BITPLANE AG IMARISCOLOC. Operating Instructions. Manual Version 1.0 January the image revolution starts here.

Transcription

1 BITPLANE AG IMARISCOLOC Operatng Instructons Manual Verson 1.0 January 2003 the mage revoluton starts here.

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3 Operatng Instructons BITPLANE AG Copyrght Ths document contans propretary nformaton protected by copyrght. No part of ths document may be reproduced, translated, or transmtted wthout the express wrtten permsson of Btplane AG, Zurch, Swtzerland. If you fnd any nformaton mssng n ths manual or need addtonal support, please contact us: Btplane AG Badenerstrasse 682 CH-8048 Zurch Swtzerland Telephone FAX Emal support@btplane.com Web address Notce Informaton n ths book s subject to change wthout notce and does not represent a commtment on the part of Btplane AG. Btplane AG s not lable for errors contaned n ths book or for ncdental or consequental damages n connecton wth the use of ths software. 2003, Btplane AG, Zurch All rghts reserved. Prnted n Swtzerland. ImarsColoc Manual Verson 1.0 Page 1

4 BITPLANE AG Operatng Instructons Contents COPYRIGHT... 1 NOTICE... 1 CONTENTS 2 WHAT IS IMARISCOLOC? 4 MAIN SCREEN... 5 PREVIEW WINDOW... 6 CHANNEL HISTOGRAMS D HISTOGRAM... 7 VOLUME STATISTICS... 8 MASK CHANNEL... 8 CONTROL PANEL... 9 SELECTION MODE... 9 AUTOMATIC THRESHOLDING HISTOGRAM MODE HISTOGRAM OPTIONS COLOC INTENSITIES COLOC COLOR BUILD COLOC CHANNEL CHANNEL STATISTICS BASIC OPERATION 13 LOADING A DATA SET SELECTING THE CHANNELS...14 SETTING THE THRESHOLDS SPECIFY SELECTION MODE SELECT HISTOGRAM MODE THRESHOLD SETTING METHODS COLOC DISPLAY COLOC INTENSITIES COLOC COLOR COLOC VOLUME STATISTICS DEFINITIONS NUMBER OF COLOCALIZED VOXELS % OF DATASET COLOCALIZED % OF CHANNEL A VOLUME COLOCALIZED % OF CHANNEL B VOLUME COLOCALIZED % OF CHANNEL A MATERIAL COLOCALIZED % OF CHANNEL B MATERIAL COLOCALIZED % OF UNMASKED DATASET COLOCALIZED % OF MASK A MATERIAL COLOCALIZED % OF MASK B MATERIAL COLOCALIZED CHANNEL CORRELATION IN COLOCALIZED VOLUME CHANNEL CORRELATION IN DATA SET VOLUME CHANNEL CORRELATION IN UNMASKED VOLUME Page 2 ImarsColoc Manual Verson 1.0

5 Operatng Instructons BITPLANE AG BUILD COLOC CHANNEL CALCULATION AND BUILDING VIEWING STATISTICS EXPORT STATISTICS VE COLOC CHANNEL ADVANCED OPERATION 21 POLYGON MODE DRAWING METHODS MASK CHANNEL AND AUTOMATIC THRESHOLD TIME SETTINGS EXAMPLES 24 VISUALIZE THE COLOC CHANNEL IN IMARIS EXAMPLE VISUALIZE THE COLOC CHANNEL IN SURPASS EXAMPLE ImarsColoc Manual Verson 1.0 Page 3

6 BITPLANE AG Operatng Instructons What s ImarsColoc? In fluorescent mcroscopy, colocalzaton descrbes the presence of two fluorochromes at the same physcal locaton. ImarsColoc provdes extended functonalty for the analyss and vsualzaton of colocalzaton n mult-channel data sets. It provdes an automated selecton of colocalzaton based on a method ntroduced by Costes & Lockett at the NIH, NCI/IC. ImarsColoc processes 2D, 3D and n conjuncton wth ImarsTme also 4D data sets. It operates on two channels smultaneously and measures the degree of overlap of the two channels. Wth Coloc, the user can determne and dsplay locatons wthn the data set wth common nformaton from all channels, e.g., to llustrate where all types of protens can be found smultaneously or to detect low-densty tssues n all channels. The desred range for sgnal analyss can be lmted by defnng ntensty thresholds or sets of ntensty values for each of the nvolved channels. All voxels fallng nsde every of these channel-specfc ntensty lmts represent a colocalzaton. They are emphaszed n the vew and statstcs are calculated and dsplayed. When the selecton s changed, the numerc and vsual feedback happens n real-tme. The result of the colocalzaton as a whole can be saved as a separate channel, whch can then be processed and vewed as any orgnally acqured channel n IMARIS. Multple colocalzaton mages are easly combned nto a sngle vew n ImarsSurpass. ImarsColoc s an optonal module wthn the IMARIS program and s not avalable as a standalone program. Page 4 ImarsColoc Manual Verson 1.0

7 Operatng Instructons BITPLANE AG Man screen When selected n the IMARIS menu bar, the ImarsColoc man screen s dsplayed. The screen conssts of the followng areas: Channel Hstograms 2D Hstogram Prevew Wndow Volume Statstcs Control Panel Mask Channel Fgure 1: ImarsColoc man screen ImarsColoc Manual Verson 1.0 Page 5

8 BITPLANE AG Operatng Instructons Prevew Wndow Ths area vsualzes the data set together wth a prevew of the colocalzed regon. It dsplays a Slce representaton of the data set,.e. a 2D vew from the top n the z-axs drecton. A sldng bar wth a handle allows nspectng and dsplayng the sngle slces n the data set along the z-axs. The prevew wndow updates - n real tme - the dsplay of the colocalzed regon based on the actual settngs of the colocalzaton parameters. The orgnal channels can be swtched on or off usng the channel vsblty edtor. The user can zoom and pan the vew wth the mddle and rght mouse buttons, respectvely. Fg. 2: Prevew wndow wth Channel vsblty and Dsplay adjustments Channel Hstograms Ths area allows the selecton of the two channels for the colocalzaton analyss. The ntensty hstograms are dsplayed for the currently selected channels. Wth most data sets, checkng the Ignore border bn and Logarthmc optons (see Hstogram optons on page 11) allows a more representatve hstogram dsplay. Page 6 ImarsColoc Manual Verson 1.0

9 Operatng Instructons BITPLANE AG The threshold for each channel can then be defned by draggng the yellow lne drectly n the hstogram or by enterng the value n the threshold feld. Fg. 3: Channel selecton and ntensty Hstograms 2D Hstogram Ths area dsplays a two-dmensonal ntensty hstogram of the selected channels. It reflects the dstrbuton of pars of voxel ntenstes occurrng n the two selected channels. The range of ntensty pars consdered as colocalzed can be defned on the hstogram ether as channel thresholds, vsble n form of a rectangle or, f selected, as an ntensty area n form of a free confgurable polygon. Fg. 4: 2D Hstogram ImarsColoc Manual Verson 1.0 Page 7

10 BITPLANE AG Operatng Instructons Volume Statstcs Ths area dsplays statstcs about the resultng colocalzed volume. The dsplay adapts nstantly to all modfcatons n the selecton. Fg. 5: Volume Statstcs Mask Channel Ths feature allows the user to defne a thrd channel as a maskng area for the entre analyss. A maskng channel s e.g. a thrd mcroscope channel acqured wth settngs that allows defnng a regon of nterest by smple ntensty thesholdng. All voxels outsde of the regon of nterest defned by the mask channel are gnored for the colocalzaton analyss. The volume excluded by the mask appears hatched n the Prevew wndow. The mask channel s used n conjuncton wth the Automatc Threshold functon. For ths functon, the selecton of an approprate regon of nterest s essental. Fg. 6: Mask Channel Page 8 ImarsColoc Manual Verson 1.0

11 Operatng Instructons BITPLANE AG Control Panel The Control panel contans several sectons wth all possble selecton and defnton tools and settngs for the hstograms, the thresholdng, and the dsplay as well as the buttons for the color defnton and the buld of the coloc channel. Fg. 7: Control panel Selecton mode Threshold The user can defne, for each of the channels separately, an ntensty value as a threshold. Only voxels wth an ntensty value above the specfed threshold wll be consdered for the colocalzaton analyss. The two thresholds are vsble and can be set n four dfferent ways: As a hatched regon wth a bolded lne on the 1D hstograms. As a hatched regon wth bolded lnes on the 2D hstogram. The regon always contans the top rght part of the hstogram. The left and lower bolded lnes defne the ntensty thresholds for the two channels. As a value n the Threshold feld of each channel. As an actve contour n the Prevew wndow. ImarsColoc Manual Verson 1.0 Page 9

12 BITPLANE AG Operatng Instructons When usng one of the methods to modfy the threshold values, the other ndcatons as well as the Prevew dsplay are updated n realtme. Polygon The user can defne a freely shaped polygonal regon whose nteror defnes the ntensty pars consdered as colocalzed. Automatc thresholdng Ths button performs an automatc threshold run on both channels based on an algorthm developed by Costes and Lockett at the Natonal Insttute of Health, NCI/IC, whch s based on the excluson of ntensty pars that exhbt no correlaton (Pearson s correlaton below zero). The automatc threshold search s done usng the 2D hstogram Startng wth the hghest ntensty value, the algorthm reduces the threshold value step by step along a lne shown n Fgure 8 and computes the correlaton coeffcent of the mage usng only voxels wth ntenstes below the threshold. The algorthm contnues reducng the thresholds untl the correlaton reaches 0, thus defnng the automatc threshold. Channel A lne along whch algorthm proceeds Correlaton = 0 on both channels. Fg. 8: Automatc Threshold Detecton Channel B For more nformaton please refer to the followng publcaton: S. Costes, E. Cho, M. Catalfamo, T. Karpova, J. McNally, P. Henkart, S. Lockett, Proceedngs Mcrosc. Mcroanal. 8 (Suppl. 2), CD, 2002 Page 10 ImarsColoc Manual Verson 1.0

13 Operatng Instructons BITPLANE AG Hstogram mode The hstogram mode optons are used to defne the calculaton bass for the colocalzaton wthn the data set. Sngle slce Sngle tme pont All tme ponts The hstograms are calculated and dsplayed for the current slce only. The hstograms are calculated and shown for the entre 3D stack of the current tme pont. Ths mode allows for faster browsng through the data set along the z-axs. The hstograms are calculated and shown for an entre tme data set,.e. for all tme ponts wthn the 4D data set. Hstogram optons The hstogram mode optons are used to defne the dsplay of the 1D and 2D hstograms. Ignore border bns Logarthmc Color coded Often a lot of voxels fall nto the bn wth the lowest ntensty. In a frequency plot ths causes a massve and unnecessary downscale of all other ntensty bns whch are of more nterest. The border bns can therefore be gnored when scalng the dsplay of the hstogram. Ignore s selected as default. The frequency plot s shown n a logarthmc scalng nstead of a lnear scalng. Apples a false color look-up table to the hstogram to mprove the vsblty of ntensty dfferences. The hgh frequences (bns n the 2D hstogram nto whch a lot of voxels accumulate) are shown n yellow-to-whte, bns wth lttle voxels are shown n blue-toblack. ImarsColoc Manual Verson 1.0 Page 11

14 BITPLANE AG Operatng Instructons Coloc ntenstes The Coloc ntenstes opton defnes the brghtness of the calculated colocalzaton dsplayed n the Prevew wndow. Source channels Constant value Uses the ntenstes of the two channels for dsplayng the ntensty of a colocalzed voxel as the square root of the product of the ntenstes of the orgnal channels. Dsplays the ntensty of a colocalzed voxel defned by the specfed value. No dstncton s made whether hgh-ntensty or low-ntensty voxels colocalze as long as they are wthn the selecton. Coloc color Coloc color allows the user to specfy hs personal color settngs for the colocalzaton dsplay n the Prevew wndow. Edt Clckng on the Edt button opens the Color wndow. The user can ether select one of the predefned colors to apply on the colocalzaton areas or defne, add, and apply hs own color defntons. Buld Coloc Channel Clckng ths button calculates the colocalzaton channel accordng to the selected settngs and adds t to the data set. The channel can then be saved and s part of the data set. Channel Statstcs After buldng the coloc channel, ts statstcs become avalable n the Image Propertes. The Channel Statstcs button dsplays drectly the Image Propertes wndow wth the statstcs for the calculated channel. Page 12 ImarsColoc Manual Verson 1.0

15 Operatng Instructons BITPLANE AG Basc Operaton The basc use of ImarsColoc n the daly workflow comprses the followng steps: 1. Loadng a data set 2. Selectng the channels 3. Settng the thresholds 4. Checkng the statstcs 5. Buldng the Coloc channel 6. Savng the Coloc channel Loadng a data set Any 2D, 3D, or 4D mult-channel and/or tme data set of the all types supported by IMARIS can be loaded and used for colocalzaton detecton: Fg. 9: Fle open dalog 1. In the man toolbar select Fle Open. The Open box s dsplayed. 2. Select fle type from the Fles of Type pull-down menu. 3. Select a fle name from the lst and clck Open or double-clck on the requested fle entry. The fle s loaded. ImarsColoc Manual Verson 1.0 Page 13

16 BITPLANE AG Operatng Instructons Selectng the channels The two channels for colocalzaton detecton must be selected frst. 1. Select channel A and channel B from the pull-down lsts. The 1D and 2D ntensty hstogram dsplays are updated. 2. Modfy the Hstogram optons f necessary n order to dsplay an expressve voxel ntensty representaton. Settng the thresholds Specfy Selecton mode In order to be able to set the thresholds, the user must check the Selecton mode Threshold n the control panel. Select Hstogram mode Check: Sngle Slce for calculatng the hstogram for current slce only. One Tme Pont to calculate the hstogram for the entre 3D tme pont. All Tme Ponts for the entre 4D mage. Threshold settng methods There are bascally four dfferent methods how the thresholds can be defned. On the 1D Channel Hstogram 1. Pont wth the cursor on the hstogram. The cursor turns nto a cross. 2. Clck on the hstogram to determne a threshold. The bolded left lne of the overlad hatched area (ndcatng the selected voxel ntensty range for colocalzaton) jumps on the marked spot. 3. Clck on the lne and drag t to another poston n the hstogram. Effects of the modfcaton are nstantly vsble n the other ndcatons as well as n the Prevew wndow. 4. Perform for both channels untl the settngs are as desred. Page 14 ImarsColoc Manual Verson 1.0

17 Operatng Instructons BITPLANE AG By enterng a value 1. Clck n the Threshold feld of one of the channels and enter a value, then press Enter. The bolded left lne of the overlad hatched area (ndcatng the selected voxel ntensty range for colocalzaton) jumps on the marked spot. Effects of the modfcaton are nstantly vsble n the other ndcatons as well as n the Prevew dsplay. 2. Perform for both channels untl the settngs are as desred. On the 2D Hstogram 1. Pont wth the cursor on the 2D hstogram. The cursor turns nto a cross. 2. Clck on the hstogram to determne the thresholds. The edge of the overlad hatched area (ndcatng the selected voxel ntensty range for colocalzaton) jumps on the marked spot. 3. Clck on the edge and drag t to any another poston n the hstogram. Effects of the modfcaton are nstantly vsble n the other ndcatons as well as n the Prevew dsplay. On the Prevew Wndow 1. Clck wth the cursor on the mage n the Prevew wndow. An actve contour s dsplayed, outlnng mage elements brghter than the locaton clcked. 2. Drag the lne on the mage to defne the threshold of channel A. The selecton works best when startng wth brghter spots and draggng towards darker areas. 3. Press and hold the SHIFT key, clck wth the cursor on the mage, then drag the lne to determne the threshold of channel B. Colocalzaton occurs where the voxels are above threshold n channel A and channel B. Fg. 10: Threshold selecton on the Prevew wndow ImarsColoc Manual Verson 1.0 Page 15

18 BITPLANE AG Operatng Instructons Coloc Dsplay Coloc ntenstes The user may determne the ntensty for the colocalzed voxels to be shown. Check: Source channel for usng the ntenstes of the two channels for dsplayng the ntensty Constant value for dsplayng the ntensty of a colocalzed voxel defned by the specfed value. Coloc Color 1. Clck the Edt button. The color box s dsplayed. 2. Select any of the colors and clck OK. The ndcaton bar shows the selected color and the colocalzaton areas n the Prevew wndow are colored accordngly. 3. If requred, clck Color defnton and use the colorwash to create and add your own colors for selecton. Coloc Volume Statstcs The values are updated n real-tme and refer to the entre volume data of the current tme pont, regardless of the settngs for the Hstogram mode. The values are calculated by the followng formulas: Defntons Regons: Data set Coloc Object A Object B all data set voxels colocalzed voxels Voxels wth channel A ntensty above threshold A Voxels wth channel B ntensty above threshold B Indcators: N avg, coloc Number of voxels Channel A ntensty of th voxel Channel B ntensty of th voxel Average channel A ntensty n the coloc regon Page 16 ImarsColoc Manual Verson 1.0

19 Operatng Instructons BITPLANE AG Number of colocalzed voxels % of dataset colocalzed % of channel A volume colocalzed % of channel B volume colocalzed % of channel A materal colocalzed % of channel B materal colocalzed Total count of colocalzed voxels N coloc. Percentage of total data set voxels colocalzed. N N coloc dataset 100% Percentage of channel A voxels above threshold A colocalzed. N N coloc objecta 100% Percentage of channel B voxels above threshold B colocalzed. N N coloc objectb 100% Percentage of channel A materal above threshold A that s colocalzed. coloc objecta 100% Percentage of channel B materal above threshold B that s colocalzed. coloc objectb 100% % of unmasked dataset colocalzed Percentage of channel A and channel B nsde the resdual mask volume that s colocalzed. N N coloc mask 100% ImarsColoc Manual Verson 1.0 Page 17

20 BITPLANE AG Operatng Instructons % of mask A materal colocalzed % of mask B materal colocalzed Channel correlaton n colocalzed volume Channel correlaton n data set volume Percentage of resdual channel A materal nsde the mask volume that s colocalzed. coloc mask 100% Percentage of resdual channel B materal nsde the mask volume that s colocalzed. coloc mask 100% Pearson correlaton of channel A and channel B nsde the colocalzed regon. (Note: The correlaton value ranges between 1 and -1. A value of 1 represents perfect correlaton, 0 no correlaton, and -1 perfect nverse correlaton.) R AB, coloc = coloc coloc avg, coloc 2 avg, coloc coloc avg, coloc 2 avg, coloc Pearson correlaton of channel A and channel B nsde the entre data set volume. R AB, dataset = dataset dataset avg, dataset 2 avg, dataset coloc avg, dataset 2 avg, dataset Channel correlaton n unmasked volume Pearson correlaton of the resdual channel A and channel B nsde the mask volume. R AB. mask = mask mask avg, mask 2 avg, mask mask avg, mask 2 avg, mask Page 18 ImarsColoc Manual Verson 1.0

21 Operatng Instructons BITPLANE AG Buld Coloc Channel Calculaton and buldng After ts buld the Coloc channel s added to the current data set. The new channel s then avalable n all IMARIS vews - most nterestngly n Surpass. 1. Clck the Buld Coloc Channel button. The colocalzaton channel s calculated whle a box s dsplayed showng the process. Vewng statstcs The calculated statstcs for each colocalzaton channel can be vewed n the Image propertes. 1. In the menu bar select Edt Image Propertes. The Image Propertes wndow s dsplayed. 2. Clck the requred channel n the menu tree. The Name of the channel, the Descrpton of the source channels and the selecton method are ndcated and the Coloc Statstcs are dsplayed. Export statstcs The statstcs can be exported. 1. In the Image Propertes wndow clck the Export button. The Export Coloc Statstcs wndow s dsplayed. 2. Select the drectory and enter the name for the fle to be saved. The statstcs are saved as.csv Excel fle and can be opened drectly wth Mcrosoft Excel. ImarsColoc Manual Verson 1.0 Page 19

22 BITPLANE AG Operatng Instructons Save Coloc Channel Before the channel s added permanently to the data set t must be saved. 1. In the menu bar select Fle Save As or clck the Save as button n the toolbar. The Save As box s dsplayed. 2. Select the drectory and enter the name for the fle to be saved or confrm the suggeston. 3. Select the requested fle format and clck OK. The data set s saved. Fg. 11: Save As dalog Page 20 ImarsColoc Manual Verson 1.0

23 Operatng Instructons BITPLANE AG Advanced Operaton Polygon Mode Instead of usng the basc threshold mode for defnng the area where a colocalzaton wll be determned, the user can defne a freely shaped polygonal regon. The polygon can be drawn on the 2D hstogram, when the Selecton mode Polygon s checked. Ths method s especally useful for elmnatng channel bleed through regons along the border of the lower ntensty bns. Drawng methods After checkng the Polygon mode, the overlad threshold rectangle n the 2D hstogram s converted nto a polygon wth four corner ponts. The user can now start to modfy ths polygon or to draw a completely new one. The ntal pont of the polygon s vsble as black flled. Settng ponts Press and hold the SHIFT key. The cursor turns nto a cross wth a lttle + sgn. Clck on the 2D hstogram wth the left mouse button to place a pont (vertex). Any addtonal pont connects wth lnes to the ntal pont and the last pont nserted. Insertng ponts n lnes Press and hold the SHIFT key. The cursor turns nto a cross wth a lttle + sgn. Pont on the lne between two vertces and clck on the lne wth the left mouse button to add a pont. Automatc pont settng Press and hold the CTRL key. The cursor turns nto a cross wth a lttle + sgn and a stack symbol. Drag the cursor around. New vertces are added automatcally at each change of cursor poston. Movng ponts Pont on one of the vertces, left-clck and drag the vertex around to the desred locaton. The polygon changes ts shape. Movng lnes Pont on a lne between two vertces, left-clck and drag the lne around to the desred locaton. The polygon changes ts shape. ImarsColoc Manual Verson 1.0 Page 21

24 BITPLANE AG Operatng Instructons Movng the polygon Pont somewhere besde of the polygon edge, left-clck and drag the polygon around to the desred locaton. Deletng ponts Pont on one of the vertces. Press the DEL key to delete the vertex. Deletng the polygon Pont somewhere besde of the polygon edge and press the DEL key to delete the entre polygon. Fore more nformaton select Help Keys Help n the menu bar. Fgure 12: Typcal polygon selecton for channel bleed through Page 22 ImarsColoc Manual Verson 1.0

25 Operatng Instructons BITPLANE AG Mask Channel and Automatc Threshold Automatc thresholdng produces only relevant results, f an adequate mask s defned (please refer to the publcaton by Costes and Lockett, mentoned on page 10). 1. In the Mask channel area select the channel to be used from the pull-down lst. 2. In the Mask channel area check the Mask dataset feld. The 1D hstogram of the mask channel s dsplayed and the Threshold feld s actvated. 3. Pont wth the cursor nto the hstogram and clck to set the threshold. A bolded lne marks the threshold n the hstogram and the correspondng value s ndcated n the threshold feld. The masked volume of the data set appears hatched n the Prevew wndow. 4. Drag the cursor n the hstogram to modfy the threshold. Effects of the modfcaton are nstantly vsble n the threshold feld as well as n the Prevew wndow. 5. When the mask s set as requred, clck the Automatc thresholdng buttons to calculate the thresholds. Buld the colocalzaton channel by clckng the Buld Coloc Channel button. Tme Settngs For a detaled descrpton of the Tme opton please refer to the IMARIS manual (ImarsTme s an optonal module of IMARIS and may be not avalable). There are no tme-dependent parameters for the colocalzaton analyss. The Prevew wndow always dsplays the currently selected tme pont of the data set. The values n the Statstcs wndow always refer to the volume of the current tme pont. However, when the Buld coloc channel button s pressed, the coloc channel and statstcal values for all tme ponts are calculated. ImarsColoc Manual Verson 1.0 Page 23

26 BITPLANE AG Operatng Instructons Examples Vsualze the Coloc Channel n IMARIS Example 1 The colocalzaton channel can be vsualzed lke any other channel n IMARIS. In the followng examples, a coloc channel was bult on the retna data set. Vsualze and adjust the Coloc channel n IMARIS Easy 3D. 1. After buldng the Coloc channel, clck on the Easy 3D button n the IMARIS menu bar. 2. Check the desred Renderng mode parameters and adjust the Settngs f necessary, then clck the Calculate button. The 3D mage of channels s calculated and dsplayed. Fgure 13: Easy 3D representaton of all three channels Page 24 ImarsColoc Manual Verson 1.0

27 Operatng Instructons BITPLANE AG As the colocalzed regon always lays nsde the ntal channels, makng the ntal channels nvsble or more transparent allows a better vew of the colocalzed regon. 3. In the IMARIS menu bar select Edt Channel Vsblty. 4. In the Channel Vsblty wndow uncheck one of the two orgnal channels, then clck the Calculate button agan. The 3D mage of the orgnal and the Coloc channel s calculated and dsplayed. 5. In the IMARIS menu bar select Edt Dsplay Adjustment. 6. In the Dsplay Adjustment wndow select the Coloc channel and adjust ts settngs. 7. In the Dsplay Adjustment wndow select the remanng orgnal channel and adjust ts settngs. In ths example the channel s set as partly opaque. 8. After each modfcaton, clck the Calculate button agan to calculate the result. Fgure 14: Easy 3D representaton of the Coloc channel wth one orgnal channel ImarsColoc Manual Verson 1.0 Page 25

28 BITPLANE AG Operatng Instructons Vsualze the Coloc Channel n SURPASS Example 2 The colocalzaton channel can be vsualzed lke any other channel n Surpass (Surpass s an optonal module of IMARIS and may be not avalable). Create an Iso Surface of the Coloc channel add the two ntal channels as volume renderng. 1. After buldng the Coloc channel, clck on the Surpass button n the IMARIS menu bar. 2. In the Object area clck the Add New Iso Surface button. 3. In the Iso Surface Setup wndow check and select the Coloc channel. 4. Select the desred parameters. In ths example, we set the threshold just below the lowest ntensty values n the hstogram. Fgure 15: Iso Surface settngs for colocalzed channel 5. In the Object area clck the Add New Volume button. Page 26 ImarsColoc Manual Verson 1.0

29 Operatng Instructons BITPLANE AG 6. In the IMARIS menu bar select Edt Channel Vsblty. 7. In the Channel Vsblty wndow uncheck the coloc channel. The Surpass Volume object dsplays only the two ntal channels. 8. In the IMARIS menu bar select Edt Dsplay Adjustment. 9. In the Dsplay Adjustment wndow select the two ntal channels and adjust ther settngs, e.g. decrease the blend opacty. Fgure 15: Iso Surface of colocalzed regon and volume renderng of ntal data set ImarsColoc Manual Verson 1.0 Page 27