Home

The Huygens Essential User Guide

1. These are the new parameters that will be used for this image after clicking Accept Microscope type widefield Numerical Aperture Lens immersion refr index 1 515 Medium refractive index 1 415 X sample size nn 100 0 Y sample size nn 100 0 Z sample size nn 100 0 Excitation wavelength nm 488 Emission wavelength nm 520 Excitation photon count The Huygens template is a simple xm1 file which can be edited by hand as well see ex amples in the common Templates directory Export template To disk multiPhoton threeChannelConf small_fluor Task info Task report Task plot Figure 12 Exporting a microscopical parameters template The time needed to deconvolve an image increases more than proportional with its volume Therefore deconvolution can be accelerated considerably by crop ping 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 pro posal the microscopical parameters are taken into account making sure that cropping will not have a negative impact on the deconvolution result Because the survey depends on accurate mi croscopical parameters it is recommended to use the intelligent cropper as final step in the prepro cessing stage but you can launch it before the restoration process from the Toots gt C
2. toration method and a rough estimation based on the appearance of the image is usually enough see Figure 29 Blacklevel Figure 30 shows the histograms of three synthetic im Read more in http support svi nl wiki SignalToNoiseRatio ages At left an im age homogen eously filled with fh tae the value 5 At the i A blacklevel Figure 30 Histogram of images with various blacklevel values middle we applied Poisson noise as if the image was build by a CCD camera At the image on the right a value of 20 was added to simu late electronic shift This shift is called blacklevel A large blacklevel value will reduce the effect ive dynamic range of your microscope but will do no harm to the deconvolution since it is auto matically accounted for in the background estimation stage However it is also possible that the blacklevel is negative In the image histogram this will show up as a spike on the left Sampling densities 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
3. ing distinct absorption prop Home setnome ugh dr erties The absorption prop m mage parameters erties can be controlled by T the user The different emis Object size 0 30 micron sion wavelengths give each Eee dye its specific color FR To excite the fluorescent matter light must traverse other matter The resulting Figure 24 attenuation of the excitation subsamp l light will bject nn A ght will cause objects J which are hidden from the Excitation f light source by other objects to be Emission weakly illuminated if at all The at tenuation of the excitation light will be visible as shadows on other ob jects To optimally use the depth per ception cues generated by these shad ows a homogeneous plane the gray table below the data volume is placed on which the cast shadows be Y come clearly visible After excitation the fluorescent mat ter will emit light at a longer wavelength Since this emitted light Table has changed wavelength it is not cap able to re excite the same fluorescent matter multiple scattering does not occur Thus only the light emitted in Figure 25 With the SFP renderer excitation and the direction of the viewer either dir subsequent emission of light of fluorescent materials is simulated Each subsequent voxel in the light beam ectly or f the semi reflectin yor by way ORINE Be ee excitation is affected by shadowing from its predecessors table is of importance By simulati
4. if the series is a numbered series like sLice001 tif slice002 tif sliceOnn tif Huygens Essential will read the series into a single 3D image Because TIFF s usually carry no additional microscopic information check the parameters carefully 42 Scientific Volume Imaging 9 Appendix 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 e000 tif eQOL ttt c031 tif If you wish to work on the complete c stack you only have to select cO00 tif while opening If you select file c020 t if the first 20 slices will be skipped Numbered series without the TIFF extension like c04 c05 c18 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 c_t00_z000 tif c_t00_z001 tif c_t00_z002 tif c_t00_z003 tif c_t01_z000 tif c_t01_z001 tif c_t01_z002 tif c_t01_z0
5. sampling distance than the critical distance to the contrary Figure 31 shows the dependency of this critical sampling distance on the NA for specific wavelengths To apply the plot of Figure 31 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 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 32 Scientific Volume Imaging 7 Establishing image parameters Creat
6. 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 amplitudes 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 al
7. trical signals must be converted to numbers processed by the computer This converting stage is 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 exceeding some value are all converted to 4095 clipping information in the clipped samples is Jost In practice be suspicious if you find in your image voxel intensity values at the extremes of the numerical range probably clipping occurred 38 Scientific Volume Imaging 8 Improving the quality of your images Undersampling 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 32 or calculated using the formulas as given in The Nyquist rate from the Huygens Deconvolution Recipes Do not undersample to
8. Backprojected means the size of the pinhole as it appears in the specimen plane see Computing the backprojected pinhole radius on page 33 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 FRheekacoevg they 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 32 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 im age parameters not only at this deconvolution stage but also at any time by right clicking on the image thumbnail and select Fle Task Visualization Tools Help original ing SHOW PARAMETERS OT CORRECT PARAMETERS Correcting will pop ver the sampling density factors found in the geonetry section of the image Change these as A necessary up the window shown on Figure en 1 1 j Y sample size nm Z sample siz
9. bit machine number into the backpro jected pinhole radius P 255 anes S min T S min r 1000 c 3 b h i M obj M system where P is the 8 bit machine number for the pinhole s jy 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 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 the replacing circular pinhole gt C d r pid Hs eo 4 Jr gt d 2r For a square pinhole shape 1 4 m 0 564 For circular pinholes C shape 9 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 34 Scientific Volume Imaging 7 Establishing image parameters 0 69 ox N Airydisks 5 b NA with N Airydisks 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 distance is
10. brightness ofthe 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 itto a TIFF file with Fue gt Save Other available options to change are render size and transparency depth accessible through the Options menu Hue Selector Ns 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 intersecting with other With the Smerte depth only the piece of surface closest to the viewer s eye screens the others behind it with its corresponding transparency level 24 Scientific Volume Imaging 4 Huygens Essential Visualization Tools With the Normat de
11. channel images 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 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 pho tomultiplier for instance Huygens Essential by Scientific Volume imaging B V le Eile Task Visualization Tools Help Multi channel images can be deconvolved in a semi auto matic fashion giving you the opportunity to fine tune the results obtained with each in dividual channel After the Bere 2 thumbnail images in the display area one by one preprocessing stage the multi Srleet 3 decovaleed chanel ta hecone channel 0 in channel image is split into Choose channe o channel 0 0 single channel images named ee channel to become channel 1 in Choose channel 1 channel 1 0 channel 0 channel 1 and so on The first of these is automatically selected for de Task info Task report Task plot convolution To deconvolve it oe proceed as follows LY nein 5 5 5 5 Figure 19 Deconvolving a two channel image Deconvolving a channel in a multi channel image The procedure to deconvo
12. for Task report Task plot Image currently no image file loaded NZ Status ready instance a file named Memon 0 Huygense280 exe Place Figure 1 The start up window on Microsoft Windows If no license this file on your desktop and string is installed the software runs in Freeware mode You can find double click its icon to start your computer s ID number by using HeLe gt ABour the installation During in stallation the directory C Program files SVI will be created by default After completion the Huygens Essential and Huygens Scripting 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 Open a 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 usr local bin Currently the Irix distribution is a single tardist file containing various components By default all components are installed Become superuser and type swmgr f dist65 2 7 0 p7 tardist Press Start in the Softw
13. 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 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 WF 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 Thermal effects Mechanical instabi
14. page 20 Use the Renper button to start the actual rendering The result can be saved as a TIFF image Fite gt SAVE Use LiGHT DIRECTION 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 21 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 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 41 To start the Surface Renderer right click on an image s thumbnail to open the contextual menu then select 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 The Huygens Essential User Guide 23 4 Huygens Essential Visualization Tools Use the threshold slider to apply different thresholds to your data channels to se
15. 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 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 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 30 Scientific Volume Imaging 7 Establishi
16. the system magnification 82 9 This value corresponds well with the largest possible system magnification for the MRC600 A supplied calibration curve If a calibration 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 7 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 37 8 Improving the quality of your images 8 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 Clipping A mismatch between the refractive index of the lens immersion medium and specimen
17. their corresponding pixel in G is above the threshold 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 certain value can be controlled by the threshold slider in the iso colocalization surface paramet ers You can control the transparency 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 24 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 one of your data chan nels to The MIP rendering of one original channel can be a good spatial reference for the objects from the colocalization map Use the threshold slider to select what voxels are considered for th
18. usr local bin essential If this still does not help then Huygens Essential has not been installed correctly In Linux KDE desktop you may also start Huygens Essential from the Application menu and in the Gnome desktop from the Main Menu The Huygens Essential User Guide 5 2 Installing Huygens Essential Irix Haygens Essential by Scientific Volume Imaging B V File Task Visualization Tools On Irix you can start Huy gens Essential by typing essential ata shell prompt which 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 Task info Task plot usr sbin essential Image currently no image file loaded V If this still does not help Figure 6 The start up window The gray area in the upper part is the then Huygens Essential has 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 Adding to the shell search dialog boxes will appear during processing Also intermediate path deconvolution results are shown here The bottom bar is a status area not been installed correctly Users of the csh or tesh shell can add the usr local bin or usr sbin directory to their shell
19. 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 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 w Widefield enables you to deconvolve WF data c Confocal enables you to deconvolve confocal data n Nipkow Disk enables you to deconvolve data from Nipkow spinning Disks p multi photon for 2 or more photon microscopes 4 4 pi for experimental microscopes lt server gt can be one of d desktop 1 or 2 CPU s s small server 3 8 CPU s m medium server 9 32 CPU s 1 large server 33 64 CPU s x extreme server 65 or more CPU s Remark for Linux and Mac OS X multi processing users d desktop 1 thread m medium server max 2 threads 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 Surface Renderer visualization option Colocalization Analyzer option Object Analyzer option 64 bit ora gc th The Huygens Essential User Guide 41 9 Appendix lt l
20. 03 tif And an image sTCh of four slices three frames and two channels sTCh_t00_z000_ch00 tif sTCh_t00_z000_ch01 tif sTCh_t00_z001_ch00 tif sTCh_t00_z001_ch01 tif sTCh_t00_z002_ch00 tif sTCh_t00_z002_ch01 tif sTCh_t00_z003_ch00 tif sTCh_t00_z003_ch01 tif sTCh_t01_z000_ch00 tif sTCh_t01_z000_ch01 tif sTCh_t01_z001_ch00 tif sTCh_t01_z001_ch01 tif The Huygens Essential User Guide 43 9 Appendix sTCh_t01_z002_ch00 tif sTCh_t01_z002_ch01 tif sTCh_t01_z003_ch00 tif sTCh_t01_z003_ch01 tif sTCh_t02_z000_ch00 tif sTCh_t02_z000_ch01 tif sTCh_t02_z001_ch00 tif sTCh_t02_z001_ch01 tif sTCh_t02_z002_ch00 tif sTCh_t02_z002_ch01 tif sTCh_t02_z003_ch00 tif sTCh_t02_z003_ch01 tif 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 32 How do I generate a debug log See http support svi nl wiki DebugMode 44 Scientific Volume Imaging 9 Appendix Addresses and URLs Where can we be reached Support and FAQ Scientific Volume Imaging b v Alex
21. 1 23 artifacts 38 average background 13 B backprojected distance 9 36 backprojected pinhole 9 34 36 37 bead 7 15 28 30 42 Biorad 17 36 38 blacklevel 12 32 33 bleaching 16 31 33 40 brick 14 31 brightfield images 8 C channel removal 12 circular pinhole 35 clipping 13 39 Colocalization Analyzer 25 42 color 25 color mode 19 color of the channels 9 compress color mode 19 compute engine 1 41 computing time 31 confocal 30 33 34 42 contrast 8 19 correction 16 35 40 43 correction for z drift 16 critical sampling 9 critical sampling distance 33 cropping 7 11 12 cyan background 9 D debug 45 deconvolving 8 31 demo image 7 12 distance 9 distributors 45 download 1 4 drift corrector 16 E email SVI 45 emission 21 23 33 34 excitation 21 23 33 35 39 F FAQ 45 Fast Classic Maximum Likelihood 46 Scientific Volume Imaging Estimation 14 faxing SVI 45 fishtank effect 39 Fluorescence Volume Renderer 21 FluVR 21 42 Frequently Asked Questions FAQ 45 G geometrical distortion 39 H histogram 7 12 13 33 hue 25 HuEss 2 42 HuPro 42 HuScript 42 I ICS file 17 ICS2 file 17 illumination instability 40 image statistics 9 Imaris Classic file 17 internal reflection 39 40 inversion of contrast 8 L Leica 36 44 Leica style numbering 44 license string 2 5 24 25 28 41 linear color mode 19 M Maximum Intensity Projections 11 Maximum Li
22. 2 4 wenp d t emnps eom2002Jun27 a9e84ad0476692f4 hans svi nl Sb4fe6c38eb6922831b7 HuScript 2 4 wep d emnps eom2002Jun27 a9e84ad0476692f4 hans svi nl fabaSdf4cch4Sdd767a2 HuEss 2 4 wcnp d t emnps eom2002Jun27 a9e84ad0476692 4 hans svi n1 74818b89c85282b2b1c7 HuBatch 2 3 venp d t ennps eon2003Ju131 39e84ad0476692f4 bert svi nl 1ef37696ee1d08b957ee Huotif 2 3 wonp d tv ennps eon2003Ju131 59e84ad0476692F4 bert svi nl 9531f48119e1e5d04fe7 f N 5 m it Add a new license Explain license Delete license Ark anaw icons Explain Reonse Doleta license oK E 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 Figure 4 The Add License dialog box 1 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 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 AIX Irix and Linux usr local svi e Mac OS X depends on where you installed the software
23. 2 5 um You can check this by stopping the disk So with an 100x lens the backprojected distance is about 2 5 um 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 confocal 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 TCS4d Range begin Range end Reported parameter P 0 255 630 Diameter micron a earlier reported as 500 Pinhole geometry square System magnification 4 5 Backprojected pinhole 233 Pg T 2309 Eq 6 radius nm M oh q Table 1 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 34 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 34 for informatio
24. 600 1024 Radiance pinhole parameters 36 Scientific Volume Imaging 7 Establishing image 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 upe with m between 1 0 and 1 56 a factor 1 25 for the fluorescence 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 2m Vet re nsin 11 where 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 appears thus to be 4918
25. 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 blue 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 1 2 this will be shown in red platforms For each product you HuEss 2 4 wenp d t emnps eom2002Jun27 a9e84ad0476692f4 hans svi n1 74818bE need to have a license string 1n There were no obvious reasons found why this license Bi Id not bi lid stalled Select a license string in the Burn R Product name HuEss License manager and press the This license product name matches the currently running product 1 Version 2 4 ExPpLain LICENSE button All details for rm BR the current license will be listed If EA Sea n w enables Widefield microscope functionality you run into licensing problems you c enables Confocal microscope functionality n enables Nipkow disk microscope functionality may
26. 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 default 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 Loa
27. Huygens Essential User Guide 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 2005 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 21 23 26 and 27 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 essential Doc 2 8 1205 Contents Table of Contents 1 What is Huygens Essential 1 2 Installing Huygens Essential 1 MacOSX 1 Microsoft Windows 1 Linux 2 SGI Irix 6 5 2 IBM AIX 5 2 2 After the installation 2 The license string 2 Obtainin
28. RedHat and SuSE distributions Since Linux versions evolve rapidly best consult SVI s http www svi nl 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 IBM 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
29. anderlaan 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 usat info svi nl http www svi nl Distributors A list of distributors can be found on our web site http www svi nl distributors Knowledge database 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 httpi supp rt svi nl 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 nl wiki The Huygens Essential User Guide 45 Index Alphabetical Index exe file 1 gif file 21 ics file 17 ids file 17 pic file 17 pkg file 1 rpm file 2 tar file 2 tardist file 2 xml file 11 2D images 8 4 pi 42 A acquisition pitfalls 38 acquisition time 33 Airy disk 33 36 38 animation 2
30. ange 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 restoration Still the extra quality gain becomes very small at higher iteration counts 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 Iteration mode In Fast mone highly recommended the iteration steps are bigger than in HicH Quauiry mode More information can be read in the Dictionary from the Herre 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 Stopping the MLE algorithm Pressing Restore starts the iterative MLE algorithm a Stop button appears Pressing Stop 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 18 t
31. are 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 Z README AIX anda shell script AIX install sh To install the software you have to run the file AIX 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 usr 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 Here 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 i
32. ata 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 threshold 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 subtracted from the voxels intensities when calculating the coefficients if the result is negative it is under stood as a zero Generally the colocalization coefficients depend much on correct estimation of the image background and resolution For these reasons we strongly recommend to compute coloc alization coefficients only on deconvolved images 6 Read more on http support svi nl wiki TwoChannelHistogram 26 Scientific Volume Imaging 5 Huygens Essential Analysis Tools Next we select what colocalization map we want to calculate Pearson Overlap Manders M K or H In the computation of Manders coefficients the background values act like a threshold to the computation of m _1_ only pixels in R contribute when
33. by the REFERENCE CHANNEL parameter After determining the z positions per frame the z positions can be filtered with a MEDIAN GAUSSIAN or Kuwanara filter of Figure 18 The z drift corrector variable width 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 positions show sudden jumps we recommend the edge preserving Kuwahara filter 16 Scientific Volume Imaging 3 The image restoration process Saving the result Saving the restored image After each deconvolution run you can save the result Select the image to be saved and do Fiz 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 18 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
34. channel multi photon confocal images To facilitate com parison ofraw 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 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 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 Volume Imaging B V Bee File Task Visualization Tools Help Go to the folder were you downloaded the distribution and double click on it It will be extracted by StuffIt Ex pander to a pkg file which will be placed in the same dir ectory Double click this pkg file and follow the in stallation wizard No valid license could be found Continuing in freeware mode without deconvolution functionality You have received an execut able file for installation
35. con 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 wenp d tv emnps eom2008Dec31 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 2 Scientific Volume Imaging 2 Installing Huygens Essential Obtaining a license string r V About x pra If you are not upgrading from a previous install ation it is likely that a license is not yet avail able To enable us to generate a license string for you we need the fingerprint of your computer Huygens compute engine 2 8 0p0 Huygens Essential 3 1 0p0 the system ID number If you have not done so re already start Huygens Essential The system ID P 0 Box 615 is displayed in the Herp gt Asour dialog Figure ee 2 Send it to sales svi nl and you will be ae nos provided with a license string To prevent any an typing error use the Cory button to save the ID to the clipboard You can print it into your mail Check for Huygens updates message with the Epir gt Paste menu item of This computer has the following sys
36. cope 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 iterations 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 31 and some examples of noisy im ages in Figure 29 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 ch
37. e 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 explained on page 31 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 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 40 Scientific Volume Imaging 9 Appendix 9 Appendix License string details You can have detailed information about your license strings by going to Herr gt License select the license string you want to inspect and click EXPLAIN LICENSE This is a License String example HuEss 2 7 wcenp4 d ft demnps 2008Sep30 33dfa680a8402167 info svi nl c338a4da57cb342151d3 As from Huygens Compute engine
38. e MIP rendering depending on their intensities As with the surface pipe you can also control the transparency and the brightness of this MIP All the obtained information can be saved to external data text or image files through the Fitz menu 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 41 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 assemble a multi channel PSF from single channel PSFs The PSF Distiller wizard leads you through four stages to carry out the following tasks The Huygens Essential User Guide 27 6 The PSF Distiller e Preprocessing P stage to check mi croscopic image para meters This stage is similar to the decon volution P stage e Stage 1 Averaging stage In this sta
39. e actual brightness of a screen pixel is usually quite non linear 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 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 The Huygens Essential User Guide 19 4 Huygens Essential Visualization Tools Time series If you open the Twin Slicer on a time series a second slider is added Both the time slider and the spatial slider have a swing option When the spa tial swing is pressed the slider original 1 Slice x y Zoom 50 Color Grey scale Gamma Lineair moves back and forth when sa ee Te __ im f x z so 200 500 True colors Compressed the time swing button is y z 100 300 600 False colors wide Field pres
40. e nm Parameters Templates Once the proper parameters have been set and verified they Task info Task report Image raid people oded faba ics 7 can be saved to a Huygens tem Se e a 99 plate file suffix hgst Those Figure 9 Parameters check stage Sampling Red coloring template files are loadable in the indicates a suspicious value and orange a non optimal value very start ofthe wizard hence the user can skip the parameters verification stage provided that an image is to be restored with the same optical properties as the ones which were recorded on the template v ENEE w alata Ele Task Visualization Tools Help v Microscopical parameters of gt N File Templates Load Save Geometry x sample size y sample size 147 602 ace z sample size Here you can load previously saved microscopical parameters from a huygens tenplate file i By pressing the skip button you will enter a wizard Optical Parameters vi ich will assist you in assigning the proper optical data In its final stage this data can be saved to a Microscope type nipkow template file for future usage en ae ER N ee Numerical aperture 0 96 Pinhole Spacing 12 500 Refractive indexes Mil widefield multiPhoton threeChannelConf Lens Water 1 3381 small_fluor Medium 1 5150 Channel parameters Select channel 1 Task info Tas
41. 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 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 WF 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 40 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
42. er 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 uniform 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 atone 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 40 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
43. g a license string 3 Installing the license string 3 Location of the license file 4 Trouble shooting license strings 4 Updating the software 4 Removing the software 4 System requirements for Huygens Essential 5 Windows and Linux 5 Mac OS X 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 16 Bleaching correction 16 Stopping the MLE algorithm 16 Finishing or restarting a deconvolution run 16 z drift corrector for time series 16 Saving the result 17 Saving the restored image 17 Saving your Task report 17 Multi channel images 17 Deconvolving a channel in a multi channel image 17 Joining the results 18 The Huygens Essential User Guide i Contents 4 Huy
44. ge all channels of the image are searched for beads that meet the selec tion criteria After each successful or un successful search there is the possibility to load in additional bead images or go to the next stage e Stage 2 Distill stage In this stage the PSF is measured from the averaged beads for all available channels Huygens Essential by Scientific Volume imaging B V File Task Visualization Tools original psf04 ic There are currently 7 beads in the accumulator Load an additional bead image or click Distill to start the PSF distiller Load Additional Task info Task plot Image ipeople theo Demolmages Beadimagesipsf05 ics Status opening image file Memory 2 Figure 28 The PSF Distiller After adding one or more bead images an average bead image is displayed in the Accumulator thumbnail After pressing the Distu button a PSF will be generated in a PSF thumbnail 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 current 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 bead
45. gens Essential Visualization Tools 18 The Twin Slicer 18 Color mode 19 Contrast mapping mode 19 Time series 20 The MIP Renderer 20 Soft threshold 20 Rendering a movie 21 The SFP Renderer 21 Summary 21 SFP fundamentals 22 Rendering a movie 23 The Surface Renderer 23 Hue Selector 25 Transparency depth 25 5 Huygens Essential Analysis Tools 25 The Colocalization Analyzer 25 Iso colocalization object analysis 25 How to use the Colocalization Analyzer 26 6 The PSF Distiller 28 Beads for PSF distillation 29 The Distiller stages 29 Starting the Distiller 29 Parameter check stage 29 Averaging stage 30 Confocal and two photon bead images 30 Widefield bead images 30 Distillation stage 30 Assembly stage 30 7 Establishing image parameters 31 Image size 31 Brick wise processing 31 Signal to Noise Ratio SNR 31 Blacklevel 33 Sampling densities 33 Computing the backprojected pinhole radius 34 Airy disk as unit for the backprojected pinhole 34 Converting from integer parameter 35 Shape factor 35 Airy disk as unit for the backprojected radius of a square pinhole 35 Computing the backprojected pinhole distance in Nipkow spinning disks 36 Pinhole radius tables 36 Leica confocal microscopes 36 TCS 4d SP1 NT 36 TCS SP2 36 Zeiss confocal microscopes 37 Olympus confocal microscopes 37 Biorad confocal microscopes 37 Checking the Biorad system magnification 38 A supplied calibration curve 38 8 Improving the quality of your ima
46. ges 38 Data acquisition pitfalls 38 Refractive index mismatch 39 Clipping 39 Undersampling 39 Do not undersample to limit photodamage 39 Bleaching 40 Illumination instability 40 Mechanical instability 40 Thermal effects 40 Internal reflection 40 Deconvolution improvements 40 Acquire an experimental PSF 40 ii Scientific Volume Imaging Contents Spherical aberration correction 41 Improve the estimated parameters 41 9 Appendix 41 License string details 41 Questions 43 Where can I find support on the web 43 What does the quality factor mean while running Huygens 43 Can I deconvolve a TIFF series 43 TIFF file series naming convention 43 Can I deconvolve a single plane widefield image 44 Can I deconvolve a single TIFF image 45 How do I generate a debug log 45 Addresses and URLs 45 Where can we be reached 45 Scientific Volume Imaging b v 45 Distributors 45 Support and FAQ 45 Knowledge database 45 The SVI wiki 45 Alphabetical Index 46 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 deconvolution of micro scopic images Its wizard driven user interface guides you through the process of deconvolving mi croscopic images Huygens Essential is able to deconvolve a wide variety of images ranging from 2D widefield WF images to 4D multi
47. han 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 The Huygens Essential User Guide 33 7 Establishing image parameters 0 61 ox N jirydisks 2 r b NA with NA the numerical aperture of the lens N the number of Airy disks and the excit Airydisks ation wavelength In principle using A 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 py Note that this relation bypasses the need to know internal system and lens magnifications Converting from integer parameter Shape factor 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 shape which takes care of the conversion from size diameter or edge size to radius The following formula can be used to translate the 8
48. he processor vendor like Intel or AMD the processor family like Pentium and the processor model For Irix machines this is the IP level 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 45 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 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
49. hed microscop ic parameters This took place off the screen and is fully transparent to the user If the size of the computer s RAM is too small to deconvolve the image as a whole it is split up in parts called bricks SVT 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 30 In this final stage the original inage will be deconvolved on the basis of the PSF and background as computed in the previous stages Below are the default values for deconvolving images If necessary you can change these values Click on Deconvolve to start the deconvolution run Maximum iterations Signal to noise ratio Quality threshold Fr Iteration mode Bleaching correction lt lt Back Task info Task report Image hosts rog u home theofimages faba64 ics Ga Status Moved to stage four of deconvolution Memory 0 Figure 17 Stage 4 starting the deconvolution The Point Spread Function PSF One ofthe 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 propert
50. icer will show the 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 20 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 Disagree the action of the slider on it the slider will then affect only the other image view see Fig ure 21 Click the name button to select an image disable the slider function or center the image Slice x y P Zoom 200 Color False colors Gamma Lineair ey 25 150 400 Grey scale Linear z 50 200 500 True colors Compressed ya 100 300 600 False colors Wide Field 32 Este o 10 20 w 40 50 60 Figure 20 The Twin Slicer applied to a 3D dataset The selected display settings are highlighted in blue 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 position You can center the image again by selecting CENTER IMAGE from the name button 18 Scientific Vol
51. ies are packed into this 3D function The PSF can be obtained in two different ways 14 Scientific Volume Imaging 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 stage one Because a theoretical PSF can be generated without any user intervention Huygens Essential does the calculation in the background without any notice 3 The image restoration process 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 38 and how to correct it on page 40 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 Fite gt Oren PSF Main menu If you load a PSF Huygens Essential will automatically use it If the measured PSF contains less 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 27 for more information on measuring a PSF A measured PSF should only be used for deconvolution if the image and the bead s were recorded with the same micros
52. 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 43 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 DECONVOLUTION 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 n st LLer and proceed with generating a Point Spread Function PSF see page 14 from measured beads See The PSF Distiller on page 27 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 bett
53. ir 1S placed in the Refractive indexes Relative background images subdirectory of Sample sizes Signal to Noise ratio SNR svi Wavelength stage P The Preprocessing _ _ semua a A a a E 3 image into a watery object total internal reflection will truncate the NA stage lo adin g an ima ge As a rule of thumb the effective NA is 1 lower than the manufacturer s specification converting data sets para meter check and cropping Figure 7 The Dictionary The Dictionary and the Questions e stage 1 Tune the paramet from Here give additional information ers This stage will be skipped when you entered from the preprocessing stage In the preprocessing 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 Restarrt 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 Open from the Fite menu to enter the file browser and move to the directory where your
54. k report Task plot 0 0 0 0 Excitation wavelength ET L Memery 075 dl Emission wavelength Ew Figure 10 Importing a microscopical parameters template paea 250 Photon count A Close Reset Accept The Import MICROSCOPIC TEMPLATE button will allow you to choose ES Rent a template from a list of pre saved template files which reside both in the common templates directory and in the user s person al template directory The Huygens common templates directory is named 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 OSX The user s personal templates directory is called svi onthe 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 nisk option Figure 11 Microscopical parameters corrector 10 Scientific Volume Imaging 3 The image restoration process The intelligent cropper Lo a imported microscopic template widefield C C gt C O The Export TEMPLATE button will allow you to either save the tem plate to the personal template directory by choosing the To DISK Option or overwrite one of the existing templates by se lecting them from the list
55. kelihood Estimation 14 15 mechanical instability 40 MIP 11 20 24 MIP Renderer 20 MLE 14 16 movie rendering 21 23 multi channel 7 12 16 17 22 28 multi photon excitation 30 33 40 42 N NA 9 33 35 36 38 40 Nipkow 9 36 42 non circular 35 numbering of TIFF series 44 numerical aperture 9 35 38 39 Nyquist rate 33 39 0 object analysis 25 Olympus 37 optical units 38 orange background 9 P photon 30 32 39 40 photon count 9 pinhole 9 33 34 36 38 Point Spread Function PSF 14 PSF 8 14 15 28 29 39 44 PSF Distiller 4 7 15 28 quality threshold 15 R red background 9 refractive index mismatch 39 Renderer MIP 20 SFP 21 Surface 23 25 rendering 20 23 24 S sampling 13 29 33 39 saturation 13 saving 17 Scientific Volume Imaging b v 45 SFP renderer 21 shape correction 35 shape factor 35 Signal to noise ratio SNR 15 31 Simulated Fluorescence Process 21 SNR 15 31 specimen moves 40 spherical aberration 15 39 41 square pinhole 35 36 statistics 9 StuffIt Expander 1 Surface Renderer 23 42 T task report 17 template 10 thermal effects 40 TIFF file 17 24 43 TIFF series 23 43 44 time series 8 16 20 21 23 42 total internal reflection 39 Transparency 23 25 Twin Slicer 14 16 18 20 two photon 30 40 42 U undersampling 39 unstable arc lamps 40 updates 3 V vibrations 40 Viewer Colocalization Analyzer 25 MIP Rendere
56. lect what voxels are considered to shape volumes Connected voxels after the threshold determine independent volumes that will be represented by the 3D surfaces containing them with different colors You can use up to two different data channels for surface rendering one in each of the two available surface graphic pipes The color of the different objects Huygens Surface Renderer original File Options inside a channel can be modi fied with a selector see Hue Selector below There is a third graphic pipe to redirect data to the rendered image the MIP pipe works projecting the voxels with maximum intensity lay ing in the path of the rays traced along the viewing dir ection see The MIP Renderer on page 20 In combination Twist 123 deg Feady Canvas size 624 x 559 Dragging on Zoom 1 20 Tat 21 deg Frame selection Frame 0 0 Primary surface Threshold 8 3 Transparency 0 00 Brightness 0 60 a os Chan 0 Secondary surface Threshold 8 3 Transparency 0 00 Brightness 0 60 CFD Chan 1 MIP Threshold 6 2 Transparency 0 60 Brightness 1 00 Chan 2 with the surface pipes you can obtain very clear repres Figure 26 The Surface Renderer entations ofthe different ob jects in your image The MIP rendering of one channel can be a good spatial reference for the objects in the other channels You can control the transparency and the
57. lity 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 deconvolution Best cause of ac 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 8 Also available on line in http support svi nl wiki NyquistRate The Huygens Essential User Guide 39 8 Improving the quality of your images Internal reflection 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 mea
58. lve 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 channel 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 The Huygens Essential User Guide 17 3 The image restoration process Joining the results 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 Fe gt Save RESTORED As menu 4 Huygens Essential Visualization Tools Huygens Essential provides different tools for data visualization The Twin Slicer 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 sucer The Twin Sl
59. n ADDITIONAL after completing a search 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 Dista to enter the 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 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 The Huygens Essential User Guide 29 6 The PSF Distiller 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 We advise you to measure the PSF of your system for each recording situation and certainly 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
60. n on how to convert to a backprojected radius The Huygens Essential User Guide 35 7 Establishing image parameters Zeiss confocal microscopes Table 2 Table 3 Zeiss LSM410 inverted Range begin Range end Reported parameter P 0 255 Diameter micron 0 1000 Pinhole geometry square System magnification Mss 2 23 Backprojected pinhole ra dius nm 992 P Eq 7 M obj 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 2m sys Eq 8 obj Zeiss LSM510 pinhole parameters Olympus confocal microscopes Olympus Fluoview Reported parameter 2 3 Diameter d micron 100 150 200 300 Pinhole geometry square System magnification Mss 3 426 3 8 FV500 Backprojected pinhole radi us nm 2 Ve M sys M obj 10 d Eq 9 Table 4 Olympus Fluoview pinhole parameters Biorad confocal microscopes Biorad Reported parameter Pg n a Diameter d mm 0 8 Pinhole geometry circular System magnification Mss 53 83 reported 60 for the Radiance 53 for the 1024 Backprojected pinhole radius nm 10 d gt q 10 m system m obj Table 5 Biorad MRC
61. nding box transparency Channel parameters cho m az Render Soft threshold 0 01 Animate You will find different render ing options on the window and also in the Options menu The configurable parameters are the rendering size and quality the appearance of the bounding box and the mode of the soft thresholds applied to the image channels Soft threshold 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 S If S lt threshold value range 2 20 Scientific Volume Imaging 4 Huygens Essential Visualization Tools Rendering a movie 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 from Harp to Sort you progressively increase the range value thus broadening the t
62. ng The transparency of the object for the emission light controls the propagation of the emitted light to what extent the viewer can peer inside the object through the matter the algorithm The light source is drawn here inside the figure but in real is computes the final intensities of all placed at infinite distance as to make the light rays parallel wavelengths the spectrum of the The renderer in Essential is in non perspective mode so light reaching the viewpoint By de called orthogonal projection i e the viewpoint is at infinity fault 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 different sceneries Since the volume rendering process is rather compute intensive a preview image is dis played see Figure 24 Apart from the viewpoint settings and the optional zooming the following sliders affect the image e Transparencies 22 Scientific Volume Imaging 4 Huygens Essential Visualization Tools 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 f
63. ng image parameters Signal to Noise Ratio SNR Figure 29 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 The Signal to Noise Ratio SNR can be estimated from the quality of the image In Figure 29 you find some examples of recordings where different noise levels were added to an original restored image 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 M SNR s If a significant blacklevel is present it should be subtracted from these values before making the quotient See 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 The Huygens Essential User Guide 31 7 Establishing image parameters
64. o inspect the result in detail Depending on the outcome of that you can choose Acain 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 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 For 3D time series the program pops up an additional tool that enables you to correct for movement in the z axial direction that Fiter type min could have been occurred for instance by iter with 5 thermal drift ofthe microscope table In Ruterunce channels One channel case of a multi channel image see p 17 Reference channel the corrector can survey ALL CHANNELS and determine the mean z position of the chan nels or it can take ONE CHANNEL as Set
65. ock gt Set of license policy or locking flags having of one or more characters d License expiry date e Email address m Hardware system 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 t
66. ojection 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 Time 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 us ing the crop tool s channels selecting tool CHANNELS gt SELECT CHANNELS Stage 1 Parameter tuning Stage 1 enables you to tune your parameter settings as set in the pre processing stage After you have fin ish 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 Figure 13 The Crop tool Stage 2 The image histogram 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 reco
67. or ESP Ghostscript 705 epswrite CreationDate 2005 02 09 10 34 11 LanguageLevel 2 Figure 31 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 backprojected radius in nanometers r Backprojected means the size of the pinhole as it appears in the specimen plane the physical pinhole radius f phys 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 a AA D M obj m system where M obj is the magnification factor of the objective and m is the fixed magnification of system the system 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 t
68. or 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 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 Frame 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
69. pth 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 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 an Object Analyzer will be available soon The Colocalization Analyzer The Colocalization Analyzer provides information about the amount of spatial overlap between structures in different data channels for 3D images and time series As this overlapping can be defined in many ways Huygens gives 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 The Colocalization Analyzer is an extended optional tool and is enabled by a C flag in the license string see License string details on page 41 Iso colocalization object analysis One ofthe features ofthe colocalization analyzer is iso colocalization object analysis It allows you to quickly determine the properties of the different colocalization regions in
70. r 20 SFP Renderer 21 Surface Renderer 23 Twin Slicer 18 violet background 9 W wavelength 9 22 28 33 36 39 44 website 4 21 widefield 30 42 Z z drift corrector 16 z stage moving 40 Zeiss 36 37
71. r 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 SHow n SFP RENDERER see Figure 24 A virtual light source produces excitation light that illuminates the object This casts shadows either on parts ofthe 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 ofthe viewer see Figure 25 The voxel values in the image are taken as the density of a fluorescent material If the voxels are multiparameter multi channel in microscopic parlance see page 17 each parameter is taken as The Huygens Essential User Guide 21 4 Huygens Essential Visualization Tools a different fluorescent dye Huygens Essent SPP Renderer erin Each dye has its specific ex S zn citation and emission wavelength with correspond amp
72. ram selected from linear logarithmic The vertical mapping mode can be or sigmoid 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 ofthe image are correct in especially the sampling distance and the microscope type The following choices are pos sible here e Lowest value De fault The image is searched for a 3D re gion with the lowest average value The axial size ofthe re gion is around 0 3 micron the lateral size is controlled by the radius parameter which is default set to 0 5 micron In near object The neighborhood around Huygens Essential by Scientific Volume Imaging B V Hle Task Visualization Tools Help Task info Image hostsirog uhomeitheofmages faba 4 ics Dl Status Moved to stage three of deconvolution Memory 0 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 Figure 16 Estimating the average background the voxel with the highest value is searched for a planar region with the lowest average value The size ofthe region is controlled by
73. ransition 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 Anmate and select a directory to save the TIFF frames to You can later load and edit 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 make 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 rendere
74. rding 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 Cro ae intensity 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 x Select the start and stop time frame 0 value and the y axis is the number of pixels in the image with C that gray value If the image is more than 8 bits gray values are collected to form a bin for example gray values from 0 9 form 3 bin 0 values from 10 19 form bin 1 etc The histogram is now the number of pixels in every bin P ty Cancel Apply OK The histogram from the demo image as shown in Figure 15 is Figure 14 Reducing the of reasonable quality The narrow peak you see at the left rep number of time frames resents the background pixels all with similar values The height of the 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 32 12 Scientific Volume Imaging 3 The image restoration process If a peak is visible at the extreme right hand side of the histogram i
75. rop ORIGINAL IMAGE Menu Once you have cropped your image during the guided restoration process you can not crop it again except 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 indicate 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 pr
76. s 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 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 Fe gt Open If your license in cludes the PSF Distiller option the Starr PSF p st LLer 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 28 Scientific Volume Imaging 6 The PSF Distiller Parameter check stage A Averaging stage Distillation stage Assembly 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 Do not use undersampled bead images If any of the entry fields for the 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
77. 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 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 45 3 The image restoration process The processing stages Huygens Essential guides you through the process of microscopic image deconvolution 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 Here gt Questions and HELP gt Dictionary Figure 7 6 Scientific Volume Imaging 3 The image restoration process The following steps and stages are to Bein a al be followed 1 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 i 1 Bleaching conection a e Opening an image A demo ee Multi channel image the Multi photon i i Numerical Apert faba64 ics ids file Nahen m m x Point Spread Function PSF pa
78. sed the slider only moves T s kis o 1 2 3 4 5 6 7 8 Fan 10 n 12 13 14 15 16 17 18 forward i e in the positive 5 Time per time direction g 1 2 3 Figure 22 The Twin Slicer with time slider The MIP Renderer 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 23 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 SHow n MIP RENDERER SX Huygens Essential MIP Renderer e959 61grb_huy o x File Options Select your viewpoint by mov ing the Tilt and Twist sliders or by dragging the mouse pointer on the large view that will be empty at first before your first render ing Also try changing the zoom You will see how the preview thumbnail changes When you have set all the ren dering options click Render to create the final view that you bei can save as a TIFF image Figure 23 The Maximum Intensity Projection MIP Renderer Zoom 1 21 Set Go Tit ua Quality Image parameters Time frame 0 Bou
79. sure 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 commendable 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 27 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 on
80. t 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 occasions they are replaced by zeros Clipping will have a negative effect on the results of deconvolution especially with WF images See page 38 The histogram stage is in cluded for examining purpose only It has no meaning for the deconvolution process that follows Stage 3 Estimate the average background in the image In this stage the average back ground in a volume image is estimated The average back ground is thought to corres pond with the noise free equi valent of the background in the measured noisy image It Huygens Essential by Scientific Volume Imaging B File Task Visualization Tools v 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 Task info Task plot Image usrflocal svi images faba64 ics Status Histogram computed Memory 0 Figure 15 The image histog
81. tem ID your mail program 13352 8350 de1d 4250 Copy In this dialog box you can also find a button to CHECK FOR HUYGENS UPDATES ON our Company Server Figure 2 The Herr gt Asour window The i system ID is shown at the bottom Installing the license string Select the license string in your email message and copy it to the clipboard using Eprr gt Cory in your mailing program Start Huygens Essential and go to Herrer 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 license string into the text field Complete the procedure using the App License 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 license 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 monp d t ennps eom2003Jun23 2372e2f612c80f3 theo svi nl 9b1 52a60373b2d7e3bd 1 u nn nn FlUVR 2 4 w d emnps eom2002Jun27 a9e84ad0476692F4 hans svi nl 064f42bf357f1aaaT2af HuPro
82. ter the colors for the differ ent image channels see Multi channel images on page 17 The color of a particular chan nel can be edited by clicking the corresponding button This opens a platform specific color editor Figure 8 Image statistics Right mouse click on a thumb nail image and select snow PARAMETERS and you will find Select a color for each channel EE ars channel 1 channel 2 channel 3 ES Cancel Apply Figure 8 The Image Color Picker Clicking on a channel button top opens a color editor right by which the color can be changed The color editor is platform specific The picture on the right shows the Mac OSX color editor besides the parameter settings Cancel OK statistical information of the particular image Amongst them are the mean sum stand ard deviation norm and position of the center of mass 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
83. 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 3 Learn more about histograms in http support svi nl wiki ImageHistogram The Huygens Essential User Guide 13 3 The image restoration process is determined by searching this region for the planar region with radius r that has the lowest value Press the Estrmate button to continue You may now adapt the value if you like to either by alter ing the value in the Estimate BACKGROUND field or in the RELATIVE BACKGROUND field Setting this last 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 iter ative deconvolution run stage 4 first carries out several pre processing steps ly 3 Huygens Essential by Scientific Volume imaging B V File Task Visualization Tools Background This value is calculated in stage 3 You can veri fy whether this value represents areas in the image which you consider background by opening the Twin Slicer see Figure 20 and moving the mouse pointer over the areas of interest The voxel values are displayed below the image Modify the value as you see fit A Point Spread Function PSF see below was generated from the establis
84. tion surface 125 Threshold 4e 07 4 Transparency 0 60 Brightness 0 80 zu off cho MIP of original image Threshold 15 6 Transparency 0 60 Brightness 0 78 Channel Twist 121 deg Frame selection Frame 070 Two channel histogram Frame colocalization coefficients M1 0 732 M2 0 676 Kl 1 859 K1 0 057 Red chan 1 bg 3 00 Green chan 0 bg 2 00 Pearson 0 203 Overlap 0 229 M1 0 777 M2 0 405 K1 1 511 K1 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 q cho chi ch2 cho Chi ch2 Colocalization map Pearson Ready Canvas size 509 x 484 Dragging on Figure 27 Colocalization Analyzer with surfaces for regions of overlapping 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 coordinate We follow the usual naming convention in colocalization theory for the two compared channels Red for the first channel Green for the second channel We can select in the lower part ofthe window which d
85. ume Imaging 4 Huygens Essential Visualization Tools 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 Color mode SX Huygens Essential Twin Sheer ial x Gray pixel values are assigned different shades of gray ran ging from black for the lowest values to white for the highest values False color pixels values are assigned ee e253 61g uy different colors ran al am 1m am an nu i x 2 50 200 500 True colors Compressed ging from black dark Io soo sum Era do Ft purple for the lowest a values to bright red 0 10 20 30 40 50 60 70 80 30 100 110 120 130 140 150 160 Value chand chan2 019 000 000 at x 9 2 503 063 050 for the highest value Figure 21 The Twin Slicer showing two slices of the same 3D image True color pixel val ues are assigned dif ferent shades of a particular color ranging from black for the lowest values to the bright est possible shade for the highest values Multi channel images are always rendered with a true color scheme otherwise the information from the different channels will result in very confusing images Contrast mapping mode 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 th
86. use this information to analyze nv enables multi photon microscope functionality the problem See License string de N tails on page 41 ox l ey Figure 5 The License details Updating the software Download the new version from the SVI website at http www svi nl 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 software 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 MacOSX Drag the installation to the waste basket e Microsoft Windows Clicking Start in your Windows desktop and select Procrams gt Huycens 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
87. 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 ofthe data p 20 The color range in which these objects are displayed can be modified using a hue selector p 24 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 Visuauization 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 ofthe data You can select the viewpoint of the MIP projection by moving the Tit and Twist slides or by dragging the mouse pointer on the object view Also try changing the zoom 5 http support svi nl wiki ColocalizationTheory The Huygens Essential User Guide 25 5 Huygens Essential Analysis Tools Huygens Colocalization analyzer a Ol File Options Zoom _Iso colocaliza

The Huygens Essential User Guide

Contents

Download Pdf Manuals

Related Search

Related Contents