Analyzer using the break tool 57 and connect tool 4 or the delete button The window at the bottom left shows the collection of tracks and the objects per track Clicking on the track or object number highlights the concerned track in the image Vice versa the track num ber is highlighted when clicking on a specific track in the image The filter tool Figure 16 6 can select a group of tracks based on common properties amp gt amp Filter Position speed Flow Select Break Boundary Connect Delete invert Number of tracks vs Corrected path length um Time 0 000 s Tine 0 m m A U o Fur va Oo uw a c z 300 200 Corrected path length um Threshold range Corrected path length um M Lower threshold 0 U
178. onal User Guide for version 4 1 Advanced Usage Render Parameters Advanced Usage Table 10 1 gives an overview of the different render parameters available in the MIP TABLE 10 1 MIP render parameters Option ANIMATION FRAME COUNT ANIMATION FRAME RATE RENDER SIZE RENDER QUALITY COLOR MODE BOUNDING BOX SCALE BAR SOFT THRESHOLD MODE TIME FRAME CENTER SCENE renderer Render Options Description Set the number of frames that will be rendered in a movie 180 frames with a frame rate of 24 fps result in a movie with a duration of 7 5 seconds Adjust the frame rate a rate of 24 frames per second is fine for smooth movies Adjust the size of the rendered image When the render size exceeds the display area then use the right mouse button to pick up and move the rendered image Set the default quality FAST or HIGH QUALITY This setting will be used for rendering animations Choose between GREY EMISSION COLORS GLOBAL PALETTE See Adjusting the Global Color Scheme on page 151 FALSE COLOR or CUSTOM COLOR Enable or disable the bounding box or adjust the line color Enable or disable the scale bar adjust the scale bar length and the line color Adjust the smoothness of the soft threshold See Thresh old on page 58 A selection of the time frame for time series Undo both the panning of the rendered image middle mouse button and of the scene canvas right mouse but ton
179. ong the length axis However because microscopic data even when it is deconvolved often shows orientation dependent imaging due to the lower axial resolu tion structures are often elongated in the axial direction Moreover in most cases the voxels themselves are much higher than they are wide causing all small objects to be elongated Clearly without correction the anisotropy in resolution would result in an overestima tion of the width To avoid this problem the rotation angles at which the caliper is held are divided in axial directions and lateral directions To measure in the axial directions several slices are taken out of the object and analyzed one by one Each of these slices is parallel to the caliper plane perpendicular to the length axis of the object In any slice there are directions more oriented towards the opti cal axis axial directions than others The largest axial width of each slice is obtained by holding the caliper in these directions and searching for the largest among them After all slices along the length axis have been examined the largest axial width of the object is reported as WiAx The lateral directions in the caliper plane are the directions closer to the xy plane The caliper measures now the width of each slice in directions near perpendicular to the optical axis Taking the largest figure among these lateral directions might again intro duce a bias due to elongation so now two values are compu
180. ot understand the watershed segmentation yet you can keep the option turned off The watershed segmentation builds watersheds at the local minima which will separate merged objects It can also be explained as a flooding algorithm where the inverse inten sityprofile is flooded with water At every position where water touches other water a watershed is build The difference between these two segmentation methods is visualized in Figure 17 5 The seed level has the same meaning for the watershed as it has for the simpel threshold segmentation method However the garbage level filtering uses a slightly different approach The garbage level will still remove the objects with a volume below the given value but if this concerns an object that was seperated from an other object by the watershed algo rithm a neighbour it will first be merged with this neighbour after which the volume of the new merged object is reviewed This merging will keep going as long as the merged object is below garbage volume and it has direct neighbours If the volume is it still below garbage level and it has no neighbours anymore it will be removed else the whole object is kept ordinary threshold watersheds low threshold Ajisuajul X FIGURE 17 5 Difference between the seed and threshold and watershed segmentation methods Huygens Professional User Guide for version 4 1 103 Interaction with the Objects The watershed segmen
181. other Slicers 46 Huygens Professional User Guide for version 4 1 CHAPTER 8 The Twin Slicer The Twin Slicer allows to synchronize views of two images measure distances plot line profiles etc In basic mode which is also available without a license image comparison is intuitive and easy while the advanced mode gives the user the freedom to rotate the cut ting plane to any arbitrary orientation link synchronize or unlink viewing parameters between the two images and more To launch the Huygens Twin Slicer select an image and select VISUALIZATION gt TWIN SLICER from the main menu To view another image in an existing slicer click the image name in the drop down menu above the left or right view port See Figure 8 1 f Huygens Twin Slicer basic mode File Linking View Plot Help original m Advanced mode Imaginal disc2 60 Value ch 0 208 ch 1 WO 7 22 px pm Zoom 7 1912x Le s Orientation Gamma Color Brightness xy Linear Greyscale 5 xz Compress Emission colors D yz Strong compression amp Global colors B 9 Widefield False colors Zoom to fit Strong widefield Time frame E Left mouse button draws a ruler Middle mouse button zooms in on bright spots Press c to center the slice FIGURE 8 1 The Twin Slicer in basic mode showing an original and deconvolved image side by side Huygens Professional User Guide for version 4 1 47 Usi
182. parameter Label Object label i i the active pipe radio buttons Comparison method is not hl Use for comparison Fixed value You have to select one of the two options fixed MONDE EET value or another parameter and enter the value or select the parameter you want to compare with Fixed value 1 Other parameter ININ Istneighbe Because the filter is based on the reported parame Filter Fiteralseies ters you may need to configure the reported parameters or select other statistics first in order to filter based on the desired property For interesting usages of this filter see for example the neighbors article in the SVI Wiki For time series another button shows to allow filtering all time frames Statistics Table 20 22 The statistics table is the place where all the objects parameters are reported after the analysis You can explore the table values in interesting ways directly on the Object Ana lyzer see below or export the contents for further analysis in other program When you move your mouse over the table rows the objects they refer to are highlighted on the rendering canvas When you point to a cell reporting a distance this distance is also plotted on the screen Label Chan Pipe Voxels CMassX C MassY CMassZ Frame 1N IN CMCM 1N CMCMx 1N CMCMy a WS Image object nalyzer test Image analyzed with HuPro 3 6 0p0 on 2010 06 03 m ft Conditions pipe 0 chan 0 thresh 366
183. parameter that is used to tune the detection filters Bag te pr ES mh FR Be La B G FIGURE 15 3 An Note that the selections are three dimensional i e spheres example of object instead of circles The x z and y z slicers can be used to view and background and modify the position of the selections along the optical selections z axis When the NEXT button is pressed the Object Stabilizer analyzes the selections and uses that information to detect objects in the first time frame The detected objects are marked by blue spheres as shown in Figure 15 4 When the detection fails then the selec tion tools can be used to select new objects and background mark detected objects as object 45 or as background 4s and delete selections When the selections are mod ified pressing NEXT will recompute the detection instead of proceeding to the next screen The histogram and sliders in the optimizing object detection screen See Figure 15 4 can be used to apply thresholds on the number of objects probability width and brightness Objects that fall outside of one of these thresholds will be ignored The probability is a statistic that reflects the certainty of the detection algorithm i e objects with a low prob ability are probably noise When the thresholds are modified pressing NEXT will recom pute the detection instead of proceeding to the next screen In the next screen the tracking parameters can be modified The d
184. parent image into n single channel images and then generate a PSF estimate the background and the signal to noise ratio for each of them Next you have to run the deconvolution algorithm channel by channel which ends you up with n restored one channel images Finally you have to join the separate images to a restored n channel image Although all tools are available in Huygens Professional it is a time consuming task Not to speak about multi channel time series Huygens Professional User Guide for version 4 1 21 22 Huygens Professional User Guide for version 4 1 The Processing Stages in the Wizard TT Deconvolution Wizard The Processin g The deconvolution wizard guides through the process of microscopic image deconvolu tion also referred to as restoration in several stages Stages in the Wizard Each stage is composed of one or more tasks While proceeding each stage is briefly described in the bottom left Help window tab The stage progress is indicated in the Wiz ard status pane below the Help tab Additional information can be found in the online help HELP gt ONLINE HELP as well as by clicking on the highlighted help questions The following steps and stages are to be followed e Loading an image e Verifying microscopic parameters e Start Stage here the possibility exists to load a deconvolution template and a mea sured PSF e Preprocessing Stage this stage goes through all preprocessing steps
185. pically below 100 the difference between successive iterations becomes insignificant and progress grinds to a halt Therefore it is a good idea to monitor progress with a quality measure and to stop iterations when the change in quality drops below a threshold At a high setting of this quality threshold e g 0 1 the quality difference between subsequent iterations may drop below the threshold before the indicated maximum number of iterations has been completed The smaller the threshold the larger the number of iterations which are completed and the higher the quality of restoration Still the extra quality gain becomes very small at higher iteration counts 4 Iteration mode In optimized mode highly recommended the iteration steps are bigger than in classical mode The advantage of classical mode is that the direction of its smaller steps is sure to be in the right direction this is not always the case in opti mized mode Fortunately the algorithm detects if the optimized mode hits upon a sub optimal result If so it switches back to the classical mode to search for the opti mum 5 Bleaching correction If this option is set to if possible then the data is inspected for bleaching 3D stacks and time series of widefield images will always be corrected Confocal images can only be corrected if they are part of a time series and when the bleaching over time shows exponential behavior 6 Bricklayout When this option is set to auto the
186. pper threshold Inf Slice orientation xy XZ yz Twist 9 6 Reset all thresholds Tit S Zoom Rotate Colors Contrast Select tracks FIGURE 16 6 The filter tab in the Track Analyzer These properties can be the number of detected objects nodes per track start time duration the path length and the average speed The upper and lower threshold sliders can be used to define a range within those properties When the SELECT TRACKS button is pressed all objects within the threshold ranges are selected The INVERT button inverts the selection to the other tracks An important thing to note is that all thresholds are applied when SELECT TRACKS is pressed Press the RESET ALL THRESHOLDS button to disable all thresholds To remove tracks with less than 4 objects for example select the detected object per track property tick the upper threshold box drag the upper threshold slider to a value just above 4 and click SELECT TRACKS Now those tracks are selected These tracks can be removed by pressing the DELETE button The Position Tab This tab provides information on the position and displacement of the tracked objects The histogram at the top as shown in Figure 16 7 gives the number of objects versus the distance to their track origins for a specific duration Example if the time frame slider is set on 3 frames and the height of the bar at a distance of 1 2 um is 9 then there are 9 objects that travelled a net distance of 1 2
187. pting the theoretical PSF to the sample depth In case the image suffers from severe spherical aberration it might be better to use a the oretical PSF with this depth dependent correction than an experimental one Improve the Deconvolution Parameters Some deconvolution parameters for example the SNR and the background level can be fine tuned to get the best out of the restoration process See the SVI Wiki for detailed steps in configuring the restoration process 8 http www svi nl PointSpreadFunction 9 http www svi nl SphericalAberration 10 http www svi nl DeconvolutionProcedure 148 Huygens Professional User Guide for version 4 1 The Point Spread Function CHAPTER 23 The Point Spread Function Quality Factor Appendix One of the basic concepts in image deconvolution is the point spread function PSF The PSF of the microscope is the image which results from imaging a point object in the microscope Because of wave diffraction a point object is imaged spread out into a fuzzy spot the point spread function In fluorescence imaging the PSF completely deter mines the image formation In other words all microscopic imaging properties are packed into this 3D function In Huygens Professional a PSF can be obtained in two different Ways 1 Generating a theoretical PSF When a measured PSF is not available Huygens Profes sional automatically uses a theoretical PSF The PSF is computed from t
188. r in creating the key frames that define the main scenes and the smooth transitions between them Interactive manipulation of the scenes is pos sible using the interfaces of the renderers or by dragging nodes in the Timeline The movies can be exported to AVI files or to TIFF series that can be combined with other software Movie projects can be saved for later editing or for usage with other 3D datasets An introductory tutorial can be found in the HELP menu at the top right This interactive tutorial guides the user step by step through the process of creating a simple movie Figure 13 1 shows the Movie Maker s user interface The numbered areas are 1 The storyboard this filmstrip shows the main elements of the movie which are the keyframes and the transitions between them 2 The preview area this mini movie player quickly creates a low resolution version of the movie 3 The timeline this interactive plot shows how render parameters change over time Use the menu button below this timeline to the render parameter to be visualized Green nodes representing render parameters at each keyframe can be dragged verti cally Huygens Professional User Guide for version 4 1 73 Creating and Adjusting Keyframes amp Movie Maker New project e959 2ch File Edit Tools Presets D uu B New Open Save Storyboard Preview 1 4 Frame rate fps 12 Timeline 0 5 Frame number FIGURE 13 1 An overview of
189. re information can be found on the SVI Wiki Throughout Huygens Professional pinhole sizes of confocal systems are specified as the backprojected radius in nm Backprojected means the size of the pinhole as it appears in the specimen plane i e the physical pinhole radius r divided by the total magnification of the detection system This total magnification is the product of the variable objective magnification m multiplied by a fixed internal magnification m 5 http www svi nl NyquistRate 140 Huygens Professional User Guide for version 4 1 Computing the Backprojected Pinhole Radius and Distance r phy gt EQ 2 obj sys The SVI Wiki has a calculator to automatically compute the backprojected pinhole radius for specific microscope models Note that the Bio Rad MRC500 600 and 1024 microscopes have a very high magnification in the detection system See Check ing the Bio Rad System Magnification on page 142 The equations that can be found in the next pages are intended to orientate the user in finding out the backprojected value for different types of microscopes but the idea is always the same given a diameter d of the real pinhole we might need to multiply it by a factor for unit conversion to obtain the radius in nm and divide the result by some other factor that takes account of the magnification of the microscope These include both the objective and the intrinsic system magnification
190. rent pipes overlap in space or they apparently do from the current point of view and when you click somewhere on the screen you are actually selecting both of them if the pipe interaction mode is PS or ALL See Active Pipe Mode 6 on page 120 In that case two rows are added to the table and this second letter lets you know which of the two was in front of the other from the current point of view By moving the mouse over the table rows the corresponding object is highlighted on the canvas See Figure 19 1 item 11 Objects in the Primary pipe are highlighted in red and objects in the Secondary pipe in green A good way to find an object in a very long table is by clicking on it on the rendering canvas while the Analyze object mode is active the table will be shifted to show its corre sponding row and it will be highlighted When you move over the table cells the current value is shown on the tooltip bar at the bottom See Figure 19 1 item 23 This combined with the selection of rows see below or columns See Figure 19 1 item 20 allows a quick exploration of the table and the comparison of different parameter values In the following example when the cursor is moved over one cell that contains the dis tance to the first neighbor the following is reported in the tooltip 38 8 1NP CMCM 4 2358 um The first part is the label of the object in the current row label 38 on the Secondary pipe Then the parameter tag INP CMCM refer
191. rizontally on its zoom 1 86 bottom to control the point of view of the scene e Zoom the number is just indicative 1 meaning that the whole dataset is shown in the canvas e Tilt the angle of rotation in degrees around the canvas x axis e Twist the angle of rotation in degrees around the image z axis By clicking on the labels you can enter numerical values manually to quickly switch to the desired scene Hide Pane Button 13 This button at the top right of the window collapses the control pane to make more room for the rendered scene Once you have defined the segmentation conditions for all pipes you don t need to interact with those controls anymore but with the objects so you can hide the pane to focus on the scene Experiment Presets 14 This button opens a preset selection dialog that allows to select an experiment preset a Select statistics Correlation inside channels collection of parameters that make sense to be reported together in the context of certain experimental needs See Figure 17 8 on page 107 Users are very much welcome to send their own suggestions to implement new presets We will gather all the feedback in different wiki articles that will explain what parameters are best for certain experiments and how to interpret them Please feel free to tell us about your experiences In this dialog you can see three columns On its left column a series of different experi mental needs are li
192. rom left to right and from top to bottom e S Analyze object lets you click on different defined objects and obtain the local statistics e Select area lets you define a 2D region on the current view of the image so you can do different things with it analyze or dis card objects below it anchor them as references or interactively define regions of interest ROI We will see what all this is useful for e 98 Discard object lets you discard irrelevant objects one by one Just select this mode and click on the disturbing objects e is Select object as anchor lets you select and deselect objects to be anchors for example to act as references to measure dis tances from other objects when asking for local statistics When you set an object as a reference anchor it will light up and change color on the screen to indicate its new status It is possible to select a group of anchor objects and you can operate with them through the Anchors menu e ba Rotate scene interacts with the full image to rotate it in the space by dragging the mouse pointer on the rendering view That can also be achieved by moving the Tilt and Twist sliders along the rendering e Pan scene interacts with the full image to move it in space laterally This means that you can pan the scene in the 2D plane of your screen not along the third dimen sion along your line of sight 104 Huygens Professional User Guide for version 4 1 Ren
193. rs Parameter Color mode Background color Compute shadow Table Table distance Table reflection Table size Table color First scene Last scene Set number of movie frames Set movie frame rate Light direction Light intensity Render size Render mode Render Options Description Adjust the color of the selected channel Adjust the color of the scene background Choose whether or not the objects shadow should be included in the scene Choose whether or not the table underneath the object should be included in the scene Adjust the distance between the object and the table Adjust the degree of reflection of the table Adjust the size of the underlying table Adjust the color of the table Set the viewpoint of the first scene of the movie Set the viewpoint of the last scene of the movie Set the number of frames that will be included in the movie Set the number of frames per second of the movie Set the position of the light source Set the intensity of the light source Set the size in pixels of the SFP scene Set whether the scene must be rendered in fast mode in high qual ity mode rendered in a movie or not rendered Table 11 2 gives an overview of the different render options that are available through the TABLE 11 2 Render options for the SFP Renderer Option SHOW SVI LOGO VIRTUAL RENDER SIZE COLOR MODE OPTIONS menu Templates Description Show or hide the SVI l
194. s using the middle button at any time You will learn this with practice if you need it but you don t have to care about it right now When a mouse mode is active and the cursor is inside the canvas the tooltip in the status bar See Figure 19 1 item 23 tells you what each mouse button can do Selection Interactive Operations 3 The rest of the buttons in the toolbar are not mouse modes they do not set new behav iors for your mouse when interacting with the objects view but execute operations on previously defined conditions for example deleting the objects inside a defined ROI They are all disabled until these conditions have been set in the example until you define a ROI to operate with After having drawn a 2D selection See The Selected Area 9 on page 121 in the Select area mouse mode you can click on e Keep all objects under the selected area discarding anything else o S Discard all objects under the selected area Set as anchors all objects under the selected area The way the drawn 2D area considers objects in its limit can be controlled with an option in the top Options menu the area can consider only objects fully under the selection or also objects partially outside it relaxed selection mode In any case the objects not affected by the selection are rendered with dimmer intensity to clearly indicate what objects are selected When you have selected certain objects as anchors you can further operat
195. s 3072 matching names were found Would you like to select a file series or justthe single file opened that appears to be part of a file test image 100 z000 ch00 tif E series Huygens Professional shows the File File pattern test image t Time Frame z Slice ch Channel tif XE oo Series Tool dialog Figure 23 1 This tool To ag im 1 jm enables the user to select a subset of a file bpm 1 m o a 1 a series and select a dimension for each the ne coun Message indices in the file name so that each image ew toad selection Leadsinglefile is assigned to the correct z plane time frame and channel FIGURE 23 1 The Huygens File Series Tool automatically scans a directory for a file series The file pattern is shown in the first row in the dialog The counters in the file name are replaced by menu buttons for selecting the appropriate dimension for each counter The options are e Slice The range of this counter becomes the z dimension e Time Frame The range of this counter becomes the time dimension e Channel The range of this counter becomes the channel dimension e Ignore the variable is ignored This is useful to omit e g the value of time stamps e The value of the counter in the selected file the value of this counter has to match the value in the selected file 150 Huygens Professional User Guide for version 4 1 Adjusting the Global Color Scheme Adjusting th
196. s This not only gives information on the speed a scalar value but also on the direction of the velocity as shown in Figure 16 9 y velocity um s vs x velocity um s y velocity um s vs x velocity um s p j Save as Export data XY XZ YZ Save as Export data XY XZ YZ FIGURE 16 9 Left An xy velocity scatter plot showing a drift in the positive y direction Right An xy velocity scatter plot of objects moving with a constant speed of about 0 35 um s in random directions The Flow Tab This tab can be used to gain insight into the amount of objects that cross a certain boundary over time Boundary surfaces can be defined as a 2D poly line in the xy slice image These lines are extruded to planes in the axial direction To start drawing one click the DRAW BOUNDARY button and then click on the first point of the bound ary A single click adds a new line segment and a double click finishes the poly line When a boundary has been defined then the plot in the Flow tab shows the number of objects that cross this boundary at a certain time frame Figure 16 10 2 E pg YT Filter Position Flow Fe SD CL lei _ Select Bresk Boundary Undo Redo Connect Delete Invert EEUU cks pur Frame number Save as Export data e Draw boundary FIGURE 16 10 The flow histogram shows that objects are crossing the boundary bet
197. s scheme CUSTOM COLORS Use the same custom color scheme for both slicers BRIGHTNESS Synchronize the brightness GAMMA Synchronize the gamma setting Some useful ways of linking the controls are e Comparison mode to configure the Huygens Twin Slicer to compare two images e g original and deconvolved it is best to link all orientation parameters i e slice posi tion time frame zoom level panning and rotation This ensures that there is always looked at the same piece of data e Orthogonal mode to view a part of an image in two orthogonal directions for instance axial xy and frontal xz do the following Select the same image for both the left and right slicer Tick ADVANCED LINKING and link the slice position time frame zoom level and panning Unlink the rotation Select the Rotate tab at the bottom of the window and select the xz and xy orienta tion Now it is possible to zoom pan and slice while the centers of the left and right slice are always aligned Note that when the cutting planes are not the same the projected mouse pointer will show a distance in um besides it If this number is positive it means that real pointer is more towards the observer in front of the screen e Overview mode An easy overview mode can be configured as follows Select the same image for both the left and right slicer Tick ADVANCED LINKING and link the slice position time frame and rotation Unlink the zoom l
198. s Distill PSF Theor PSF Image Stats Estimate Bg Slicer Operations Parameters m Generate a microscopic PSF Zoom 2 83 Output Destination psf M D Dimensions Automatic M Min XY slices Manual 100 Z position micron 0 0 Tilt 00 deg 0 Q XY XZ i YZ Twist 000 dea u Voxel data Linking A J Position V Orient V Center Listen Time Zoom Other Slicing Slice center at voxel coordinate 0064 0064 038 lt gt I gt lt gt View options J Link channels Stretching Global Gamma Linear Tel Cmd faba128 genpsf gt psf Color Contrast Time Run Command Ready Canvas size 440 x 400 Interactive FIGURE 3 7 The operations window where Theoretical PSF is selected Various operations can be selected from the menu bar or the quick access buttons The Operations Window In the former steps all operations were performed without any operation parameters i e variables or options applicable to a particular function For example if one wants to know the image statistics no extra information is needed but the name of the image All these types of operations are accessible from the main window However most opera tions like adding some constant value to an image need extra input data This can be done in the operations window shown in Figure 3 7 This window can be accessed via DECONVOLUTION2 OPERATIONS WINDOW in the main menu In the operations window an operation may be selected from the
199. s per unit area volume 3D or area 2D It is a microscopic parameter that describes the conditions of the image acqui sition established by the way the microscope is configured usually by the zoom factor The ideal sampling density depends on the system optics and is determined by the Nyquist rate It is recommended to sample as close to the Nyquist rate as possible Note that the actual sampling distances from the acquisition must be used in the deconvolu tion The SVI Wiki has an on line tool that computes the Nyquist rate for any widefield con focal spinning disc and 4 Pi microscope Sampling according to the Nyquist rate makes sure that all information generated by the optics of the microscope is captured in digital form It can be shown that if the sampling distance is smaller than the so called critical sampling distance no new information about the object is captured Apart from practical problems like bleaching acquisition time and data size there is no objection at all against using a smaller sampling distance than the critical distance on the contrary 3 http www svi nl BlackLevel 4 http www svi nl NyquistCalculator Huygens Professional User Guide for version 4 1 139 Computing the Backprojected Pinhole Radius and Distance Figure 21 3 shows the dependency of this critical sampling distance on the numerical Lateral WF Axial WF Axial Confocal Lateral Confocal 1000 100 Sampling Distanc
200. s to the CM to CM distance 4 http www svi nl ObjectAnalyzerROI 128 Huygens Professional User Guide for version 4 1 Main window components between this object and the nearest object in the other pipe Then comes the distance itself 4 23 microns 38 1 5 14 10279 92715 13 935 o 2636 93 2 1 7649 81495 5 2257 8 1499 3 1 S 7 14458 16371 14 0 131635 8 30245 27858 35824 2 0866 M 0 103053 8 42350 45 366 4955 2 0866 39 1 s 1 32902 78999 15 019806 3 1273 15083 10844 17127 40 1 S 40 77 396 94 902 15224 0 8238 28 2 22204 17 244 4 0386 9 4394 Ready rendered Canvas size 878 x 577 Dragging on 38 S 1NP CMCM 4 2358 um That tooltip region can show not only the information of the cell pointed by the mouse which would not be really useful but also other values that can be set as reference Try this while keeping the Ct r1 key pressed on the keyboard select a couple of rows by clicking on the row number at the very left of the table The selected rows will turn green If you now hover the mouse over a cell on any other row you will get the value not only of that cell but also those in the corresponding cells of the selected objects rows This is a quick way to compare results for different objects that can be distant in the table m 33 1 9 13923 13045 12681 0 161024 98 22042 33217 256158 076713 45 4 1 2 59195 72512 14466 0 506534 9 082808 11211 43561 1 17
201. sen to run multiple jobs at the same time However it is not necessarily true that concurrent execution of tasks is faster than sequential execution because in the former case mul tiple tasks will compete for the available memory deconvolution demands a lot of memor y If the available memory is insufficient a slowdown will occur Huygens Professional User Guide for version 4 1 43 44 Huygens Professional User Guide for version 4 1 Color CHAPTER 7 Color The Slicer The Slicer see Figure 3 2 on page 13 allows you to quickly inspect an image but also to compare multiple images e g a deconvolution result with the original or view the same image in multiple orientations simultaneously To start the Slicer select VISUALIZATIONSSLICER from the menu in the main window or double click the image s thumbnail You can open as many Slicers as you like on the same image or on different images and multiple Slicers can be linked and listen to other Slicer windows The Slicer enables the user to show a single 2D plane extracted from a 3D volume There are controls to select any plane orientation in space zoom and scroll through the avail able planes For time series a separate control is available to scroll through the available time frames Pixel intensity values for the cursor position on the image are displayed in the Voxel data panel You can move the image by clicking the middle mouse button and keep
202. sets 14 on page 123 HISTORY e Undo the last operation or Redo it again e Reload original data after cropping the image or discarding objects e View the whole operations history up to the current point e Load and save analysis templates so that the current view and parameters can be stored and retrieved or a whole operations history re executed with other data This is also useful to store your analysis steps and reproduce them e Setanalyzer as in any other open instance of the tool ROI operations to define a region of interest in complex ways and to modify and save the currently defined ROI 116 Huygens Professional User Guide for version 4 1 Main window components Set Set to extruded selected area uses the interactively defined 2D area to set the ROI to the volume below it Set using MIP threshold use the threshold and data channel of the MIP pipe to set a 3D ROI Make coincide with objects use the currently segmented objects to define a 3D ROI Objects from the Primary Secondary or both pipes can be used depending on the active pipes and the pipe mode See Active Pipe Mode 6 on page 120 Make coincide with anchors use the currently selected anchors to define a 3D ROI This may leave holes in the interior of the ROI if the anchor is not a solid object Make coincide with intersection this is interesting to do object analysis with colo calizing volumes only When you have two pipes active and som
203. sing the Orthogonal Slicer Maximum intensity projections MIP of multi channel images in any orientation Volume rendering using the SFP renderer The Simulated Fluorescence Process SFP algorithm computes high quality 3D images based on the physics of light Iso surface rendering Export of sophisticated 3D animations combine high quality MIP SFP and surface renderings use predefined animation presets animate the viewpoint zoom and any other visualization parameter uselinear or smooth interpolation in transitions export to AVI and Tiff series Huygens Professional User Guide for version 4 1 Huygens Professional User Guide for version 4 1 Microsoft Windows CHAPTER 2 Microsoft Windows Mac OS X Linux Debian Linux RPM Installation Huygens Professional can be downloaded from the SVI website Double click on the Huygens installer executable e g huygens 410p1 exe Double click its icon to start the installation During installation the directory C Program filesNSVIN will be created by default After completion the four Huygens icons appear on the desktop Double clicking on the Huygens Professional icon starts the pro gram Double click the package file for instance huygens 4 1 0 p1 Leopard 1386 pkg tar gz The archive manager expands it to a pkg file which will be placed in the same directory Double click this file and follow the installation wizard Debian
204. sted When you click on each of them a different list of parameters is listed in the middle column and a description is shown on the right column Even more when you hover with your mouse over the listed parameters you get a tooltip text explaining each parameter with more detail By selecting a particular preset all the listed parameters apart from the basic ones will be reported and calculated Moreover all the listed parameters will also be available for filtering the objects See Filtering Objects on page 125 The check boxes allow a few of these parameters to be also reported on the screen See Figure 19 1 item 8 for the cur rent object The magnitude of the distance that is plotted on the screen is followed by a triple dash Only one distance can be plotted at a time by clicking on an object but many can be reported on the table and explored interactively there by simply moving the cursor over the cells Only one preset can be selected at a time and all its parameters will be reported Advanced and more flexible configuration of the parameters is always possible through OPTIONS CONFIGURE STATISTICS REPORT in the menu In this configuration dialog users can also store any set of reported parameters as a new preset Huygens Professional User Guide for version 4 1 123 Time frame selector 15 Frame selection When time series are loaded in the OA this slider Frame 0 1 controls which time frame is take for visualiza
205. sualization of the chromatic shifts A view of the Chromatic Shift Corrector at start up with a loaded two channel bead image is shown in Figure 14 1 Huygens Professional User Guide for version 4 1 79 Estimation of the chromatic shifts Visualization of the chromatic shifts Huygens Chromatic Shift Corrector File View Plot AJ D l SEE amp Estimation method Correlation cShift 2010 12 14 Chromatic shift templates CM alignment Reference channel b Shift vectors microns 4 Edit vector e Channel 1 Help Start by choosing an estimation method and a reference channel then click on the Estimate shifts button below The estimated chromatic shift vectors will be reported for each channel jon the Shift vectors table A plot will be shown to compare the reference channel with the other channels Brightness Brightness per channel 0 v Estimate shifts Gamma Linear Zoom FIGURE 14 1 The Chromatic Shift Corrector at start up prior to estimating the chromatic shifts For the automatic estimation of the chromatic shifts the following two methods can be chosen e Cross correlation This can be considered an all round method The software searches for the best alignment across channels by maximizing the overlap e Center of mass alignment This method works best if the image contains a single object The object should not touch the image borders and the contras
206. sult 78 Huygens Professional User Guide for version 4 1 Starting the Chromatic Shift Corrector CHAPTER 14 Starting the Chromatic Shift Corrector The Chromatic Shift Corrector The Chromatic Shift Corrector is a post deconvolution tool that can estimate and cor rect for chromatic shifts removing the existing misalignments across different channels The result of this correction is a channel aligned image free of chromatic shifts The support for templates included in this tool allows to apply the chromatic shift cor rection of one image to other images This is particularly interesting when the estimation carried out on a beads image is suitable for other sets of images The Chromatic Shift Corrector shows its Help content dynamically to guide the user through the process of estimating and correcting for chromatic shifts e Launch Huygens Essential or Huygens Professional Load a multichannel image to be corrected for chromatic shifts e Select the image thumbnail and in the top menu go to DECONVOLUTION gt CHRO MATIC SHIFT CORRECTOR The Chromatic Shift Corrector will open and show the image on an orthogonal slicer where the existing chromatic shifts can be seen in a 3D view Below the orthogonal slicer a Z slicer a time slicer and other visualization tools such as contrast colour scheme channel selection and zoom tools can be found These tools are useful to enhance the view of the image for a better vi
207. surements on the SVI Wiki If your license includes the PSF Distiller option select DECONVOLUTION gt PSF DISTILLER to start the Distiller In the opening window Figure 5 2 different panes show the input beads field the Help field the report field the wizard fields and the PSF and Accus field F Q Huygens PSF distiller e m sg Help File Input beads Help Reports PSFs amp Accus Welcome to the Huygens PSF distiller wizard Microscopic parameters loaded This tool allows you to derive a PSF from images of fluorescent beads Starting stage Beads E See also How should beads be recorded AEE See also What is a PSF Wizard start stage Welcome to the PSF distiller wizard Distiller status Press Nextto start the wizard and to enter the stage x in which the bead image parameters can be Total beads averaged 0 inspected Per image averaged beads 0 Restart Ready Dragging on FIGURE 5 2 The PSF Distiller window Different panes show the input beads field the Help field the report field the wizard fields and the PSF and Accus field e The Input beads pane Shows the selected file with the bead images for the PSF distiller process You can import and use multiple files with beads for the distillation of a PSF With the cursor you can hover over the thumbnail and with a right mouse click you can roll down a menu to open the image in one of the slicers The edit parameters function is
208. t image Cell nucleus FISH stained recorded at the Nuclear organization Group SILS University of Amsterdam head Prof Roel van Driel under the 3D Genome research project The image in Figure 17 1 is a MIP projection of the test 3D dataset Notice that there are a few bright objects in the red channel against a more or less homogeneous background it is actually a quite flat cell nucleus and lots of scattered objects of different sizes and intensities in the green channel The red channel is in general very dim with the excep tion of the inner bright objects and some increase in intensity in its periphery making something like a border When the image is opened in the OA this is what we first see what is shown in Figure 17 2 The bright objects in the red channel are recognizable now from a top view The image was automatically rendered with some default settings The intensity range was explored to set a convenient threshold to segment the objects in the first red channel that are shown as iso surfaces Every independent object gets a different color ranging from red to green How were these objects separated from the background isolated from the rest of the intensities in the image so that they could be represented on the rendering as indepen dent entities They are said to be segmented l http www svi nl ObjectAnalyzerTutorial 100 Huygens Professional User Guide for version 4 1 Segmenting the Objects S
209. t Spread Function PSF cece eee cece eee eee 17 Step 5 Estimate the Average Background Value cece ee eee eee eee 18 Step 6 Estimate the Signal to Noise Ratio SNR ce eee eee eee eee eee 18 Step 7 Perf rm Deconvolution RUNG sev vooeocuveserv ERE E OUS PEE ewe 19 Step 8ssaviriP your IMAGE 25s eee es RR CREE Ged E D ER Ed VNDC EE ER 21 Deconvolution Wizard scececcccccccccececececes 23 The Processing Stages in the Wizard oes a eto en b ea eio eoi seman 23 Huygens Professional User Guide for version 4 1 Loading ae KA 24 Verifying Mictoscopic Parameters vas veo Vert PE eb tue waded NES 25 Usine d Measured PSP aae gara ERA CURES REPRE E QUE RI OE STA TA ARS 27 The Intelligent Cropper a oae vt S eet Pe Rab A eda due 27 Themsen vrede PE 28 Estimating the Average Backeround is arrest 29 The Deconvolution SJ se 30 Finishing or Restarting a Deconvolution Run eee 31 Milti hannel Images aus p er deron bor bare ec e edes ale sr utr 31 Z d tt Correcting for Die SS qoem voee eR enden 32 Savin the Ressurs 32 CHAPTER 5 The PSF Distiller e 0o 0 090 0 0 0 09 09 09 09 09 09 09 09 0 0 09 09 09 09 09 0909 09 09 09 09 09 co o 0 33 beads suited for PSP Distillation zer ep CERES sd 33 The PSF Distiller Window 3 54449 vea x te P WIR CORR AER RA arae DS 34 The Processing Stages in the Wizards bac seat ne ie aii eee 35 AVERSA iuo ace echt Mee TS 36 Dilla 37 Bias ER ERE SUE vvs 37 CHAPTER 6
210. t between object and background should be high The chromatic shifts will be quantified by vectors indicating how much a channel is shifted with respect to a given reference channel The channel to act as reference no shift can be selected by the user via the Reference Channel selection box Because this channel will have no chromatic shift its shift will not be reported If an estimation method and a reference channel have been selected the chromatic shifts will be estimated and reported upon clicking on the ESTIMATE SHIFTS button The estimated chromatic shifts will be reported on the shift vectors table The shifts of each channel but the reference will be listed The length unit of the shift vectors is set to micrometers The user can select any channel but the reference for editing This can be done by using the EDIT CHANNEL selection box under the vector table A vector will be drawn on the orthogonal slicer showing the estimated shift vector of the edited channel so that the estimated shift can be easily assessed At the same time a plot 80 Huygens Professional User Guide for version 4 1 Editing the chromatic shift vectors Editing the chromatic shift vectors will be displayed showing the intensity profiles of the edited channel and the reference channel along the direction of the estimated shift vector The plot also shows the intensity profile of the edited channel as if it were corrected with the ex
211. tation light usually a laser line The wavelength in nm of the light emitted by the sub ject The number of photons used in multi photon micros copy The width of the beam relative to the aperture The default for this value is 2 meaning that the aperture has a diameter of 20 where c is the standard deviation of the Gaussian distribution in the beam Using the parameter wizard or the parameter editor one can modify the parameters that are explained in Table 4 1 If values are displayed in a red background they are highly suspicious An orange back ground indicates a non optimal situation See Figure 4 1 Oversampling is also indi cated with a cyan background that becomes violet when it is very severe 25 Huygens Professional User Guide for version 4 1 r Parameter wizard Help File Help Fields with an orange background indicate an seriously undersampling condition sure to be review these what can be considered as optimal This is not a problem but it may may lead to high memory usage Purple fields indicate extremely dense sampling Again not a problem but it may lead to unnecessary high memory usage See also All about microscopic parameters Wizard status Obtained initial parameters from image golgi4subMini The following entry needs attention Z dimension undersampled please check value View and check geometrical parameters Sampling density undersampling condition red fi
212. tation method uses an extra input namely the sigma setting This sigma setting is used to smooth the intensities by a gaussian filter before doing the watershed segmentation to avoid oversegmentation Especially with very noise images local minima occur quite often resulting in a numberous small objects The Object Ana lyzer can handle at most 32768 2293 objects When you want to use the watershed segmentation it is advisable to first use the simple seed and threshold segmentation When the threshold is set at the correct position you can turn on the watershed Due to its complexity the watershed segmentation may take some time especially in comparision with the ordinary seed and threshold segementa tion Mode d e d ae Objects Apply to SEE On the left of the window you can find a column of buttons Many of the buttons are deactivated at this moment they will be activated when it makes sense later But almost all in the first group are always available They control the Mouse mode i e what the mouse does when you left click on the image When you hover with the mouse over these buttons you get a tip on what they are for If they have a keyboard shortcut to activate them it is also shown here For example the first mouse mode is intended to Analyze objects and you can always activate it from within the rendering view by pressing the keyboard key 1 These are the currently available mouse modes f
213. ted on the canvas because that is the only one selected on the preset selection dialog but all the interesting information is now included in the table too 108 Huygens Professional User Guide for version 4 1 Object Statistics Other Measuring Parameters There are two basic parameter presets this one we have used here to explore the nearest neighbors and another one to retrieve morphological parameters about objects called SMALL PARTICLES GEOMETRY This other parameter preset can report object information like length width aspect ratio and sphericity Details on how these parameters are defined can be found on page 111 The presets are organized in basic and advanced modes The basic modes refer to presets on which you don t have to do much before starting measuring you click on the objects and you get the desired information The advanced modes require that you define some conditions first to define a ROI for example They are not intrinsically more complex but they demand from the user to know how to set these reference conditions first For more details about any parameter preset just click the Help button you can find at the lower right part of the preset selection dialog and follow the on screen tooltips during the interaction with the module Exploring the Table Let s try another thing click on the ANALYZE ALL button 6 at the top right of the table so the data of all objects are gathered When the computations
214. ted per slice the largest and the smallest width along the lateral directions After exploring all slices some global relevant figures are reported The largest lateral width obtained while sliding the caliper plane along the length axis is reported as WiLat The largest of the smallest widths is reported as WiLatC In case of small objects and moderate to high ratios between the axial and lateral resolution this last value is likely to suffer least from the orientation dependent imaging The waist the smallest of the smallest widths in the lateral directions is reported as WaistLat What about objects with a vertical length axis In that case the caliper plane perpendic ular to the length axis will be horizontal parallel to the xy plane In a horizontal caliper plane all directions in it are perpendicular to the vertical z axis As a result there is no most axial direction in that plane The software then orients the lateral width towards the x axis and the axial direction towards the y axis The sphericity is reported in two ways in the Object Analyzer 1 The axial sphericity is defined in general as the ratio of the volume of an ellipsoid with axes length L width W and thickness T to the volume of a sphere circumscribed around the segmented object defined by its length 112 Huygens Professional User Guide for version 4 1 Aspect Ratio Because it is based on three axes it gives an idea of the 3D aspect ratio of t
215. tely The chromatic shift estimation of a four channel image is shown in Figure 14 3 The channels can be edited one by one and their shifts modified with the EDIT CHANNEL tool Huygens Chromatic Shift Corrector File View Plot Estimation method Chromatic shift templates Correlation Shift 2010 12 14 CM alignment Reference channel D D Shift vectors microns 0 57 0 34 0 0 0 05 0 0 0 03 Edit vector e N Channel 1 Help The plot shows a profile of a specific channel with the shift vectors from the table dashed line applied Prior to correcting the image the shift vectors can be modified for each ichannel with the Edit vector tool Click on the Correct image button below to correct the image for chromatic shift as defined by the shift vectors in the table A new corrected image will be created Brightness Brightness per channel 0 v Correct image Gamma Linear Contrast FIGURE 14 3 Using the Edit Channel tool of the Chromatic Shift Corrector The estimated vector has been lengthened and the dashed line plot is consequently slightly moved to the left Alternatively any customized shifts can be typed in the vector table for each channel Upon clicking on the CORRECT IMAGE button the image will be corrected with the chro matic shifts listed in the vector table A new corrected image will be created The estimated shift vectors can be saved to a template by using the template too
216. the Storyboard The Huygens Movie Maker Storyboard allows the user to mix key A EEMEMEMEMSMSMMMESNEUNNUMNUNUULULULN LL LB LBL BL frames from different render ers but transitions can only be Frame 0 Frame 12 Frame 13 D Frame 25 made between keyframes from 7 HUM the same renderer as shown in Figure 13 2 UR RERBA RR RCRCS RCRUM BR RC FIGURE 13 2 The Movie Maker s storyboard showing two surface renderer keyframes followed by two MIP renderer keyframes Editing Keyframes To edit an existing keyframe double click it or select the frame and choose EDIT gt EDIT KEYFRAME from the menu This will load the keyframe s settings in the corresponding renderer The renderer s con trols can now be used to adjust the 3D scene To submit the changes to the Movie Maker press the add keyframe button again Because the original keyframe is still selected the Movie Maker will ask if the original frame should be replaced Usi ng the Rearranging Keyframes S toryboard The storyboard allows the user to copy delete and rearrange keyframes The cut 4 copy 53 delete and edit buttons are activated whenever a keyframe is selected If there is no keyframe selected the paste button will append the copied or cut key frame to the last frame in the storyboard If one of the keyframes is selected the Movie Maker will ask where the frame should be inserted All these operations can be undone by cli
217. the interest probably is in regions where colo calization exceeds typically a value of 20 of the maximum With the background settings voxels are in or excluded based on their voxel intensities intensities higher then the background are kept while lower intensities are disregarded for the calculation Note the intensity value of voxels do not relate to the level of colocal ization In fact it may well be that high colocalization levels occur in regions with low voxel intensities Therefore background settings should not be used to remove signal that can still have some colocalization level Ideally work with deconvolved images and consider all the sig nal then study the colocalization levels using thresholds to split regions of high colocal ization from regions with low colocalization something that can not be know beforehand Setting the background is used for removing really constant background signals Thresholds do not affect colocalization but only the way colocalization maps are repre sented on the screen and objects are defined Depending on the locally calculated colo calization objects may merge or split What affects colocalization as explained in the colocalization theory at the SVI Wiki are the backgrounds In the computation of Huygens Professional User Guide for version 4 1 135 RBNCC Manders coefficients the background values act like in this example to the computation of M1 only pixels in R con
218. the number of frames changing these will affect Number of frames 121 the duration of the movie The stretch tool tries to Duration 10 00 seconds redistribute the total number of frames over the whole Cancet j Accept movie in such a way that the relative length of each transition does not change FIGURE 13 7 The stretch dialog helps increasing or decreasing the number of frames in a complex movie Huygens Professional User Guide for version 4 1 77 Synchronizing Transitions in Time and Slice Plane Transitions Storyboard Most render parameters like the en ili twist and zoom can be set to non integer values The time frame in case of a time series and the slicer z position however are fixed to integer values When the transi tion of such a parameter is not lin ear or when the change in value does not match the number of frames then this parameter is out of sync In that case the Movie Maker Frame 0 Frame 27 Surface Surface Timeline will show a warning symbol 4 on the transition arrow See Render parameter Timeframe Figure 13 8 In the final result these asynchronous transitions may FIGURE 13 8 A transition in which the fime frame parameter is out of sync with the show irregularities number of frames In this example the transition counts 27 frames while the time To restore the synchronization frame parameter increases linearly from 0 to 30 Note the irr
219. tion l and analysis Changes to the current frame like discarding objects or selecting anchors are remem bered when you change the time frame But voxel editions using the ROI to crop the dataset are lost unless you export them first with Export the applied ROI to original amp You can analyze all frames in a time series accumulating the data in the table through the FILE2ANALYZE TIME SERIES menu entry Render Pipes 16 17 Object Segmentation The Huygens Object Analyzer has two pipes named send iP Primary and Secondary pipes for you to put image seed threshold on histogram fil data through for object analysis and another pipe to simultaneously visualize a MIP of one of the channels See Figure 19 1 item 17 Secondary does not mean here less important it is just that you can use the first pipe Primary and the second pipe Secondary abbreviated P and S in some places We keep numbers to refer to image channels which is something different because in each of these pipes one can put any image channel in any order One can even segment the same channel twice with different conditions in each of the pipes if necessary This pane controls what data channel goes through each pipe how its objects are seg mented with a seed and a threshold and how the data is rendered transparency and brightness A garbage volume in voxels can also be set to get rid of little spurious objects with a number
220. tion Instability Some widefield systems are equipped with unstable arc lamps which can cause amongst others jitter These are irregular deviations from the average intensity in time Huygens Professional will correct this instability but when the instability is severe it cannot do so sufficiently Mechanical Instability Mechanical instability can take many shapes for example e Vibrations sometimes seen in confocal images They may seriously hamper deconvo lution e Thez stage moves irregular or with sudden jumps This deforms the data along the z axis and is fatal for widefield and confocal deconvolution e Thespecimen moves If in widefield data the object can clearly be seen moving when slicing along over a few um in z This will cause problems for the deconvolution Best cause of action apart from speeding up acquisition is limiting the z range of the data as much as possible Confocal data of moving specimen causes less problems Thermal Effects Thermal effects are known to affect calibration of the z stage especially if piezo actua tors without feedback control are used In particular harmful for widefield data In time series the effect can be seen as a drift of the z position or even a periodic movement induced by e g an air conditioning system switching on and off The z drift corrector is able to correct this in most situations though Internal Reflection At high NA the angle of incidence of the most oblique rays ca
221. tora tion of such images is likely to further increase the dynamic range resulting in the large structures becoming even dimmer In such cases use the compress display mode to increase the contrast of the low valued regions and reduce the contrast of the high valued regions Another way to improve the visibility of dark structures is the usage of false colors as mentioned before e Strong compression Same as above with stronger effect e Widefield In restoring widefield images it sometimes happens that blur removal is not perfect for instance when one is forced to use a theoretical point spread function in sub opti mal optical conditions In such cases the visibility of blur remnants can be effectively suppressed e Strong Widefield Same as above with stronger effect Time Slices of a time series can be selected by entering a frame number or using the slider Linkin g When you open two ore more Slicers on the same image then the Slicers are automati cally linked This means the position and orientation of both views are linked by default A right click on one of the images will change the center in the other Slicers Besides centering and orientation the Time Zoom and Twist and Tilt sliders can be linked by setting the appropriate option in the Linking panel If you would like to link the Slicers of two different images e g a deconvolved image and its original select Other in order to let the current Slicer listen to
222. tribute when their corresponding pixel in G is above the back ground More information can be found at the SVI Wiki In Huygens version 4 0 and higher the colocalization analyzer is extended with Repli cate Based Noise Corrected Correlation RBNCC Noise causes difficulties around colocalization analysis since noise also introduces colocalization This can be handled by using a background threshold which is explained in the previous subsections but when the noise is quite severe a threshold may not be sufficient With the RBNCC algorithm one can correct for noise that contributes to the underesti mation of the colocalization coefficients Pearson and Spearman coefficients The RBNCC method has been developed by J Adler S N Pagakis and I Parmryd from Uppsala University Journal of Microscopy 230 121 133 2008 More information can be found at the SVI Wiki Colocalization coefficients are calculated and directly corrected with RBNCC when this option is activated To use the RBNCC method a license with the RBNCC option is required When RBNCC is activated in the license then within the RBNCC frame a button shows red when the RBNCC is disabled and green when it is enabled If the button shows grey the license is insufficient to work with RBNCC RBNCC With RBNCC enabled a refer Reference image Coloc Reference ence image can be selected by Correlation Correction S 1 051 P 1 013 OS 1 042 OP 1 016 hovering over and clic
223. tware package tailored for deconvolu tion of microscopic images It enables you to deconvolve and analyze a wide variety of images ranging from 2D widefield WF images to 4D multi channel two photon confo cal images or from spinning disk confocal microscopes Also people who use experi mental set ups like 4pi microscopes may benefit from the software The Huygens Professional toolbox contains the following features Image Deconvolution Functions e Accelerated Maximum Likelihood Estimation restoration algorithm optimized for low light level images e Iterative Constrained Tikhonov Miller restoration algorithm e Quick Tikhonov Miller and Quick MLE restoration algorithms e PSF Distiller and Point Spread Function PSF measurement tool box to derive a microscopic PSF from finite sized micro bead images containing Automatic alignment and averaging procedure to combine the signal from differ ent micro beads in one or more images PSF reconstruction tool to correct for the finite size of micro beads e Generates a theoretical Point Spread Function for widefield confocal and two pho ton microscopes based on electromagnetic diffraction theory e Automatic bleaching correction of 3D and 4D widefield images and 4D confocal and multi photon images Huygens Professional User Guide for version 4 1 1 z drift corrector tool for time series that enables you to correct for movement in the z axial direction that could have occurr
224. ty of the darker structures Restoration of such images is likely to further increase the dynamic range resulting in the large structures becoming even dimmer In such cases use the compress display mode to increase the contrast of the low valued regions and reduce the contrast of the high valued regions Another way to improve the visibility of dark structures is the usage of false colors See Changing Display Colors on page 49 e Widefield in restoring widefield images it sometimes happens that blur removal is not perfect for instance when one is forced to use a theoretical point spread function in sub optimal optical conditions In such cases the visibility of blur remnants can be effectively suppressed Huygens Professional User Guide for version 4 1 49 Using the Slicer in Advanced Mode Automatic Panning Slicing and Zooming When the middle mouse button is clicked the Twin Slicer will automatically center and zoom in on the brightest spot in a 3D neighborhood around the cursor The button centered at the top of the window offers switching between basic and advanced mode The advanced mode allows independent control of the left and right slicer In thid mode all controls are available in twofold and accessible through the tabs in the bottom of the window Changing Time Frames Drag the slider in the Time frame tab to change the time frame or press the play button to animate the time series The time frame can be
225. uality threshold 01 Iteration mode Optimized Z Bleaching correction If possible z Wizard status g P Obtained initial parameters from image decon final Ape e Image decon final is undersampled gt Deconvolve FIGURE 4 5 The Deconvolution Stage in the wizard inishi When a deconvolution run is finished the result is shown in the right viewer where you nisnin g or 8 p Restartin ga can inspect the result in detail Depending on the outcome one can choose to RESTART RESUME or ACCEPT the restoration Deconvolution Run e Restart discards the present result and returns to the very first stage where the microscopic parameters can be entered Now the process can be restarted with differ ent microscopic and or deconvolution parameters This can be selected for the entire process or for the last channel e Resume keeps the result and returns to the stage where the deconvolution parame ters can be entered The software will ask to continue where it left off or to start from the raw image again A new result will be generated to compare with the previous one This can be repeated several times e Accept proceeds to the final stage or if the data was multi channel to the next chan nel If several results are generated by resuming the deconvolution it will be asked to select the best result as the final one which will be renamed as decon final When the final
226. um erste eee 145 Deconvolution IMprovemeniS Ledere been 148 iv Huygens Professional User Guide for version 4 1 CHAPTER 23 CHAPTER 24 JZIDDOHU ETE EEE d The Point Spread Pine ode me 149 Ol 149 Fe See eee 150 Adjusting the Global Color Schemes nauem eat nardo were man ede A cdi 151 Moe Selector EE Evian Care am o a excog b a d n e roe Poo Ud 151 mase Statistics vassere 152 Setting the Coverslip POSION ues koa Socr ee GA 152 Excitation Beam Overtill Facto sus ies ace cgo ho re cR ee a d 154 Brightiicld magse Saudi bero pe 155 Support and Contact Information 157 Contact Infor HatlOm soos oa ptos RA S QR ERR te E NC EOS RATED S S Ma ca BC dq 157 SUPPO daa tabac a ori vs eame edo qu eme p dondida ctam iere 157 License string Details vaa esee pre eee 158 Huygens Professional User Guide for version 4 1 vi Huygens Professional User Guide for version 4 1 What is Huygens Professional CHAPTER 1 What is Huygens Professional Introduction This guide is useful for the beginner in deconvolution and for the expert that starts with the Huygens Professional toolkit In this introductory chapter we explain the structure of the guide and provide general information installation instructions license details system requirements contact addresses conventions used throughout the manual and the status of the current release of Huygens Professional Huygens Professional is an image processing sof
227. um in 3 time frames The second and third plot in this tab are closely related This second plot shows the aver age displacement for a certain duration as given in Equation 1 In this equation t is the duration in frames and N is the number of objects x is the position vector of object i at time frame t N lu x MD t ND Pata Xo EQ 1 i 1 Huygens Professional User Guide for version 4 1 95 9 p b 2988 3 T7 Select Break Boundary Undo Redo Connect Delete Invert T me 0 000 s Time frame 0 EI P Filter Position Speed Flow Number of tracks T 2 5 5 Distance from track origin um Bu Save as Export data Number of tracks vs Distance from track origin um RRIA et GN SEE vs e 75 10 Time frame l FIGURE 16 7 In case of a large number of objects that show Brownian motion the shape of the histogram in the position tab will be a half normal distribution The third plot shows the mean squared displacement or MSD as given in Equation 2 Note that the x axis of this plot shows the duration f in seconds instead of frames MSD t Avg distance from track origin um vs Frame number gt o L W m A o m U Fir P2 10 Frame number Save as Export data Mean squared displacement um vs Time s Mean squared displacement um Save as Export data N 1
228. upon releasing the SFP scene Threshold The Soft threshold slider in the Channel parameters panel at the right affects the thresh old level The application of a threshold is a preprocessing step that reduces the back ground in the image i e voxels with intensity values below the threshold value become transparent Contrary to a standard threshold which is all or nothing values above the threshold are kept values below it are deleted the soft threshold function handles images in a different way It makes a smooth transition between the original an the deleted value Object Size The characteristic object size can be set by the Object size slider in the Image parameters panel at the right side This parameter affects both the excitation and the emission trans parency While traveling through the object the light intensity is attenuated to some degree This enables us to define some definition for penetration depth at which the light intensity is decreased to some extent for instance 10 96 of its initial value This penetra tion depth should be in line with the object size A transparent object is small with respect to the penetration depth Thus for the same physical properties of the light one object can be transparent while the other is opaque due to its size To find a reasonable range in transparencies the object size may be altered The initial object size is computed 62 Huygens Professional User Guide for version 4 1
229. ure a PSF from beads the diameter of the beads should be in the order of the half intensity width HIW also referred to as full width at half maximum FWHM of the expected PSF Larger beads will reduce the accuracy of the Distiller while smaller beads yield insufficient signal for accurate stacking in the averaging procedure resulting also in 1 We advise to measure the PSF after a change in the recording setup and certainly after each maintenance job in which the optics or scanning device was serviced 2 http www svi nl HalflIntensityWidth Huygens Professional User Guide for version 4 1 33 The PSF Distiller Window reduced accuracy Beads ranging from 120 to 250 nm can be used Typically beads with a diameter of 160 nm perform very well for many types of microscopy Confocal and two photon bead images from 160 nm beads should look like smooth fuzzy blobs with hardly visible noise Use the default SNR settings If available it is a good idea to average 2 to 5 beads Two photon bead images may look slightly noisy If so set the SNR to 20 and average 4 to 10 beads Widefield images taken from 160 nm beads should look like smooth fuzzy blobs with no visible noise The default SNR settings can be used It is not necessary to average any more beads Beads should be recorded with the same microscopic parameters that you will use later to image your specimens Please find more practical information about beads for PSF mea
230. w Hide options EY Ready Image size 320x346x78 Ratio not locked Extract Crop FIGURE 3 5 The cropper window Huygens Professional User Guide for version 4 1 Step 3 Inspect your Image To adjust this crop region manually draw the corners or sides of the cropbox to the desired position or use the entries in the Specifications panel Accept the new borders by pressing the CROP or EXTRACT button The latter one will create a new image instead of adjusting the original Do not crop the object too tightly because you would remove blur information relevant for deconvolution See The Intelligent Cropper on page 27 for more details about the intelligent cropper and its options The Image Histogram Select the image faba128 in the main window and display its histogram via ANALY SISS IMAGE HISTOGRAM in the main menu The histogram enables you to visually inspect the intensity distribution of the image visually The distribution of faba128 as seen in the left window in Figure 3 6 is of reasonable quality i e there are no sharp peaks at both sides of the histogram that may indicate clipping The right window in Figure 3 6 shows an example of the histogram of a clipped image Clipping occurs when input signals that are too high are mapped to the highest value available or when negative input signals are mapped to zeroes by the CCD camera Clipping at the right hand side of the
231. w images that still have all the measured background See Backgrounds vs thresholds in colocalization on page 135 Next we select what colocalization map we want to calculate Object Pearson Spearman Overlap Manders M or K or Intersection Notice the difference between maps and coefficients the colocalization coefficients parametrize the degree of colocalization of the full image while a colocalization map parametrizes the colocalization locally In a map a single colocalization value is calcu lated per voxel creating a 3D distribution that is represented in a 3D image by iso sur faces In other words the colocalization map shows the contribution of each voxel to the colocalization coefficient The Colocalization Analyzer computes only the map selected by the user but it always computes the selected coefficients When the COMPUTE button is pressed the pane colo calization coefficients will show the selected colocalization coefficients for the selected time frame If you have checked the COMPUTE ALL option next to the timeframe slider the coefficients will be calcuted for all time frames sequentially Your choice of the colocalization map is needed for the functionality in the second tab See Figure 20 2 In this tab an iso colocalization surface is generated based on the colo calization map This iso surface rendering is only possible if a colocalization map exists Huygens Professional User Guide for version 4 1 133
232. ween time frame 4 and 24 Exporting Data The plot graphics as well data in the plots and the track data can be exported to a file The buttons below each of the plots SAVE AS and EXPORT DATA save the plot as an Encapsulated PostScript file EPS and export the data as comma separated text CSV respectively The track data of the selected tracks can be exported to a CSV file using the menu FILE gt EXPORT gt SELECTED TRACKS AS CSV Huygens Professional User Guide for version 4 1 97 The EPS format can be imported by most office programs including Microsoft Office and OpenOffice The CSV format is plain text and can be parsed by most spreadsheet programs and other software like Matlab and Mathematica 98 Huygens Professional User Guide for version 4 1 Starting the Object Analyzer CHAPTER 17 Starting the Object Analyzer Introduction to the Object Analyzer The interactive Object Analyzer OA tool allows you to obtain statistics of individual objects by clicking on them or analyzing all objects with a single button press In this context an object is a distinct group of interesting voxels that are spatially con nected one to another Interesting voxels are distinguished from the background by using a seed and threshold criterion Therefore defining objects in an image implies 1 Segmentation Separating interesting voxels from the background according to a given criterion 2 Labeling Grouping the
233. y conflictive point is setting the signal to noise ratio Mind that this is not a number describing the image but something that can be tuned to achieve different deconvolution results In practice this is not difficult at all please see The Deconvolu tion Stage on page 15 and the Set the Signal to Noise Ratio article in the SVI Wiki Saving Templates The microscopic and deconvolution templates are by default saved in the SVI Tem plates folder in the user s home directory where Huygens can find them The next time he Batch Processor is used the saved templates will be found in the wizard to set up the batch task There are some sample templates in a global location where the system administrator can also store templates for everybody to use This global location is a subdirectory Templates ofthe Huygens installation directory See Table 2 1 on page 8 Adding the Task After selecting the deconvolution template the DONE button appears By pushing it the task is created and shown in the Tasks field The task can be deactivated by clicking the READY TO RUN button in the status column setting the status to DEACTIVATED Marked tasks can be deleted by clicking the DELETE button amp or selecting EDIT2 DELETE TASK from the menu Tasks can still be added to the queue before starting the computations or start comput ing right away and add new tasks afterward Duplicating Tasks The DUPLICATE TASKS button 3 is very convenie
234. yer and Apple Quicktime 2 http en wikipedia org wiki Mjpeg Huygens Professional User Guide for version 4 1 65 66 Huygens Professional User Guide for version 4 1 CHAPTER 12 The Surface Renderer An iso surface is a 3D surface representation of points with equal intensities in a 3D stack it is the 3D equivalent of a contour line See Figure 12 3 The Huygens Surface Renderer is a powerful 3D visualization tool that enables the visualization of these sur faces and thus the representation of the data in a convenient way to clearly see separated volumes Shading enhances the perception of 3D shapes and texture See Figure 12 1 VI view point light direction surface normals projection plane determine shading data volume FIGURE 12 1 schematic overview of surface rendering The orientation of the surface determines the shading Besides iso intensity surfaces this renderer is able to generate MIP projections which are blended with the surfaces to be used as a reference to the original microscopic data See Chapter 10 on page 57 Because the Surface Renderer is based on fast ray tracing that runs efficiently on multi core computers there is no need for any special graphics card as would be necessary for conventional polygon based techniques To start the Surface Renderer right click on a thumbnail and select VIEW gt SURFACE RENDERER from the pop up menu or choose VISUALIZATIONS SURFACE REND
235. zation settings screen there is the option to select the quality of the inter polation filter that is used to resample the images The high quality mode is not available when cor recting for rotation because it uses Fourier trans forms to shift the image FIGURE 15 6 Effect of filters in the Stabilization settings screen The top shows the measured displacement the curve in the middle is noise filtered and the bottom is drift filtered Push the NEXT button to apply the stabilization and proceed to the cropping screen Huygens Professional User Guide for version 4 1 87 Alignment of Slices in 3D Stacks Cropping the Result Because there is some extra space needed for the translated and rotated frames the stabi lized image will be larger than the original one The cropping screen See Figure 15 7 IE Huygens Object Stabilizer H2B apoptosis Bunka o m File Edit View Help Position 85 7 0 08 Cropping ike 1 p f d A m Veg The stabilized result is now shown The image dimensions were HSE bd increased to make the translations and rotations fit If you would like to crop this image then drag the corners of the red box to modify the cropping region or select original size from the list below Select the cropping method Full 724x731x1x53 no cropping Original size 512x512x1x53 Manual 193x196x1x53 Select the reference frame First frame Middle frame Automatic mi

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