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The Huygens Essential User Guide

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1. System magnification My 2 23 Backprojected pinhole ra dius nm 992 P pel obj Eq 7 Zeiss LSM410 inverted pinhole parameters Zeiss LSM510 Diameter micron d Pinhole geometry circular System magnification Mss 3 33 Backprojected pinhole ra dius nm 10 d 2 M sys obj Eq 8 Zeiss LSM510 pinhole parameters 7 Establishing image parameters Olympus confocal microscopes Olympus Fluoview Reported parameter 1 2 3 4 5 Diameter d micron 0 100 150 200 300 Pinhole geometry square System magnification Msys 3 426 3 8 FV500 3 Backprojected pinhole radi 10 d gt en 7 Eq 9 us nm 2 mm m sys obj Table 4 Olympus Fluoview pinhole parameters Biorad confocal microscopes Biorad Reported parameter Po n a Diameter d mm 0 8 Pinhole geometry circular 53 83 reported 60 for the System magnification Mys Radiance 53 for the 1024 109 d Backprojected pinhole radius nm Ai Eq 10 system obj Table 5 Biorad MRC600 1024 Radiance pinhole parameters Checking the Biorad system magnification The Biorad MRC500 600 1024 microscopes have a very high magnification in the detection sys tem The fixed system magnification is according to Pawley 53 x m with m between 1 0 tube tube and 1 56 a factor 1 25 for the fluorescence
2. Primary Send MIP ROI surface Oo Off HA Twist 0 deg Object statistics ROI manipulation Channel mode Pipe 0 ch 1 2 obj Pipe 1 ch 0 4 obj db No objects selected Analyze all Och 1 Man Surf ch Label voxels Isovol Surface Sum Min Max Length WiAx WiLat C Mass Delete Inside Outside Conditions pipe 0 chan 1 thresh 109 9 seed 137 4 garb 1 objects 2 P B 1 1 5351 4 525 23 946 7 484e5 109 940 270 261 4 21 2 51 2 16 115 9 241 8 17 5 Original 1 0 P B 1 2 6036 5 073 24 736 8 188e5 109 960 246 786 4 63 2 26 2 50 332 6 284 7 18 8 onditions pipe an 1 thresh 109 9 seed 137 4 garb 1 objects 2 pipe 1 chan 0 thresh 261 2 seed 326 5 garb 1 objects 4 S F 0 3 121 0 091 1 160 3 509e4 261 480 347 627 1 08 0 48 0 49 192 6 152 2 19 9 S F 0 2 178 0 119 1 085 5 468e4 262 430 391 175 0 73 0 68 0 69 214 0 184 0 15 0 S F 0 1 508 0 382 2 461 1 833e5 261 370 516 437 1 15 0 90 0 88 261 6 209 1 14 6 S F 0 4 246 0 167 1 404 7 427e4 261 270 367 705 0 92 0 63 0 70 339 7 237 8 20 7 Ready Canvas size 610 x 500 Dragging on Figure 28 The Object Analyzer with segmented objects represented as isosurfaces An area is selected to be extruded and define a 3D ROI Mouse mode Depending on the selected mode the mouse has different behaviors e Rotate object interacts with the full image to rotate it in the space by dragging the mouse pointer on the object view That can also be achieved by moving the Tilt and Twist sliders e Measure object le
3. Qhan1 ia nel can be a good spatial refer Brightae ence for the objects in the other channels an 2 Twist 123 deg Ready _Can va 640 x 480 Dragana off fi MIP Threshold 6 1 J Figure 27 The TE Renderer You can control the transparency and the brightness of the rendered surfaces with the correspond ing sliders independently in each graphic pipe Select the viewpoint by moving the Tilt and Twist sliders or by dragging the mouse pointer on the large view Also try changing the zoom When you have your rendering ready save it to a TIFF file with Fie gt Save Other available options to change are render size and transparency depth accessible through the OPTIONS menu Hue Selector The hue selector is a component that allows you to select the color range actually the hue prop erty of the color in which the different objects of each channel are displayed by the Surface Ren derer or the Colocalization Analyzer Thus objects belonging to different channels can be repres ented with very different hue ranges to make them clearly distinct but also with some gradual dif ferences inside the selected range to distinguish independent objects You can also collapse a range to have all objects in a channel displayed with exactly the same color Transparency depth This option controls how different surfaces are seen through the others The effects are mainly vis ible when you have some objects int
4. 52 Can I deconvolve a single TIFF image 52 How do I generate a debug log 52 Addresses and URLs 53 Where can we be reached 53 Scientific Volume Imaging b v 53 Distributors 53 Support and FAQ 53 Knowledge database FAQ 53 The SVI wiki 53 Starting points 53 Quick reference 54 Alphabetical Index 55 The Huygens Essential User Guide iii Contents iv Scientific Volume Imaging 1 What is Huygens Essential 1 What is Huygens Essential Huygens Essential is an image processing software package tailored for restoration visualization and analysis of microscopic images Its wizard driven user interface guides you through the pro cess of deconvolving images from light microscopes Huygens Essential is able to deconvolve a wide variety of images ranging from 2D widefield WF images to 4D multi channel multi photon confocal images To facilitate comparison of raw and deconvolved data or results from different deconvolution runs the Essential is equipped with a dual 4D slicer tool You can also render 3D images and animations with its powerful visualization tools Post restoration analysis is possible using the interactive analysis tools Based on the same image processing engine the compute engine as Huygens Professional Huy gens Essential combines the quality and speed of the algorithms available in Huygens Professional with the ease of use of a wizard driven intelligent user interface Huygens Essential is based
5. Airydisks ation wavelength In principle using is not correct because the Airy diffraction pattern is formed by the emitted light However we suspect microscope manufacturers prefer to use the ex citation wavelength because it is better defined and does not depend on settings of devices like ad justable band filters For this reason in the formula above we too use Note that this relation bypasses the need to know internal system and lens magnifications Converting from integer parameter Unfortunately quite a few commercial microscopes do not report the physical pinhole size or the Airy disk size Instead often an integer size parameter is specified with a range 0 255 8 bit Matters are further complicated by the use of non circular pinholes To compensate this we intro duce a shape factor Chape which takes care of the conversion from size diameter or edge size to radius The following formula can be used to translate the 8 bit machine number into the backpro jected pinhole radius P3 255 SuSE ax min min shape 3 r 1000 c m m obj system where Pg is the 8 bit machine number for the pinhole s j is the maximal pinhole size in micro meters S in is the minimal pinhole size in micrometers The factor 1000 is to convert from mi crons to nanometers Shape factor The shape correction from a square to a circular pinhole is based on setting the area of the square pinhole equal to the area of t
6. and fit the bricks together in a seamless fashion Brick wise processing is an automatic feature of Huygens Essential 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 best one Signal to Noise Ratio SNR The Signal to Noise Ratio SNR can be estimated from the quality of the image In Figure 31 you find some examples of recordings where different noise levels were added to an original restored image Figure 31 Examples of different SNR values Same image different SNR values Top left original image Top right image with SNR 30 Bottom left image with SNR 15 Bottom right image with SNR 5 If you want to calculate this SNR with more detail you must remember that we define the SNR as the square root of the number of photons in the brightest part of the image Estimating the SNR in noisy images is surprisingly 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 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 Thus the SNR is The Huygens Essential User Guide 39 7 Establishing image parameters M SNR 4 S If a significant blacklevel is present it should be subtracted from these values before making the quotient See
7. but a fair trade off can be often found It is better to record 10 separate noisy slices 100 nm apart than slices on 1000 nm each averaged 5 times in order to reduce noise See also A typical example in Sampling an image from the Huy gens Deconvolution Recipes or in the above mentioned Internet link Bleaching is a practically unavoidable phenomenon in fluorescence microscopy Because the im age 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 photon images But in WF decon volution bleaching is more of a problem Fortunately usually the bleaching in WF images can be corrected quite easily Huygens will do so automatically However if the bleaching is strong the correction might not be perfect resulting in lower quality deconvolution results Illumination instability Some WE systems are equipped with unstable arc lamps Huygens 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 deconvolution e z stage moves irregular or with sudden jumps Fatal for confocal or WF deconvolution e Specimen moves If in WF data the object can clearly be seen moving when slicing along over a few micron in z this will cause problems for the deconv
8. 2 Scientific Volume Imaging 2 Installing Huygens Essential Obtaining a license string If you are not upgrading from a previous installa tion it is likely that a license is not yet available To H te engine 2 8 0p0 enable us to generate a license string for you we PR ace as J Huygens Essential 3 1 0p0 need the fingerprint of your computer the system ID number If you have not done so already start Ee rac Vee v Huygens Essential The system ID is displayed in Dt Bas Sls the Hep gt Asout dialog Figure 2 Send it to ge sales svi nl and you will be provided with a Fa ROBES i URL http w svi nl license string To prevent any typing error use the Cory button to save the ID to the clipboard You can print it into your mail message with the Eprr gt _ Check for Huygens updates Paste menu item of your mail program This computer has the following system ID 3352 d350 deld 4250 Copy OK Figure 2 The Herr gt Asout window The system ID is shown at the bottom In this dialog box you can also find a button to CHECK FOR HUYGENS UPDATES ON our company Server Installing the license string Select the license string in your email message and copy it to the clipboard using Epir gt Cory in your mailing program Start Huygens Essential and go to Herre gt License a dialog box pops up Figure 3 Then press the App a NEW License button and a new window will pop up Figure 4 Paste the strin
9. 24 SNR 15 39 specimen moves 47 spherical aberration 14 46 48 square pinhole 42 44 statistics 9 StuffIt Expander 1 Surface Renderer 26 50 T task report 17 template 10 thermal effects 48 threshold 30 35 TIFF file 16 27 51 TIFF series 26 51 time series 8 15 16 22 24 26 50 total internal reflection 46 Transparency 25 27 Twin Slicer 14 16 21 22 two photon 37 47 49 U undersampling 47 unstable arc lamps 47 updates 3 V vibrations 47 Viewer MIP Renderer 23 SFP Renderer 24 Surface Renderer 26 Twin Slicer 21 violet background 9 W wavelength 9 24 25 36 40 42 44 47 51 website 4 24 widefield 38 49 Z z drift corrector 16 z stage moving 47 Zeiss 43 44 exe file 1 gif file 24 ics file 17 ids file 17 pic file 16 pkg file 1 rpm file 2 tar file 2 tardist file 2 xml file 11 2 2D images 8 4 4 pi 49 The Huygens Essential User Guide 55
10. RELoap button The Export button takes the image data inside the currently defined ROI and exports it to over write the original image in Huygens Essential that was not modified up to this moment Data out side the ROI is removed from the image that can now be saved in a new file This makes the Ob ject Analyzer a powerful cropping tool useful as a independent preprocessing tool for visualiza tion or for example colocalization analysis The Retoap button always reloads the image in the main window That means that in will reload the result of your last Export if you did any Data analysis When the measure object mouse mode is active local statistics are returned in the lower window pane when clicking on different objects As these properties and the number of objects vary with the selected thresholds first a report of the current conditions is shown Conditions pipe O chan 1 thresh 136 7 seed 170 8 garb 1 objects 4 pipe 1 chan 0 thresh 256 1 seed 320 1 garb 1 objects 4 That reports the active isosurface pipes what data channels they are using the current seed and threshold levels in absolute values the garbage level in pixels and the total number of found ob jects per pipe Notice that the object statistics pane is a text box Output will be placed in the current cursor posi tion Make sure to put it at the end again after selecting text for copying The following statistics are reported for each object e Object
11. Volume Imaging original 50 Color Grey scale original 1 Gamma Lineair fo 150 400 Grey scale Linear 200 500 True colors 300 600 False colors Compressed Wide Field Figure 23 The Twin Slicer with time slider 12 13 14 15 16 17 18 2 3 4 Huygens Essential Visualization Tools The MIP Renderer Soft threshold Rendering a movie The Maximum Intensity Projection MIP Renderer is part of the Huygens Essential since release 2 6 0p4 and enables you to obtain a spatial projection of your data from a given viewpoint see Figure 24 The renderer projects in the visualization plane the voxels with maximum intensity that fall in the way of parallel rays traced from the viewpoint to the plane of projection Notice that this im plies that two MIP renderings from opposite viewpoints show symmetrical images To start the MIP Renderer right click on an image s thumbnail to open the contextual menu then select Snow in MIP RENDERER NG Huygens Essential MIP Renderer e959 blgrb huy 0 x File Options Select your viewpoint by moving the Tilt and Twist sliders or by dragging the mouse pointer on Zoom 1 21 the large view that will be empty at first before your first render Set Go Tilt 31 deg Quality ing Also try changing the zoom You will see how the preview Image parameters Time frame 0 thumbnail changes Whe
12. are highly suspicious An Orange background indicates a non optimal situation Oversampling is also indicated with a cyan background that becomes when it is very severe Figure 9 shows settings which do not fulfill the criterion for the critical sampling distance versus numerical aperture See Sampling densities on page 40 The Huygens Essential User Guide 9 3 The image restoration process Huygens Essential 32bit by Scientific Volume Imaging B V You can see and correct the image File Task Visualization Tools parameters not only at this decon volution stage but also at any time original by right clicking on the image thumbnail and selecting SHow PARAMETERS Or CORRECT PARAMETERS Correcting will pop up the window reprocessing Stage Parameter C Below are the sampling density factors found in the em ee r geometry section of the image Change these as shown on Figure 11 necessary x sample size nm Y sample size nm Z sample size nm Parameters Templates Once the proper parameters have been set and verified they can be Task info saved to a Huygens template file in tore bor apie e severe undersamplin 2 a 2 suffix hgst Those template Memory 2 ae ae files are loadable in the very start Figure 9 Parameters check stage Sampling Red coloring of the wizard hence the user can indicates a suspicious value and orange a non optimal value skip the parameters verificati
13. attachment and also a factor 1 25 for the DIC attach ment The factor of 53 includes the 8x eyepiece just below the scan head but does not include that variability in magnification due to the variations in tube length that are result from the align ing the system The high system magnification allows you to view the diffraction pattern Airy disk at the pin hole plane directly by eye To enable you to verify the correctness of equation 10 in the table above for your instrument we outline the way the system magnification was derived In a Biorad MRC600 with a 1 3 60x objective the Airy disk has a diameter of around 2 2 5 mm at the pinhole plane The diameter of the first Airy zero ring is 7 6 lateral optical units 0 u us ing the following transformation to express a distance x in dimensionless 0 u OTe VER SEN 11 where 11 SIN amp is the numerical aperture NA In the system above an o u is 0 3 0 033 mm At the specimen plane backprojected a lateral o u is in this case around 61 nm The total magnification 9 Pawley J B Handbook of biological confocal microscopy 2 ed 1995 Plenum Press New York and London ISBN 0 306 448262 Page 30 The Huygens Essential User Guide 45 7 Establishing image parameters appears thus to be 4918 the system magnification 82 9 This value corresponds well with the largest possible system magnification for the MRC600 A supplied calibration curve If a calibrati
14. everything is ready you just click the Run button to start the jobs The progress of the Batch Processor and the report for each individual task are shown on the text tabs of the Batch Processing Overview The status of each task on the task list changes accord ingly to the evolution of the process The restored images are saved on the selected destination directory as soon as they are ready Menus File menu By using this menu you can CLose the current process create a New task list Open previously saved processes and Save them to the disk or Appenp other task lists to the current one You can also Exit the Processor Task menu By using this menu you can Run the tasks and Save Barca Procress or SAVE Barca Report to text files on the disk More information Updated information about the Batch Processor can be found in HELD SuppoOrt sVi nl wikis BalLcnProcessor 20 Scientific Volume Imaging 4 Huygens Essential Visualization Tools 4 Huygens Essential Visualization Tools Huygens Essential provides different tools for data visualization The Twin Slicer Color mode This allows you to compare a deconvolution result with the original but also different deconvolu tion results obtained from the same original Open the Twin Slicer by double clicking on one of the thumbnail images in the main window or by right clicking on the image thumbnail then SHow on TWIN SLICER The Twin Slicer will show t
15. exceeding some value are all converted to 4095 clipping information in the clipped samples is lost In practice be suspicious if you find in your image voxel intensity values at the extremes of the numerical range probably clipping occurred Read more in http support svi nl wiki ClippedImages One of the rules of measurement that is often overlooked is that one takes too few xy slices from the microscopic object In that case the sampling distance is too large too few samples under sampled which leaves you with a 3 D stack with hardly any relation between the adjacent planes It is important to know how the sampling conditions should be established in order to recover an image from the sampled values How you should sample your object depends on your microscope type WF or confocal and on the microscope parameters used like the numerical aperture and wavelength of the light The correct sampling distances can be estimated as explained on Sampling densities page 40 or calculated using the formulas as given in The Nyquist rate from the Huygens Deconvolution Recipes Do not undersample to limit photodamage Bleaching Some times undersampling is done to limit photodamage to live cells If photodamage plays a role it is better to distribute the available photons over more pixels resulting in an apparently noisier image than putting the photons in fewer pixels to get a low noise but undersampled image Of course there are limits
16. icons appear on your desktop Double clicking on the Huygens Essential icon starts the program the start up window will be displayed Figure 1 The Huygens Essential User Guide 1 2 Installing Huygens Essential Linux SGI Irix 6 5 IBM AIX 5 2 The Huygens Essential Linux distribution is a rpm file for instance huygens 2 7 0 p7 rpm Opena Unix shell go to the directory were this file is located become superuser and type rpm ivh force huygens 2 7 0 p7 rpm After installing the software type essential ina shell to start the software A directory usr local svi will be created initialization scripts will be installed in jusr local bin Currently the Irix distribution is a single tardist file containing various components By default all components are installed Become superuser and type Syvmgr E dist6b 2 11 0 pl it rdist Press Start in the Software Manager window After installing the software type essential ina shell to start the software The program will display the start up window Figure 1 A directory usr local svi will be created the ex ecutables will be installed in usr local bin Log in as root on your workstation or ask your system administrator to do so Huygens for AIX is distributed in a tar file Go to the directory where you downloaded it to After unpacking the distri bution file with tar xvf my_aixfile tar you will find three new files svi tar zZ README AIX and a shell script ATX inst
17. on cross platform technology It is available on various Microsoft Win dows operating systems Linux for Intel or AMD based systems MAC OS X IBM AIX and SGI s Irix 6 5 For AIX and Irix 64 bit multiprocessing versions are also available 2 Installing Huygens Essential Mac OS X Microsoft Windows You can download Huygens Essential from the SVI website http www svi nl i Huygens Essential by Scientific Yolume Imaging B Y File Task Visualization Tools Help Go to the folder were you down l loaded the distribution and double click on it It will be ex tracted by StuffIt Expander to a pkg file which will be placed in the same directory Double p ane by Doy gt No valid license could be found Continuing in freeware mode without click this ce conte urd aa NSR pkg file and follow the install ation wizard You have received an executable file for installation for instance a Task report Task plot Image currently no image file loaded Status ready file named Huygense280 vr exe Place this file on your Figure 1 The start up window on Microsoft Windows If no license desktop and double click its icon string is installed the software runs in Freeware mode You can find to start the installation During your computer s ID number by using HELP gt ABOUT installation the directory C Program files SVI will be created by default After completion the Huygens Essential and Huygens Scripting
18. recorded at a given fixed wavelength independently of what device made the acquisition Thus it is a logical channel of stored data and not necessarily a physical channel as all the image channels could have been measured by a single photomulti plier for instance Multi channel images can be de convolved in a semi automatic fashion giving you the opportun ity to fine tune the results ob tained with each individual chan nel After the preprocessing stage the multi channel image is split into single channel images named channel 0 channel 1 and so on The first of these is automatically selected for decon volution To deconvolve it pro ceed as follows Huygens Essential by Scientific Volume Imaging BV File Task Visualization Tools channel 0 0 Join the deconvolved channels together in a multi channel result image by clicking on the thumbnail images in the display area one by one Select a deconvolved channel to become channel 0 in e result image Choose channel 0 channel 0 0 Select a deconvolved channel to become channel 1 in e the result imag Choose channel 1 channel 1 0 Task info Task report Task plot Image Mosts rog u home theo Demolmages zorigSmall ics Se Status All stages of deconvolution done Memory 1 The procedure to deconvolve a channel in a multi channel data set is exactly the same as for a single channel image You can therefore do multiple reruns on the chan
19. reference Surface pipe primary or secondary position front or back data chan nel and label number e Voxel count e Volume enclosed by the local isosurface e Surface of the local isosurface Sum minimum and maximum of all object voxels intensities e Length of the object e Caliper width in the axial and lateral direction of the microscope e Center of mass of the object It may happen that clicking at one point of the screen produces an ambiguous selection of objects because to have two of them one behind the other belonging to different graphic pipes That will make the analyzer to report information about the two pointed objects In cases like that is when the position information front or back is more relevant There is no ambiguity when the two overlapping objects belong to the same pipe in that case information is always reported about the object closer to the viewer If they belong to the same pipe the overlapping is only apparent and due to the perspective projection distinct objects inside a pipe are never in contact in space There is an ANALYZE ALL button to obtain information about all the presently defined objects on the primary pipe without having to click on them one by one The width of the objects is measured using a virtual caliper held perpendicular to the length axis The caliper is moved along the length axis to find the largest width while being rotated around The Huygens Essential User Guide 31 5
20. remove the task from the list Adding multiple tasks Now that you have learnt how to add one task to the job list and explored its capabilities we can speed things up and add many tasks at the same time 5 Seehttp support svi nl wiki RestorationMethod for more information The Huygens Essential User Guide 19 3 The image restoration process You can use the button with the two plus signs for that That will open a new dialog where you can select multiple images that will be deconvolved with the same microscopic and restoration templates as specified You can add all image files present in a directory or add multiple files In the second alternative multiple files can be selected by holding down the control key apple in the Mac Once you have accepted a list with all the images select the templates to be used with them and click App to generate all the task lines All of them will use the selected templates Repeat the procedure to add more image or use different templates Click Cancer to finish When you change one task s template you see that the template name is also changed indicating that some parameters were modified locally These changes do not affect the template as it is used by the other tasks Running the batch job Make sure you selected the desired location to save the results When you have your batch process configured you can save it for future reference in the menu do Fre gt SAve When
21. select SHOW PARAMETERS right by which the color can be and you will find besides the ee The rag eas is tatinal in Platform specific The picture on a parameter settings statistical in ve formation of the particular image color editor Amongst them are the mean sum standard deviation norm and po sition of the center of mass Right mouse click on a thumbnail Verifying the microscopic parameters Next to the basic voxel data the Huygens Essential also tries to read as much as possible informa tion about the microscopic recording conditions However depending on the file type this inform ation may be incomplete or incorrect In this first stage all parameters relevant for deconvolution are displayed and can be modified Optical parameters first page e Microscope type e Lens and medium refractive index e Numerical Aperture NA Optical parameters second page e Backprojected pinhole radius in nm Backprojected means the size of the pinhole as it appears in the specimen plane see Computing the backprojected pinhole radius on page 41 e Backprojected distance between the pinholes in microns only visible if the microscope type is Nipkow e Excitation and emission wavelengths e Photon count number of excitation photons involved in the fluorescence e Voxel sizes in the three directions x y z third page e Summary of all parameters now in effect If values are displayed in a PERSEN they
22. the paragraph Blacklevel below for more details In low noise images it is much more difficult as you no longer see single photon events so easily Fortunately in such cases the establishment of the precise SNR is not very important for the res toration method and a rough estimation based on the appearance of the image is usually enough see Figure 31 Read more in http support svi nl wiki SignalToNoiseRatio Blacklevel Figure 32 shows the histograms of three synthetic images At left an image homo geneously filled with the value 5 At the middle we applied A Ablacklevel PG Figure 32 Histogram of images with various blacklevel values image was build by a CCD camera At the image on the right a value of 20 was added to simulate electronic shift This shift is called blacklevel A large blacklevel value will reduce the effective dynamic range of your microscope but will do no harm to the deconvolution since it is automatic ally accounted for in the background estimation stage However it is also possible that the black level is negative In the image histogram this will show up as a spike on the left Sampling densities The sampling density is the number of recorded samples voxels per unit volume when acquiring the image It is a microscopic parameter that describes the conditions of your image acquisition established by the way you configure your microscope usually by the zoom factor The ideal sampl
23. the removed object are not considered anymore even when you change the thresholds again Regions of interest ROT You can define a region of interest ROJ in the data to discard objects out of it for further analys is 1 Select the define ROI mouse mode 2 Draw an area on the isosurface pane keeping the mouse button pressed down When you release it the area selection will be automatically closed The selected area is extruded perpendicular to the screen to create a 3D selection 3 Select on what data channel you what to operate Your options are 1 One channel as selected in the Primary graphic pipe 2 All channels 4 Click on the appropriate button what do you want to delete objects Ins pe or Oursme the selected area You will see all the excluded objects disappear and the volume enclosed in the ROI become high lighted The way the ROI is represented is controlled by another graphics pipe whose controls are grouped in the ROI surface pane As you can define different ROIs for different channels you can use different visualization parameters for each of them Switch the highlighting off to leave just the data 6 For more explanations see http support svi nl wiki SeedAndThreshold 30 Scientific Volume Imaging 5 Huygens Essential Analysis Tools You can repeat the select delete procedure in this or another view to refine your ROI definition as much as you want To recover the original data again press the
24. threshold 0 02 fluorescent dye Each dye has its Object size 0 30 micron specific excitation and emission Traps rca wavelength with corresponding SSe Excitation 0 50 distinct absorption properties The absorption properties can Twist 0 deg ander Ready be controlled by the user The Figure 25 The SFP volume renderer Up right the preview different emission wavelengths 6 PHS p subsampled image that acts directly on the slider position The give each dye its specific color actual rendering is started by pressing the RENDER button 24 Scientific Volume Imaging 4 Huygens Essential Visualization Tools To excite the fluorescent matter light must traverse other matter The resulting attenuation 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 homogen eous plane the gray table below the data volume is placed on which the cast shadows become clearly visible After excitation the fluorescent matter will emit light at a longer 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 w
25. were you left off keeping improving the image quite recommendable or to start from the raw image again A new result will be generated to compare with the previous one for instance using the Twin Slicer You can repeat this several times Accept proceeds to the final stage or if the data was multi channel to the next channel see page 17 If you generated several results by resuming the deconvolution you will be asked to select the best result as the final one that will be renamed to deconvolved The other results will remain as well in case you want to save them z drift corrector for time series Saving the result For 3D time series the program pops up an additional tool that enables you to correct for movement in the z axial direction that could pner pe have been occurred for instance by thermal nn drift of the microscope table In case of a Reference channels l One Channa multi channel image see p 17 the corrector Reference channel 0 Ea can survey ALL CHANNELS and determine the mean z position of the channels or it can take ONE CHANNEL as set by the REFERENCE CHANNEL parameter After determining the z positions per frame the z positions can be filtered with a MEDIAN GAUSSIAN Or Kuwaunara filter of variable width Figure 18 The z drift corrector When the drift is gradual 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 posi
26. BM AIX Huygens Essential and Huygens Scripting run on all SGI equipment running Irix 6 5 on a MIPS R5000 processor or higher IBM AIX 5 2 equipment with a Power4 processor or higher The re commended RAM size is 512 MB or larger Running the program Mac OS X You can find the program icon in the installation directory Clicking it will start Huygens Essen tial and open the main window Figure 6 You can also run the program by typing essential at a shell prompt Windows The Windows installation procedure has automatically placed an Huygens Essential icon on your desktop Clicking it will start Huygens Essential and open the main window Figure 6 Linux On Linux you can start Huygens Essential by typing essential ata shell prompt It will start Huygens Essential and opens the main window Figure 6 If the shell is unable to find this command then typing the full path should help usr local bin essential If this still does not help then Huygens Essential has not been installed correctly The Huygens Essential User Guide 5 2 Installing Huygens Essential In Linux KDE desktop you may also start Huygens Essential Huygens Essential by Scientific Volume imagingBV Q File Task Visualization Tools from the Application menu and in the Gnome desktop from the Main Menu Irix On Irix you can start Huygens Essential by typing essential ata shell prompt which will start Huygens Es s
27. Huygens Essential User Guide we J 1 F ar y l he i 2 i Scientific Volume Imaging Huygens Essential User Guide Scientific Volume Imaging b v Alexanderlaan 14 1213 XS Hilversum P O box 615 1200 AP Hilversum The Netherlands http www svi nl Copyright 1995 2006 by Scientific Volume Imaging b v Alexanderlaan 14 1213 XS Hilversum P O Box 615 1200 AP Hilversum The Netherlands All rights reserved Cover illustration Macrophage recorded by Dr James Evans White head Institute MIT Boston MA USA using widefield microscopy as deconvolved with Huygens At the right the same dataset again macrophage fluorescently stained for tubulin yellow green actin red and the nucleus DAPI blue Left part original data right part as deconvolved with the classical Maximum Likelihood Estimation method MLE The image was visualized using the Simulated Fluorescence Process SFP volume rendering package from Scientific Volume Imaging Image in figures 22 24 27 and 29 isolated Rat Hepatocyte couplet re corded by Dr Permsin Marbet at the Department of Anatomy of the University of Basel Switzerland head Prof Lukas Landmann as de convolved with Huygens Image in figure 28 FISH stained cell nucleus recorded at the Nuclear Organization Group SILS University of Amsterdam The Netherlands head Prof Roel van Driel This document was typeset with OpenOffice org
28. Huygens Essential Analysis Tools Because microscopic data even deconvolved microscopic data often shows orientation dependent imaging due to the lower axial resolution a distinction is made between width measurements where the measurement direction is in the axial direction vs the lateral direction The caliper ro tates 180 in space along the length axis to find maximum widths but this rotation is split in two 90 ranges one where the component along the optical axis is dominant and another one where lateral components are larger Both values are reported When an anchor object was previously defined in the set measurement anchor mouse mode dis tance to the anchor is also reported at the top of the object statistics pane for the last pointed ob ject It is possible to select a group of anchor objects The location of the anchor is then the center of mass of all anchor objects A button is available at the statistics pane to remove all the anchor pos itions Visualization parameters You can select the viewpoint of the rendering by moving the Tur and Twist sliders or by drag ging the mouse pointer on the object view when the rotate object mouse mode is active Also try changing the Zoom slider You can control the transparency and the brightness of the isosurface pipes independently with the correspondent sliders or just set them off The color range in which the objects are displayed can be modified using the hue select
29. ION to begin processing your image or you can convert your data set with the Toots button If you have loaded a bead image you also can proceed selecting Start PSF pistiLLEr and proceed with generating a Point Spread Function PSF see page 14 from measured beads See The PSF Distiller on page 36 A A special license is needed in order to launch the PSF Distiller You can OPEN ADDITIONAL images for reference purposes but only the one named original will be deconvolved during the guided restoration The Huygens Essential User Guide 7 3 The image restoration process Preprocessing Converting a data set optional Before you press the START DECONVOLUTION button you can convert a 3D stack into time series im ages Convert XYZ to XYZT or vice versa or you can convert a 3D stack into a time series of 2D images XYZ to XYT or vice versa These functions can be found in the Toots Hint If you have a data stack that is poorly sampled in z not fulfilling the Nyquist criterion you better interpret the different planes as independent i e as 2D images and do 2D deconvolution planewise while taking the optimal Nyquist criterion for z as imposed by the optical parameters see the diagram in page To do this in one run for all the planes convert the 3D stack to a 2D time series do the deconvolution run and convert back from 2D time to 3D Time series A time series is a sequence of images recorded along time at unif
30. SHow IN SURFACE RENDERER Let the renderer initialize You can find three graphic pipes to redirect your image data channels to two surface pipes and one MIP pipe These can be activated independently Use the threshold slider to apply different thresholds to your data channels to select what voxels are considered to shape volumes Connected voxels after the threshold determine independent volumes that will be represented by the 3D isosurfaces containing them with different colors You can use up to two different data channels for surface rendering one in each of the two avail able surface graphic pipes The color of the different objects inside a channel can be modified with a selector see Hue Selector below 26 Scientific Volume Imaging 4 Huygens Essential Visualization Tools Huygens Surface Renderer original m file Options There is a third graphic pipe to redirect data to the rendered im age the MIP pipe works project om Frame selection Frame 070 Render pipes Primary surface Threshold 8 2 ing the voxels with maximum in tensity laying in the path of the rays traced along the viewing direction see The MIP Renderer on page 23 In combination with the surface pipes you can obtain Transparency 0 00 Brightwe Qian 0 _ i Secondary surface eg Threshold 8 2 Transparency 0 00 very clear representations of the different objects in your image The MIP rendering of one chan
31. ackprojected radius in nanometers r Backprojected means the size of the pinhole as it appears in the specimen plane the physical pinhole radius P divided by the total magnification of the detection system This total magnification is the product of the variable ob jective magnification times a fixed internal magnification r hys ra 1 M obj M system where m is the magnification factor of the objective and m is the fixed magnification of system the system The Huygens Essential User Guide 41 7 Establishing image parameters The equations that you can find in the next pages for different microscopes are intended to orient ate the user in finding out the backprojected value but the idea is always the same given the dia meter of the real pinhole d we apply a factor for unit conversion to obtain the radius in nm and some dividing numbers that take account of the magnification of the microscope These include both the objective and the intrinsic system magnification In some microscopes with pinhole shapes other than circular a geometrical correction will also be needed Airy disk as unit for the backprojected pinhole Some confocal microscopes report the pinhole size diameter with the Airy disk diameter as unit The backprojected pinhole radius can then be computed with 0 61A N PR NA Airydisks 2 with NA the numerical aperture of the lens N the number of Airy disks and the excit
32. all sh To install the software you have to run the file ATX install sh by typing AIX install sh This script will unpack the svi tar Z file under usr local and then will do some post installation tasks and verifica tion After a successful installation it will print the message OK to the screen A directory usr local svi will be created the executables will be installed in Fusr7 Local bin After the installation The license string After a first time installation there is not yet a license available Still you can start the software Without a license it will run in Freeware mode Among others this gives you access to the Li cense tools in the Heir menu The next section explain how to obtain and install a license string On AIX Irix and Linux start the software by clicking its icon or by typing essential intoa Unix shell On Mac OS X and Windows click its icon The software will open in Freeware mode and display the start up window Figure 1 The license key used by all SVI software is a single string per licensed package It may look as fol lows HuEss 2 7 wecnp d tv emnps eom2008Dec3l e7b7c623393d708e frank svi nl 4fce0dbe86e8ca4344dd At startup Huygens Essential searches for a license file huygensLicense which contains a li cense string This license string is provided by SVI via e mail Installing the license string is the same for all platforms though on Linux Irix and AIX only the superuser can do this
33. alue Default The image is searched for a 3D region with the lowest average value The axial size of the re gion is around 0 3 mi cron the lateral size is controlled by the radius parameter which is de fault set to 0 5 micron 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 i Huyge ns Essential by Scientific Volume imaging BV pes File Task Visualization Tools original Stage Three Background Estimation In this section the background value of the image will be estimated For widefield images best select widefield wf for other images the safest is lowest Estimation mode lowest Area Radius micron 0 5 Estimate gt Task info Task report Task plot Image Mmosts rog uhome theo images fabab4 ics Status Moved to stage three of deconvolution l Memory 0 DOO p ADA AA ha Figure 16 Estimating the average background controlled by the radius parameter e WF 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 background is determined by searching this region for the planar region with radius r that has the lowest value Press the Estimate button to continue You may now adapt the value if you like to either by alter ing the value in the E
34. and cropping e stage 1 Tune the parameters This stage will be skipped when Figure 7 The Dictionary The Dictionary and the Questions you entered from the prepro from Her give additional information cessing stage In the prepro cessing stage you have already checked the parameter settings for the intelligent cropper intelligent since it uses a priori knowledge for setting the optimal cropping boundaries automatically You will enter this stage from the latest one when clicking the lt lt REsTart button This is useful if you wish to fine tune your parameters for the best deconvolution result in particular when you like to set your parameters slightly different when using multi channel images see page 17 e stage 2 Inspecting the image histogram e stage 3 Background estimation e stage 4 The deconvolution run e saving the result The different stages will be explained below Loading an image Select Oren from the Fi menu to enter the file browser and move to the directory where your im ages are stored Select the image to be deconvolved e g the faba64 ics ids file pair in the images subdirectory of svi Several formats from microscope vendors are supported If you have TIFF images to be processed please read TIFF file series naming convention on page 51 for the naming convention in order to be able to read a multi dimensional image as a whole When the file is read successfully you can either press START DECONVOLUT
35. areas of NE interest The voxel val bind Ge ues are displayed below Figure 17 Stage 4 starting the deconvolution the image Modify the value as you see fit 2 A Point Spread Function PSF see below was generated from the established microscop ic parameters This took place off the screen and is fully transparent to the user 3 Ifthe size of the computer s RAM is too small to deconvolve the image as a whole it is split up in parts called bricks SVI s Fast Classic Maximum Likelihood Estimation MLE algorithm runs on the image or on all the bricks and fits the deconvolved bricks seamlessly together see Brick wise processing on page 39 OR eene The Point Spread Function PSF One of the basic concepts in image deconvolution is the Point Spread Function The PSF of your microscope is the image which results from imaging a point object in the microscope Due to wave diffraction a point object is imaged spread out into a fuzzy spot the PSF In fluorescence ima ging the PSF completely determines the image formation In other words all microscopic imaging properties are packed into this 3D function The PSF can be obtained in two different ways 1 Generating a theoretical PSF When a measured PSF is not available Huygens Essen tial automatically uses a theoretical PSF The PSF is computed from the microscopic parameters that come with your image and which you have double checked in the prepro cessing P stage or in stag
36. ast mapping mode Time series e Linear default In this mode the pixel values are mapped to screen buffer color intensit ies 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 Where an image contains a few very bright spots and some larger darker structures using Linear mode will result in poor visibility of the darker structures Restor ation 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 in crease 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 col ors see above e Widefield WF mode In restoring widefield images it sometimes happens that blur re moval 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 If you open the Twin Slicer on a time series a second slider is ad ded Both the time slider and the spatial slider have a swing op tion When the spatial swing is pressed the slider moves back and forth when the time swing button is pressed the slider only moves forward i e in the positive time direction 22 Scientific
37. at infinity e Excitation The transparency of the object for the excitation light The less trans parency the more shadow is casted on the subsequent voxels and on the table e Emission The transparency of the object for the emission light The lower the transparency for the emission light the more difficult it is to peer inside or trough the object e The Characteristic object size affects both the excitation and the emission transparency 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 say 10 of its initial value This penetration 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 oblique due to its size To find a reasonable range in transparencies the object size may be altered At start up the object size is computed from the microscopic sampling sizes and number of pixels the image is composed off If your image has not the The Huygens Essential User Guide 25 4 Huygens Essential Visualization Tools correct parameters for example a TIFF series the object size is set according to the de fault parameters as set by the Huygens Essential software and may not be related to the actual object size e F
38. ault SNR settings If available it is a good idea to average 2 5 beads Two photon bead images may look slightly noisy If so set the SNR to 20 and average 4 10 beads To load additional bead images press Loan ADDITIONAL after completing a search The Huygens Essential User Guide 37 6 The PSF Distiller Widefield bead images Images from 160 nm beads should look like smooth fuzzy blobs with no visible noise Use the de fault SNR settings It is not necessary to average any more beads After you have added the last bead recording press Distt to enter the Distillation stage Distillation stage The distillation stage usually requires no user intervention though in some cases a popup will be displayed with a question or a warning All channels will be processed automatically Assembly stage In the assembly stage you can add a previously obtained PSF image file as a channel before or after the current result Press App CHANNEL if you want to do this The Distiller will compare the microscopic image parameters of the selected PSF image and check its content In case there are problems the software will ask you to decide between ignoring the differences and discarding the selected file If you do not want to add channels press Next and then Done This leaves you with a loaded meas ured PSF for immediate use in a subsequent deconvolution run O We advise you to measure the PSF of your system for each recording situation and ce
39. ay of the semi reflecting table is of importance By simulating the propagation of the emitted light through the matter the al gorithm computes the final intensities of all wavelengths the spectrum of the light reaching the viewpoint By default the first channel ch 0 is the red object the second channel is ch 1 is the green object and the third ch 2 is the blue object The properties of the interaction between object and light transparency both for excitation and emission as well as the viewpoint can be adapted interactively by the user to produce dif ferent sceneries Since the volume ren dering process is rather compute intens ive a preview image is displayed see Figure 25 Apart from the viewpoint settings and the optional zooming the following sliders affect the image e Transparencies amp A aa Excitation Emission Table Figure 26 With the SFP renderer excitation and subseguent emission of light of fluorescent materials is simulated Each subsequent voxel in the light beam excitation 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 The light source is drawn here inside the figure but in real is placed at infinite distance as to make the light rays parallel The renderer in Essential is in non perspective mode so called orthogonal projection i e the viewpoint is
40. ble a multi channel PSF from single channel PSFs The PSF Distiller wizard leads you through four stages to carry out the following tasks e Preprocessing P stage to check microscopic im age parameters This an stage is similar to the de convolution P stage e Stage I Averaging stage In this stage all n multiple bead There are currently 7 beads in the accumulator S and one or ne or channels of the image 44 nels one ee Load an additional bead image or click Distill to start the PSF distiller are searched for beads that meet the selection criteria After each suc cessful or unsuccessful search there is the pos Load Additional Distill sibility to load in addi Task info p l Image people theo Demolmages Beadimages psf05 ics tional bead images or go S Status opening image fie Memory 2 to the next stage Figure 30 The PSF Distiller After adding one or more bead e Stage 2 Distill stage In images an average bead image is displayed in the Accumulator this stage the PSF is thumbnail After pressing the Dist button a PSF will be generated in a PSF thumbnail measured from the aver aged beads for all available channels e Stage 3 Assembly stage In case you want to combine results from earlier Distill runs with the current result to obtain a multi channel PSF you can add the earlier result here It is also possible to add single or multi channel earlier results to a cu
41. cal microscopes TCS 4d SP1 NT The size of the square pinhole is given as an 8 bit number which maps to the physical pinhole ra dius given in the following table The Huygens Essential User Guide 43 7 Establishing image parameters Table 1 TCS4d Range begin Range end Reported parameter P 0 255 630 ae Teron a earlier reported as 500 Pinhole geometry square System magnification 4 5 Backprojected pinhole ENN SSS SS Eq 6 radius nm fits Ov Leica TCS4d pinhole parameters This table is believed to be valid for the SP1 and NT models too If the pinhole is specified in Airy disk units see Airy disk as unit for the backprojected radius of a square pinhole on page 43 for information on how to convert to a backprojected radius TCS SP2 The Leica TCS SP2 has a system magnification of 3 6 However the size of its square pinhole is usually specified in Airy disk units making it independent of the actual overall magnification It is dependent though on wavelength and NA of the objective See Airy disk as unit for the backpro jected radius of a square pinhole on page 43 for information on how to convert to a backprojected radius Zeiss confocal microscopes Table 2 Table 3 44 Scientific Volume Imaging Zeiss LSM410 inverted Range begin Range end Reported parameter P 0 255 Diameter micron 0 1000 Pinhole geometry square
42. chO00 tar sich COO 2000 chO1 tar STN 00 _ 7001 GRO Eat SION C00 _ 200k CHUL tar STN 00 2002 _ cno tir siCh 200 2002 cno0l tat SICH E00 2005 NOV Lat STN EO 2000 NOLTE sich CUTI 2000 ND Ba STOCK CUI 2000 chill CiT siCh CUIL ZO0L choc ak SIN FULL 2001 Ong eat STCh Ol 2002 cho0 yt ae SICH EOL 72002 chol tat STN EL 200 NON AE SICH COI 2003 oNOL fit STCh 202 2000 cho0 tat SICH 202 7000 ONUL tit SICh EUZ 2001 choo CIE STN t02 7001 chol tat SICH E02 2002 cho0 tat gren EN 20 FN STON E02 2003 CNI0 118 STC CUZ 2003 CHULL CiT Can I deconvolve a single plane widefield image Yes Single plane WF deconvolution works because the data is extrapolated into a region above and below the plane spanning typically between 10 20 planes of 100 300 nm sampling in z The software generates an appropriate PSF Can I deconvolve a single TIFF image Yes Huygens Essential treats the image as the only known plane of a 3D stack and proceeds as usual Set the z sampling distance to the Nyquist value see Sampling densities on page 40 How do I generate a debug log See http support svi nl wiki DebugMode 52 Scientific Volume Imaging 9 Appendix Addresses and URLs Where can we be reached Support and FAQ Scientific Volume Imaging b v Alexanderlaan 14 1213XS Hilversum The Netherlands You can call us directly by phone 31 35 6859405 or 653 345445 or fax us at 31 35 6837971 or email us at
43. d according to a given criterion and 2 labeling grouping them together 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 isosurface renderer An isosurface is a 3D surface representation of points with equal values in a 3D data distribution Is the 3D equivalent of a 2D contour line O The Object Analyzer is an extended optional tool and is enabled by a A flag in the license string see License string details on page 49 How to use the Object Analyzer To start the Object Analyzer in Huygens Essential right click on an image s thumbnail to open the contextual menu then select SHow IN OBJECT ANALYZER Alternatively you can select an image s thumbnail then in the menu bar select VISUALIZATION gt LAUNCH OBJECT ANALYZER Wait for the ana lyzer to initialize and to compute the first image based on the default parameters It is recommen ded to use a deconvolved image 28 Scientific Volume Imaging 5 Huygens Essential Analysis Tools EG Huygens Object Analyzer original Zoom 2 05 Mouse mode k amp db Frame selection Frame 0 0 Render pipes Primary surface amp ROI control Threshold 110 40 Tilt Seed thr 25 jarbage vo 0 deg Transparency 0 60 Brightness 1 00 Chan 1 B
44. d microscopic template and a deconvolution template which can all be updated once faskline is added Adding Task Lines can be done one image at time or more images at the same time You can use the two buttons with ihe plusses for thal The progress of the Batch Processor and the report form each individual task is shown in the Batch Processing Overviina info Progress Repon Ready Figure 20 Batch Processor main window e Save location is the location where the resulting images will be placed during the batch run With the two folder buttons you can respectively select a location or create a new one in the currently selected folder 18 Scientific Volume Imaging 3 The image restoration process Usage Current task lines 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 micro scopic 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 Add Task Line s to add tasks to the list one by one or many at the same time The text tabs Info Progress and Report give information about the whole process in its different stages Run button to launch the process when everything is configured Adding one task First let us try adding a single task fuid task line s Select an image file micro template decon
45. d on page 39 Subsequently you do a restor ation run and inspect the result for artifacts and residue background If you are confident all is fine you resume the restoration with a higher SNR setting say 30 50 and perhaps a higher back ground The software will ask you to continue were you left off keeping improving the image quite recommendable or to start from the raw image again with the new parameters A new result 48 Scientific Volume Imaging 8 Improving the quality of your images will be generated to compare with the previous one for instance using the Twin Slicer You can repeat this several times and at the end you will be asked to select the best result as the final one If you have done this a previous time for a similar image then of course you just use the values es tablished then Find a detailed example in the following wiki article http support svi nl wiki DeconvolutionProcedure 9 Appendix License string details You can have detailed information about your license strings by going to HELP gt License select the license string you want to inspect and click EXPLAIN LICENSE This is a License String example HuEss 2 7 wecnp4 d ft demnps 2008Sep30 33dfa680a8402167 into svi nl ces3caddas ch342151d3 As from Huygens Compute engine version 2 4 the license strings have the following format lt prod gt lt vers gt lt micr gt lt server gt lt flags gt lt lock gt lt date
46. different data channels for isosurface rendering one in each of the two avaliable surface graphic pipes Use the threshold sliders to apply different threshold levels to your pipes to select what voxels are considered to form objects based on their intensities The remaining adjacent voxels are grouped together to determine independent volumes that will be represented with different colors 1 The image is segmented into objects by an effective seed and threshold level and 6 con nection technique The image is first explored for features that have intensities above the seed level then the selection is expanded around this seed to capture all the neighbor voxels down to the threshold If the seed is set to zero then the segmentation is done by a standard threshold level Voxels connected by one face are grouped together 2 To remove too small objects in an early stage from the analysis a garbage volume in pixels can be set below which objects are discarded 3 The segmented and automatically labeled image is shown as a colored isosurface image Each object is assigned a different hue from a range of hues selectable with the hue se lector see p 27 Because the isosurface renderer can recompute a new isosurface rapidly the thresholds can be ma nipulated interactively Object removal Uninteresting objects can be deleted one by one by clicking on them in the delete object mouse mode explained above All the voxels in a channel inside
47. e ox Figure 3 The License manager dialog Left as displayed on Mac OS X right as displayed on Irix The License manager allows you to add delete and troubleshoot licenses Enter your new license here Cancel Add license Figure 4 The Add License dialog box Location of the license file The license string is added to the file huygensLicense in the svi directory On the different supported platforms this is located in I Use your operating system s generic copy paste operations Mac OS X apple c apple v Windows control c control v Linux Irix Aix on most common desktops copying is done simply by marking the text area with the mouse and pasting by either middle mouse click or control v The Huygens Essential User Guide 3 2 Installing Huygens Essential e AIX Irix and Linux usr local svi e Mac OS X depends on where you installed the software A typical example is Applications SVI e Windows C Program Files SVI On AIX 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 Restart Huygens Essential to activate the new license This will enable the deconvolution or PSF distiller functionality Trouble shooting license strings License details explained The license string as used by SVI has Here all details will be listed for the license which is shown in blu
48. e Help You also can reduce the number of time frames by selecting Time gt SELECT FRAMES from the Crop menu as shown in Figure 14 This applies to time series see Time series on page 8 Removing channels You can remove channels from a multi channel image see p 17 using the crop tool s channels selecting tool CHANNELS gt SELECT CHANNELS Stage 1 Parameter tuning Stage 1 enables you to tune your para meter settings as set in the pre pro cessing stage After you have finish cropping stage 1 is skipped and you will directly jump into the image histogram stage 2 Still you may wish to tune your parameter settings afterwards You can enter stage 1 by pressing the RESTART button in the latest stage Stage 2 The image histogram Figure 13 The Crop tool The next stage shows you the image histogram The histogram image is an important statistical tool for inspecting your image It is included to let you spot problems that might have occurred during the recording It has no image manipulation options as such it just may prevent you from future recording problems The histogram shows the number of pixels as a function of the in tensity gray value or groups of intensities If your image is an 8 bit image gray values from 0 255 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 bits gray values are collected to form a bin for e
49. e after this paragraph Each license element will be shown on a separate line the Same appearance on all supported If a license or license element is not valid for the current product then this will be shown in red platforms For each product you need to HuEss 2 4 wonp d t emnps eom2002Jun27 adebdadl476692f4 hansesvi nl 74818bE have a license string installed Select a license string in the License manager There were no obvious reasons found why this license i would not be valid Product name HuEss and press the EXPLAIN LICENSE button All This license product name matches the currently running product 1 1 1 1 Version 2 4 details for the current license will be lis FE et ted If you run into licensing problems Ks Ge en s W enables Widefield microscope functionality you May use this information to analyze e enables Confocal microscope functionality i n enables Nipkow disk microscope functionality the problem See License string details n enables multi photon microscope functionality on page 49 Updating the software Figure 5 The License details Download the new version from the SVI website at http www svi n1l Proceed with the installation as explained above A Do not uninstall the old version as this will delete your license string On Mac OS X make sure you make a backup of the license string in a safe place before you remove the previous installation Removing the softwa
50. e one Because a theoretical PSF can be generated without any user intervention Huygens Essential does the calculation in the background without any notice O Images affected by spherical aberration S A due to a refractive index mismatch are better re stored through the use of theoretical depth dependent PSF s Read about S A on page 46 and how to correct it on page 48 2 Measuring a PSF By using the PSF Distiller or the tools in Huygens Professional you can derive a measured PSF from images of small lt 200 nm fluorescent beads You can load a previously measured PSF with Fe gt Oren PSF Main menu If you load a PSF Huygens Essential will automatically use it If the measured PSF contains less 14 Scientific Volume Imaging 3 The image restoration process channels than the image a theoretical PSF will be generated for the channels where there is no PSF available See The PSF Distiller on page 36 for more information on measuring a PSF D 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 settings as the bead image s The Classical Maximum Likelihood Estimation CMLE algorithm Huygens Essential uses the Classical Maximum Likelihood Estimation for the deconvolution pro cess This method is an extremely versatile algorithm applicable for all types of data sets The following option values may be set Number of iterati
51. ential and opens the main win dow Figure 6 Task info If the shell is unable to find this PE command then typing the full L Memory 7 sel path should help Figure 6 The start up window The gray area in the upper part is the work area where the thumbnail representation of the original image and its individual channels will be placed The blue window in the left will show help text task reports and supporting information during the various processing stages In the gray bottom right field different If this still does not help then dialog boxes will appear during processing Also intermediate deconvolution results are shown here The bottom bar is a status area usr sbin essential Huygens Essential has not been installed correctly Adding to the shell search path Users of the csh or tcsh shell can add the usr local bin or usr sbin directory to their shell search path by adding the following line to the cshrc file in their home directory set path usr local bin Spath orset path usr sbin Spath You can inquire your shell by typing echo S shell Support on installation If you find any problem in installing the program or the licenses that you could not solve with the guidelines here included please search the knowledge database or contact SVI on the addresses on page 53 3 The image restoration process The processing stages Huygens Essential guides you through the process of microscopic image deconvo
52. ersecting with other With the Simp te depth only the piece of surface closest to the viewer s eye screens the others behind it with its corresponding transparency level With the Normat depth up to two pieces of surface are considered to screen other objects Thus one object B inside the surface A will appear less screened than a third object C behind A B is only screened by the piece of surface A closer to the viewer s eye while the object C is screened by two pieces of surface A The Deep option will consider many more screening levels making the final rendering more com plex The Huygens Essential User Guide 27 5 Huygens Essential Analysis Tools 5 Huygens Essential Analysis Tools Huygens Essential is extended with new tools for interactive analysis of 3D and 4D microscopic images The Colocalization Analyzer is present in Huygens Essential since version 3 0 and the Object Analyzer since version 3 1 The Object Analyzer The interactive Object Analyzer tool allows you to obtain statistics of individual objects by click ing 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 connected one to another Interesting voxels are distinguished from the background by using a seed and threshold criterion Therefore defining an image s objects implies 1 segmentation separating interesting voxels from the backgroun
53. essential Doc 2 9 0406 Contents Table of Contents 1 What is Huygens Essential 1 2 Installing Huygens Essential 1 MacOSX I Microsoft Windows 1 Linux 2 SGI Irix 6 5 2 IBM AIX 5 2 2 After the installation 2 The license string 2 Obtaining a license string 3 Installing the license string 3 Location of the license file 3 Trouble shooting license strings 4 Updating the software 4 Removing the software 4 System requirements for Huygens Essential 5 Windows and Linux 5 Mac OSX 5 SGI Irix IBM AIX 5 Running the program 5 Support on installation 6 3 The image restoration process 6 The processing stages 6 Loading an image 7 Preprocessing 8 Converting a data set optional 8 Time series 8 Adapting the image 8 Processing brightfield images 8 Setting the image channel colors 9 Image statistics 9 Verifying the microscopic parameters 9 Parameters Templates 10 The intelligent cropper 11 Cropping animageinxyz 11 Cropping an image in time 12 Removing channels 12 Stage 1 Parameter tuning 12 Stage 2 The image histogram 12 Stage 3 Estimate the average background in the image 13 Stage 4 Deconvolution 14 The Point Spread Function PSF 14 The Classical Maximum Likelihood Estimation CMLE algorithm 15 Number of iterations 15 Signal to noise ratio 15 Quality threshold 15 Iteration mode 15 Bleaching correction 15 Stopping the MLE algorithm 16 Finishing or restarting a deconvolution run 16 z drift corrector fo
54. focal shift is dependent on the depth of the focus in the specimen If the mismatch is large e g when going from oil immersion into a watery medium the PSF will become asymmetric at depths of already a few micron Especially harmful for WE deconvolution Workaround keep the z range of the data as small as possible Solu tion use a water immersion lens e Total internal reflection When the lens numerical aperture NA is larger than the medium refractive index n total internal reflection will occur causing excitation light to be bounced back into the lens and limiting the effective NA If S A is unavoidable you can still improve the image during restoration using an adaptive point spread function See Spherical aberration correction on page 48 Clipping The light intensities from the microscopic object are converted to electrical signals that pass an adjustable amplifier Also an electrical DC component can be added or subtracted by the micro scope operator The electrical signal may thus range from negative to highly positive These elec trical signals must be converted to numbers processed by the computer This converting stage is 46 Scientific Volume Imaging 8 Improving the quality of your images Undersampling done in the CCD camera and its electronics Most CCD cameras have an 12 bit converter limiting the output numbers to a range of 0 to 4095 Negative input signals are usually converted to 0 while positive input values
55. g into the text field using your keyboard Complete the procedure using the App Licens to add the string to the huygensLicense file Please try to avoid typing the license string by hand any small typing error will invalidate the li cense With an invalid license the software will remain in Freeware mode Your current license is desktop system The license options are widefield confocal nipkow disk multi photon time Your license file Users hans huygensLicense Your current license is desktop system The license options are widefield confocal nipkow disk multi photon time Yourlicense file contains the following licenses Your license file contains the following licenses HuEss 2 5 wonp d t emnps eon2003Jun23 2372e2 612080 3 theofsvi nl 9blf52a60373b2dA7e3bd Flu R 2 4 w d emnps eom2002Jun27 a9e84ad0476692f4 hans svi nl O64f42bf357flaaat2af HuPro 2 4 wenp d t emnps eom2002Jun27 a9e84ad0476692f4 hans svi nl S8b4febc38eb6922831b7 HuScript 2 4 wep d emnps eom2002Jun27 a9e84ad0476692f4 hans svi nl fabaSdf4cch45dd767a2 HuEss 2 4 wenp d t emnps eon2002Jun27 a9e84ad0476692f4 hansesvi nl 74818h89c85282h2ble 7 HuBatch 2 3 wenp d t emnps eom2003Ju131 a9e84ad0476692f4 bert svi nl lef37696ee1d08b95Tee HuMotif 2 3 wenp d tv emnps eom2003Jul31 a9e84ad04 76692f4 bert svi nl 9531f48119eleSdl4fe7 Add a new license Explain license Delete license Add a new license Explain license Delete licens
56. gt lt sysid gt lt email gt lt checksum gt Where lt prod gt can be one of HuEss HuScript HuPro FluVR lt vers gt is the version number which is 2 4 or higher lt micr gt consists of one or more characters representing microscope types Widefield enables you to deconvolve WF data Confocal enables you to deconvolve confocal data Nipkow Disk enables you to deconvolve data from Nipkow spinning Disks multi photon for 2 or more photon microscopes 4 pi for experimental microscopes Bm Daa lt server gt can be one of desktop 1 or 2 CPU s small server 3 8 CPU s medium server 9 32 CPU s large server 33 64 CPU s extreme server 65 or more CPU s x fF 38 Hn O Remark for Linux and Mac OS X multi processing users d desktop 1 thread m medium server max 2 threads The Huygens Essential User Guide 49 9 Appendix 1 large server max 4 threads x extreme server 5 threads and more lt extra flags gt which enables additional functionality Distiller option enables obtaining PSF s from measured beads Time series option enables deconvolution of time series either 2D time or 3D time t v Surface Renderer visualization option C Colocalization Analyzer option A Object Analyzer option lt lock gt Set of license policy or locking flags having of one or more characters d License expiry date e Email address m Hardware s
57. he replacing circular pinhole 42 Scientific Volume Imaging 7 Establishing image parameters 4 _ d m r and thus r Ap gt d 2r aS For a square pinhole shape 1 yr 0 564 For circular pinholes shape 0 5 to just convert from diameter to radius Airy disk as unit for the backprojected radius of a square pinhole The relation between the edge size of square pinhole in Airy disk units and the backprojected radi us is a combination of equations 2 and 4 Taking into account that equation 2 already converts a diameter into a radius we get 0 69 oy N Airydisks gt MA 5 with N Ads the number of Airy disks Computing the backprojected pinhole distance in Nipkow spinning disks As is the case for the backprojected pinhole diameter the distances between the pinholes in spin ning disks must be divided by the system magnification For the most used Yokogawa for ex ample the pinhole physical distance is 250 um You can check this by stopping the disk So with an x100 lens the backprojected distance is about 2 5 um If an extra zoom lens is placed between the disk and the sample its magnification must be also considered Pinhole radius tables In the case of the Leica TCS the Biorad MRC the Olympus Fluoview and the Zeiss LSM micro scopes the pinhole geometry and system magnification is known resulting in the conversion for mulas from the following tables Leica confo
58. he selected image on the left By clicking on the menu bar below the image you can select a dif ferent image Likewise the bar below the right display field gives access to one of the other im ages currently present in Huygens Essential see Figure 21 Currently only two images with the same dimensions can be displayed at the same time If you select the same image twice you may compare different slices from the same image For this first move the slider until the desired position then click on one of the image s name to DisaB_e the action of the slider on it the slider will then affect only the other image view see Fig ure 22 Click the name button to select an me image disable the original 0 slider function or center image center the image disable original Slice x y i Zoom 200 NE i Color False colors 7 SETTE Lineair k F 1 2 ure 2 The T win MEN 25 150 400 Greyscale Linear Slicer applied to a 3D x z 50 200 500 True colors Compressed dataset The selected o yz 100 ll 300 600 False colors Wide Field disp lay cai tings ire highlighted in blue 32 win p O ee 0 10 20 30 40 50 60 Pixel intensity values for the cursor position on the image are displayed at the bottom of the win dow You can move the image by clicking the left mouse button and keeping it pressed while moving the image to the desired po
59. he way colocalization maps are represented on the screen and objects splitted appart from each other based on the locally calculated colocaliza tion What affects colocalization as explained in the colocalization theory at the SVI wiki are the backgrounds In the computation of Manders coefficients the background values act like in this ex ample to the computation of m only pixels in R contribute when their corresponding pixel in G is above the background Read more More information is available at http support svi nl wiki ColocalizationAnalyzer The Huygens Essential User Guide 35 6 The PSF Distiller 6 The PSF Distiller Starting from version 2 5 7 Huygens Essential is optionally equipped with the PSF Distiller to al low you to measure your microscope s PSF Measured PSFs allow you to improve deconvolution results and may also serve as a quality test for your microscope It is a calibration of the micro scope in the sense of relating a physical known object with what the microscope actually meas ures A The PSF Distiller is an optional component enabled by a f flag in the license string Please refer to the License string details on page 49 The PSF Distiller is an intelligent wizard based tool that 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 PSFs from information collected from multi wavelength beads or assem
60. hing image parameters 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 res olution 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 with negligible loss of information In cases were the pinhole is much larger the lateral imaging properties much resemble those of a WE 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 Read more on the SVI wiki http support svi nl wiki NyquistRate axial WE axial confocal lateral WF 1000 lateral confocal 100 Sample density nm 10 0 4 0 6 0 8 2 1 4 Numerical Aperture Figure 33 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 Computing the backprojected pinhole radius A Throughout the Huygens Professional and Huygens Essential pinhole sizes of confocal systems are specified as the b
61. iPhoton three ChannelConf lt Task info Task report Task plot small fluor Image fabab4 ics SOS Status Imported microscopic template widefield a C2 e C2 o Memory 0 Figure 12 Exporting a microscopical parameters template The intelligent cropper The time needed to deconvolve an image increases more than propor tional with its volume Therefore deconvolution can be accelerated considerably by cropping the image Huygens Essential is equipped with an intelligent cropper which automatically surveys the image to find a reasonable proposal for the crop region In computing this initial proposal the mi croscopical 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 para meters it is recommended to use the intelligent cropper as final step in the preprocessing stage but you can launch it before the restoration process from the TooLs gt Crop ORIGINAL IMAGE menu Once you have cropped your image during the guided restoration process you can not crop it again ex cept after closing the image and reloading it again Cropping an image in x y z After you have verified your image parameters the Crop tool is launched if you press the Yes but ton to the question Launch the cropper The cropper will look as in Figure 13 the image will be in gray scale mode if it is a single channel Red lines ind
62. icate the borders of the proposed crop ping region This is computed from the image content and the microscopic parameters at launch time of the cropper The cropper allows manual adjustment of the proposed crop region To adjust the crop region put the cursor inside the red boundary press the left mouse button and keep it pressed to sweep out a volume Accept the new borders by pressing the Crop button Do not crop the object too tightly because you would remove blur information relevant for deconvolution Do not crop the image to make it too large along the optical axis Z an aspect ratio close to 1 1 1 or less than 1 for Z is much better The three views shown are Maximum Intensity Projections MIP s along the main axes The pro jections are computed by tracing parallel rays perpendicular to the projection plane through the data volume each ray ending in a pixel of the projection image The maximum intensity value found in each ray path is projected For example each pixel in the xy projection image corres ponds with the maximum value in the vertical column of voxels above it By default the projections are over the whole dataset including all the frames in time series but this might be confusing sometimes The small colored triangles can be used to restrict the projec tions within a specific range of slices The Huygens Essential User Guide 11 3 The image restoration process Cropping an image in time File Channels Tim
63. if Huygens Essential will read the series into a single 3D image Because TIFF s usually carry no additional microscopic information check the parameters carefully TIFF file series naming convention If you have TIFF images to be read into the Huygens Professional or the Huygens Essential you should know about the naming convention used If you select a file from a numbered series the selected file and the following files will be inter preted as x y planes of a 3D stack and read into a 3D image of suitable size and channel configur ation A one channel 3D images only go with numbers As an example a dataset called c with 32 slices numbers as follows OK eas i ob be If you wish to work on the complete c stack you only have to select c000 tif while opening If you select file c020 tif the first 20 slices will be skipped Numbered series without the TIFF extension like c04 GOD cig are not read in as a series Huygens Essential and Professional read and write TIFF series with Leica style numbering if there are more channels different wavelengths slices or frames in time An image of four slices and two frames is named with Leica style numbering as follows onto 32 000 er Cot 00 7001 SEE C2200 20072 ETE PN ZO0S sc LE C EOL 22 000 SAG ae eT OL 2001 trr GEL JOE os 200 sear The Huygens Essential User Guide 51 9 Appendix And an image sTCh of four slices three frames and two channels SICH t00 2000 C
64. info svi nl http www svi nl Distributors A list of distributors can be found on our web site http www svi nl company distributors php Knowledge database FAQ It is good to know that an extensive support base is available where you may find answers to ques tions that come to front while reading this document FAQ s are available for many items such as deconvolution and general microscopy installation memory management visualization file formats platforms and reported bugs httipyy support syi unl On this web site you will also find a form to submit questions to SVI s support team The SVI wiki The SVI wiki is a rapidly expanding public knowledge resource on 3D microscopy and deconvolu tion Based on the WikiWikiWeb principle it is open to contributions from every visitor In addi tion it serves as a support medium for SVI customers and relations to discuss different aspects of SVI s Huygens Software The SVI wiki can be found in http support svi wiki Starting points Here you can find a selection of good starting points in the SVI wiki to learn more about the Huy gens Software and microscopical imaging e On the parameters describing your imaging conditions sampling numerical aperture pinholes seehttp support svi nl wiki MicroscopicParameters The Huygens Essential User Guide 53 9 Appendix Quick reference On the restoration parameters signal to noise ratio backgrounds quality crite
65. ing density depends on the system optics and is determined by the Nyquist rate it is recom mended that acquisition is done according to it as much as possible If this was not achieved the actual sampling distances must be used in any case as parameters when doing deconvolution Imaging with one sampling density but using different values for deconvolution will produce wrong image restorations It is very important for the quality of a deconvolution result that all information generated by the optics of the microscope is captured in digital form It can be shown that if the sampling density is higher than a certain value all information about the object is captured We will call this value the critical sampling distance corresponding to the Nyquist rate 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 to the contrary Figure 33 shows the dependency of this critical sampling distance on the NA for specific wavelengths To apply the plot of Figure 33 to different wavelengths you can simply scale the ver tical axis with the wavelength Example you are working with a WF microscope with NA 1 3 at emission wavelength 570 nm From the plot you read that the critical lateral Nyquist sampling distance at 500 nm emission is 95 nm so in your case this becomes 570 500 x 95 nm 108 nm 40 Scientific Volume Imaging 7 Establis
66. ion process that follows Stage 3 Estimate the average background in the image In this stage the average back ground in a volume image is es timated The average background is thought to correspond with the noise free equivalent of the back ground in the measured noisy a Ris a E EEE File Task Visualization Tools original Workspace Computing histogram Done The histogram is displayed in the taskplot window on the left You can use the histogram image to check whether there is no saturation of the pixel values Also you can see whether a significant background is present If you want to generate the histogram with a different vertical mapping function you can select Again otherwise choose Accept to proceed Histogram lin scale Taskreport MSAN Image fusrflocal sviflmages fabab4 ics Gr Status Histogram computed Memory 0 Figure 15 The image histogram The vertical mapping mode can be selected from linear logarithmic or sigmoid image It is determined by searching the image first for a region with low values Subsequently the value for the background is determined by searching in this region for the area with radius r which has the lowest average value It is important for the search strategy that the microscopic parameters of the image are correct in especially the sampling distance and the microscope type The following choices are possible here e Lowest v
67. ke an animation along time frames The SFP Renderer Summary SFP fundamentals Starting from Essential 2 5 a simplified version of SVI s high end volume renderer FluVR Fluor escence Volume Renderer is available for visualizing your volumetric object from a selectable viewpoint Like FluVR this renderer is based on taking the volume image as a distribution of fluorescent material simulating what happens if the material is excited and how the subsequently emitted light travels to the observer The computational work is done by the Simulated Fluores cence Process SFP algorithm The ray tracing technique does not require a special graphical board as the polygon based tech niques do To start the SFP renderer right click on an image s thumbnail to open the contextual menu then select Snow In SFP RENDERER See Figure 25 A virtual light source produces excitation light that illuminates the object This casts shadows either on parts of the object itself or on a table below it The interaction between the excitation light and the object provokes the emission light that also interacts with the object before it reaches the eye of the viewer see Figure 26 The voxel values in the image ee elves are taken as the density of a fluorescent material If the voxels are multiparameter multi channel in microscopic Home Sethome Light dir parlance see page 17 each Ree aici parameter is taken as a different Soft
68. l data text or image files through the Fire menu Backgrounds vs thresholds in colocalization Backgrounds are for removing signal prior to the calculation In an ideally restored image that would not be necessary because all the signal present in the image is good signal The back ground would have been removed during the image restoration Thresholds are 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 Most of the times you have some non zero colocalization every where but you are probably interested in regions where colocalization exceeds let us say a value of 20 of the maximum With the backgrounds you discard data based on voxels intensities high intensity regions are left low intensity regions are disregarded But that says nothing about colocalization You can very well have high colocalization levels in regions with low voxel intensity Therefore you should not use backgrounds to remove signal that can still have some colocalization level Ideally you consider all the signal then study the colocalization levels using thresholds to split regions of high colocalization from regions with low colocalization something that you can not know beforehand Backgrounds are for removing really constant background signals Thresholds do not affect colocalization but only t
69. lapping Backgrounds are for removing signal that would lead to spurious colocalization prior to the calcu lation This is intended for minor tuning or just in case you really need to calculate colocalization in raw images that still have all the measured background See Backgrounds vs thresholds in colocalization below Next we select what colocalization map we want to calculate Pearson Overlap Manders M K or H 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 colocal ization locally In a map a single colocalization value is calculated per voxel creating a 3D distri bution that is represented in a 3D image by isosurfaces The Colocalization Analyzer computes only the map selected by the user but it always computes all the available coefficients Finally we click on Compute The Frame colocalization coefficients pane will show all available colocalization coefficients for the whole frame The obtained colocalization map is represented in the renderer window by iso colocalization sur faces regions in which the degree of colocalization exceeds a certain value become objects This 34 Scientific Volume Imaging 5 Huygens Essential Analysis Tools certain value can be controlled by the threshold slider in the iso colocalization surface paramet ers You can control the transpa
70. litudes Taking the absolute square des troys all phase information 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 es pecially if one is dealing with a high contrast image In practice one goes about deconvolving brightfield images by inverting them using Toots gt INVERT IMAGE and processing them further as incoherent fluorescence widefield images Still 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 de convolution run you may reverse to the original contrast setting 8 Scientific Volume Imaging 3 The image restoration process Setting the image channel colors The Color picker tool Toots gt SET CHANNEL COLORS allows you to alter the colors for the different image channels see Multi chan channel 1 J nel images on page 17 The color channel ET of a particular channel can be ed ited by clicking the corresponding channel 3 B button This opens a platform spe a cific color editor Figure 8 Cancel Apply Image statistics Figure 8 The Image Color Picker Clicking on a channel button top opens a color editor image and
71. lution also re ferred to as restoration in several stages Each stage is composed of one or more tasks While proceeding each stage is briefly described in the bottom left Task Info window pane The stages progress is indicated at the right side of the status bar see Figure 15 on page 13 Additional in formation can be found in the HELP gt Questions and HE tp gt Dictionary Figure 7 6 Scientific Volume Imaging 3 The image restoration process The following steps and stages are to be x followed Welcome to the Dictionary of the Huygens Essential This help item contains often used words related to microscope imaging and the use of the Huygens Essential e Opening an image A demo im eshngcaneelien age the faba64 ics ids file pair is placed in the Point Spread Function PSF ima ges subdirectory of SV Refractive indexes Relative background e stage P The Preprocessing Sample sizes Signal to Noise ratio SNR stage loading an image con pala 8 The Numerical Aperture of an objective is the refractive index of the immersion medium times the sine of the half aperture angle Because the sine lt 1 0 the NA is always lower than the medium refractive index If 1 4 N oil immersion lens is used to image into a watery object total internal reflection will truncate the NA s a rule of thumb the effective NA is 1 lower than the manufacturer s specification verting data sets parameter check
72. mbly stage 38 7 Establishing image parameters 38 Image size 38 Brick wise processing 39 Signal to Noise Ratio SNR 39 Blacklevel 40 Sampling densities 40 Computing the backprojected pinhole radius 41 Airy disk as unit for the backprojected pinhole 42 Converting from integer parameter 42 Shape factor 42 Airy disk as unit for the backprojected radius of a square pinhole 43 ii Scientific Volume Imaging Contents Computing the backprojected pinhole distance in Nipkow spinning disks 43 Pinhole radius tables 43 Leica confocal microscopes 43 TCS 4d SP1 NT 43 TCS SP2 44 Zeiss confocal microscopes 44 Olympus confocal microscopes 45 Biorad confocal microscopes 45 Checking the Biorad system magnification 45 A supplied calibration curve 46 8 Improving the quality of your images 46 Data acquisition pitfalls 46 Refractive index mismatch 46 Clipping 46 Undersampling 47 Do not undersample to limit photodamage 47 Bleaching 47 Illumination instability 47 Mechanical instability 47 Thermal effects 48 Internal reflection 48 Deconvolution improvements 48 Acquire an experimental PSF 48 Spherical aberration correction 48 Improve the estimated parameters 48 9 Appendix 49 License string details 49 Questions 50 Where can I find support on the web 50 What does the quality factor mean while running Huygens 50 Can I deconvolve a TIFF series 51 TIFF file series naming convention 51 Can I deconvolve a single plane widefield image
73. n Still the extra quality gain becomes very small at higher iteration counts Iteration mode In Fast mone highly recommended the iteration steps are bigger than in Hisn Quarry mode More information can be read in the Dictionary from the HELP menu Bleaching correction The data is inspected for bleaching 3D images and time series of WF images will always be cor rected Confocal images can only be corrected if they are part of a time series and when the bleaching over time shows exponential behavior 4 Huygens Professional also has Quick MLE time Quick Tikhonov Miller and Iterative Constrained Tikhonov Miller The Huygens Essential User Guide 15 3 The image restoration process Stopping the MLE algorithm Pressing Restore starts the iterative MLE algorithm a Stop button appears Pressing Stor halts the iterations and retrieves the result from the previous iteration If the first iteration is not yet com plete a empty image will result Finishing or restarting a deconvolution run When a deconvolution run is finished use the Twin Slicer page 21 to inspect the result in detail Depending on the outcome of that you can choose AGAIN REsuME or ACCEPT Acan discards the present result and re runs the deconvolution possibly with different paramet ers Resume re runs the MLE procedure without discarding the result and with the possibility to change the deconvolution parameters The software will ask you to continue
74. n you have set all the rendering options click Render to create the final view that you can save as a TIFF Bounding box transparency Channel parameters cho Chi ch2 Soft threshold 0 01 Render Twist 122 deg Animate image Ready Figure 24 The Maximum Intensity Projection MIP Renderer You will find different rendering options on the window and also in the Options menu The configurable parameters are the ren dering size and quality the appearance of the bounding box and the mode of the soft thresholds applied to the image channels A soft threshold is a preprocessing tool that reduces the background in the image so voxels with intensity values below the threshold value become more transparent Contrary to a standard threshold that 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 smooth transitions between the original an the deleted values If the original value S of a voxel is S gt threshold value range 2 then the final filtered value D does not change D 5 If S lt threshold value range 2 then the voxel is deleted D 0 For the values in between a smooth function is applied if threshold value range 2 lt S lt threshold value range 2 then D f S according to a shape function which in this case is a sinusoidal By changing the parameters in Options gt Sort THRESHOLD mode fr
75. nd Twist slides or by dragging the mouse pointer on the object view Also try changing the zoom At the beginning the iso colocalization surface sliders are deactivated as we have to calculate a colocalization map first First we select the data to analyze For time series the frame selector selects the time coordin ate 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 overlapping the histogram pixels trend to concentrate along the diagonal y x line On the other side total absence of over lapping would produce a 2D histogram with values only on the coordinate axes By moving the colored background lines in the histogram or by changing the numeric values in the input fields we specify the backgrounds for the two selected channels These values are sub tracted 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 estima
76. ndable to measure an experimental PSF and if it is very different from the ideal one use it for deconvolution instead of the theoretical one Obtaining an experimental PSF is based on recording the image of a well know probe as close as the ideal subresolution light source as possible and distilling a PSF from it using The PSF Distil ler as explained on page 36 Spherical aberration correction The Huygens software automatically adapts the theoretical point spread function PSF to the sample depth to correct for spherical aberration in case of a refractive index mismatch For that the program considers that the coverslip is placed at the bottom of the image at lower Z coordin ates as in an inverted microscope If your image suffers from a refractive index mismatch you should adapt the image to this condition before the restoration the Huygens Essential has a MIrror ALONG Z tool to flip the image in case your coverslip is on the top In case your image suffers from high spherical aberration it might be better to use a theoretical PSF with this depth dependent correction than an experimental one Read more in http support svi nl wiki SphericalAberration Improve the estimated parameters This is an usual procedure to improve the estimated parameters for deconvolution you start out with a signal to noise ratio SNR derived either from an estimate of the number of photons in the image or simply from a visual inspection as explaine
77. nel at hand just as you can with single channel data When you are done press Accept in the last stage 4 screen This will cause the next channel to be selected for restoration Proceed as usual with that channel and the remaining channels If you do not want to process all the channels in an image you may skip one or more channels When you press Accept for the last channel you enter a screen which allows you to select the res ults which you want to combine into the final deconvolved multi channel image This means that up to this point you can still change your mind as to which of the results you want to combine even in what order Once you press Accept a multi channel image named Restored is created To save it go to the FiLE gt SAVE RESTORED AS Menu The Huygens Essential User Guide 17 3 The image restoration process Batch processor Start up Once you know how to deal with a particular kind of dataset and are sure of the restoration para meters see Improve the estimated parameters on page 48 you can restore a couple or more of similar datasets automatically This is called batch processing A batch process is made of independent image restoration tasks one per image that are executed one by one until all are finished Depending on the multithread capabilities of the computer mul tiple tasks can be executed in parallel You can for example program batch scripts using Huygens Scripting that enables you t
78. o run scripts written in Tcl using the extensive set of Tcl Huygens image processing commands You can also configure batch processes easily using the interactive Huygens Batch Processor The Batch Processor is the tool to do large scale deconvolution of multiple images within the Huygens Essential To launch the Batch Processor first open Huygens Essential then click on the menu DECONVOLUTION gt BATCH PROCESSOR Window description These are the different elements that form the window Huygens Essential Batch Processor File Task Save location usr people josef svifBatch sl Ea Current task lines SS EN MiA 266_22VARRA Ism F o O emie Listed avsph green ICs cme Listen green Jsm cmle Listed LeidenAR1 max dec_psfics Paranti cmle Listed E LeidenART max dec mstics Paranti cmle Liss tend D LeidenAR1 max dec met V 328 07 Paranti cmile Listed a LeidenAR1 maxs der mstByledcs Parami cmle Lis tid D Faid task line s Select an image file micro template Batch processing decon template How to use Ine Balch Processor quickly Save Location is the location where the resulting images will be placed during the baich run With the bwo folder button you can respectively select location or create a new one in the current selected folder Currant Task Lines is the list of tasks Tasks are small jobs thal vill be processed by the Baich Porcessor one by one Each task line consists of an image an
79. of the processor board which can be found in the first line of the output of the hinv command The s flag is only meaningful for PCs It enables additional matching on the processor core step ping number which is a notation from Intel like a revision number that indicates the changes or improvements inside the CPU s instructions set Questions Where can I find support on the web See Addresses and URLs on page 53 What does the quality factor mean while running Huygens Deconvolution as it is done in Huygens Essential hinges around the idea of finding an as good as possible estimate of the object that is imaged by the microscope To assess the quality of an estim ate Huygens Essential computes the image of each estimate as it would appear in the microscope 50 Scientific Volume Imaging 9 Appendix and compares it 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 estimate will yield a better quality factor The quality factor as reported by Huygens Es sential is a measure relative to the first estimate and therefore a number greater or equal to 1 If the increase in quality drops below a threshold the iterations are stopped See also Quality threshold on page 15 Can I deconvolve a TIFF series Yes if the series is a numbered series like slice001 tif slice002 tif sliceOnn t
80. olution Best cause of ac 10 Also available on line in http support svi nl wiki NyquistRate The Huygens Essential User Guide 47 8 Improving the quality of your images Thermal effects Internal reflection tion 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 are known to affect calibration of the z stage especially if piezo actuators without feedback control are used In particular harmful for WF data At high NA the angle of incidence of the most oblique rays can be close to 70 degrees When a ray has to cross the cover glass to medium interface at such an angle total reflection may occur To be precise total reflection occurs when the NA of your lens is higher than the refractive index of the embedding medium This will reduce the effective NA of the lens Deconvolution improvements Acquire an experimental PSF A point spread function PSF is the image of a single point object The degree of spreading blur ring in the image of this point object is a measure for the quality of an optical system and 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 problems inside the optical path Therefore it is always re comme
81. om Harp to Sort you progressively increase the range value thus broadening the transition from the original to the deleted values You can apply different soft thresholds to the different image channels With the MIP Renderer you can also make an animation of your image changing the viewpoint in different frames Select the viewpoint coordinates for the first frame then click Ser gt Home Select now the viewpoint coordinates for the last frame and click Ser gt Enp You can now go to the last or the first frame by clicking Go gt Enp or Go gt Home Select all the rendering parameters in cluding the total number of rendered frames for the movie Options gt ANIMATION FRAME COUNT Fi nally click Animate and select a directory to save the TIFF frames to You can later load and edit The Huygens Essential User Guide 23 4 Huygens Essential Visualization Tools these TIFF images with your favorite animation tool For instance you can use the convert tool from ImageMagick http www imagemagick org to make a GIF animation using convert delay 20 animatedMip tiff animatedMip gif You can now place this single file GIF animation directly on your web page as most of the Inter net browsers currently available can handle this kind of movie files With the appropriate codec you can also use convert to make a MPEG animation See the Im ageMagick website for more details If your image is a time series you can also ma
82. on stage provided that an image is to be restored with the same optical properties as the ones which were recorded on the template gHuygens Essential 32bit by Scientific Volume Imaging BV s lt lt CS i CSSS lt C Ci CS S S Kj microscopical parameters of i File Task Visualization Tools original Templates Load Save Geometry x sample size y sample size z sample size Optical Parameters Microscope type nipkow data In its final stage this data can be saved to a template file for future usage Import microscopic template Numerical aperture 0 96 Pinhole Spacing 2 500 Refractive indexes Lens Water 1 3381 Medium 1 5150 widefield multiPhoton three ChannelConf small fluor Channel parameters E Select channel 1 Excitation wavelength la Emission wavelength Task info Task report Task plot Image fabab4 ics KZ Status Entered preprocessing stage Memory 0 Figure 10 Importing a microscopical parameters template Pinhole radius Photon count dose Reset _ Accept template from a list of pre saved template files which reside both in warning emission wavelength suspiciously high the common templates directory and in the user s personal template directory The Huygens common templates directory is named The Import MICROSCOPIC TEMPLATE button will allo
83. on curve was supplied with your microscope best use that curve to convert the dis played setting to a physical size and from there convert to the backprojected radius 5 Improving the quality of your images In this chapter you can find some basic suggestions on how to acquire better microscope images They are based on frequent problems that we find on users data These recommendations go on the direction of obtaining the greatest quality images from your microscope from the point of view of acquiring as much information as clean as possible This alone worths the effort but it will also be very valuable for the deconvolution afterwards Some basic guidelines to improve the deconvolution results are also included You can find more information at the SVI wiki at http support svi nl wiki Data acquisition pitfalls Some of the most common problems arising when acquiring images are addressed in this section Refractive index mismatch A mismatch between the refractive index of the lens immersion medium and specimen embedding medium will cause several serious problems e Geometrical distortion the fishtank effect Objects will appear elongated in the microscope Huygens Essential takes this into ac count when calculating the theoretical PSF but it will not modify the image geometry e Spherical aberration S A S A will cause the oblique rays to be focused in a different location than the central rays The distance in this
84. onal tool and is enabled by a C flag in the license string see License string details on page 49 Iso colocalization object analysis One of the features of the colocalization analyzer is iso colocalization object analysis It allows you to quickly determine the properties of the different colocalization regions in your 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 ob jects local colocalization parameters are computed and reported To relate the iso colocalization objects to the original data the surface objects can be blended with a MIP projection of the data p 23 The color range in which these objects are displayed can be modified using a hue selector p 27 How to use the Colocalization Analyzer To start the Colocalization Analyzer in Huygens Essential right click on an image s thumbnail to open the contextual menu then select SHOW IN COLOCALIZATION ANALYZER Alternatively you can se lect an image s thumbnail then in the menu bar select VISUALIZATION gt LAUNCH COLOCALIZATION ANALYZER The image must be multi channel see p 17 as the colocalization is based on the over lapping of different channel intensities Wait for the analyzer to initialize and to compute the first MIP projection of the data You can select the viewpoint of the MIP projection by moving the Tit a
85. ons MLE based deconvolution uses an iterative process that never stops if no stopping criterion is giv en This stopping criterion can simply be a maximum number at which the process will stop This value depends on the desired final quality of your image For an initial run you can leave the value at its default To achieve the best result you can increase this value Another stopping criterion is one based on the Quality change of the process see Quality threshold below Signal to noise ratio You have to make an estimation of the SNR from your recorded image Inspect your image and decide if your image is noisy SNR lt 10 has moderate noise 10 lt SNR lt 20 or is a low noise image SNR gt 20 See Signal to Noise Ratio SNR on page 39 and some examples of noisy im ages in Figure 31 Quality threshold Beyond a certain amount of iterations typically below 100 the difference between successive iter ations 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 sub sequent iterations may drop below the threshold before the indicated maximum number of itera tions has been completed The smaller the threshold the larger the number of iterations which are completed and the higher the quality of restoratio
86. orm time intervals Every recor ded image is a time frame The Huygens Essential is capable of automatic deconvolution of 2D time or 3D time data There are some tools that are intended only for time series as the confocal bleaching corrector or the z drift corrector page 16 Adapting the image In the Toots menu you can find a contrast inverter helpful for the processing of brightfield images see below A Crop tool is also available but its use is recommended only after properly tuning the image parameters and will be explained in a later stage In the Toots menu you can also find a Mirror ALONG Z tool to flip the image when the coverslip is in the top This is specially important in case of a refractive index mismatch see Spherical aber ration correction on page 48 Processing brightfield images Brightfield imaging is not a linear imaging process In a linear imaging process the image forma tion can be described as the linear convolution of the object distribution and the point spread func tion PSF see page 14 hence the name deconvolution for the reverse process So in principle one cannot apply deconvolution based on linear imaging to non linear imaging modes like brightfield and reflection One could say 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 the absolute squared values of the amp
87. ors see p 27 Other available options to change are render size and transparency depth see p 27 accessible through the Options menu The render size can be set to fit the size of the canvas which is the pane where the rendering is displayed and whose size depends on the size of the window Save the results All the obtained information can be saved to external data files through the Fue menu The exported image as viewed on the screen will have the size determined by the render size visualization parameter mentioned above Notice that if the render size is too big it might not fit the display pane but it will still be saved as rendered You can shift the rendering by dragging it with the middle mouse button The exported text file includes the calculated object statistics as shown in the lower data pane Read more More information is available at http support svi nl wiki ObjectAnalyzer 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 32 Scientific Volume Imaging 5 Huygens Essential Analysis Tools As this overlapping can be defined in many ways Huygens gives you the colocalization coeffi cients most commonly used in literature Pearson Overlap and Manders M and K see Colocaliz ation Theory in the SVI wiki O The Colocalization Analyzer is an extended opti
88. r time series 16 Saving the result 16 Saving the restored image 16 Saving your Task report 17 Multi channel images 17 Deconvolving a channel in a multi channel image 17 Joining the results 17 Batch processor 18 The Huygens Essential User Guide i Contents Startup 18 Window description 18 Usage 19 Adding one task 19 Adding multiple tasks 19 Running the batch job 20 Menus 20 File menu 20 Task menu 20 More information 20 4 Huygens Essential Visualization Tools 21 The Twin Slicer 21 Color mode 21 Contrast mapping mode 22 Time series 22 The MIP Renderer 23 Soft threshold 23 Rendering a movie 23 The SFP Renderer 24 Summary 24 SFP fundamentals 24 Rendering a movie 26 The Surface Renderer 26 Hue Selector 27 Transparency depth 27 5 Huygens Essential Analysis Tools 28 The Object Analyzer 28 How to use the Object Analyzer 28 Mouse mode 29 Frame selector 29 Render pipes 30 Object removal 30 Regions of interest ROI 30 Data analysis 31 Visualization parameters 32 Save the results 32 Read more 32 The Colocalization Analyzer 32 Iso colocalization object analysis 33 How to use the Colocalization Analyzer 33 Backgrounds vs thresholds in colocalization 35 Read more 35 6 The PSF Distiller 36 Beads for PSF distillation 36 The Distiller stages 37 Starting the Distiller 37 Parameter check stage 37 Averaging stage 37 Confocal and two photon bead images 37 Widefield bead images 38 Distillation stage 38 Asse
89. rame Time series frames can be handled For a 3 D image this slider is inactive and set to Frame 0 e Object brightness Intensity of the virtual light source e Soft threshold Preprocessing tool that reduces the background in the image Voxels with gray values below the threshold value become more transparent See a detailed explana tion in the MIP Renderer section on page 23 Use the Renper button to start the actual rendering The result can be saved as a TIFF image FILE gt SAVE Use LIGHT pirection to alter the direction of the light You can open as many SFP windows as you like Rendering a movie With the SFP Renderer you can also make an animation of your image changing the viewpoint or the time coordinate in different frames The procedure is analogous to the one explained for the MIP renderer on page 23 The Surface Renderer The Surface Renderer is available from the Huygens Essential version 3 0 onwards and enables you to represent your data in a convenient way to clearly see separated volumes Because this Sur face Renderer is based on fast raytracers there is no need for any special graphic card as would be necessary for conventional polygon based techniques O The Surface renderer is an extended optional tool and is enabled by a v flag in the license string see License string details on page 49 To start the Surface Renderer right click on an image s thumbnail to open the contextual menu then select
90. re e Irix Open the Software Manager select MANAGE INSTALLED SOFTWARE and mark the pack ages you wish to remove e Linux To remove Huygens Essential type as root rpm e huygens e Mac OS X Drag the installation to the waste basket e Microsoft Windows Clicking Start in your Windows desktop and select ProGrams gt HUYGENS ESSENTIAL gt UNINSTALL A Removing the software will also cause your license string to be removed If you prefer to uninstall your current version prior to installing a newer one be sure to store your license string in a safe place 2 Huygens Essential Huygens Professional and Huygens Scripting 4 Scientific Volume Imaging 2 Installing Huygens Essential System requirements for Huygens Essential Windows and Linux Huygens Essential and Huygens Scripting run on the following Windows operation systems Win dows 2000 NT 2003 Server and XP Linux RedHat and SuSE distributions Since Linux versions evolve rapidly best consult SVI s http www svi n1l web page to see which Linux distributions are currently supported A standard Ethernet card is required to provide your computer with a system ID e Processor Pentium III or IV Intel or Athlon AMD e Recommended RAM memory 512 MB or larger to run larger images like 512 768 50 voxels e Graphics card any fairly modern card will do Mac OS X Mac OS X 10 2 or higher running on a G4 processor or higher 512 MB or more RAM SGI Irix I
91. rency and the brightness of this surface pipe with the correspond ent sliders or just set this pipe off The color range in which the objects are displayed can be mod ified using a hue selector see page 27 Some modes generate two channel colocalization maps colocalization of Red respect to Green and vice versa e g in case of the Manders M1 and M2 coefficients In these cases the iso colocal ization surface parameters will offer the possibility of rendering 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 There is one Maximum Intensity Projection MIP pipe available to redirect your data channels to The MIP rendering of one original channel maybe none of the ones used for colocalization or the two channels under analysis can be a good spatial reference for the objects from the colocalization map When you select an original channel you can use the threshold slider 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 you select both R and G channels to be rendered their correspondent backgrounds as selected in the histogram will be used as projection thresholds As with the surface pipes you can also control the transparency and the brightness of this MIP All the obtained information can be saved to externa
92. ria used by the deconvolution algorithms see http support svi nl wiki RestorationParameters A step by step example on how to tune these parameters to achieve the desired restora tion results http support svi nl wiki DeconvolutionProcedure Very important factors sampling clipping on image acquisition and restoration hetp s Support svi nl wiki Imporlantractors Typical acquisition pitfalls spherical aberration undersampling bleaching ex plained http support svi nl wiki AcquisitionPitfalls On recording beads to measure a PSF http support svi nl wiki RecordingBeads Tutorials and detailed information on using the different software tools restoration visualization analysis programming can be found in httip support svi niywiki s Tutorials If you are sending your images to SVI to get further assistance please follow the instruc tions detailed in http support svi nl wiki SendImagesToSvi Do you want to edit the wiki contributing to enhance its contents Read http support svi nl wiki HowToUseWiki With the search tools you can find much more information on the topic you want http support svi nl wiki FindPage Write here your support data in order to have it always available SVI support number Login for the wiki and the image database do not write your secret password For further information see http support svi nl wiki SupportNumber and http supper s
93. rrent multi channel result In the section The Distiller stages below the stages will be discussed in detail Beads for PSF distillation The diameter of the beads should be in the order of the Half Intensity Width of the expected PSF Larger beads will reduce the accuracy of the Distiller smaller beads yield insufficient signal for accurate stacking in the averaging procedure also resulting also in reduced accuracy Typically beads with a diameter of 160 nm perform very well for many types of microscopy 36 Scientific Volume Imaging 6 The PSF Distiller O Beads should be recorded with the same microscopic parameters that you will use later to image your specimens Please find more information about beads for PSF measurements on the SVI wiki athttp support svi nl wiki RecordingBeads The Distiller stages Starting the Distiller After launching Huygens Essential open the first bead image with Fitz gt Open If your license in cludes the PSF Distiller option the Start PSF pister button appears in the workspace press it One or more Accumulator images will be created into which later on the averaged beads will be kept You now enter the P stage Parameter check stage This stage is similar to the deconvolution P stage and allows you to check all relevant microscop ic parameters in particular the sampling density A Do not use undersampled bead images Averaging stage If any of the entry fields for the
94. rtainly after each maintenance job in which the optics or scanning device was serviced Read more on the SVI wiki at http support svi nl wiki PsfDistiller 7 Establishing image parameters The deconvolution algorithm needs to know some of the parameters describing your image acquis ition These are not too many but you should carefully determine them They are explained in this chapter Image size The amount of computing time involved in deconvolving images is more than proportional to the image size It is therefore sensible to limit the data size as much as possible With widefield im ages we recommend to not record planes below and above the object which only contain blur Huygens Essential does not need these planes to restore your object Since the blur in these planes might be affected by hard to correct bleaching they might even reduce the quality of the deconvo lution result In any case never crop your objects of interest As a rule of thumb leave about one extra micron above and below your objects 38 Scientific Volume Imaging 7 Establishing image parameters Brick wise processing Deconvolving images requires much computer memory because all computations are done in 32 bit floating point format and because several extra hidden images are needed to store intermedi ate results To reduce the memory requirements Huygens Essential will split your images into bricks deconvolve the bricks sequentially
95. sampling density turn orange or worse red the data in unusable The parameters of bead images loaded at a later stage should match the ones you establish in this stage if they do not you will be warned After setting the diameter and estimated Signal to Noise Ratio SNR of the beads the image is searched for beads which meet all selection criteria e A bead should not be too close to another bead If a bead it too close to another bead their images will interfere with each other In widefield bead images this is quite problematic due to the large size of the blur cone Fortunately widefield PSF can be derived from single bead images e A bead 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 an 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 un usable If for some reason no beads are found an explanation and some advice will be displayed in a popup The software will try to find beads first with ideal selection criteria If this does not yield a single bead then it will automatically retry with reduced inter bead distance criteria Confocal and two photon bead images Images from 160 nm beads should look like smooth fuzzy blobs with hardly visible noise Use the def
96. sition You can center the image again by selecting CENTER IMAGE from the name button With the slider you can slice your images along the three axis depending on what is the selected slice view You can also change the zoom factor the color mode and the contrast mapping mode e Gray pixel values are assigned different shades of gray ranging from black for the lowest values to white for the highest values The Huygens Essential User Guide 21 4 Huygens Essential Visualization Tools e False color pixels values are assigned different colors ranging from black dark purple for the lowest values to bright red for the highest value e True color pixel values are assigned different shades of a particular color ranging from black for the lowest values to the brightest possible shade for the highest values Multi channel images are always X Huygens Essential Twin Slicer e959 61grb_huy Slice x y Zoom 100 Color True colors x y 25 150 400 Grey scale 50 200 500 True colors y z 100 300 600 False colors 91 0 10 20 30 40 50 60 70 80 90 100 alue chan0 chan2 013 000 000 at x y z 503 063 050 e959 61grb_huy Gamma Compressed Linear Compressed Wide Field 110 120 130 140 150 160 Figure 22 The Twin Slicer showing two slices of the same 3D image rendered with a true color scheme otherwise the information from the different channels will res ult in very confusing images Contr
97. stimate BACKGROUND field or in the RELATIVE BACKGROUND field Setting this last 3 Learn more about histograms in http support svi nl wiki ImageHistogram The Huygens Essential User Guide 13 3 The image restoration process to 10 for example lowers the estimated background with 10 If you are done press accept to start the last stage the true deconvolution process Stage 4 Deconvolution Before starting the actual iterat Huygens Essential by Scientific Volume Imaging B V ive deconvolution run stage 4 File Task Visualization Tools original first carries out several prepro cessing steps 1 Background This value is calculated in stage 3 co 0 1G In this final stage the original image will be You can verify whether deconvolved on the basis of the PSF and background as n eco ution ne image takes computed in the previous stages hi I plac don ious Below are the default values for deconvolving images If necessary you can change these values t IS Value represents olution is done in an he Click on dekor er to pears the deconvolution run ntin ither a m lumber of y z areas in the image which me quality measure Maximum iterations 40 elt Signal to noise ratio 10 you consider background ing point If the Quality threshold 0 L Cep 0 18 Ge g 1 T 0 Iteration mode fast by opening the Twin Bleaching correction If possible Slicer see Figure 21 and moving the mouse DE pointer over the
98. template i di Rh Click on SELECT AN IMAGE FILE and browse to select one raw image that you want to de convolve End by clicking Open Then click on micro TEMPLATE to load on the microscopic parameter set that you use in Huygens Essential If none exists you must Create one The Create actually opens a tem plate editor where you can load and save many different templates overwriting the exist ing ones or creating new ones e Tip prepare your templates in the Huygens Essential main window before select ing them in the Batch Processor see page 10 This will allow you to use the parameters saved inside an image Repeat the same procedure with the DECON TEMPLATE to establish a set of restoration para meters In the Batch Processor appart from the wizard CMLE restoration algorithm you can also use the QMLE a quicker version that works very well with low noise images Finally the icon with the single green plus sign will be enabled click on it to add the task You will see the task included in the list of tasks By now it is the only one we will add more later You can now click on the different task elements the image name to change the data file the task is executed with the microscopic template to change the microscopic parameters You can modify them or load a new template the deconvolution template to change the restoration parameters You can modify them or load a new template the trash can icon to
99. tion of the image background and resolution For these reasons we strongly recommend to compute colocalization coefficients only on deconvolved images 7 Http support svi nl wiki ColocalizationTheory 8 Read more on http support svi nl wiki TwoChannelHistogram The Huygens Essential User Guide 33 5 Huygens Essential Analysis Tools Huygens Colocatization analyzer a O File Options Zoom s0 colocalization surface 1 25 Threshold 4e 07 4 Transparency 0 60 Brightness 0 60 Tit Off cho 007 deg MIP of original image Threshold 15 6 Transparency 0 60 Brightness 0 76 Channel Chan 2 Twist 121 deg Frame selection Frame Of O Two channel histogram Frame colocalization coefficients hohe lete fae Sula Gale alll tale E Red chan 1 bg 3 00 Green chan 0 bg 2 00 Pearson 0 203 Overlap 0 229 Mi 0 777 M2 0 405 Kl 1 511 Ki 0 035 Object colocalization statistics Object 63 found at position 132 177 75 Red chan 1 bg 3 00 Green chan 0 bg 2 00 Colocalization map type Pearson Selected threshold 4e 07 Centre of mass voxels 146 0 149 6 76 8 Volume 24631 voxels Surface 110 micron 2 Sum 0 0178 Min 4e 07 Max 2 92e 06 Red channel Green channel Background 3 00 Background 4 Cho Chi Ch2 cho Chi Ch2 Colocalization map Pearson Ready Canvas size 509 x 484 Dragging on Figure 29 Colocalization Analyzer with surfaces for regions of over
100. tions show sudden jumps we recommend the edge preserving Kuwahara filter Saving the restored image After each deconvolution run you can save the result Select the image to be saved and do Fitz gt SAVE IMAGENAME AS in the menu bar You can save the image as an Image Cytometry Standard ICS or ICS2 image file a TIFF file series an Imaris Classic file or a Biorad pic file Only the ICS and ICS2 file type save all the microscopic parameters For information on how to proceed with multi channel data see Joining the results on page 17 16 Scientific Volume Imaging 3 The image restoration process The ICS file format actually uses two separate files a header file with ics extension and other much bigger and with the actual image data with ids extension On the other hand the newer ICS2 file format uses only one single ics file with both the header and the data together Saving your Task report Select from the Main menu bar Task gt SAVE TASK REPORT to Store the information as displayed in your Task report tab deck Multi channel images Deconvolving a channel in a multi channel image Figure 19 Deconvolving a two channel image Joining the results Fluorescent microscopes can usually register different emission wavelengths almost simultan eously allowing you to image different dyes on the sample In the terminology of the Huygens Software one channel in an image refers to the intensity distribution
101. ts you click on different defined objects and obtain the local statistics Define ROI lets you define different regions of interest ROD discarding objects out of them e Delete object lets you delete irrelevant objects one by one Both this and the ROI selector act only on the current window the original image is not changed Set measurement anchor lets you select and unselect objects to be anchors references for distances to other objects when asking for local statistics It is possible to select a group of anchor objects The location of the anchor is then the centre of mass of all an chor objects Independently of the mouse mode you can at any time use the right mouse button middle in the Mac to drag the image and recenter the objects The different window elements are explained with a short text tip on the bottom of the window when the mouse passes over them First we have to select what is the data to be analyzed Frame selector In time series you can select the interesting time frame with this slider The Huygens Essential User Guide 29 5 Huygens Essential Analysis Tools Render pipes You can find three graphic pipes to redirect your image data channels to two isosurface pipes and one MIP pipe These can be activated independently by selecting a channel data from the im age Objects as defined above are represented in the isosurface pipes The MIP pipe is used as a reference You can use up to two
102. ue 27 HuEss 2 49 HuPro 49 HuScript 49 I ICS file 16 ICS2 file 16 illumination instability 47 image statistics 9 Imaris Classic file 16 internal reflection 46 48 inversion of contrast 8 isosurface 28 L labeling 28 Leica 43 44 51 Leica style numbering 51 license string 2 4 26 28 33 36 49 linear color mode 22 M Maximum Intensity Projections 11 Maximum Likelihood Estimation 14 15 mechanical instability 47 MIP 11 23 26 MIP Renderer 23 MLE 14 16 movie rendering 23 26 multi channel 7 12 16 17 24 36 multi photon excitation 37 41 47 49 N NA 9 40 42 44 46 48 Nipkow 9 43 49 non circular 42 numbering of TIFF series 51 numerical aperture 9 42 45 47 Nyquist rate 40 47 O object 28 object analysis 33 Object Analyzer 28 Olympus 45 optical units 45 orange background 9 P photon 37 39 47 photon count 9 pinhole 9 41 45 Point Spread Function PSF 14 PSF 8 14 15 36 46 52 PSF Distiller 4 7 14 36 QMLE 19 quality threshold 15 R red background 9 refractive index mismatch 46 region of interest 29 30 Renderer MIP 23 SFP 24 Surface 26 rendering 23 25 27 restoration algorithm 19 ROI 29 30 S sampling 13 37 40 47 saturation 12 saving 16 17 Scientific Volume Imaging b v 53 seed and threshold 30 segmentation 28 SFP renderer 24 shape correction 42 shape factor 42 Signal to noise ratio SNR 15 39 Simulated Fluorescence Process
103. vi nl Wiki Wn Login 54 Scientific Volume Imaging Alphabetical Index Alphabetical Index A acquisition pitfalls 46 acquisition time 40 Airy disk 41 45 Analyzer Colocalization Analyzer 32 Object Analyzer 28 anchor 32 animation 23 26 artifacts 46 average background 13 B background 34 35 backprojected distance 9 43 backprojected pinhole 9 41 45 batch processing 18 bead 7 14 36 38 50 Biorad 16 43 45 blacklevel 12 40 bleaching 15 38 40 47 brick 14 39 brightfield images 8 C caliper 31 channel removal 12 circular pinhole 42 43 clipping 12 46 47 CMLE 19 Colocalization Analyzer 28 32 50 colocalization coefficients 34 colocalization map 34 color 27 color mode 21 color of the channels 9 compress color mode 22 compute engine 1 49 computing time 38 confocal 37 41 49 contrast 8 21 22 correction 15 42 47 51 correction for z drift 16 critical sampling 9 critical sampling distance 40 41 cropping 7 11 12 31 cyan background 9 D debug 52 deconvolving 8 38 39 demo image 7 12 distance 9 distributors 53 download 1 4 drift corrector 16 E email SVI 53 emission 24 25 40 41 excitation 24 25 41 42 46 F FAQ 53 Fast Classic Maximum Likelihood Estimation 14 faxing SVI 53 fishtank effect 46 Fluorescence Volume Renderer 24 FluVR 24 49 Frequently Asked Questions FAQ 53 G geometrical distortion 46 H histogram 7 12 13 40 h
104. w you to choose a Figure 11 Microscopical l parameters corrector Templates and resides in the Huygens installation directory namely usr local svi Templates on Unix systems C Program Files SVI Templates on Windows and Applications SVI Templates on the Mac OS X The user s personal templates directory is called svi on the Unix platforms and SVI on Windows and it can be found in the user s home directory on Unix and in C Documents and Settings user_name on Windows You can also choose to load a template file from a different location by pressing the From DISK option 10 Scientific Volume Imaging 3 The image restoration process The Export TEmMp ateE button will al low you to either save the template to the personal template directory by choosing the To pisk option or overwrite one of the existing tem plates by selecting them from the e Preprocessing Stage Parameter Check list These are the new parameters that will be used for this image after clicking Accept Microscope type widefield Numerical Aperture 1 4 Lens immersion refr index is out of no ot trustec The Huygens template is a simple be highligh L K ampte size oa sample s ze NM ample size nm xml file which can be edited by y 5 I Excitation wavelength Kg hand as well see examples in the l i Excitation photon count Export template To disk common Templates directory mult
105. xample gray values from 0 9 form bin 0 values from 10 19 form bin 1 etc The histogram is now the number of pixels in every bin The histogram from the demo image as shown in Figure 15 is of reasonable quality The narrow peak you see at the left represents the background pixels all with similar values The height of the Select the start and stop time frame 0 Cancel Apply OF Figure 14 Reducing the number of time frames peak represents the amount of background pixels Because in this particular image there are many voxels with a value in a narrow range around the background the peak is higher than the other In this case there is also a small black gap at the left of the histogram This signifies an electronic offset blacklevel in the signal recording chain of the microscope see page 40 If a peak is visible at the extreme right hand side of the histogram it indicates saturation or clip ping Clipping is caused by intensities above the maximum digital value available in your micro scope Usually all values above the maximum value are replaced by the maximum value On rare 12 Scientific Volume Imaging 3 The image restoration process occasions they are replaced by zeros Clipping will have a negat ive effect on the results of decon volution especially with WF im ages See page 46 O The histogram stage is included for examining purpose only It has no meaning for the deconvo lut
106. ystem ID n Number of processors p Processor type s Processor details lt date gt is the end of license or end of support date in the format eomYYYYMMMDD lt sysid gt is 16 hexadecimal digits of system identification lt email gt is the email address of the customer who bought this license in the format email address lt checksum gt is 20 hexadecimal digits of license checksum When the d flag license expiry date is specified then after the locking flags comes the expiry date Without this flag the expiry date will be preceded with the letters eom which turns the date into an end date of the current support contract Currently the e flag is always present If it is after the system ID an email address surrounded by braces is given This is the email address of either the creator of the license or the sales person in case of a temporary license or the customer to whom the license was sold This is used for informational purposes only It does not limit the li cense in any way If the m flag is present then the hardware system identification ID must match the one from the running system Similar for the n flag It requires that the number of processors as specified by the system ID matches the number in the current system The p requires a match on processor type For PCs this consists of the name of the processor vendor like Intel or AMD the processor family like Pentium and the processor model For Irix machines this is the IP level

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