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TissueQuest 3.0 User Manual

1. Figure 138 Channels list running the algorithm While the algorithm is running the following splash screen will be displayed informing you about the progression of the process elapsed time number of processed FOVs a eel Elapsed time 00 00 04 acing of 18 0 Cancel Figure 139 Run Detection splash screen TissueQuest 3 0 User Manual doc Page 104 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 17 Analysis Results After analyzing the samples and or fields of view the user will obtain for each channel a set of image results Labeled a pseudo coloring of the events will be displayed The colors are chosen randomly and do not indicate different staining intensities They simply help showing the segmentation results Sample Neg UV irr Field of View FOV 005 s p i30 eis 1 1 Figure 140 Image Results Labeled e Overlay the contours of the events will be visible on the displayed image TissueQuest 3 0 User Manual doc Page 105 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Sample Neg UV in Field of View FOV 005 Jal i50 piss i Delete Events a Split Events Merge Events Create Events Figure 141 Image Results Overlay right For Various Shapes 1 0 the following output parameters are available for each detected event Area the area of the segmented object Minimum of Intensity minimum
2. Neg normal_ Region 001_FOV 01 13 January 2010 Neg normal_Region 001_FOY 03 16 February 200 13 January 2010 Neg normal_Region 001_FOV 03 16 February 200 13 January 2010 Neg normal_Region 001 FOY 03 16 February 200 13 January 2010 Neg normal_Region 001 FOVO3 16 February 200 13 January 2010 Neg normal_Region 001_FOV 04 16 February 200 13 January 2010 Leave images into Neg normal Region 001_FOV04 16 February 200 13 January 2010 r r thair folder Neg normal Region 001_FOV 05 16 February 200 13 January 2010 Neg normal Region 001_FOV05 16 February 200 13 January 2010 Neg UV rr_Region 001_FOW 005 16 February 200 13 January 2010 Neg UV rr_Region 001_FOV005 16 February 200 13 January 2010 SISSIES SIS Here you can specify a folder that contains the images you want to use within your project Figure 4 New Project Step 2 On the second page you must choose the folder that stores the images you want to use in your project Select the images to be included in the new project by following the next steps Where are your images Choose the folder that contains the images by using the Browse button Do you want to filter out some images If you wish you can apply a filter to the names of the files in order to select only those images you need to use in your project For example by using the filter Region1 DAPI and applying the
3. Sample Neg UWV irr Field of View FOV 005 2 s 8 is0 PEET 1 1 Delete Events Split Events Merge Events Create Events Figure 56 Detail Window split events To merge events follow the steps below TissueQuest 3 0 User Manual doc Page 51 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 1 Select the overlay image of the master channel simply click on it 2 Press Merge Events button 3 Create groups of events to be merged Click on each event using left mouse button to include them in the group Only neighboring touching events can be selected in the same group for merge Click using right mouse button in order to close the group 4 Press Apply button to merge the events Sample Neg normal Field of View FOV 032 lt Hal E o0 Le 1 1 H Delete Events J Split Events Merge Events Create Events H Figure 57 Detail Window merge events 8 5 Create events To create events follow the next steps 1 Select the overlay image of the master channel simply click on it 2 Press Create events 3 Holding the left mouse button pressed draw each individual event that will be created 4 Press Apply button to create the events TissueQuest 3 0 User Manual doc Page 52 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Sample Neg normal Field of View FOV 032 eo es 8 300 PEEN 1 1 Delete Eve
4. Figure 101 Modify gate step 4 b After finishing the modifications press again the Edit gate button to disable the function 10 5 4 Delete gate e Click the Edit gate button caj in the scattergram toolbar TissueQuest 3 0 User Manual doc Page 77 of 116 Internal TissueQuest 3 0 User Manual www tissuegnostics com a0 a E m 5 E ma Mean Intensity F Figure 102 Delete gate step 1 The corner points of the gate are highlighted by a small red rectangle Figure 103 Delete gate step 2 e Select the gate that you want to delete by clicking one of its corner points In this case it doesn t matter which of the corner points you choose for selection When the gate is selected the lines are highlighted u E m E m im C z Gli E m A P Page 78 of 116 TissueQuest 3 0 User Manual doc Internal www tissuegnostics com TissueQuest 3 0 User Manual Figure 104 Delete gate step 3 Press Delete on your keyboard The selected gate will immediately disappear Q You can also delete a gate from Gate management See Chapter 10 5 5 TIP 10 5 5 Gate Management To delete gates or to modify the gates display settings select Gates Manage from the application s main menu File View Project Samples Scattergrams Algorithm Admin Help Mmg Figure 105 Gate management menu The Gate management dialog will appear
5. To make some fields of view representative follow the next steps e Select the desired fields of view in FOVs in project list e Press button next to the right of FOVs in project list to make the selected fields of view representative e Press gt gt utton next to the right of FOVs in project list to make all the fields of view in the project representative To remove some fields of view from representative set follow the next steps e Select the desired fields of view in Repr FOVs list E ees e Press button next to the left of Repr FOVs list to remove the selected fields of view from the representative set Press l set button next to the left of Repr FOVs list to remove all the fields of view from the representative To remove some fields of view from the project follow the next steps e Select the desired fields of view in FOVs in project list e Press button next to the left of FOVs in project list to remove the selected fields of view from the project e Press button next to the left of FOVs in project list to remove all the fields of view from the project 7 All the fields of view removed from the project will be automatically removed from the vf Note representative set TissueQuest 3 0 User Manual doc Page 25 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual FOVS in project FO FOVS in project Fov 014 v03 FOV 014 FOV 032 FOV 036 FOV 043 FOV 054 FOVSs in project Repr FOVs
6. click on Set a Filename Sample __ 7 23 F ename sample Mark a part of the Filename example then press the respective button below it Sample Field of View Channel For each part of the filename you may indicate if the part has or has not a fixed size by checking or unchecking the fixed size checkbox The fixed size checkbox might improve working with filenames where only a part of the name is used to identify the three relevant parts i e sample field of view channel or where one particular part might be characterized by terms of different length in the individual channels e g filter names For example in the image above the Channel doesn t have a fixed size We have 49 DAPI channel with size of 7 and 20 Rhodamine with size of 12 In this case you must uncheck the fixed size of the Channel Press Next to continue 4 4 Step 4 In the next step samples markers and channels can be associated with each other Please note The term sample refers to a tissue section or cell culture monolayer which has to be analyzed as one entity and will give one set of results A sample usually consists of several channels characterized by the fluorochromes used for labeling channels usually have the name of the filter or fluorochrome they represent e g Cy2 As a fluorochrome can be used with different markers e g antibodies different samples might have different markers in the same channel e g CD3 Cy2 and CD45 Cy2
7. FOS in JVs in project FOV 014 dFovois 014 dFovoia 014 Figure 16 Include exclude one FOV from project representative list FOVs in project TissueQuest 3 0 User Manual doc Page 26 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Sample FOVs FOVSs in project FOVS in project FOV 043 FOV 054 Sample FOYS FOVSs in project FOY FOVs in project FOV 014 FOY 014 OV 014 4 1 g FOV 032 FOV 032 Fov 032 gt FOV 036 FOV 036 FOV 036 FOY 043 rr FOV 043 FOV 054 227 Keoy ose A povos4 eGR Foy 054 ee Sample FOS FOVS in project epr FOV sample FOY FOWVs in project FOV 014 FOV 032 FOV 036 FOV 043 FOV 043 FOV 054 FOV 054 Figure 17 Include exclude all FOVs from project representative list FOV size Select predefined template Select a predefined template for FOV size from the Predefined combo box Manage templates To manage the templates for FOV size use button next to the right of the Predefined combo box A dialog will appear in which you can manage the templates for FOV size For more details see Chapter 4 4 1 Manually specify the size You can manually specify the size of the FOV by writing its width and height in the Width and Height edit boxes TissueQuest 3 0 User Manual doc Page 27 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 4 4 1 Templates for the FOV size scaling In this dialog box you can define new templates in order to adjust th
8. Pow O12 Representative POs Pow Oi Q send all FOVs to non representative list Send one FOV to non representative list Figure 124 Project Properties Representative Non representative FOVs moving all FOVs 12 3 Markers In this section are listed all the markers i e antibody staining chemical reagent type of molecule visualized by means of immunofluorescence in the project and you can change their settings Markers Figure 125 Project Properties Markers section TissueQuest 3 0 User Manual doc Page 90 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual e You can select a color for each marker by clicking in its corresponding Color area see picture below Markers j TELLEF SEER EENET E A 5 2 wl 71713 1715 17 Figure 126 Project Properties choosing marker color e You can choose the master marker by marking the corresponding Master checkbox of the desired marker If you change the master channel all results belonging to the affected samples will be Prot lost including mask and overlay images numerical results cutoff and gates 12 4 FOV Size In this section you can set the dimensions of the FOVs You can do this in two ways Predefined option Use the Predefined option by choosing an existing template from the dropdown menu TissueQuest 3 0 User Manual doc Page 91 of 116 Internal www tissuegnostics com TissueQ
9. Such images will not be available The events in the scattergrams will remain available Do you want to continue Figure 23 Clean Temporary Files warning message 6 3 Group of Channels By choosing Group of Channels from the Project menu the Group of Channels dialog will appear normal skin UV treated DAPI ee i Neg normal DAPI Neg UV irr DAPI 5 normal skin DAPI i UW4treated DAPI E Rhodamine Neg normal Rhodamine Neg UV rr Rhodamine normal skin Rhodamine UV treated Rhodamine Figure 24 Group of Channels dialog Grouping channels means including in a group more channels that share same parameters Auto group channels You can group channels automatically by checking Auto checkbox The channels will be automatically grouped based on their associated marker All the channels having associated the same marker will be included in the same group of channels TissueQuest 3 0 User Manual doc Page 33 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Manually group channels To start grouping channels yourself uncheck Auto Group Channels In the Sample Editor select the desired channels by clicking on each channel hold pressed Ctrl key for multiple selection and then right click on them A popup menu will be displayed Under the Group submenu select how you want to group the channels e All channels with Name marker this option will include in the
10. they both have the same channel Cy2 but two different markers CD3 and CD45 TissueQuest offers to define markers independently of the fluorescence channels used for acquisition and allows assigning specific names to these fluorescence channels The same name for a channel can be applied to all samples in the project if the same marker antibody has been stained on all samples or you can apply different markers to the same channel in different samples e g if there are four samples stained with CD3 Cy2 and 3 samples stained with CD45 Cy2 the Cy2 channel of samples 1 4 can be named CD3 while the Cy2 channel of samples 5 7 can be named CD45 That means that the corresponding scattergrams will be labeled CD3 in samples 1 4 and CD45 in samples 5 7 If the marker names are not associated to channels all corresponding scattergrams in samples 1 7 will be labeled Cy2 and the user has to know what Cy2 actually stands for in each of the samples TissueQuest 3 0 User Manual doc Internal Page 16 of 116 www tissuegnostics com TissueQuest 3 0 User Manual Sample Editor Marker Family Check markers and samples sia DAPI _ _ _ _ 4 S i Ag Ti 40 T i fea i a H p i A E Link all samples UV treated Mark the Fields of View FOV and move them where you need them Define group of channels Auto Sample Fos FOS in project Repr FOVs H Meg nor
11. 19 New Project Step 5 This overview allows the user to check up if the settings are correct Press Back to change previous settings or Finish to accept the current project settings and create the project TissueQuest 3 0 User Manual doc Page 29 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 5 Open Existing Project To open an already existing project press Open an existing project button Gh from the main toolbar of the application In the dialog that appears browse for the desired project and then press Open UV Inradiated Skin t e ii Pr Mame Date modified Type ay J Cache 1 13 2010 11 35 AM File folder Recent Places N images 1 13 2010 11 28 AM File folder Samples 1 13 2010 11 26 AM File folder ME WitresistesSkintgprej 1A3 20101251PM_ TPRI Fie Desktop Libraries LY Computer IET E UV Inadiated Skin Files of type TissueQuest Project Files tqproj Figure 20 Open an existing project I You may also open a project by going to File menu of the application and choose Q tp Open 5 1 Save Project at To save the project press Save current project button The project will be saved I You may also save a project by going to File menu of the application and choose Save Q TIP Experiment TissueQuest 3 0 User Manual doc Page 30 of 116 Internal www tissu
12. 5 1 Add rectangular gate Gre lf you add gates before adding the cutoffs the statistics of the gates will not appear TIP e Click Add gate button d in the scattergram toolbar e After clicking on the button move the mouse into the chart area of the scattergram The cursor changes into a cross TissueQuest 3 0 User Manual doc Page 72 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 200 Rhodamine gee Mean Intensity Figure 90 Add gate step 1 Press the left mouse button inside the chart area to define the starting point of the region of interest Holding the left mouse button pressed resize the rectangular region to the desired size ri Rhodamine _je Mean Intensity 2 Figure 91 Add gate step 2 e Release the left mouse button to finally determine the gate All the events inside the gate will change their color to a predefined hue The gate is labeled with a default text e g Gate 1 The color the label and the visibility of each gate can be modified in the gate management see Chapter 10 5 5 x b hi Re Rhodamine y Mean Intensity Leal ca 0 100 20 255 Page 73 of 116 TissueQuest 3 0 User Manual doc Internal www tissuegnostics com TissueQuest 3 0 User Manual Figure 92 Add gate step 3 10 5 2 Polygon gating method e Click on the Add gate button Ler in the scattergra
13. 83 Move cutoff step 2 Finish the operation by releasing left mouse button 2 Rhodamine ee Mean Intensity 1 100 20 255 F DAPI Mean Figure 84 Move cutoff step 3 10 4 3 Delete cutoff Move the mouse cursor over the cutoff that you want to delete As a result the cursor changes to a pointed arrow shape Page 69 of 116 TissueQuest 3 0 User Manual doc Internal www tissuegnostics com TissueQuest 3 0 User Manual 2 Rhodamine ge Mean Intensity Figure 85 Delete cutoff step 1 e Click on the cutoff using the right mouse button to see the contextual menu Select Delete cutoff entry in the menu The cutoff in the scattergram will immediately disappear Eg Rhodamine ___jee ro Mean Intensity Delete cutoff Copy image to clipboard Copy cutoff Paste X Paste Y Paste both X amp Y DAPI Mean Intensity Rhodamine Mean Intensity View backward data around this point Figure 86 Delete cutoff step 2 note The cutoff will be deleted from all the scattergram in which it appears 10 4 4 Copy cutoff e Move the mouse over the cutoff that you want to copy As a result the cursor changes to a pointed arrow shape or J TissueQuest 3 0 User Manual doc Page 70 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 2 Rhodamine ge Mean Intensity Figure 87 Copy cutoff
14. Copy image to clipboard Copy cutoff Paste Y Paste both X amp Y DAPI Mean Intensity DAPI Area View backward data around this point Figure 112 Scattergrams contextual menu TissueQuest 3 0 User Manual doc Page 82 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 11 Forward and backward connection One of the most fascinating and instructive features of TissueFAXS is the ability to connect image objects with data space i e the user can visualize any particular cell of an image in the scattergram and vice versa The forward connection is a feature that allows you to access the numerical results for a specific event To view the respective data follow the next steps e Inthe Detail Window that displays the image of a channel just double click on an event That event will be highlighted by a red and white mark like a little flashing square Sample Neg UV in Field of View FOV 005 a f 180 PET 1 1 Delete Events Split Events Merge Events Create Events Figure 113 Forward connection step 1 TissueQuest 3 0 User Manual doc Page 83 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual In all the scattergrams corresponding to the sample the results will be highlighted by a red and black mark like a little flashing square 200 Rhodamine _ qe Mean Intensity Figure 114 Forward connection step 2
15. adjusting the size of the FOV press Update button TissueQuest 3 0 User Manual doc Page 92 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 13 Evaluation 13 1 Experiment evaluation Evaluation is realized on two levels During the process of setting the parameter for the pattern recognition the results obtained by those image processing procedures have to be optically evaluated for principal adequacy After the image analysis process is accomplished the obtained data need to be evaluated by appropriately setting cutoff values based on controls and or setting gates in order to define subpopulations The quadrant statistics contains the statistical evaluation for each of the four quadrants in each scattergram 13 2 Scattergram evaluation The scattergram shows two parameters out of the measurement data set They can be shown on a linear e g DNA staining intensity or a logarithmic e g mean relative fluorescence of antibodies scale Depending on what the user is investigating new scattergrams may be created at any time and the parameters on one or both axes of existing scattergrams may be changed Existing scattergrams may be hidden or deleted see Chapter 10 1 3 Each point in a scattergram represents a single measure event nucleus cell cellular compartment or any other structure depending on the respective marker used for labeling in that channel In other words each point represents the correlation of
16. ae ee ae ee eee eee ee eee eee ree ee eee ee 72 VOD Z Polygon gating method essersi e a E a E a a E EEA a a aA 14 TOS o M dN GA ee ne ee E E E E E E E A ee E E 15 18 oe Delete gale ee ae a ee er eee eee eee eee eee eee TT Lors e AUS eee Ea L A E T lati aes etn ae een ts E E A E A D E A N E eee ueec aete 79 106 Scatergoram parame UCN Scrios en EE TEE este EE TEE EEEa 81 1O VIEW DAC VV AIG nn E E OE E E A a 82 11 Forward and backward CONNECTION wisn cnisccacennasscta sniecenanddieanicins onledeinsin vnc Seiebu eulbdvioumndeaaete odedveuncundtiebaseeewneendatebsededadece 83 tei FONC ORIG CHOI sates pecs ntese sees secneeeces E ee aetna A vet oasecioei edna aba cuece conned A E S 83 Thes IK Wal Ch COMING CMON xe aceactenncee sets e E E E E E nace AE E ES 84 12 PRISO DEO RSIS Soeria E E E E AE E A E E E 87 i 24 ANO ee a E E E E E ee E ee E 88 12 2 Representative Non representative FOVS ccccccsccccceececceceseeececeeececeeseeseueeeseucesseeeessaeeesseeessaeeeseeeees 88 Me MAKO aei E E seen avaccecetesccassarsceseeunesdeancnscet aneeccaageessedeese se ueaestaneeaneeicassnieoneasee 90 Ied gO o ee E eee ee cee ae ee ee eee eee een eee ae eee 91 13 Ey UO GN acc precstmeca cre narteateanceamne aot unccemertaas E E 93 13 1 ExpenimentevalualiOi eisrean aiaa EEEE RAAR 93 13 2 Scatergram CV AU AU OWN ssporesirsiriritenn aa E iiaa DEEE ETERN 93 14 SPECIA TEAU ES aesae es iaa EE EE EE OE E aE EAEE E ee ene ees ee ere 94 tate PONO e
17. e measures the marker intensity of each cell as well as other parameters related to intensity texture and morphology The starting point for cell identification is the segmentation of nuclei e g object recognition and background rejection The segmentation algorithms require different parameters for every type of channel The detection parameters for the channels are actually adjusted for the entire group of channels to which the channels belongs In the figure below a comparison is shown of the default parameters for a master channel side and a non master channel right side Nuclei Size Discrimination by Area Discrimination by Gray Autodetect Background Threshold Threshold Level left Ring Mask Interior Radius Exterior Radius Identified Cell Mask Max Growing Offset From Nuclei utodetect Background Threshold Minimum Level Maximum Level Threshold Level Include Nuclei Label Figure 133 Default parameters for a master channel left and a non master channel right To change the default value of a parameter simply modify it in the corresponding field TissueQuest 3 0 User Manual doc Page 98 of 116 www tissuegnostics com TissueQuest 3 0 User Manual 15 1 Changing Segmentation Method 15 2 Various Shapes 1 0 TissueQuest provides a method especially created to detect structures with various shapes and sizes Various Shapes 1 0 For tuning this method a
18. en E a a en eee te EE E 112 19 BMS E a es eee S E E E E E EEO E E E E OE 114 20 FST SSN SS eeepc ep atest ada emivic ia oeaeiee bane nadeepn conde ecdeadaancenteennteedaaeeds Fehler Textmarke nicht definiert 21 DOCUMENL HIStory ccccccseecccceeeeceeceeeceeceesceeseeeecessueecesseeeeeesaaeeesaeeees Fehler Textmarke nicht definiert 22 Document Quality Assurance 2 0 eccccceeccccceeecceceeecceeeeeeceeseseceesaeeeeesaeaeees Fehler Textmarke nicht definiert TissueQuest 3 0 User Manual doc Page 3 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Disclaimers 1 TissueFAXSplus is a microscope based cell analysis system for cells in cryocuts paraffin sections and TMAs It consists of the software modules TissueFAXS TissueQuest and HistoQuest and is used for acquisition of images in the fluorescence and brightfield mode for counting the number of positive and negative cells and for quantification of staining intensities TissueFAXSplus is used for the standardization of tissue analysis in combination with immunohistochemical and immunofluorescence staining The system does not give any direct diagnosis and there is the possibility that the samples do not contain enough information to give a clear diagnosis The results of the analysis are purely statistical values Users must reevaluate images and likelihood of the statistical data Pure interpretation of statistical data is a high risk Observ
19. group all channels containing a certain marker e Only selected channel this option will include in the group only the channels you have selected ne Neg normal DAPI ATT Neg UV4rr DAPI normal skin DAPI Right Click a damine 0 Rhodar i UV treated DAPI har All channels with Rhodamine marker a Neg normal Rhodamine Only selected channel Neg UV rr Rhodamine et fe normal skin Rhodamine UV treated Rhodamine ST a a A ale OO ae men ere Figure 25 Group of Channels dialog grouping channels The parameters for detection algorithm are adjustable individually for each group of Q TIP channels You cannot group master and non master channels Rename group of channels Click twice on the name of the group A text editor will be displayed Type the text here and press Enter to save the new name Press Escape in order to cancel E DAPI Neg normal DAPI Neg UV rr DAPI mormal skin DAPI UV treated DAPI cimma D i Meg normal Rhodamine Neg UVrr Rhodamine normal skin Rhodamine UV treated Rhodamine Figure 26 Rename group of channels TissueQuest 3 0 User Manual doc Page 34 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 6 4 Virtual Channels By choosing Virtual Channels from the Project menu the Virtual Channels dialog will appear 40 0A BT DARTI Figure 27 Virtual Channels dialog Here you can choo
20. normal_Region 001_FOV 032_20 R Neg normal FOV 032 20 Rhodamine OK If some problems Neg normal_ Region 001 FOV 032 490 Neg normal FOV 032 49 DAFI OK appeared you may Neg normal_Region 001_FOY 036_20 R Neg normal FOY 036 20 Rhodamine OK change the meaning Neg normal_ Region 001 FOV 036 490 Neg normal FOV 036 49 DAPI OK or the problematic Neg normal_Region 001_FOV 043_20 R Neg normal FOV 043 20 Rhodamine OK filename Neg normal_Region 001_FOV 043 490 Neg normal FOV 043 49 DAPI OK Neg normal_Region 001_FOV 054 20 Neg normal FOV 054 20 Rhodamine OK Neg normal_Region 001_FOV 054 490 Neg normal FOV 054 49 DAPI OK Neg UV4rr_ Region 001 FOVO005 20R Weg UV rr FOV 005 20 Rhodamine OK Neg UVrr_ Region 001 FOV005 49D Neg UV rr FOV 005 49 DAPI OK Please mark the parts of the Filename example Figure 5 New Project Step 3 TissueQuest needs to know exactly which image file belongs to which sample to which field of view within a certain sample and to which channel within a certain field of view To define the sample the field of view and the channel follow the next steps TissueQuest 3 0 User Manual doc Page 15 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual What s the meaning of the filename By default the first image file in the list is selected as filename sample This can be changed however To choose the filename to be used as a pattern right click on any file name in the list and
21. see displayed a splash screen containing the main information about TissueQuest version in use File View Project Samples Scattergrams Gates Algorithm Admin o amp nal ie F About TissueQuest iy ka eu 9 aa Soll tilt Figure 154 Help menu TISSUE WD GNOSTICS MeEOic AL Ee BiAaATECH FAOtuUuTIO H amp TISSUE QQUEST GELL ANALYSIS So FF T WARE liv CE 44 ATTENTION CONSULT OPERATING INSTRUCTIONS FOR USE Copyright 2005 2011 TissueGnostics GmbH All rights reserved Figure 155 Help menu Splash screen TissueQuest 3 0 User Manual doc Page 114 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual TissueQuest 3 0 User Manual doc Page 115 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 20 TissueQuest 3 0 User Manual doc Page 116 of 116 Internal
22. step 1 e Click on the cutoff using the right mouse button to see the contextual menu Select the Copy cutoff entry in the menu The cutoff in the scattergram is copied and can be pasted into another scattergram Rhodamine gee ro Mean Intensity Delete cutoff Copy image to clipboard Copy cutoff Paste X Paste Y Paste both X amp Y DAPI Mean Intensity Rhodamine Mean Intensity View backward data around this point Figure 88 Copy cutoff step 2 10 4 5 Paste cutoff 4 If you want to paste a cutoff it must be already copied To copy a cutoff see Chapter roe 10 4 4 TissueQuest 3 0 User Manual doc Page 71 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual e Move the mouse cursor inside the diagram area of the scattergram where you want to paste the cutoff and right click to see the contextual menu e Select the Paste both X amp Y if you want to paste both cutoffs horizontal and vertical select Paste X only the vertical cutoff select Paste Y only the horizontal cutoff 0 00 19 87 Rhodamine p Po Mean Intensity a Delete cutoff Copy image to clipboard Copy cutoff Paste X Paste Y Paste both X amp Y DAPI Mean Intensity Rhodamine Mean Intensity View backward data around this point Figure 89 Paste cutoff e The cutoff immediately appears in the scattergram 10 5 Set gates 10
23. to create the new user TissueQuest 3 0 User Manual doc Page 109 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual New User TRF an US oe Please type in the name for the new user and a password Username Password Repeat password Figure 146 New User dialog You can also add a description for each user in the Description dialog box Open the Description dialog box by pressing the Description button L_bescription h Description a tae ae Ad User Administrator Real name Description Figure 147 User Description dialog To delete a user select it then press Delete user button _ Delete user A warning message like the one below will appear asking you if you really want to delete that user TissueQuest 3 0 User Manual doc Page 110 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Figure 148 Delete User dialog To change the password of a user select it then press Change password button Change password The Change password dialog will appear New Password 1 eo Define a new password Current password New password Repeat password Figure 149 Change Password dialog Administrator If you change the password of the Administrator you must enter its current password If you change the password of a standard user its current password is not required Page 111 of 116 TissueQuest 3 0 User Manual
24. two parameters of a single measure event For each parameter that is represented on each axis a cutoff value may be established in order to define the level of specificity for each marker i e what is a specific staining and what is back ground Single cells with a specific staining reactivity for a given marker are referred to as positive for that marker Cells lacking such a specific staining are referred to as negative for that marker The exact value for each cutoff has to be determined by appropriate controls e g isotype matched negative controls for antibody based labeling These two cutoffs per scattergram define four quadrant regions LL lower left UL upper left UR upper right and LR lower right which may contain different populations of measured events in the respective scattergram e LL Marker X negative Marker Y negative e UL Marker X negative Marker Y positive e UR Marker X positive Marker Y positive e LR Marker X positive Marker Y negative Beyond the basic distinction of positive and negative event populations there may be less obvious differences in the samples analyzed In order to acknowledge these differences the gate tool may be applied see Chapter 10 5 Gates allow distinguishing between positive populations e g all measure events above a certain cutoff value that are low intermediate or high for a given marker The advantage is that the result of this procedure is an ex
25. will appear Default Link of Samples Globa aa E Neg Uv irr Uv treated samples to be All Samples eae 5 Neg normal 7 MB Neg LY irr a Mi normal skin E treated Linked samples Figure 64 Sample linking dialog e Inthe left column you will find the list of available samples e Mark the checkbox for the samples you want to link then press button to move the respective samples into the right column in order to link them TissueQuest 3 0 User Manual doc Page 57 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual If you cannot mark the checkbox for a sample it means the sample is not compatible Q TIP with the previously selected samples e Use the button to move all samples to the right column e Use the button to send a sample back to the left column or the button to send all samples to the left column As you may notice each link of sample has associated a color This color is displayed Q TIP in front of the names of link and samples This helps you easily identify the samples belonging to each link of the sample F All Samples W Default Link of Samples Globa Neg normal sefault Link of Samples Global Fe W eg Wy irr Mi normal skin WB UV treated Dropdown list with existing links Figure 65 Sample linking dialog dropdown list Manage links Press the J button to access Sample Links dialog Here you can visualize details about the existing lin
26. 11 2 Backward connection The backward connection is a feature that allows you to access the events that have specific numerical results Backward connection can be used for a single numerical value or for an entire gate To view the events corresponding to a single numerical value follow the next steps e Within a scattergram right click on a dot In the context menu that appears choose View backward data around this point Delete cutoff Rhodamine ____jee Mean Intensity jai ci Copy Image to clipboard 0 5 Copy cutoff DAPI l Inten Paste X Faste Y Faste both X amp Y DAPI Mean Intensity Rhodamine Mean Intensity N 7 F View backward data around this point 2 Figure 115 Backward connection single numerical value a e After backward data is processed the corresponding events can be followed up in Detail window TissueQuest 3 0 User Manual doc Page 84 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Backward data around point Sample UV treated Scattergram DAPI Mean Intensity DAPI Area A FOW 012 EH FOW 016 Figure 116 Backward connection single numerical value b To check the events corresponding to an entire gate follow the next steps e Within a scattergram right click on the border of a gate e Inthe context menu that appears choose View backward data for Gate name 255 Right click on the edge
27. Duo Quad Processor 3 GHz or more 4 GB RAM Internet connection USB Port for dongle 1000 GB hard disk space for storing your projects Dual monitor system with a resolution of 1680x1050 or higher 2 6 2 Software Requirements Windows XP Windows XP 64 bit Windows Vista 32 bit Windows Vista 64 bit Windows 7 32 bit Recommended Windows 7 64 bit 7 By default TissueQuest needs to be run by a user member of the Administrators note group In this case all users will share some settings e g institution data system data users TissueQuest can be also run by a user member of the Users group in which case every user will have its own settings Please contact your Support team in order to get the desired configuration for your workstation Once all the components are successfully installed the application is ready to be used Notice regarding virtual machines TissueGnostics software is not meant to work on virtual machines Also TissueGnostics does not offer support for these configurations as the software directly uses advanced CPU features and therefore any third layer between the software and the CPU will affect performance and might affect the results of the analysis in an unpredictable way TissueQuest 3 0 User Manual doc Page 10 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 3 Login to TissueQuest After starting the application a login panel will appear In order to login to Tiss
28. SampleName RawData_ of Each row corresponds to an event and it contains all parameters for all channels e Export raw data of dots to CSV this option creates a csv file for each sample and channel named SampleName ChannelName csv Each file exports the following data for dots one dot per row Field Of View Event ID Dot ID Dot Area Dot Min Intensity Dot Mean Intensity Dot Max Intensity Dot Sum Intensity e Export raw data of individual dots to excel this option creates an x s file containing a sheet for each sample and channel named SampleName Channe Name Each sheet exports the following data for dots one dot per row Field Of View Event ID Dot ID Dot Area Dot Min Intensity Dot Mean Intensity Dot Max Intensity Dot Sum Intensity TissueQuest 3 0 User Manual doc Page 97 of 116 Internal www tissuegnostics com 15 Segmentation Methods TissueQuest 3 0 User Manual TissueQuest application offers unique and proprietary algorithms for recognition of individual cells in a tissue context or a cell culture monolayer An algorithm is a sequence of mathematically realized processing steps applied to an image or part of an image with the aim of improving image quality and or segmenting relevant objects in this case cells in an image e discriminates background from specific cell objects e recognizes individual cells where does one cell end and the neighboring cell begin
29. The following options of exporting data are available e Print output as images this feature allows you to save all the printing data as images instead of printing it using a printer e Measurement data as ASCII this feature allows you to export statistical data and raw data into ASCII files text files e Measurement data to Excel this feature allows you to export statistical data and raw data into Excel files The following information will be exported Statistics M Raw data Select destination file Cilexport xls Figure 132 Export Data to Excel In Exporting data to Excel you may choose what to include in export Statistics mark this checkbox if you want to include statistical data in export Raw data mark this checkbox if you want to include raw data in export e Export Raw data of a Gate to Excel this option is available only for TissueQuest projects where all samples are linked meaning the displayed selected gates are propagated to all samples If the samples are not all linked this option will be disabled For each gate in each sample one or more sheets are created If all the events of the gate can be exported to only one sheet its name will be GateName SampleName RawData TissueQuest 3 0 User Manual doc Page 96 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual If the events of the gate can not be exported to only one sheet its name will be GateName
30. act measurement with full statistical information rather than a rough estimation as obtainable by just visual evaluation The quadrant statistics provides information about the mean x and y labeling intensity about the absolute and relative number of events per quadrant and if the user can provide information about the scales in the microscope used to acquire the images also the number of events per mm TissueQuest 3 0 User Manual doc Internal Page 93 of 116 www tissuegnostics com TissueQuest 3 0 User Manual 14 Special features 14 1 Print project x To print the project press Print current project button The following dialog will appear Print Samples Only Visible All Custom 3 5 Print Fields Of View All Representative and Expanded gt All Representative None Print Scattergrams Only Visible and Expanded C All Visible Figure 129 Printing Information Collector In the Printing Information Collector dialog you can make the following settings e For Samples you can print only visible samples all samples or only the samples you want to select e For Fields of View you can print all representatives and expanded FOVs all representative FOVs or none e For Scattergrams you can print only visible and expanded scattergrams or all visible scattergrams Before printing a project you may want to see a Print Preview available in File Print Preview TissueQue
31. ations TissueFAXS is similar to TissueFAXSplus but is for fluorescence samples only and must not be used with brightfield immunohistochemical samples TissueFAXS consists of the software modules TissueFAXS and TissueQuest HistoFAXS is similar to TissueFAXSplus but is for brightfield immunohistochemical samples only and must not be used with fluorescence samples HistoFAXS consists of the software modules TissueFAXS and HistoQuest 2 Each and any product should be used only after training performed by TissueGnostics or authorized distributors of TissueGnostics A list of authorized distributors is available here http www tissuegnostics com index php load xml en home company distributors xml amp lang en 3 The names of actual companies and products mentioned herein may be the trademarks of their respective owners 4 In order to use TissueQuest application it is essential for the users to have sufficient knowledge of PC and Microsoft Windows operation system usage 5 The information from this document is subject to changes without notice 6 The information contained in this document is the proprietary and exclusive property of TissueGnostics GmbH except as otherwise indicated No part of this document in whole or in part may be reproduced stored transmitted or used for design purposes without the prior written permission of TissueGnostics GmbH 7 The information in this document is provided for informat
32. cal reagent e g CD3 FITC stain antibody genetic probe that allows the identification of defined cell types tissue types cellular or tissue compartments cellular functions disease status etc Project A set of tissue samples which are analyzed together with individual settings for each channel and sample linking Quadrant Statistics Shows the following values X Mean i e mean value for the parameter shown on the x axis Y Mean i e mean value for the parameter shown on the y axis Scattergram An x y diagram showing two measurement parameters for all data units or a selection thereof in linear or logarithmic scale Within the scattergram cutoffs can be set in order to define a threshold for each measurement parameter TissueQuest 3 0 User Manual doc Page 7 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 1 4 Conventions In this section you can find listed and explained the conventions used throughout this manual ff Note v Note s describes important features or instructions Y TIP y Y Tip s highlights features or hints that can help you save time or avoid difficulties CAUTION v Caution s warns readers about possible damage to equipment or to data or about potential problems in the outcome of what they are doing y Italics are used in order to highlight a title and also new or unfamiliar terms usually at their first appearance in the text y Bold are used in ord
33. ce respectively This is accomplished by applying the algorithms of the TissueQuest software to the images of tissue sections Channel An image corresponding to a specific marker from a Field of View Count number of events in the respective unit sample gate ROI quadrant Percentage i e how many events are in the respective quadrant region relative to the number of total events for each of the four quadrant regions Cutoff value A numerical parameter used as a threshold value in a scattergram in order to distinguish between specific and unspecific staining patterns allowing determining positive and negative measure events Event A data unit obtained by TissueQuest algorithms representing a quantum of information about a defined object nucleus cell An event is identified by one or several image processing and pattern recognition algorithms An event contains information about area mean intensity Field of View FOV The area of a sample tissue section visible in the microscope and captured at one x y z position on the slide and a with a given optical magnification An image of one Field of View may contain several markers Gate A subset of events within a scattergram that is selected by defining a region of interest ROI Gates may have any polygonal shape and contain any number of cells The gates in TissueQuest do not support mutual exclusion Marker A molecular labeling component chemical or biologi
34. doc Internal www tissuegnostics com TissueQuest 3 0 User Manual New Password ee ee ee ee FF Define anew password Current password New password Repeat password Figure 150 Change Password dialog standard user 18 2 Institution Data Institution Data Per Itt es ee Se er et A ee Enter the institution s data Instituton name Department Laboratory Figure 151 Institution Data dialog In this dialog you can provide data concerning your institution e Institution name e Department e Laboratory 18 3 Change Password To change the password you have two options e Goto application main Menu then choose Admin Change Password TissueQuest 3 0 User Manual doc Page 112 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Institution Data Change Password Figure 152 Change Password Admin Menu The following dialog will appear New Password a a Define a new password Current password New password Repeat password Figure 153 New Password dialog To change the password you must enter the current password into the Current password field then enter the new password into the New password field and also for confirmation into the Repeat password field TissueQuest 3 0 User Manual doc Page 113 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 19 Help menu By going to Help About TissueQuest you will
35. e 1 4 and create a separate group for the CD45 Cy2 channel in samples 5 7 That means that the parameters used for analysis of CD3 Cy2 will be the same and the parameters used for analysis of CD45 Cy2 will also be the same but different from those used to analyze CD3 Cyz2 Associate markers to channels In Marker Family Editor select a marker by clicking on it Then click on a channel in Sample editor You will be asked whether the marker should be associated to all channels with the same name in the entire project e f you choose Yes then the marker will be associated to all channels with the same name f you choose No then the marker will be associated only to the channel on which you clicked Use samples In Sample Editor right click on the name of the sample in the header A popup menu will be displayed Here you can select to use or not to use the sample If you choose to not use the sample then the sample will not be included in the final project Use channels In the Sample Editor select the desired channels use CTRL key for multiple selection and then right click on them A popup menu will be displayed Here you can select to use or not to use the channels If you choose to not use the channels then the channels will not be included in the final project Select sample In the Sample Editor click on the name of the sample in the header The FOVs belonging to the sample will appear in the lists below Gr
36. e E EEA E O EEA ee eee EE 94 Me OIG ei eE A E E A AE EENAA A E AAE E ENEE E E S A E A 95 15 Segmentation Methods cccccccsscccsecceececececeeeeceeeeeueeceueecsueesaeeecgeeseusecueesseeesseeseueecsueesseeeesusessueesseesseesseas 98 15 1 Changing Segmentation Method ccccccccccccecccseeeeeeeeceeeeseeeese cess eeseeeeseeeesaeeesaeeseeesaueeseeesseeeseneeseneesaees 99 15 2 Vanous Shapes LO seriean ne ee eee 99 15 2 1 Master Channel Parameters ccccccccccseccescecececseeccseeceucecsueesaueeseeseuceceueesueeeueessueecsueesseeesaeesaueessaees 99 15 2 2 Non Master Channel Parameters ccccccccceccceececeeceseeeeseeeaeeeseeeeseeeeseeeseeeseeeeseueesaeeeseeeseeeeseueeseeeaes 100 Tao DOTFDI eee e e E NA E EE EA E EE A E A E EE 101 gaye eu DOT ai 16 gt gt gee E AE ER NENE AE AEE ee EA 101 16 CAN IYE aaa scene A E EA E E E E A A EE 103 17 Anayo RESOUN aes ee E EE EEE R AAE NAE AA EAEE AEA ee 105 ok TMe Ra a aa E a E NA E E EA E E A EEE EAE EE 105 Ta R WO a A E E ae A E EE E E AE A AE 106 17 2 1 Raw Data for Various Shapes 1 0 0 0 ccccceccceeeeceeeece ceca eeeeeeeseeeeseeeeseeeeseeeseeeeseueeseeesseeeseueeseeeaeeeaes 106 Ml 252 RaW Data for DOT FINGEF sorersindinereenie iie ai aeaa Ea NEEE i aa aa aaa aE E ANAA N aaae 108 18 A UNS e E E E E FESE E S E 109 IG MO a E E E E E E E E E 109 WZ a o i re E sense A E A E dere we E E E E T E E A T 112 163 Change Password ee ner naien a seen ED E e Sa Aaa ne
37. e dots present in a specified event Dot Sum Intensity the sum of the intensities of the dots present in a specified event Dot Total Area the total area of the dots present in a specified event These output parameters are used in order to associate data to specific axis of the scattergram To do this access the Scattergram properties dialog from Scattergram toolbar i see Chapter 10 3 Scattergram properties dialog is also available when adding a new scattergram from note Scattergram management see Chapter 10 1 1 ocattergram properties Change parameters AXIS Y Axis Channel 49 DAPI ba Parameter Select input gates Change numt Dot Mean Intensity Dot Sum Intensity Dot Total Area Figure 143 Scattergrams Scattergram properties dialog DOT Parameter list TissueQuest 3 0 User Manual doc Page 108 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 18 Admin menu You can find the Admin menu in the Menu bar of the application Institution Data Change Password Figure 144 Admin menu 18 1 Users User management option is available only for the Administrator The standard user is allowed only to change its password Users Administrator New user Change password Figure 145 Users dialog In order to create a new user for TissueQuest press New user then enter the username into the Username field respectively the password into the Password field Then press OK
38. e of the new marker e g when a project has different markers for the same channel compare the example with CD3 Cy2 and CD45 Cy2 above Then press Enter The newly added marker appears in the Marker Family Editor Marker Negative Color Master etme E e i E hl Rhodamine DAPI DHA Hoechst ELME GFP 106 4660 A4es6 Texas Red CD45 PE IqgGi PE cD14 Ape IqGi APC Figure 7 New Project Step 4 Add new marker lf you want to use a marker that is not present in the dropdown list you can manually add the desired marker by typing its name in the edit box then press Enter Marker Negative Color Master Rhodamine DAPI Figure 8 Manually add new marker TissueQuest 3 0 User Manual doc Page 18 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Add negative marker A negative marker corresponds to a negative control A negative control works like this if the marker sticks on a certain area of tissue from the sample the negative control will actually stick on the complementary tissue area that is not covered by the marker To insert a negative marker go to Marker Family Editor and double click on the empty rectangle on the right side of the marker A combo box will be displayed Here you can select a known negative marker or you can type the name of the new negative marker Then press Enter The n
39. e sample tissue sample or cell culture monolayer Over the preceding decades an ever increasing amount of specific molecular markers has been established helping pathologists clinicians and researchers to appropriately characterize diseases For such a characterization however a simple look and conclude investigation is not sufficient For many markers a positive negative decision does not represent a qualified approach Rather the question has to be addressed how much of a certain marker is present where in cells and tissues Moreover measurements of simultaneously determined markers patterns and functions are required In order to optimally fit therapies to individual patients needs specimens have to be analyzed for an entire set of markers and or marker combinations in an observer independent manner In order to meet these requirements computer aided processing of histological samples is required 1 1 Purpose The purpose of this document is to help the user to understand how this application works It presents TissueQuest capabilities and guides the user through the steps required to obtain a good analysis The document covers the installation of the application system requirements and main features In the next section you may read more about the goals of the application and its place in a larger system so you will understand the purpose and advantages of this application 1 2 Goals The TissueQuest software i
40. e size scaling of the fields of view You can also change or remove an existing template Template Name Mi ix 0 5 C Mount Side M1 10x 0 5x C Mount Sid M1 20x 0 5 C Mount Sid M1 40x Oil 0 5 C Mount M1 63x Oil 0 5 C Mount Creaton Date 06 12 08 13 42 06 12 08 13 42 06 12 08 13 42 06 12 08 13 42 06 12 08 13 42 Last Modified 06 12 08 13 46 06 12 08 13 46 06 12 08 13 47 06 12 08 13 49 Template Name Mi ix 0 5 C Mount Side Mi 10x 0 5x C Mount Sid Mi 20x 0 5x C Mount Sid M1 40x Oil 0 5 C Mount Mi 63x Oil 0 5x C Mount Creaton Date 06 12 08 13 42 06 12 08 13 42 06 12 08 13 42 06 12 08 13 42 06 12 08 13 42 Last Modified 06 12 08 13 46 06 12 08 13 46 01 13 10 12 07 06 12 08 13 47 06 12 08 13 49 Template Info Template Info eae M1 20x 0 5x C Mount Side Port PCO ose lt lt New Template gt gt feiss Axio Imager M1 20x Objective 0 5x C Mount Side Port PCO PixelFly Video Camera at 1392x1024 Description Description FOV Width 0 698 7893460 0 14648000000 FOV Height FOV Width 0 6611783700 FOV Height 0 1484800000 Remove See or edit details of a template Create new template Figure 18 Templates for FOV size To create a new template follow the next steps e Enter the name and the description of the template Then enter the size of the field of view e Pre
41. e you won t be able to use it some types of Windows users have restricted rights Please ensure the selected drive has enough free space available to store your project Please note that TissueQuest will need additional space in order to temporarily store as well as binary mask images Important Avoid storing projects on Desktop My Documents or in any other folder located on the system drive usually C If the drive is nearly at its full capacity the system might behave erratically or even stop working if the drive completely runs out TissueQuest 3 0 User Manual doc Page 13 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual of space Usually systems delivered by TissueGnostics have a dedicated storage medium for projects it is advisable to use it in order to increase TissueGnostics products performance and avoid issues with low disk space Also in case the system needs to be reinstalled virus infection system files being deleted you might lose data located on the system drive After filling in the above fields press Next 4 2 Step 2 Where are your images Folder contai ning F Projects issueFAXS UV Irradiated Skin Images your images Do you want to filter out some images The filename must contain Regioni DAPI All words C Any word Use File Date modified Creation date Preview Copy images to project folder k Neg normal_Region 001_FOV OL 16 February 200 13 January 2010
42. egnostics com TissueQuest 3 0 User Manual 5 2 Print Project L To print the project press Print current project button For more details please see Chapter 14 1 TissueQuest 3 0 User Manual doc Page 31 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 6 Project Menu To access the Project menu go to TissueQuest menu bar and choose Project The following menu will appear Project samples Scattergrams Clean Clean Temporary Files Group of Channels Virtual Channels Properties Figure 21 Project menu 6 1 Clean Choose Clean option if you want to remove from your project all results detected events and temporary images mask and overlay images A warning message like the one below will appear Figure 22 Clean Project warning message 6 2 Clean Temporary Files Choose Clean Temporary Files option if you want to remove from your project only temporary images mask and overlay images This helps to save space on your hard disk especially for archiving TissueQuest projects Note by re running the analysis all the temporary files can be created again at any time in the future data and scatterplots remain unchanged as long as the settings for analysis remain unchanged A warning message like the one below will appear TissueQuest 3 0 User Manual doc Page 32 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual This will delete the results per channel
43. enu Gates Algorithm Admin Help Scattergrams Manage File View Project Samples Display Settings Figure 74 Scattergrams menu Page 64 of 116 TissueQuest 3 0 User Manual doc Internal www tissuegnostics com TissueQuest 3 0 User Manual From the menu that appears choose Display settings and a dialog will appear Display settings select dot style Select dot size Rectangle J 0 Circle z amall large C Cross a a Figure 75 Scattergrams Display settings dialog Here you can set the dot size and also select one of the three available dot styles e rectangle circle e cross as E 5 E100 E m T Rhodamine s Rhodamine pe 20 255 Rectangle Circle Cross Figure 76 Scattergrams dot styles examples When changing the dot styles the size of the events in the scattergrams can be altered l CAUTION TissueQuest 3 0 User Manual doc Page 65 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 10 3 Scattergram Toolbar In each scattergram the scattergram toolbar is placed above the chart area Figure 77 Scattergrams toolbar Actions in a scattergram For each scattergram you can perform the following operations 1 Change parameters by pressing Scattergram properties dialog will appear In this dialog you can change the parameters for the x y axis select the input gates and change the numb
44. er of shown events Scattergram properties Change parameters X AXIS Y Amis Channel 49 DAPI Channel 49 DAPT 28 Select input gates Change number of shown events Figure 78 Scattergrams Scattergram properties dialog TissueQuest 3 0 User Manual doc Page 66 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual e To change the parameters for the x and y axis select from the dropdown list corresponding to the axis the channel and the measurement For more details see Chapter 17 2 1 and Chapter 17 2 2 e Optionally you can set the input data for the scattergram by selecting input gates If no input gate is selected all events are taken and shown inside the plot e Optionally you can set the number of shown events percent This tool is useful when the number of events is very high and the dot clouds become too dense to be interpreted For example by setting the slider to 50 percent only every second event will be shown 2 Set Cutoff by pressing kh for details see Chapter 10 4 1 3 Add gate by pressing Eh for details see Chapter 10 5 1 4 Edit gate by pressing L amp p for details see Chapter 10 5 3 10 4 Cutoffs A cutoff is a numerical parameter used as a threshold value in a scattergram in order to determine positive and negative measure events e g events with staining intensities exceeding or not the staining intensity of a negative control 10 4 1 Add cutoff e Click Set cutoff bu
45. er to highlight important terms or to mark the names of the features GUI elements throughout the text of this manual y the arrow indicates a menu choice For example choose File New means you have to choose New from the File menu TissueQuest 3 0 User Manual doc Internal Page 8 of 116 www tissuegnostics com TissueQuest 3 0 User Manual 2 Installing TissueQuest 2 1 Application Dependencies There are some special requirements concerning the installation of TissueQuest You may check below for the components required to be installed on your computer It is recommended to have all these components installed before you install the TissueQuest product Install them in any order but make sure TissueQuest is the last one to be installed TissueQuest software will not run and operate properly if any of the components is not vf Note installed on your TissueQuest workstation Installing software programs and Windows system components will require Administrator privileges on your computer You might need to ask for support at your IT department if you do not have privileges to install software 2 2 Windows 3 1 Installer for Windows XP SP2 The Microsoft Windows Installer is an application installation and configuration service 2 3 Microsoft Visual C 2005 Redistributables for Windows XP The Microsoft Visual C 2005 Redistributable Package x86 installs runtime components of Visual C Libraries re
46. ergram X Mea Y Mea Events No m 9 UL 0 00 0 00 0 0 00 0 00 UR fof 32 92 11 4 63 0 06 LL 0 00 0 00 0 0 00 0 00 LR 66 61 2847 273 335 5 99 14 66 58 28 08 1284 540 17 100 00 09 14 T C E E C ma ma Rhodamine Figure 53 Detail Window scattergram TissueQuest 3 0 User Manual doc Page 48 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual FOV Viewer Toolbar Sample Neg UV irr Field of View FOV 005 250 Bris Wa i i Delete Events Split Events Merge Events Create Events Figure 54 Detail Window FOV Viewer Toolbar Zoom in Zoom out EH Fit this button scales the image to the size of the window preserving aspect ratio 1 1 Original size this button displays the image at its original size Delete Events Delete Events by pressing this button you enter the Delete Events mode Split Events Split Events by pressing this button you enter the Split Events mode Merge Events Merge Events by pressing this button you enter the Merge Events mode Create Events Create Events by pressing this button you enter the Create Events mode Invert selection Invert selection by pressing this button you can invert an existing selection This option is available in Delete Events mode when some events are already selected TissueQuest 3 0 User Manual doc Page 49 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Apply Apply by pressing this bu
47. eshold Level Include Nuclei Label Figure 135 Non Master Channel parameters Ring Mask if you check this parameter a ring mask around the objects detected in the master channel is created and included in the segmentation result Interior Radius and Exterior Radius the Interior radius and Exterior radius parameters allow the user to define in which direction outwards inwards or both and to what extent this ring should be created The value represents the number of concentric pixel layers As this ring construction takes care of neighboring objects and statistical growth process it stops when the growing environment of a neighboring cell is reached e g when cells touch each other The values for radius represent the maximum growth The interior radius ring growth process stops after the specified distance is reached or if the entire nucleus is covered Identified Cell Mask by selecting Identified cell mask as measurement strategy the nuclei are used as seeds for example starting points of image analysis and cell recognition This algorithm searches for specific staining patterns in the other channels of one field of view and causes binary mask objects to grow along those specific structures Max Growing The user may limit this growth process on demand by setting this parameter Offset from Nuclei in order to reach the marker staining some additional growing steps might be required In this case the growing process
48. ewly added negative marker appears in the Marker Family Editor DAPI ODMA Hoechst Rhodamine FITC GFP 104 A660 Agg Texas Red CDO43 PE IgG1 PE CO14 APT lgGil APC Figure 9 Add negative marker lf you want to use a marker that is not present in the dropdown list you can manually add the desired marker by typing its name in the edit box then press Enter TissueQuest 3 0 User Manual doc Page 19 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Figure 10 Manually add negative marker Benefits of using negative markers Considering that an experiment has Sample 7 with the markers DAPI and CD14 APC and Sample 2 with the markers DAPI and IgG1 APC the IgG1 APC will be considered as being the negative of CD14 APC Negative marker for First sample Second sample CD14 APC contains CD14 APC contains lgo1 APC Figure 11 Using negative markers a If lIgG1 APC is set as negative of CD14 APC the samples can be linked As a result the cutoffs and the gates defined on a scattergram are automatically propagated across the linked samples TissueQuest 3 0 User Manual doc Page 20 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Results per Sample MiTstT The cutoff is automatically set Ra COL4 APC Mean Intensity ENEG By setting the cutoff on negative control the cutoff will be _ automatically set on the marker Mean Intens
49. filter as All words all the files containing the words Region1 and DAPI will be displayed in the file list TissueQuest 3 0 User Manual doc Page 14 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual p An empty character Space is used as separator between terms If the search term ote contains a space like in My Sample 1 the entire term has to be set between quotation marks By selecting an image from the list its content will be displayed in the Preview area Select a specific type of transfer of images to the project folder copy or leave By default the images will be automatically copied into the project folder If the Leave option is selected and the original source images e g the TissueFAXS rote project are removed then the TissueQuest project will lack the source images as well results will remain however Press Next to continue 4 3 Step 3 Whats the meaning of the filename rr Freer Mark parts of the filename and explain their meaning by clicking on one of the three buttons below deal INeg normal Region 001 fReVAOe Pib Wrcriiints tif gf gr C fixed size fixed size fixed size Here you can check Filename Sample Field of View Channel Valid if all the images were Neg normal Region 001 FOV 014 20R Neg normal Foy 014 20 Rhodamine OK recognized correctly Neg normal_ Region 001 FOV 014 49D Neg normal FOV 014 49 DAPI OK Neg
50. from the context menu that appears TissueQuest 3 0 User Manual doc Page 44 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Results per Channel Channels Fh Mi Neg normal A DAPI Right Click Copy to Clipboard Save S H Rhodamine Figure 50 Channels list save or copy to clipboard 7 6 Scattergram list In the Scattergram list there are displayed the numerical results of the algorithm as scattergrams having on their axes output parameters of the algorithms corresponding to channels For more details please see Chapter 10 TissueQuest 3 0 User Manual doc Page 45 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 8 Detail Window The Detail Window offers a detailed view for e A single field of view sample normal skin Field of View FOV 016 e F io prs i i Figure 51 Detail Window single field of view e Multiple fields of view when backward connection is applied TissueQuest 3 0 User Manual doc Page 46 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual y Backward data around point Sample Neg UV irr Scattergra a Bi00 Bis 2 1 Figure 52 Detail Window multiple fields of view backward connection e Ascattergram TissueQuest 3 0 User Manual doc Page 47 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual ss Allevents in scatt
51. gray level expressed by the pixels of the object Maximum of Intensity maximum gray level expressed by the pixels of the object Range of Intensity the gray level interval max min Sum Intensity sum of all gray level intensities expressed by the pixels of the object Mean Intensity the average gray level expressed by the pixels of the object Equivalent Diameter the diameter of the disk which has the same area as the object Variance of Intensity the measure of the amount of variation of all the gray level intensities expressed by the pixels of the object STD of Intensity the average difference of the gray values from the mean of intensity TissueQuest 3 0 User Manual doc Page 106 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Perimeter the perimeter of the segmented object Compactness it measures how much the segmented object shape resembles a disk The more likely it resembles a disk the expressed compactness Is closer to 1 If not its value will be closer to O Eccentricity it measures how much the object shape resembles a circle or a line segment When it is closer to 0 the object resembles a circle when it is closer to 1 the object is more elongated Minimum Width minimum value a micrometer would express taken into account all possible measurements positions orientations Maximum Length maximum value a micrometer would express taken into account all possible measure
52. i Detection Run Detection for Representative Images Run Detection for All Images Figure 137 Algorithm menu In this menu you can choose one of the two detection options e Run Detection for Representative FOVs of a sample e Run Detection for All Images Co The same two options are available by going to the main toolbar and choosing one of Y TIP the two buttons 1 Run detection for representative images only 2 Run detection for all images 2 To run the algorithm for a specific sample or field of view follow the next steps Right click on the sample name or on the field of view name A context menu will appear Choose the desired option from the context menu e Run detection for Representative FOVs for this sample only right click on the sample name e Run detection for ALL FOVs for this sample only right click on the sample name e Run detection only for this Field of View right click on the FOV name TissueQuest 3 0 User Manual doc Page 103 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Right click Right Click Channels i Results per Channel Meg ne _E___ Results per Channel EH Neg T Fo Run detection For Representative FOVs For this sample only Run detection For ALL FOVs For this sample only Channels et ee Run detection only For this Field of view EH Rhodamine H Rhodamine
53. ia aiai aeaa aE nan e aiae ainada EEan 10 2 6 1 Hardware Requirements cccccsccccescccseeceucecenceceueeceeecueeceuseseueesausesaueseueessueessueesaueesueessueessusessaeesseeenees 10 2 6 2 Software Requirements ws iesisaviescewvailesecenswinewasnalvlocecieauiensindseeandndviweadvslulenectuarwetessd vaseadmauieneslesblocusune ered sduedsssdvexton 10 6 aT 18198100 oe oc 16 21 0 gt ae ee eo ne ee ee eee ee eee 11 4 Create new projects x ssdecisteosecdiozocesenvsavcbesssansecabeccendsonaceausveasdbedesevennsisac sactonecesasbredsbucdsoceanstewseedaubengedosesscusueedsbaces 12 4 1 SLE e E A OEA E RE E ENEE TETE E E E E E E E E nels ET O EN 13 4 2 SLE 8 L EPOE EA E E O I A E A EA E A O A E E A A A E O E 14 4 3 S L E EA O ee eee eee E 15 4 4 SIL S E PANE EEE EAA TTA A AEE OAE A EA EA EE 16 4 4 1 Templates for the FOV size SCAIING cccccceeecceeeeeeeeeeaeeeeeeaeeeeeeseeeeeeseeeeeeeseeeeeeseeeeeeseeeeeessaneeeesaeeeeeeaees 28 4 5 SIL 0 a PAE ENET N A EE AE E E O EEE E EE A EAE 29 9 OPen EXSUNG Proj ensieme ne aE Eaa E EEEE EEE EEEE EE 30 5 1 Se E mg 8 E 6 EE E S E I A TAE E EE EA N A ee ENEE E E eee S eee 30 5 2 FE ON ca ees EE avis deed eee E eee sade EEE aren ssuzovee sede coeetsuetet cee EE oes usveessescdecieetavestetiees 31 6 Mc 1 0 9 ee eee E e ee ra ne eee eee eee eee eee eae 32 6 1 CN AIAN asso gs PA E A T oo socicte ance ane ods T saree TN I A ules aes see vee uses eca ee seete 32 6 2 Clean Tempora
54. idth 33 99 Madmum Length 46 60 Feret Ratio 0 70 Figure 60 Detail Window View event data While visualizing event data forward connection is activated For more details see Chapter 11 1 TissueQuest 3 0 User Manual doc Page 55 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 9 Samples 9 1 Select Sample to be Displayed Press Select Samples button from the main Toolbar to access a dropdown list where you can select the samples you want to have displayed in the Main window Jn TissueQuest allows you to have simultaneously displayed a maximum number of 10 OTe samples File View Project Samples Scattergrams Gates Algorithm Admin Help r eer 3 oo E Select Samples Figure 61 Select Samples button In the dropdown list simply mark the checkbox corresponding to the sample s you want to display To display the previous or next block of samples use lt lt and gt gt buttons Select Samples ow Select samples Neg normal WS Neg Uy irr Wi normal skin B treated Figure 62 Select Samples dropdown list 9 2 Sample linking Linking samples means grouping two or more samples so they will share the same properties e They have the same scattergrams a scattergram added to a sample is automatically propagated to all the samples belonging to the link of samples e The scattergrams have the same properties The same cutoff val
55. ing on it then press Delete button L_Delete__ 10 6 Scattergram parameters By right clicking on the scattergram a contextual menu will appear From this contextual menu the scattergram parameters can be adjusted For each scattergram parameter the following properties can be adjusted e Autoscale e Maximum value if the Autoscale is not used e Scaling type Results per Sam ple E Neg UV irr Copy image to clipboard Copy cutoff Paste X Paste Y Paste both X amp Y i DAPI Mean Intensity vw Autoscale Maximum value Scaling type d Logarithmic Liniar Figure 110 Scattergram parameters TissueQuest 3 0 User Manual doc Page 81 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 10 7 View backward data By right clicking on the border of a gate a contextual menu will appear Hee pee a ra ae If you select View backward data for gate Gate Name you will see the backward data for that gate For details see Chapter 11 Rhodamine Mean Intensity z l 3 ul g z ie z B a iT Ez amp m c Figure 111 View backward data for a gate e By right clicking outside the border of a gate a contextual menu as shown below will appear If you select View backward data around this point you will see the backward data for that numerical value For details see Chapter 11 Delete cutoff
56. ional purposes only 8 TissueGnostics GmbH specifically disclaims all warranties express or limited including but not limited to the implied warranties of merchantability and fitness for a particular purpose except as provided for in a separate software license agreement 9 Copyright 2006 2011 TissueGnostics GmbH All rights reserved 10 Release date of this document 16 February 2012 11 Document Version 2 2 TissueQuest 3 0 User Manual doc Page 4 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Notice regarding anti virus applications e All the TissueGnostics products are shipped with the Kaspersky Anti Virus in order to ensure optimal functionality e Therefore TissueGnostics only guarantees for Kaspersky Anti Virus to work with its products e The user may decide to use other anti virus software only upon own responsibility e Any problem that arises due to other anti virus software installed will void TissueGnostics warranty and every support or service action required by the client in order to restore any of TissueGnostics damaged instruments will be billed to the customer TissueQuest 3 0 User Manual doc Page 5 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 1 Introduction Multicolor immunofluorescence is a major method of cell based research In order to determine molecular interdependencies it is critically important to measure several markers simultaneously on the sam
57. isabled until you create or open a project Create New Project i select Samples v Open an Existing Project button Figure 2 TissueQuest main toolbar To start the wizard press Create new project button from the main toolbar of the application The first step of the wizard will open You may also create a new project by going to File menu of the application and r TIP choose New TissueQuest 3 0 User Manual doc Page 12 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 4 1 Step 1 What s the name of the project 7 mrm A suggestion T EA change it if you want Where will the project be stored Para Tr Ve Project folder F Projects TissueQuest Please define a path where project will be stored Figure 3 New Project Step 1 On the first page of the wizard you must enter the project name and the folder where the project will be stored What s the name of the project Give a name to the project Automatically the name of the project will be composed of LoginName CurrentDate CurrentTime You can change this name according to your interest Where will the project be stored Choose a folder where to store the TissueQuest projects by using Browse button In the future all the new projects will be stored in this folder unless you will choose another location 2 Please ensure the selected folder exists and that you have enough rights on it rote otherwis
58. ity IgG 1 APC Figure 12 Using negative markers b If lIgG1 APC is not set as negative of CD14 APC the samples cannot be linked As a result the cutoffs and the gates defined on a scattergram are not propagated on the other sample In this case the cutoff and the gates have to be set manually at the desired location TissueQuest 3 0 User Manual doc Page 21 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Results per Sample The cutoff must be set manually CO LAAPE Mean Intensity The cutoff set on the negative control will not be Mean Intensity pan S 3 pa o i oO m is w gt ah IgG 1 APC Figure 13 Using negative markers c Change a marker or Add Change negative marker In Marker Family Editor double click on a marker or its negative A combo box will be displayed Here you can select a known marker or you can type the name of the new marker Then press Enter The newly added marker appears in the Marker Family Editor TissueQuest 3 0 User Manual doc Page 22 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual __ Marker Negative Color Master _ Marker Negative Color Master Rhodamine DAFI DHA Hoechst FITE GFP Figure 14 New Project Step 4 Change marker Change the color of the markers In Marker Family Editor click on the color corresponding to the markers A Co
59. ks modify them or create new links e To modify existing links select the desired link in the Link Name section then edit the corresponding field s Name Description Color then press Update button To create a new link fill the Name and Color fields mandatory and Description optional then press Add button To remove a link click on the desired link in the Link Name section then press Remove button TissueQuest 3 0 User Manual doc Page 58 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Link Name Default Link of Samples Global Link Info Name Default Link of Samples Global Description UuTtoO matic Update EEE Eee ee Pee Uf ion f ian ff apopes f imi ie OBOBOBOO More Colors Figure 67 Sample links dialog edit color TissueQuest 3 0 User Manual doc Internal Page 59 of 116 www tissuegnostics com TissueQuest 3 0 User Manual 10 Scattergrams 10 1 Manage scattergrams To manage scattergrams go to Scattergrams option in the Menu bar then choose Manage Scattergrams Gates Algorithm Display Settings Figure 68 Manage scattergrams menu The following dialog will appear Scattergram management ae aaa eal a Samples B normal skin DAPTI Mean Intensity DAPI Area DAPI Mean Intensity Rhodamine Mean Intensity a Rhodamine Mean Intensity Make visible all scattergrams for this sample Delete Figu
60. le to be Displayed cccccscccsscccseccceeecceeeceuceceueecaueesseeceueeceucessueesaueeseeseueeseusesueeseeessaeeseas 56 9 2 re 18 0 411 01419 6 PEA AE A E A N E A AE AA EE A E E E oer 56 10 SEEE KE L e AAAA E NE AEE E E E E A E E E E AI E A A E A 60 10 1 Manage SCAMSN ORANG ic coicsicc cndesanadiodaneasieasitelsamnmncasied eadelsciadoeinendiuasied sanimnnacied easleideaadlonelisadiemaudceuhpotnsicdisanicinnd eadleissaadontlne 60 fC ea Ia ey co ol scatergi AN nen eee en ee ee eo ee ee 60 10 1 2 Delete Scattergram cetera cite ane dese nice snore cts int aa dec neta des oatemactldoemmnnts pede deldeieutio dob antaedelsanmmnensasiendeceacmendesetoatie 61 10 1 3 Show Ride scatergr atiaeina ee nee ee a eee ec eee ee eee eee 62 MZ NIG SINS oer ose eats E A ne uameise EE EA EREE EA E TER 64 TissueQuest 3 0 User Manual doc Page 2 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 103 GAS AIT TOODA rerien iai E A E E Ea E EE N EEEa ea 66 10A OO S Er EE EE ENEE OE EET EEE EEEE EEEE EEEE EEEE EAN E EG 67 ATACO eee EE EE EE E EE OEE EE E E E EERE 67 10A 2 MOa CUO eean EEE EEE EEE EEEE OEE E E E EEEE 68 MA GIO VC CUO serea EEE E EE E EEEE EEE E EE 69 MA OC O EEE EEEE EE EE EEE EOE E EE E E EE E EERE 70 MAO S O CUNOUE PA E curate doce ecu cewecceedec eaves coset eveeeuecseassyeedecdade aces eave eveedsueenseeeyecseisunedeetadeaset save ovsedeer 71 6 FS OE e ee ee eens eee eee ee ee 72 10 5 1 Add dovere gt qe f
61. lean Temporary Files Group of Channels Virtual Channels Properties Figure 40 Project menu Samples See Chapter 9 Figure 41 Samples menu Scattergrams See Chapter 10 Manage Display Settings Figure 42 Scattergrams menu Gates See Chapter 10 5 Figure 43 Gates menu Algorithm See Chapter 16 W Use Various Shapes for Nuclei Detection Run Detection for Representative Images Run Detection for All Images Figure 44 Algorithm menu Admin See Chapter 18 TissueQuest 3 0 User Manual doc Page 41 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Users Institution Data Change Password Figure 45 Admin menu Help See Chapter 19 Figure 46 Help menu 7 3 Toolbar TissueQuest toolbar File View Project Samples Scattergramy Gates Algorthm Admin Help wi G Select Samples Figure 47 TissueQuest Toolbar Create a new project see Chapter 4 Gy Open an existing project see Chapter 5 Save current project see Chapter 5 1 E a Print current project see Chapter 5 2 Run detection for all images see Chapter 16 Run detection for representative images only see Chapter 16 Select Samples to be displayed see Chapter 9 1 TissueQuest 3 0 User Manual doc Internal Page 42 of 116 www tissuegnostics com TissueQuest 3 0 User Manual 7 4 Parameters of algorith
62. lor dialog will be display Here select the desired color and then press OK a a a a 3 8 17 1713 11 SEE S887 ll OEE Ee ll HERES ll EEEH i Figure 15 New Project Step 4 Change color Change the purpose of the markers In Marker Family Editor use the check box in the Master column in order to change the purpose of the markers TissueQuest 3 0 User Manual doc Page 23 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual If the Master is checked then the channels that have these markers associated will become master channels in their samples If the Master is not checked then the channels that have these markers associated will not be master channels in their samples for example cytoplasmatic channel Sample editor The Sample editor offers several functions which are used to group channels and samples If two channels are grouped there will be only one control box shown in the main window of TissueQuest to set the segmentation parameters In other words if different settings are required for two channels e g a ring mask for the Cy2 channel and a nuclear mask for the Cy3 channel these two channels must not be grouped but have to remain independent In the case that different settings of the segmentation parameters are required within the same channel e g the Cy2 channel in different samples the user can create groups for certain samples e g group the CD3 Cy2 channel in sampl
63. m Parameters Nuclei Size Discrimination by Area Discrimination by Gray Autodetect Background Threshold Threshold Level Ring Mask Interior Radius Exterior Radius Identified Cell Mask Max Growing Offset from Nuclei Autodetect Background Threshold Minimum Level Maximum Level Figure 48 Parameter list The Parameters list displays the parameters available for each group of channels master channel parameters and non master channel parameters and allows you to adjust them For more details see Chapter 15 2 TissueQuest 3 0 User Manual doc Page 43 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 7 5 Channel list _ Channels TE Results per Channel E Rhodamine Figure 49 Channels list The channel list displays the representative Fields of View Each Field of View has two columns e Channels in this column the original images are displayed e Results per Channel in this column the mask and the overlay images are displayed fn The mask and overlay images are results of running the detection algorithm fi ote Running the algorithm To run the algorithm for a specific sample or field of view see Chapter 16 Save to file or copy on the clipboard To save to file or to copy to the clipboard an image of a channel follow the next steps Right click on the image from the channel e A context menu will appear e Choose Save as or Copy to Clipboard
64. m toolbar 200 Rhodamine Mean Intensity Figure 93 Add polygonal gate step 1 e After clicking on the button move the mouse into the chart area of the scattergram The cursor changes into a cross e Click the left mouse button inside the chart area to define the starting point of the region of interest e Now a red line appears and connects the last added point with the current mouse position e To define the next point of the polygon click the left mouse button in the chart area The red line continues to connect the last added point with the current mouse position 200 a u ES oe ic EE D 1009 200 255 DAFI P Figure 94 Add polygonal gate step 2 TissueQuest 3 0 User Manual doc Page 74 of 116 Internal TissueQuest 3 0 User Manual www tissuegnostics com Rhodamine ge Mean Intensity Figure 95 Add polygonal gate step 3 e Repeat the last two steps until all the points are defined The last step in setting a polygonal gate is to close the polygon To do this click the right mouse button inside the chart area This will define the last point of the polygon and will connect the last point to the first point All events inside the gate change their color to a predefined hue The gate gets labeled with a default text e g Gate 1 You can change the color the label and the visibility of each gate by using the gate managemen
65. mal DAPI Preview FOV 002 FOY 002 Neg UWVrr DAPI PENTE normal skin DAFI FOV 016 i UV treated DAPI FOV 038 f I Rhodamine FOV 046 i Neg normal Rhodamine Define FOV size 2 predefined Manac i 0 8987893460 Width Height 0 6611783700 ee Check a sample to define which FO s you want to use within the project lt Back Next gt Figure 6 New Project Step 4 Page 4 of new Project Wizard contains the following sections Marker family editor Sample editor Link all samples Fields of View Group of channels FOV size Marker family editor In the Marker Family Editor you can edit the markers present in the new project By default TissueQuest creates the markers based on the Channel part of the filename as indicated in Step 3 All individual channels in the project have associated unique markers The Marker Family Editor is organized in lines each line is referred to as Marker Family A Marker Family contains A marker The negative of the marker if any is used The color associated with the markers The purpose of the markers master or non master channel s This determines the channel used for primary cell identification master channel TissueQuest 3 0 User Manual doc Page 17 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Add new marker In Marker Family Editor double click on A combo box will be displayed Here you can select a known marker or you can type the nam
66. ments positions orientations Feret Ratio the ratio between minimum width and maximum width These output parameters are used in order to associate data to specific axis of the scattergram To do this access the Scattergram properties dialog from Scattergram toolbar EI see Chapter 10 3 Scattergram properties dialog is also available when adding a new scattergram from vf note Scattergram management see Chapter 10 1 1 scattergram properties Change parameters xX Axis Y Axis Channel 43 DAPI Channel 49 DAFI Parameter arti Minimum of Intensity oe Sena Select input Mainm of inierity Change number of shown events Range of Intensity sum Intensity Mean Intensity Equivalent Diameter Variance of Intensity STD of Intensity Perimeter Compactness Eccentricity Minimum Width Maximum Length Feret Ratio Figure 142 Scattergrams Scattergram properties dialog Parameter list Press the arrow button from the Parameter section to see the dropdown list containing the raw data for Various Shapes 1 0 parameters available after running the algorithm TissueQuest 3 0 User Manual doc Page 107 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 17 2 2 Raw Data for DOT Finder For DOT detector the following output parameters are available for each detected event Dot Count the number of dots present in a specified event Dot Mean Intensity the mean intensity gray value of th
67. nts a Split Events Merge Events Create Events Apply 4 a Figure 58 Detail Window delete events 4 You cannot create events on images obtained as a result of backward data vf note computation When an image of the channel is displayed you can right click on any event to see the contextual menu This contextual menu has two options Delete event e View event data TissueQuest 3 0 User Manual doc Page 53 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Sample Neg UV irr Field of View FOV 005 8230 Pes 1 4 Delete Events a Split Events Merge Events Create Events Delete event Right Click View event data Figure 59 Detail Window contextual menu e You can delete that event by choosing Delete event e You can view the numerical results raw data for that event if you choose View event data see also Chapter 17 2 TissueQuest 3 0 User Manual doc Page 54 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Sample Neg UV irr Field of View FOV 005 Delete Events Split Events Merge Events Create Events JM Event 39 on 49 DAPI Area 1304 00 Minimum of Intensity 47 00 Maxdmum of Intensity 91 00 Range of Intensity 69 00 Sum Intensity 76723 00 Mean Intensity 58 84 Equivalent Diameter 40 75 Variance of Intensity 334 28 STD of Intensity 18 28 Perimeter 139 20 Compactness 0 85 Eccentricity 0 74 Minimum W
68. of a gate Rhodamine gee i Mean Intensity S a View backward data for gate Gate 3 0 0 100 200 255 DAPI Mean Intensity zi Figure 117 Backward connection gate a e After backward data is processed the corresponding events can be followed up in Detail window TissueQuest 3 0 User Manual doc Page 85 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual HF FOW 032 Figure 118 Backward connection gate b TissueQuest 3 0 User Manual doc Page 86 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 12 Project properties To access Project properties go to the Menu toolbar and choose Project Properties Clean Clean Temporary Files Group of Channels Virtual Channels Properties Figure 119 Project Properties menu The Project Properties window will appear MAL Mie D Se Ceu T CBSE reese aL Figure 120 Project Properties window Q mei To hide the overview of the FOV and of the channels press Hide button Ls hide J in the upper right corner of the window TissueQuest 3 0 User Manual doc Page 87 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 12 1 Samples Samples Neg UV rr B UV treated Figure 121 Project Properties Samples section In the Samples section you can choose for what samples you want to adjust settings Simply click on the desired sam
69. on e The FOVs will immediately appear in the Representative FOVs list To send the FOVs back to the Non representative FOVs list select the desired FOVs and then press the button FOV becomes representative Select desired Non epresen tive FOVs Representative FOVs Non tepresentative FOVs Represental re FOVS FOV 005 Bring FOV to representative FOVs list Non tepresentative FOVs Representative FOVs Non tepresentative FOVs Representative FOVs FOV 005 Remove FOV from representative FOVs list Send representative FOV back to non representative list Figure 123 Project Properties Representative Non representative FOVs moving one FOV Moving all FOVs e Tomove all FOVs from the Non representative FOVs list to the Representative FOVs list press the button e The FOVs will immediately appear in the Representative FOVs list e To send all FOVs back to the Non representative FOVs list press the button TissueQuest 3 0 User Manual doc Page 89 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Mon tepresentative FOVs FOV 005 FOV 007 FOV 012 FOV 017 Representative FOVs Non epresentative FOVs Representative FOVs Bring all FOVs to representative FOVs list hon fepresentaiwne Foe Represeniaive Powe Non representatws Pirs heoresceniaove POWs amase aa Oo o POV 008 p POW 012 FOW O47 hen epresentatve Fog Reoreseniaive Fig
70. oup channels Uncheck Auto Group Channels In the Sample Editor select the desired channels use CTRL key for multiple selection and then right click on them A popup menu will be displayed Under the Group submenu select how you want to group the channels The parameters for detection algorithm are adjustable individually for each group of channels wf Note Master channels and non master channels cannot be grouped together Link all samples Check Link all samples check box if you want the samples to be automatically linked See Chapter 9 2 for more details TissueQuest 3 0 User Manual doc Page 24 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Define group of channels Auto group channels Check Auto check box The channels will be automatically grouped based on their associated marker All the channels having associated the same marker will be included in the same group of channels Rename group of channels Click twice on the name of the group A text editor will be displayed Type the text here and press Enter to save the new name Press Escape in order to cancel To include some fields of view in the project follow the next steps e Select the desired fields of view in Sample FOVs list e Press button next to the right of Sample FOVs list to include the selected fields of view in the project e Press button next to the right of Sample FOVs list to include all the fields of view in the project
71. parameter set is available 15 2 1 Master Channel Parameters Nuclei Size Discrimination by Area Discrimination by Gray Autodetect Background Threshold Threshold Level Figure 134 Master channel parameters Nuclei Size specifies the size of the nuclei This is an arbitrary unitless value and it should be increased if your nuclei are oversegmented and decreased if the nuclei are undersegmented Discrimination by Area this parameter removes the smallest objects from the measure mask i e the result of the image segmentation By increasing this parameter more small events are removed from the result of the segmentation Discrimination by Gray this parameter removes the objects with lowest staining intensity By increasing this parameter more events with low staining intensity are removed from the measure mask i e the result of the image segmentation Autodetect Background Threshold specifies if automatic detection of the background level should be used by marking the checkbox or can be set manually by unselecting the checkbox and providing the desired value in the Threshold Level field TissueQuest 3 0 User Manual doc Page 99 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 15 2 2 Non Master Channel Parameters Ring Mask Interior Radius Exterior Radius Identified Cell Mask Max Growing Offset From Nuclei 4ubodeteck Background Threshold Minimum Level Maximum Level Thr
72. ple to make it available 12 2 Representative Non representative FOVs Even with modern computer technology image analysis is a time consuming task For optimizing the settings for image segmentation certain fields of view can be declared as representative fields of view This allows faster evaluation of certain settings Once the settings have been defined on the representative FOVs the analysis can be run on the entire samples all FOVs in the project Representatives FOVs are FOVs that include a significant amount of information in order to generate concluding results The results of the analysis of the representative FOVs should be similar to the analysis of all FOVs You can move your FOVs from one list to another according to your needs In this section you will learn how to select your representative FOVs Two lists of FOVs are available e Non representative FOVs to the left e Representative FOVS to the right Non tepresentative FOVs Representative FOVs FOV 014 FOV 032 Figure 122 Project Properties Representative Non representative FOVs section TissueQuest 3 0 User Manual doc Internal Page 88 of 116 www tissuegnostics com TissueQuest 3 0 User Manual Moving one or more FOVs e To move one or more FOV from the Non representative FOVs list to the Representative FOVs list select the FOVs you want to move by clicking on them hold Control key pressed for multiple selection then press the butt
73. press the Apply and then the Close buttons In the application at the Parameters section the selection will look like this TissueQuest 3 0 User Manual doc Page 36 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual High Intensity Dots Low Background High Intensity Dots Medium Background High Intensity Dots High Background Middle Intensity Dots Low Background Middle Intensity Dots Medium Background Middle Intensity Dots High Background Low Intensity Dots Low Background Low Intensity Dots Medium Background Low Intensity Dots High Background Figure 32 DOT settings dropdown box You can select now the desired settings in order to get the right DOTS detection Different Settings for Individual DOT Channels If you want to have separate settings for individual DOT channels you have to go to Project from the menu of the application and then choose Virtual Channels option In the dialog box that appears you will have to select one of the DOTS channels to be introduced After the selection is made press the Apply and then the Close buttons see image bellow Alexa Fluor Figure 33 Virtual Channels selecting DOT channel In order to introduce the second DOT channel you have to go again to Project from the file menu of the application and choose the Virtual Channels option In the dialog box that appears you will have to select the second DOT channel that you want to introduce After
74. quired to TissueQuest on Windows XP 32 bit or 64 bit 7 If you have installed previous pre release versions of Visual C 2005 or Visual Studio rote 2005 such as Beta 1 Beta 2 or Community Technical Preview CTP builds then you must uninstall these versions via Add Remove Programs in Control Panel before installing the final released version For further information about this package please visit the Microsoft homepage 2 4 Intel Performance Primitives 5 3 Intel Integrated Performance Primitives Intel IPP is a software library that increases performance of Intel s latest microprocessors Incorporating these functions into the TissueQuest software will ensure that the potential of your Intel based PC can be fully exploited for analysis of your samples 2 5 MARX Dongle Drivers TissueQuest uses a hardware based security solution from MARX Software Security When purchasing TissueQuest you will also receive an USB dongle that must be inserted in an USB port In order to successfully use the dongle you must install the corresponding driver for the operating system TissueQuest 3 0 User Manual doc Page 9 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 2 6 System Requirements 2 6 1 Hardware Requirements Minimum e Intel Pentium 4 Processor 3 GHz e 1GBRAM e USB Port for dongle e 100 GB available hard disk space e Single monitor system with a resolution of 1280x1024 Recommended Intel Core2
75. re 69 Scattergram management dialog 10 1 1 Add scattergram e Press Add new button from Scattergram management dialog Page 60 of 116 TissueQuest 3 0 User Manual doc Internal www tissuegnostics com TissueQuest 3 0 User Manual e Set the properties of the new scattergram in the Scattergram properties dialog box Press Ok to create the new scattergram Scattergram management Samples Scattergram properties I normal skin h gt gt W DAPI Mean Intensity DAPI Area Change parameters 2 FJ DAPI Mean Intensity Rhodamine Mean Intensity X Axis Channel 49 DAPI v Channel 49 DAPI X Parameter Parameter Select input gates Change number of shown events Y Axis v E oz Mean Intensit V Make visible all scattergrams for this sample Figure 70 Manage scattergram create scattergram 10 1 2 Delete Scattergram e Select the scattergram you want to delete in Scattergram management dialog On the right of the dialog box you can see the image of the selected scattergram e Press Delete to delete the scattergram TissueQuest 3 0 User Manual doc Page 61 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual scatterg Select scattergram eed you want to delete WW normal skin DAPI Mean Intensity DAPI Area DAPI Mean Intensity Rhodamine Mea 1 Intensity Rhodamine Mean Intensity Make visible all scattergrams for this
76. ry cc en ee eee ee 32 6 3 Group of Channels ssiri eee ne oe ee ee eee 33 6 4 Viua Channels cee ccs et ci ete nc aie tcl ccleaner snl cesses a oad i emcee yee i panes 35 6 5 MAOGA DO SS UNOS esgan dacism lt e seen cane E E N E EEA E E EE 36 6 6 POPO sr E E E EE E sae A EA E NOA 38 Ce MANON eaer E EE E E E aes 39 al Components of the Main Window cccccscccseccceeeccseeceuceceueecaueecaeeceueeceucessueesaueesueeseacessusesueeseeseseeeseas 39 7 2 DNS NO eesti estas ete sens se sates pa eet wend wane arse cise pa ene ates ae croc ete osteo E Waren aie N A A A E 40 7 3 TON A arta csp tet tesa wie E ie asec pase cme asst gs se ee ects cetio A dees otedeeseese ween eaes 42 1 4 Parameters Or IC ONG senese EEEE EAE E EE 43 7 9 TUS NS C sce see anes cannes ss E sane cane uoasaeaeacgencn puaceaina saan E E E EE 44 7 6 SNS I AN NS ss cee can sercessaricces E cane cant uoasadae scuencn punceainae saan E EE 45 O CAI I esse a senses es cate nsec eas EAE ase ncuencn E sacs dass ooacaneeanaeuntecenaneronceimeeieenaeenananaees 46 8 1 Detail Window FOV Viewer TOOIDAP ccccccscccsceceseeceececueeceuceceueecaueeseeseucecsueesaueeeeessusessueessueeseeeseas 49 8 2 DOLE CV Cll oorr vaceapacscae anne E E E E 50 8 3 SPICCO S oea E a O E E E eee ne 51 8 4 MOr GOVORIO cresio E EE ee ee E E 51 8 5 EAEE e E E TE E ee E eee A ee E eee eee eee ee 52 8 6 COMextUal MONU rns a E Ea eT E E 53 P SAO a E E E E E E T E A E 56 9 1 Select Samp
77. s a product for cytometric analysis of tissue sections tissue cytometry including TMAs and biopsy material as well as cytological preparations The initial point of the process chain in tissue analysis using the TissueQuest software is importing images to a new project Analysis parameters for sophisticated image processing and pattern recognition algorithms have to be optimized for your specific needs After optimization of analysis parameters segmentation is done for all images in the project which results in a set of numerical data These data are visualized in scattergrams and are subject to further processing and finally statistical analysis 1 3 Definitions Acronyms and Abbreviations Algorithm A sequence of mathematically realized processing steps applied to an image or part of an image with the aim to improve image quality and to extract specific information or segment identify image objects What an algorithm might do 1 Separate color images 2 Identify colored regions 3 Discriminate background from specific cell objects TissueQuest 3 0 User Manual doc Internal Page 6 of 116 www tissuegnostics com TissueQuest 3 0 User Manual 4 Recognize individual cells where does one cell end and neighboring cell begin 5 Identify cytoplasmatic areas Analysis An analysis is the process of identifying individual nuclei cells cellular compartments and other structures of histological pathological relevan
78. sample Figure 71 Manage scattergram delete scattergram 10 1 3 Show Hide scattergram You have two options to hide a scattergram 1 From Scattergram management uncheck the corresponding checkbox for the scattergram you want to hide then press Apply If you want to display all the scattergrams from the sample check Make visible all scattergrams for this sample Press Apply The hidden scattergram s will be displayed again TissueQuest 3 0 User Manual doc Page 62 of 116 Internal www tissuegnostics com ocattergram management a a ae ae a r CT A i F TT Tr Samples WW normal skin M DAPI Mean Intensity DAPI Area DAPI Mean Intensity Rhodamine Mean Intensity Check the scattergrams you want to be displayed _ Check this to make visible all scattergrams a Rhodamine Mean Intensity TissueQuest 3 0 User Manual Figure 72 Manage scattergram show hidden scattergram step 2 2 Use Hide scattergram button 1 in the scattergram toolbar TissueQuest 3 0 User Manual doc Internal Page 63 of 116 www tissuegnostics com TissueQuest 3 0 User Manual Results per Sample W Neg UV irr Fhodamine ge Mean Intensity Pi Ea mi E E m T i i oH Mean Intensity lad Figure 73 Manage scattergram hide scattergram 10 2 Display settings To change the scattergram display settings select Scattergrams from the application s main m
79. se the sample and the channel in order to apply DOT finder detection please see Chapter 15 3 Press the button to access the dropdown menu and to choose the desired sample WS Neg UV rr i normal skin UV treated Figure 28 Virtual Channels dialog dropdown list For each original channel you can mark the checkbox from the Dot column in order to create a virtual channel on which to measure the dots TissueQuest 3 0 User Manual doc Page 35 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Figure 29 Virtual Channels dialog mark Dot checkbox Press Apply button to apply the changes A new virtual channel is created it shares the original image with its original channel 6 5 Individual Dots Settings Same Settings for all DOT Channels If you want to have the same settings for all DOT channels you have to go to Project from the menu of the application and then choose the Virtual Channels option File View Project Samples Scattergrams Gates Algornthm Admin Help Clean Clean Temporary Files Group of Channels Virtual Channels X Properties Figure 30 Project Menu Virtual Channels option A dialog box will appear where the user can select all desired DOT channels please see image bellow Alexa Fluor Figure 31 Virtual Channels selecting DOTS channels After selecting the desired DOT channels you have to
80. ss Update button to create and store the template If a template with the same name already exists that template will be updated A new template will not be created To modify an existing template follow the next steps Select a template from the list Change the description of the template and the size of the fields of view Press Update to store new information If you also change the name of the template a new template will be created The old template will not be updated To remove an existing template follow the next steps Select the template from the list Press Remove button TissueQuest 3 0 User Manual doc Page 28 of 116 www tissuegnostics com TissueQuest 3 0 User Manual 4 5 Step 5 Here is the summary of your settings __ On the right you have a i summary of the settings Project name UV Irradiated Skin you defined in the previous steps A Basics This is an overview to A check if everything is set Cell identification strategy as you like Press Back to change Sto rage r ect i Finish to accept the Project is stored in F Projects TissueQuest V Irradiated Skin SELES RTD ech oe nee Your images in the folder F Projects TissueFAXS LYV Irradiated Skin Images Will be copied to F Projects TissueQuest V Irradiated Skin Images Samples Neg normal Channel Marker Master 20 Rhodamine Rhodamine 49 DAFI DAPI This is the overview of the settings you defined before Figure
81. st 3 0 User Manual doc Page 94 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Institution Hospital Namel Departmen Laboratory User Administra tor A 5 j E i m OE Date of analyss Monday 18 January 2010 16 43 33 TIBSUE SNOSTICS Detection method Various Shapes MEOIGAL amp SIGTEGH SOLUTIOW amp 49 DAFI 20 Ritodamine D CAPT Oot des are 24 ming mask 1 J spat 1 0 200 0 50 Gecmm mation by ares 1 max growing X tempga 4 9 Gece ination by gray 0 offset from meter 0 apia 01000 Geckground iire 30 Geckground Gweshoid so 0 5 MOE Tee no FOV 038 Color channel overlay DAPI M aster C hannel Figure 130 Print Preview example You may also print a project by going to File menu of the application and choose r TIP Print 14 2 Export To access the export options in TissueQuest select File Export in the menu bar A dropdown menu like in the image below will appear TissueQuest 3 0 User Manual doc Page 95 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Samples Scattergrams Gates Algorithm Admin Help Ctri N Ctri 0 Select Samples w oChannels h M Region 001 Save Experiment Ctrl 5 H FOV 00001 Export Print output as images Print Ctrl P Measurement data as ASCI Measurement data to Excel Raw data of a Gate to Excel Raw data of Dots to CSV Print Prewiew Exit Figure 131 Export project
82. t see Chapter 10 5 5 255 200 ue Z E S10 q E 2g CE 0 0 100 200 255 alam Figure 96 Add polygonal gate step 4 10 5 3 Modify gate Click the Edit gate button E in the scattergram toolbar Page 75 of 116 TissueQuest 3 0 User Manual doc Internal www tissuegnostics com TissueQuest 3 0 User Manual Pi a Rhodamine Mean Intensity F Figure 97 Modify gate step 1 The corner points of the gates are highlighted by a small rectangle showing the gate is ready to be moved mr x 25 20 a Cg ES sr on EE 0 100 200 755 DAPI Mean Intensity Figure 98 Modify gate step 2 Move the cursor over the corner that you want to move The cursor changes into a cross Press the left mouse button hold it pressed and move the point to the new position During the movement the new shape of the gate is highlighted in red lines y pd Re Rhodamine y Mean Intensity Figure 99 Modify gate step 3 TissueQuest 3 0 User Manual doc Page 76 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual When the new shape of the gate meets your needs release the left mouse button w E m E 0 Mean Intensity Figure 100 Modify gate step 4 a e This procedure can be repeated for every corner of the gate Rhodamine gee Wi a iT i
83. t you will find the new virtual channel displayed at the end of the Parameters list Nuclei Size Discrimination by Area Discrimination by Gray Ring Mask Interior Radius Exterior Radius V Identified Cell Mask Max Growing Offset from Nuclei Z Autodetect Background Threshold Minimum Level Maximum Level Threshold Level Include Nuclei Label TEN A Los _ Default Small Dots Large Dots Large Conic Dots Small Cylindric Dots Small Cylindric Dots Figure 136 DOT Parameters TissueQuest 3 0 User Manual doc Internal Page 101 of 116 www tissuegnostics com TissueQuest 3 0 User Manual From the dropdown list available by pressing the button select the template which best fits your needs 7 If no suitable template is available in the dropdown list please contact TissueGnostics J Note support After selecting the desired template run the algorithm in order to obtain the results A series of output measurements will be available they are described in Chapter 17 2 2 TissueQuest 3 0 User Manual doc Page 102 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 16 Image Analysis When working with TissueQuest software you have different possibilities to run detection depending on your analysis needs 1 Go to the menu bar of the application and choose Algorithm A menu like in the image below will appear Algorthm Admin Help Use Various Shapes for Nucle
84. the selection is made press Apply and then Close buttons please see image bellow TissueQuest 3 0 User Manual doc Page 37 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Figure 34 Virtual Channels selecting DOTS channels After performing these operations there will be two DOTS markers in the application at the Parameters section see image bellow High Intensity Dots Low Background High Intensity Dots Medium Background High Intensity Dots High Background Middle Intensity Dots Low Background Middle Intensity Dots Medium Background Middle Intensity Dots High Background Low Intensity Dots Low Background Low Intensity Dots Medium Background Low Intensity Dots High Background Figure 35 DOT settings dropdown box lt frote You can have different settings for detection for each DOT marker 6 6 Properties For details concerning Properties option please see Chapter 12 TissueQuest 3 0 User Manual doc Page 38 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 7 Main Window 7 1 Components of the Main Window detection Representative with numerical algorithms FOVs results 2 ST Teeter LE 7 aana aie Fila Wira Preqect 1 5 Heka or Seen ple ir erTi Deere bey rc Teoria be BAT _ Birit Background Threshold Thread Lidl x tang P De Face Ere Faris af Leche ta Sal Pekar O fea rates a Z e
85. tton you can effectively apply the following operations delete split merge and create events To delete events from your image follow the steps below 1 Select the overlay image of the master channel simply click on it 2 Press Delete Events button 3 Select the events you want to delete To select multiple events to be deleted Holding Control key pressed click on the events by using the left mouse button Holding left mouse button pressed draw a region that includes the events only the events that are totally inside the region will be selected and deleted 4 Use Invert Selection button if you want to invert your selection 5 Press Apply button to delete selected events sample Neg UV irr Feld of View FOV 005 a 8 180 eis ay i i Delete Events Split Events Merge Events Create Events ae Invert selection Apply 2 4 oelect multiple events Select events one by one Figure 55 Detail Window delete events TissueQuest 3 0 User Manual doc Page 50 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual To split events follow the steps below 1 Select the overlay image of the master channel simply click on it 2 Press Split Events button 3 Holding the left mouse button pressed draw lines across the events you want to split 4 Press Apply button to split the events fee You cannot split events on images obtained as a result of backward data computation
86. tton in the scattergram toolbar x Rhodamine ge Po E fe 5100 E mj i 0 0 109 20 255 DAPI Mean Intensity Figure 79 Add cutoff step 1 e Move the mouse to the position where you want to have the cutoff During this operation the mouse cursor is captured inside the diagram area TissueQuest 3 0 User Manual doc Page 67 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Mean Intensity Rhodamine ge Figure 80 Add cutoff step 2 e Finally the cutoff is determined by clicking the left mouse button on the desired position in the diagram 255_ P Rhodamine y Mean Intensity ca 0 10 20 255 DAFI Mean F Figure 81 Add cutoff step 3 10 4 2 Modify cutoff Move the mouse cursor over the cutoff you want to modify As a result the cursor changes to a pointed arrow shape e It is possible to move both cutoffs horizontal and vertical only the vertical cutoff only the horizontal cutoff Rhodamine p Mean Intensity TissueQuest 3 0 User Manual doc Page 68 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Figure 82 Move cutoff step 1 e Press left mouse button on the cutoff Holding left mouse button pressed move the cutoff to the desired new position Pi Rhodamine y Mean Intensity na S Figure
87. u keds boc Beacing cured Theret Figure 36 TissueQuest Main window The main window of the application the window that is the focus of the user s actions consists of five parts as seen in the picture above the menu bar the tool bar on the left you can adjust the parameters for the detection algorithms displayed in the middle there are the representative fields of view displayed on the right there are the scattergrams TissueQuest 3 0 User Manual doc Page 39 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 7 2 Menu In this section you can get familiar with the TissueQuest menu bar in a short glimpse You can find explained all the menus graphically presented below in the corresponding sections of this manual please follow given references TissueQuest menu bar Help File View Project Samples Scattergrarmns Figure 37 TissueQuest Menu File See Chapter 5 Chapter 14 New Ctrl M Open Ctrl 0 Close Save Experiment Ctrl 5 Export Print Ctrl P Print Preview Exit Figure 38 File menu View Check any of the listed elements from this menu to have it displayed x Toolbar C4 Status Bar Detail Window Algorithm Parameters Figure 39 View menu Project See Chapter 6 Chapter 7 5 Chapter 12 TissueQuest 3 0 User Manual doc Page 40 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Clean C
88. ueQuest you must enter a username into the User Name field and type the proper password into the Password field Press Login to be authenticated If the username and password are correct the login dialog will close and you can start using the application Otherwise the application will prompt again for a valid username and password combination If you press Cancel the login dialog and the application will close The login dialog is used to restrict access to the application There is one Administrator and any number of users who may be managed only by Administrator when logged on for more details please see Chapter 18 TISSUE a GNOSTICS MEOOIiC AL Be BiaTECH SoOtuTIO H amp TISSUE QUEST GELL ANALYSIS S 0uF TT WARE ivo CE IN ATTENTION CONSULT OPERATING INSTRUCTIONS FOR USE Login User name Administrator Password Copyright 2005 2011 TissueGnostics GmbH All rights reserved Figure 1 Login dialog TissueQuest 3 0 User Manual doc Page 11 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual 4 Create new projects To help you create a new project TissueQuest software offers a five steps wizard that will guide you in a user friendly manner Now take a look at the main toolbar of the application in the picture below You will notice that only two buttons are enabled Create a new project and Open an existing project The rest of the buttons are d
89. ues the cutoff values set for a scattergram of a sample are automatically propagated to the same scattergram in all the samples belonging to the link of samples The same defined gates the gate inserted in a scattergram of a sample is automatically propagated to the same scattergram in all the samples belonging to the link of samples The same input gates TissueQuest 3 0 User Manual doc Page 56 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Sample linking can link only compatible samples A set of channels are compatible if e They have the same number of channels e Their channels have associated the same markers or markers belonging to the same marker family For example let s consider the following Sample contains two channels CH and CHp with markers DAPI and Rhodamine respectively Sample2 contains two channels CH and CHa with markers Rhodamine and DAPI respectively In this case the two samples are compatible because their channels have associated the same markers CH and CH4 DAPI CH and CH Rhodamine As you may notice the order of the channels is not important Access Samples Linking from the main Menu toolbar in order to use the Linking option File View Project Samples Scattergrams J Linking mO Select Samples ibis Gates Algorthm Admin Help Figure 63 Samples Linking option The Sample Linking dialog
90. uest 3 0 User Manual FOV Size Predefined Width M1 10x 0 5x C Mount Si M1 20x 0 5x C Mount Si Height M1 40x Oil 0 5x C Moun mm Update M1 63x Oil 0 5x C Moun Figure 127 Project Properties FOV Size section e By pressing the ia button the Templates for FOV Size dialog will appear In this dialog you can see details about the existing templates or create a new template e You can also modify a template by changing its name description and the Width and Height values of the FOV e To create a new template you must specify a name a description and the Width and Height values of the FOV in mm e You can also delete a template first select it in the list by clicking on it then press Remove button e After each operation don t forget pressing the Update button a If wrong dimensions are used the computed cell densities i e cells per mm will be J Note WRONG Other parameters as well as original measurement values are not affected For more details concerning Templates for FOV size also see Chapter 4 4 1 Manually adjust Width and Height values of the FOV To manually edit Width and Height values of the FOV the scaling or image dimension simply type the desired values in the corresponding fields FOV Size ee es law Width 0 1484800000 arr Height 0 1484800000 mm Update _ Figure 128 Project Properties manually edit Width and Height values After
91. where you can do the following operations Select sample from dropdown list Gate Management k B UV treated EER EE eee fon ian fa mi img f imi ie mi minimi miN More Colors Rhodamine e Mean Intensity Figure 106 Gate management dialog e Select desired sample from dropdown list by pressing the button TissueQuest 3 0 User Manual doc Page 79 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual WW Neg normal Neg normal WY Neg UY irr B normal skin B UV treated Figure 107 Gate management select sample By default every gate has a name for example Gate 1 Gate 2 You can change this name by double clicking on the actual name of the gate in the Caption field and typing the desired new name Color Show label Figure 108 Gate management rename gate You can set any color for the events inside the gate by double clicking on the Color field of the desired gate and choose a color from the color picker You can display or hide the name of the gate on the scattergram by checking unchecking Show label option for the desired gate Caption Rhodamine _______ gt s Mean Intensity pe E m T D ma W T 5 m pr Page 80 of 116 TissueQuest 3 0 User Manual doc Internal www tissuegnostics com TissueQuest 3 0 User Manual Figure 109 Gate management Show label e To delete a gate from the list select it by left click
92. will not start immediately at the border of the nucleus but outside at a distance specified by this parameter Autodetect Background Threshold specifies if automatic detection of the background level should be used by marking the checkbox or can be set manually by unselecting the checkbox and providing the desired values in the Minimum Level Maximum Level and Threshold Level fields Include Nuclei Label By selecting Nuclei mask the area of the binary objects in the nucleus mask is also used as measurement mask in all other channels TissueQuest 3 0 User Manual doc Internal Page 100 of 116 www tissuegnostics com TissueQuest 3 0 User Manual 15 3 DOT Finder 15 3 1 DOT Parameters To detect specific dot like features present in a sample e g FISH Fluorescent In Situ Hybridization TissueQuest application provides an algorithm for dot detection This algorithm will detect dots present in the virtual channel mask specified by the user The DOT Finder can be applied to nuclear and or cytoplasmatic masks FISH is a typical example for a nuclear application dots in the cytoplasm might come from certain markers like signaling molecules or markers associated with mitochondria or markers associated with endoplasmatic reticulum First go to Project Virtual Channels and in the dialog that appears check a virtual channel see Chapter 6 4 The name of the new virtual channel will look like this Channel Name DOT At this poin
93. www tissuegnostics com TissueQuest 3 0 User Manual TissueQuest 3 0 User Manual TissueQuest 3 0 User Manual doc Page 1 of 116 Internal www tissuegnostics com TissueQuest 3 0 User Manual Table of Contents T SUS SS I O obras acca A E E A E Fehler Textmarke nicht definiert ko odU CiO eaen E A E EE were 6 1 1 BUI ae acess peer E EE E E E E E 6 1 2 GOO e e aE mae A E E E E ee eer 6 1 3 Definitions Acronyms and ADbDreviatiONS cccccecceecceseceeeceeeeseeseeseeeseeeseeeseesseecseeseeeeseeesueesueesueeseeeaaes 6 1 4 C ONVONUON S asteren ri ee ee a e eee re oe eee eee 8 2 Metdling VS SUN SS aiena a e A A A A O EE T E E EEA 9 2 1 Application Dependencies ccccccsccceeccesecesecceeccecceececcceeecececaeessueseesaeessucssuesseesauesaeesseesseesceeecaeesenesans 9 2 2 Windows 3 1 Installer for Windows XP SP2 cccccecccceecceeeeeee cece eeceeeeseeeeseeeeeeeeseueeseueeseeeeaeeeseneeseeeeseeesaes 9 2 3 Microsoft Visual C 2005 Redistributables for Windows XP cccccccceeceseeeeeeeeseeeseueeseeeeaeeesseeesaeeeseees 9 2 4 Intel Performance Primitives 5 3 20 0 cccccccccsceccscecseeeceeeeceeecceeceueecseeseueecaeessueecsueessueesaeessaeessaeessueeseeeeseeessas 9 2 5 MARX Dongle Drivers oa sence sctestiecanee ac aaucnstesianesaccsentess nave neadaanendtiedaneasesedemnd anata saacaanceatinnsvebaceioseactaededoaeaecencneax 9 2 6 System ReguireMeEN S open enceceeect asant eden dadecscenave iai aiaa a

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