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TissueFAXS 3.0 - TissueGnostics

1. Hame Acquiring Step 15 24 Estimated remaining time 00 00 02 Figure 90 Estimation of acquisition duration 5 4 Standard acquisition vs Time Lapse acquisition Time lapse acquisition consists of multiple acquisitions for a region or a set of regions The multiple acquisitions will be performed after a certain time interval specified by the user Page 80 of 195 www tissuegnostics com TissueFAXS 3 0 Time lapse feature is ideal for observing cell cultures How to acquire A region can be acquired in two modes Standard normal or Time Lapse You can specify the acquisition type in the Acquisition Information dialog that is displayed before the actual acquisition process is started You are about to perform Acquisition Please read the following summary You are about to perform Acquisition Please read the following summary before proceeding before proceeding You have 1 region to be acquired with a total number of 2 FOVs al You have 1 region to be acquired with a total number of 2 FOVs Actions Actions Beep after finish _ Time lapse acquisition Beep after finish Time lapse acquisition Shut down after finish L Acquire correction image _ Shut down after finish _ Acquire correction image Time lapse acquisition Number of runs 5 Time lapse 0 00 00 15 Maximum estimated time per run 0 0 11 Format Days Hours Minutes Seconds Figure 91 Acquisition Information Standard left image and Time
2. Please type the name and the description for the channel profile PixeLINK PL A622C 6220116 20x Transmission _Demol Description Figure 155 Save Profile dialog e Load an existing intensity profile can be loaded for a camera You must select any profile you want from the existing list Page 108 of 195 www tissuegnostics com TissueFAXS 3 0 Description cana Figure 156 Load Profile dialog e Delete if an existing intensity profile is no longer needed it can be deleted by selecting it and then pressing the Delete button Description Figure 157 Delete Profile dialog Move by One FOV With these buttons you can move the live image exactly one field of view up down or left right only available after stage calibration Page 109 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 158 Move one FOV controls Double click to center Double clicking on any point in the live image will center the image at that point Adjust camera settings To adjust the camera settings click on the title of the settings page Advanced Basic Adwanced Exposure Time ms Exposure Time ms sbu385 of Alexi ood 56019985 9710229 Deeoy Td ANT Tl Display Information on Camera Display information on camera Seer ae _ Show RGB value El settings for PixeLINK Settings for PCO PixelFly Page 110 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 1
3. Quality the quality of the compression can be adjusted using the slider 7 If choosing as extension the tiff with multiple tiles the following options will not be note available anymore Mark Crop Area Scale bar Include Categories e want to stitch thumbnails choose this to export a low resolution of the region e want to specify a custom size for images to stitch choose the desired size of the resulting export image Be aware that for larger images there is a memory restriction that comes from the operating system and hence this operation may not work if a system is low of memory e Mark Crop Area it marks the contour of the region on the final image you can select its color e Scale bar please see Chapter 4 1 2 of the current manual e Include Categories in this case the selected categories will appear on the final image Here the user can choose the categories to be included in the region overview image by pressing the browse button le TT In the selection list only those categories will be listed that contain regions on their vf note overview image Page 164 of 195 www tissuegnostics com TissueFAXS 3 0 Select Categories Select All Figure 235 Export Region Overview Categories included e Opacity of the categories adjust the Opacity parameter in order to choose the transparency of the tissue area name that appears on the region e Apply Illumination Correction this option is enabled on
4. button is disabled because there is only one image for each Field Of View Zoom in and Zoom out buttons allow zooming the image Another option would be to type the desired zoom value in the zoom editor Keep in mind however that the Region Viewer is designed to give the user an overview of the entire region and therefore is limited in giving detailed magnified Images After a region is acquired the user can double press the left mouse button on the region to open the Acquired Image tab and to see the acquired image splitted into Fields of View The toolbar contains the following buttons e Export Fields of View Region Overview e Plugins e Zoom in e Zoom out e Map shows hides region map e Add subregions and tissue areas belonging to categories e Subregions Show Defined Regions All Categories Manage Categories e Crop it marks the images that have been acquired outside the defined region e Gridlines default value is Off e Scalebar default value is Off e Measure e Annotations e lllumination Correction e Reacquire Flag e Add Flag it adds Fields of View to the region e Remove Flag it removes Fields of View from the region e Time lapse acquisition button if it is the case To view a more detailed high resolution portion of the acquired image double click on it This will create a new window called the Field of View Viewer see next section 5 17 1 Subregions Subregions are normal reg
5. www tissuegnostics com a A x sy Y i Tissue Detec Slide Preview ini on S ee Ja ITISSUEGNOSTICS Run Selection Parameters codo CGAI Zabo 60000 a0006 POLOS acuda OTT 60609 Beqgoo Figure 247 Automatic detection of TMA structures panel Background Automatic Threshold TMA Block 01 AOL E TMA Block 01 A02 TMA Block 01 A03 TMA Block 01 A04 TMA Block 01 405 TMA Block 01 801 1 TMA Block 01 802 TMA Block 01 B03 TMA Block 01 804 TMA Block 01 B05 TMA Block 01 C01 TMA Block 01 c02 Ho TMA Block 01 003 TMA Block 01 C04 TMA Block 01 CO5 TMA Block 01 D01 TMA Block 01 D02 16 TMA Block 01 D03 TMA Block 01 DO4 VE TMA Block 01 005 TMA Block 01 E01 TMA Block 01 E02 TMA Block 01 E03 TMA Block 01 04 TMA Block 01 E05 TMA Block 02 A01 TMA Block 02 A02 TissueFAXS 3 0 E x In order to refine the results of the automatic detection of TMA structures TissueFAXS provides a set of parameters that can be adjusted according to your needs in order to obtain the desired results Background Use this to eliminate the high background and ununiform illumination Its value should be equal to the average size of the spot radius Median filter Use this to improve detection when images containing some artifacts have to be used for detection Basically the algorithm will ignore the pixels brighter darker than the medium value
6. About TissueFAXS splash screen Page 191 of 195 www tissuegnostics com 9 Modularization TissueFAXS application requires license for some of its features that you can find listed below gt Please note that the behavior of TissueFAXS viewer is not depended on any vf note dongle or any other licensing mechanism Licenses that have a date time as expiry date will be checked each time the feature is used Licenses that have a limited number of uses will be incremented as described in the table In the vast majority of cases the use counter will be incremented after an acquisition is done with the feature enabled When we are saying acquisition we are taking into account reacquisition also When we are saying region we are referring to one of the following based on context TMA Spot Normal Region and Time Region So if we acquire with stitch same region for 20 times the stitch counter will be increased by 20 Number of fluorescence channels Projects created with a higher number of channels will be opened but acquisition will not be possible If the user owns a predefined number of uses the counter will be incremented after each successful or canceled acquisition The counter will not be incremented if acquisition fails Checked only at experiment creation and acquisition Transmission in fluorescence experiments Project created with Transmission in the list will not be acquired If the user owns a predefined number of
7. Figure 166 Creating groups context menu Right clicking on items in the menu allows you to directly acquire or remove them from the experiment Acquire experiment item Clicking on this option allows you to directly acquire the selected experiment item Remove Clicking on this option allows you to directly remove the selected item from the experiment Remove Regions Clicking on this option allows you to directly remove all items from the selected slide group from the experiment Build Clear Cache for Slide Builds clears cache for the selected slide Remove Acquired Images This option will erase all the acquired images and associated files from the storage area Notice the fact that this option is available only for acquired regions Propagate region slide to slide This option copies selected regions from a slide to other slide slides that you can select in the dialog below Page 116 of 195 www tissuegnostics com TissueFAXS 3 0 JE Slides Selection Slide 1 Slide 2 Slide 4 Slide 5 _ Slide 6 Slide 7 All items Figure 167 Slide selection for propagation checkbox M You can select any slide to propagate regions except for the slides that appear Y TIP disabled in the Slide Selection dialog A slide appears disabled because It is the slide containing the original regions to be propagated afterwards you cannot propagate a slide to itself The content type is different from
8. Save as template menu For saving the template you must select a path for the file in the dialog that appears The template file is saved as aqtpl 5 1 3 Experiment Identification New Experiment Experiment Identification Experiment Name Demo Template Experiment Comment Experiment Type Brightfield Fluorescence Content Type 2 Generic O Tissue Microarray Experiment Storage Folder ait Figure 74 Experiment Identification In this page of the wizard the user will establish data that will define the characteristics of the experiment Page 70 of 195 www tissuegnostics com TissueFAXS 3 0 e Experiment Name mandatory the corresponding TissueFAXS experiment file will have this name and it will be stored in a folder having by default the same name as the experiment it can be modified later e Experiment Comment optional you can add a short description of the experiment e Experiment Type mandatory you can choose what type of experiment you will create Brightfield Experiment or Fluorescence Experiment If you have created a new experiment from a template existing experiment the experiment type will already be selected and cannot be modified e Content Type mandatory you can choose a Generic Experiment or a Tissue Microarray TMA Experiment e Experiment Storage Folder mandatory to change the experiment storage folder just click on the label and then browse for the desired fold
9. TissueFAXS 3 0 Page 87 of 195 www tissuegnostics com TissueFAXS 3 0 When the mouse is over a reflector an information dialog is automatically displayed It aa Y TIP contains brief information as abbreviation full name and description Reflectors Description 01 DAPI Figure 108 Reflector Info bubble 5 5 3 TL Shutter for transmitted light TL Shutter is used to block or unblock the light coming from the halogen or white light LED lamp TL Shutter The TL Shutter is closed Figure 109 TL Shutter control a TL Shutter The TL Shutter is opened Figure 110 TL Shutter control b Click on the shutter icon to open the TL shutter Page 88 of 195 www tissuegnostics com TissueFAXS 3 0 5 9 4 TL Lamp Use the light switch icon to turn on off the TL Lamp Right to the switch the current state of the TL Lamp is i El On i displayed On Off Check the 3200K check box to turn on the color temperature of the TL Lamp directly to 3200K Gh Note The color temperature can only be set for halogen lamps This checkbox has no relevance for white light LED Figure 111 TL Halogen Lamp control Adjust the light intensity by moving the slider response of the lamp For instance when the lamp is turned on it takes some time fr There is a small delay between changing a value on the control and the actual ae ote until it reaches the luminosity corresponding to the control value 5 5 5 RL S
10. separate disposal of electrical and electronic equipment C CE mark Date of Manufacture eel Manufacturer Notice regarding anti virus applications e Allthe 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 Page 6 of 195 www tissuegnostics com TissueFAXS 3 0 1 Introduction 1 1 Purpose The purpose of this document is to help the user understand how TissueFAXS works It describes TissueFAXS software capabilities and also guides the user to the steps required to acquire high quality images The document covers the installation of the application system requirements and main features In the next section you will read more about the goals of the application and its place in a larger system You will also understand the purpose and the advantages of this application 1 2 Goals The user should use this manual as his primary source of instruction when using the application The functionality of all panels menus and tools are exp
11. Acquire Slide this item starts the acquisition process lts target is the selected slide only Acquire Region starts the acquisition process for the selected region only Label Up Label Down The Label Up and Label Down entries allow you to specify the position of the slide on the stage Usually all the slides are inserted in the same way but if this is not the case the label position can be customized for individual slides on the stage Show position on slide us You have the option of viewing the objective s position on the slide in relation to your preview by selecting Show position on slide in the menu An orange box appears indicating the size of one field of view along with cross interrupted lines that mark the center The FOV size is determined by the objective in use If the FOV is not calibrated for the selected objective the box will have the default size TissueFAXS 3 0 Page 130 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 188 Show position on slide x e Show preview info By pressing this button you will be able to see the location of the preview area common for all slides on the slide and the position of the focus point for each individual slide for preview operation You can also access this option from the contextual menu that appears when right clicking in the slide editor e Setas preview focus point Use this option to set the focus point to the desired position by right clic
12. because they were taken before the stage has completely stopped after rapid movement motion blur Default values are 1 or 2 higher values may be suitable if you are working with cell culture dishes Camera adapter magnification this is an adapter used to connect the camera to the microscope having its own magnification factor written on it if this information is missing or 0491 5 x is wrong in TissueFAXS the total magnification of the experiment will be wrong Press Save amp Exit to save the settings and close the Options dialog TissueFAXS 3 0 If your camera is already used in TissueFAXS it is possible not to be listed even if you press Reload Page 94 of 195 www tissuegnostics com TissueFAXS 3 0 5 7 2 Change Default Camera You can change the camera you are using any time you need To perform this operation follow the steps described below e From the Tools menu select Options to open the Options dialog box e Inthe Options dialog choose Hardware Settings Camera Manager e Select the desired camera from the combo box If the camera is not listed there please see the steps described in Chapter 5 7 1 e Press Save amp Exit to save the settings and close the Options dialog 5 7 3 Operations with Current Camera The camera you are currently using allows you the following actions e Enable or disable the live image for the current camera depending on what your are working at you may want t
13. low brightness the exposure time range is 0 01 65 ms In Video Mode high brightness the exposure time range is 1 10000 ms Increase this parameter when the illumination condition are poor or when the markers on the samples are dim in fluorescence experiments Page 95 of 195 www tissuegnostics com TissueFAXS 3 0 E Due to the large value 10000 allowed for exposure time this control starts with the rote maximum value of 500 in order to increase usability If you require higher exposure time do a right click on this control and access the Maximum menu Exposure Time ms Step Maximum Figure 126 Exposure Time control Old value 500 Figure 97 Changing the maximum exposure time allowed combo box Gain Controls the intensity of the image by artificially increasing the values of pixels by using software multiplication Use this when you don t want to increase the exposure time in order to increase the acquisition speed Figure 127 Gain control Lower Upper These parameters specify the interval which will be scaled to 256 gray levels for display Lower Sensitivity Threshold by increasing this the low intensities like background in the image will be lost Upper Sensitivity Threshold by decreasing this value the shades in the image will become brighter Z lf you want to manually adjust these parameters you should set them in such a way rote that the lower parameter limit equa
14. 0 5 TissueFAXS PEA TissueFAXS software will not run and operate properly if any of the components is not vf Note installed on your TissueFAXS 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 NET Framework 2 0 Runtime The TissueFAXS product is entirely built over the version 2 0 of NET Framework which can be also found at the Microsoft official website The Microsoft NET Framework version 2 0 x86 redistributable package installs the NET Framework runtime and associated files required to run applications developed to target the NET Framework v2 0 TissueFAXS is such an application For best results at least NET Framework SP2 must be installed For further information about this package please visit the Microsoft homepage 2 3 MIB RDK 1 6 0 5 You find this kit in the Installation Kit Folder IKF of TissueFAXS TissueFAXS requires this installation to communicate with the microscope Below are some features of the Micro Toolbox MTB e Gives an identical interface for Carl Zeiss microscopes under different operating systems Has high level functions for controlling the components e Gives information about the equipment e g which objectives and reflectors are mounted e May simulate devices programming and demos
15. 2 3 Definition of the second TMA block on slide The second TMA block on slide is defined by indicating the center of the TMA spot in the most upper left corner of the TMA block e hold the SHIFT key e click the left mouse button in the center of the upper left corner of the TMA block e release the SHIFT key The second and subsequent TMA blocks will have the same size as the last TMA block manually added using the first method Page 178 of 195 www tissuegnostics com TissueFAXS 3 0 Preview Slide Conc z cal TEH kA mt Figure 253 Manual definition of the second TMA block on the slide 6 2 4 Selection of TMA blocks and spots The TMA blocks and spots can be selected as below e select an individual TMA spot by clicking on it with the left mouse button e select an individual TMA block by clicking in the free area between the TMA spots with the left mouse button e select portions of the TMA block by drawing a rectangle that surrounds the desired TMA spots and blocks e select multiple TMA spots by holding CTRL key e select the entire content of the slide by pressing the CTRL A keys Page 179 of 195 www tissuegnostics com TissueFAXS 3 0 A ut a EE u Figure 254 Multiple selections with Figure 255 Multiple selections with Figure 256 Select all rectangle CTRL 6 2 5 Resize the TMA spots The TMA spots can be resized as follows e select the desired TMA blocks or spots e use CTRL key
16. Finish Experiment Ready After you have defined your experiment settings your experiment is ready to be created The Finish Experiment Ready window allows you to review a summary of your settings before proceeding TissueFAXS 3 0 Changes can be made by simply clicking the lt lt Back button and returning at the previous pages in order to modify the settings Page 73 of 195 www tissuegnostics com TissueFAXS 3 0 F New Experiment E A k si Finish Experiment Ready Here is the summary of your settings Experiment Name Demo FL Experiment Type Fluorescence Content Type Generic Storage Directory C TissueFAXxS Projects Preview Objective 2 5x Air Acquisition Objective 20x Air Acquisition Reflectors DAPI FITC Cy5 Acquisition Camera PCO PixelFly 0 Figure 78 Last page from wizard when creating a fluorescence experiment 5 2 Preview Settings Preview Settings can be accessed by clicking on the Preview tab in the upper left corner of the main TissueFAXS window If the Preview tab is not visible you can make it visible by pressing the Settings button from the application s toolbar Preview Objective 10x Air se Acquisition Figure 79 Preview tab This feature of TissueFAXS covers the settings for the preview operation such as the objective which must be a low magnification objective channels for instance the reflectors in use and individual settings for
17. In case of Bigh tied experiments Filter Channel name wil not be used Directories Store each slide in a separate directory Store each region in a separate directory Default Directory F Projects Raw Data L Save raw data Figure 22 Storage Settings dialog TissueFAXS 3 0 e Name Parts select the parts that will generate the filename Experiment Region Field Of View Filter e Parts Separator select the separator between the parts of the filename e Image Format select the image file format e g jog for small file size and good quality bmp for large file size and high quality e Store each slide in a separate directory select how to organize image files in directories e Store each region in a separate directory select the default directory in which the projects will be stored e Raw Data check Save raw data if you want to save the raw data image corresponding to your file jf Note 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 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 nee
18. Name Reflector Profiles f Focus Channel cquistion gt DAPI DAPI i Experiment Profile m Rhodamine Rhodamine FITC i Transmission Transmission Add Remove Channels Figure 84 Acquisition Settings panel Focus Channel The Focus Channel is the channel for which TissueFAXS will perform the auto focus in fluorescence experiments The focus for Focus Channel will be memorized by the application and then used in acquiring the other channels The default Focus Channel in TissueFAXS fluorescence experiments is DAPI If DAPI is absent from the experiment TissueFAXS will automatically set as Focus Channel the first channel listed in the Acquisition Channel section Channels e For Brightfield Experiment Only Transmission is available e For Fluorescence Experiment choose the desired reflector including transmission Lote After choosing the reflectors you must readjust the camera settings for each reflector y The name of the channel is editable Objective Lens Select the desired objective from Acquisition Acquisition Objective 7 After choosing the acquisition objective you must readjust the camera settings for rote each combination of acquisition objective and acquisition channel Camera Settings To edit camera settings press View button For more information please see Chapter 5 7 of the current manual You may also want to use settings from intensity profiles if you have previously created an
19. Row Insert Column Acquire New FOVs Remove Subregion Figure 218 Reacquire Flags contextual menu Add Flags By adding flags the size of the region will be enlarged in order to include new tissue area that could be relevant for analysis As previously mentioned FOV Matrix Size property of the region represents the number of FOVs per row and column Based on the shape of the region a part of these FOVs are not acquired Adding flags without changing the FOV matrix size property e Check the Add flag button E then click on an empty position outside the region shape you might be interested in next to already existing FOVs but inside the crop area To remove the added flag click again on the same empty position After finishing this operation uncheck the Add Flag button Page 151 of 195 TissueFAXS 3 0 m Fl AC sE wy Apply Unde ECRI ee O A E a AAA As E EA PE CYP DP Y A A ES a y oe fot e fa P de Sur A TAPIA RO TITR TACNA WEY NAT i i tee A Figure 219 Adding flags without changing the FOV matrix size a E AN r J i F ik Apply Undo GIIA NAS Y OLY z 7 a4 oe Fs ATA Wi TA T Paa FEA F Page 152 of 195 www tissuegnostics com Figure 220 Adding flags without changing the FOV matrix size b Adding flags by changing the FOV matrix size property If you
20. TMA block is acquired or not Two possible values are present Yes or No Objective the objective lens used for acquisition of the current region Rows the number of rows for the current region Columns the number of columns for the current region e TMA Spots Count the number of spot items Page 190 of 195 www tissuegnostics com TissueFAXS 3 0 8 Help Menu Figure 265 Help menu Help press this button or F1 key in order to open the TissueFAXS User Manual About this option will display a splash screen containing the main information about the TissueFAXS version in use and also the Disclaimer section scroll to read TISSUE Da GNOSTICS MEDICAL amp BIOTEC H 5a LU TiO N amp MSSUE if FAxS CELL ANALYSIS SYSTEM CE DN ATTENTION CONSULT OPERATING INSTRUCTIONS FOR USE TissvefAXsalus amp a migosaope based cel analesis system jor cele in ovocuts parari sectons and TMAS It const of the softaere modules TissuelAXxS TrssueQuest 2 HistoQuest and is used for acguisibon of images in the Auorescence and brightietd mode for wuning the number of positive and negative cells and for quantficabon of staining intensities TissuefAXSolus used for the standardization of tissue analysis in combination wii immuneh ipaenicl and immunofiverescence staining TissueFAXS Copyright 2006 2011 TissueGnostics GmbH All rights reserved www Essuegnostics com Figure 266
21. This option yields a submenu which contains four types of drawing tools Page 123 of 195 www tissuegnostics com TissueFAXS 3 0 Custom Remove cquired Images Circular 7 Remove Selected Items LI Rectangular O Go To This Slide Custom with Joystick Go To This Point Acquire Slide 1 Acquire Selected Items Build Cache for Region 006 Clear Cache for Region 006 Select All Preview Label Up Label Down Show Position on Slide Export Slides Images Copy Paste Compute Stitch Figure 175 Add region context menu There are four different methods for drawing your regions of interest Custom This method allows the user to create a custom shaped region with a rubber line tool The tips of the line are made static by left clicking on the image Simply right click anywhere to close your region a a ors eT e rit a e k E Fi l i AE i a no a 1 a ae A i a i Ep gt i A j pie i we ARA Figure 176 Creating custom region Rectangular This method allows the user to simply drag a box over the image to create a rectangular region A start point and an end point are chosen by left clicking on the image Page 124 of 195 www tissuegnostics com TissueFAXS 3 0 O 7311 FOYS ma s a u 7 Ts y Y i e ote T ine EN Figure 177 Creating rectangular region e Circular This method requires two left clicks to create a circular region The first left click determines the center while th
22. TissueFAXS 3 0 4 Options The Options panel is very useful in managing the main utilities of the application Here by accessing the listed items you can visualize the available settings and also modify them according to your needs Acquistion These actions will be started on the finish of the acquisition Licensing Beep after acquisition Measurements L Shut down after acquisition pS _ Acquire Correction Image Support Move to a safe distance When swinging in or out the checked objectives the stage will be moved to a safe distance Preview IN Reflectors 10x Air Storage scan Strategy Stitching Focus Calibration Advanced Focus Please select the objective to be used during calibration Speed 3 none Cache Camera Manager L Use default cache action for regions without cache Contrast Manager Dan Default Cache Action Build Cache Stage Manager Load Settings L Build cache during acquisition Fabichna s enabled he cache wil be Suit after acquisition and sito if stitching is set to Manual no cache is Suit Preview Figure 6 Options Panel 4 1 Application Options 4 1 1 Actions This section contains several important actions concerning the interaction between TissueFAXS and the other components of your system the computer and the microscope Page 16 of 195 www tissuegnostics com TissueFAXS 3 0 Acquistion These actions will be started on the finish of the acquisition Licensing Beep after
23. a smaller area will not be taken into account e Tissue Closing Radius try to increase this parameter if your region is split into multiple small subregions The result will be a single region containing smaller subregions e Exclude border connected regions the regions touching the border will be ignored e Regions number of regions expected to be detected e Default this dropdown button has two options Save 45 Tissue Detection Default Parameters Restore Tissue Detection Default Parameters Figure 193 Tissue Detection Default Parameters Save as Tissue Detection Default Parameters it allows you to store this set of parameters values for future detection Restore Tissue Detection Default Parameters it restores all the parameters to the last saved default parameters Advanced Settings e Thresholds Automatic if this is checked the RGB red green blue channels values are automatically determined within three intervals given by the user These intervals are established by increasing the lower default value is 5 and upper default value is 255 values If Automatic option is unchecked the tissue detection algorithm will use three values given by the user for Red Green and Blue channels Apply same value for all channels by checking this option all channels will have identical values or intervals you must set the value or interval for the red parameter this value or interval will appear automatically for
24. a fi i i Fa a Pe Pe do A PARA E i ad a ae et oF ot A a pa dE y A f FAR ea ER NA oes A a Figure 224 Remove flags b e You can also click Flag to remove entire row column from the contextual menu that appears by right clicking on the border row column To reverse this operation choose Unflag entire row from the contextual menu e Toremove all added flags click Clear Remove Flags A from the Region Viewer toolbar or choose Clear All Remove Flags from the contextual menu Apply Apply Pressing the Apply button will effectively modify the region shape by adding removing FOVs will remove images files too Undo Pressing the Undo button A will discard all changes insert rows columns Add Remove Flags Acquire New FOVs This option from the contextual menu will acquire all newly added FOVs The Flags feature is not compatible with the Time Lapse acquisition and therefore it is J Note available only for Standard acquisition mode Page 156 of 195 www tissuegnostics com TissueFAXS 3 0 To view a detailed high resolution section of an acquired image double click on it within the Region Viewer window This will create a new window called the Field of View Viewer FOV Viewer Slide 11 Region 002 3 3 FOV 00153 ios a El Overlay Export J O pa Figure 225 FOV Viewer Fluorescence Experiment The features in the FOV Viewer ar
25. acquisition C Shut down after acquisition Measurements LJ Acquire Correction Image support Move to a safe distance Default Experiment Settings When swinging in or out the checked objectives the stage will be moved to a safe distance 10x Air Scan Settings Hardware Settings Experiment Options Calibration Please select the objective to be used during calibration 3 none Cache _ Use default cache action for regions without cache Default Cache Action Build Cache _ Build cache during acquisition Jf stitching is enabled the cache wil be buit after acquisition and sto if siding is set fo Manual no cache is built Figure 7 Actions dialog Acquisition After finishing the acquisition three actions can be performed e Beep after acquisition TissueFAXS will announce the finish of the acquisition process by repeated beep sounds e Shut down after acquisition the computer will automatically shut down after acquisition e Acquire Correction Image before the acquisition process a correction image will be acquired Safe distance Safe distance means the Z position to which the stage is lowered in order to ensure that no objective lens collides with the slides or any part of the stage Safe distance is used in the following situations e calibration of the stage e when moving between slides By default for any objective with magnification higher than 20x TissueFAXS will lower Q TIP
26. and columns To view acquired regions double click on the region from the experiment editor on the left side of the main window The region viewer panel which was empty before will be updated with the regions image The Region Viewer is shown in the next screenshot Page 137 of 195 www tissuegnostics com TissueFAXS 3 0 Microscope Camera Acquired Images Export Plugins AL 4 E Subregions baa 3 Z EJ AA L Sten ar e q ir ki dl at A ae Y i e qe PS A o Slide 4 Region 003 i E A i im _ a ss AAA AAA MM ae A os Figure 199 Acquired Images viewer Fluorescence experiment The slide name and region name are displayed at the bottom left corner of the region viewer Region Overlay In fluorescence experiments the Region Overlay window as shown below Tiea une eny DE EEE 0 0 255 Rhodamine 100 MN 255 0 0 button is enabled Clicking on it yields a new Cancel Figure 200 Adjusting channel intensity and color This window allows you to choose which channels to view in your acquired image Here you can adjust the color and light intensity for each channel If more than one channel is selected clicking Apply will yield an overlay image which is composed of the selected channels according to the set algorithms Page 138 of 195 www tissuegnostics com In brightfield experiments the Region Overlay
27. at TISSUEGNDS Figure 99 Example of Status Bar 5 5 Microscope In this section of the manual you will find a description of those components of the microscope that can be controlled by using TissueFAXS and you will learn how to use them Page 84 of 195 www tissuegnostics com 5 5 1 Objectives 99999 l E E g Dx 25x 10 20x 40x Figure 100 Objective Turret control a Objectives 09 ee Ox 25x 10 Figure 101 Objective Turret control b 5 5 2 Reflectors Figure 102 Reflector Turret control a Reflectors Figure 103 Reflector Turret control b Edit Reflectors TissueFAXS 3 0 The Objectives panel displays the objectives available in the microscope s Objective Turret SJ Note The first listed missing objective is displayed as 0x The rest of the missing objectives are not displayed Select an objective from this panel by clicking on it This will switch the current objective to the selected one SJ note The objective in use is always shown in red The Reflectors panel displays all the reflectors available in the microscope s Reflector Turret ff Note The first listed empty position in the reflector turret is displayed as Transmission The rest of the missing reflectors are not displayed Select a reflector from this panel by clicking on it This will switch the current reflector to the selected one jf Note The current refl
28. click on the first last row you can select Insert row before after If you click on the first last column you can select Insert column before after Check the Add flag button vi then click on an empty position outside the region shape you might be interested in next to already existing FOVs To remove the added flag click again on the same empty position After finishing this operation uncheck the Add Flag button You can also click Flag to add entire row column from the contextual menu that appears by right clicking on the newly inserted row column To reverse this operation choose Unflag entire row from the contextual menu AMEN a E L Y Apy undo Fa Figure 221 Adding flags by changing the FOV matrix size a TissueFAXS 3 0 Page 153 of 195 www tissuegnostics com TissueFAXS 3 0 AZ UT OR JZ ane HAS Figure 222 Adding flags by changing the FOV matrix size b e To remove all added flags click Clear Add Flags A from the Region Viewer toolbar or choose Clear All Add Flags from the contextual menu Remove Flags e Check the Remove flag button M then click on a border FOV you don t need To uncheck the added flag click again on the same FOV After finishing this operation uncheck the Remove Flag button Page 154 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 223 Remove flags a Page 155 of 195 www tissuegnostics com TissueFAXS 3 0 EPR J a F
29. control the defined slide inserts or to change the active one you must choose Tools Options Stage Manager Using stage configurations Drop down box Herault vl is used to select the desired insert configuration The area below contains information about the current configuration Example 1 The default configuration of the stage Page 42 of 195 www tissuegnostics com TissueFAXS 3 0 Data Configuration parameters Template used Identification Id Mame Default Misc Farams Collection Figure 34 Stage Manager Default Configuration section Example 2 Information for a well plate Current Stage Select configuration For stage Data Configuration parameters Alpha O Stage X OriginOiFrset 100 Stage Y OriginCiFfset 100 Template used 4x6 Identification Id 4x6 Name 4x6 Misc Params Collection Figure 35 Stage Manager Well plate section After you have selected the desired configuration you must click on the Active checkbox near the dropdown list Once you re done you must press Save and Exit Any existing experiment will be saved and closed The selected configuration will apply only to newly created experiments Creating new configurations In the drop down list with the available configuration click on the entry New Current Stage Select configuration For stage Figure 36 Creating new configurations combo box At this point the Create template dialog will
30. dialog to be displayed when you reset the preview focus points otherwise check this option e Do not show slide renaming dialog again this option checked by default will prevent the slide renaming dialog to appear each time you create a new experiment e Time Lapse Acquisition check this option in order to set by default multiple acquisitions for a region or a group of regions You must set desired values for the following parameters Number of runs how many times the acquisition will run Time lapse the interval between runs 4 1 5 Support When using TissueFAXS as in any other software application you might occasionally encounter different kinds of errors or you might simply need support and answers to your TissueFAXS related questions The Support section was designed to help you in these situations Page 24 of 195 www tissuegnostics com TissueFAXS 3 0 1 Options Application Options Actions Licensing From Address Measurements Remember Submit Options Support Address Dit Deen se as auto eportittissuegnostics com Scan Settings SMTP Server mail tissuegnostics 0 com 587 Hardware Settings Experiment Options LI Auto Save Report Test Connection Generate Exception Open Reports Folder Send Saved Reports Figure 16 Support dialog Enter the following information for dynamic error reporting The real name of the user who bought the software e g institution name The email addre
31. dl Version 6 1 7600 16385 win _rtm 090713 1255 C Windows SYSTEM321M5COREE DLL Version 2 0 50727 4927 NetFXspW7 050727 4900 C Windows lsystem32WKERNEL32 dll Version 6 1 7600 16385 win7_rtm 090713 1255 C Windows system32 KERNELBASE dll Version 6 1 7500 16385 win _rtm 090713 1255 C Windows lsystem32Wmsvwcrt dll Version 7 0 7600 16385 win7_rtm 090713 1255 C Windows SYSTEM32Wsechost dll Version 6 1 7600 16385 win7_rtm 090713 1255 C Windows system32 RPCRT4 dll Version 6 1 7600 16385 win7_rtm 090713 1255 CiWindowslsystem3215HLWAPI dll Version 6 1 7600 16385 win7_rtm 090713 1255 C Windows system32 GDI32 dll Version 6 1 7600 16385 win7_rtm 090713 1255 C Windows lsystem32YUSER32 dll Version 6 1 7600 16385 win7_rtm 090713 1255 Read Product Improvement Program Terms TissueFAXS 3 0 Figure 18 TissueFAXS Error dialog Error description here you can see a short description of the error Error detail here you can find detailed data related to the error Open Reports Folder if a report is not sent it will be automatically stored in a local folder press Open Reports Folder to open that folder Send Saved Reports press this button to send all unsent reports if any After effectively sending saved reports they will be automatically deleted from the local folder where they were stored Product Improvement Program Product Improvement Program che
32. is not recommended Be also aware that regions acquired with overlap can t be properly handled by HistoQuest Use internal stitch TissueFAXS will determine based on the settings from Stitch options see below how to stitch the FOVs in order to obtain region overview without any discontinuities HistoQuest will be able to properly handle this kind of regions Overlap settings the external and internal stitching will properly work if the regions are acquired with an overlap that will allow compensating hardware errors It is recommended using a value of at least 15 Avoid acquiring regions with large empty areas when using stitching Width Height Overlap are used to specify the percentage of width height that will overlap neighboring FOVs Stitch options Page 33 of 195 www tissuegnostics com TissueFAXS 3 0 Stitch options Compute stitch mode DuringFocus Min allowed stitch score After Acquisition A Figure 25 Stitch options Compute stitch mode e Manual speeds up the acquisition process However the user will have to manually call the Compute Stitch menu e During Focus slows down the acquisition process the user will be able to see the acquisition results in real time e After Acquisition this option will call the stitch after each region has been acquired ra ie If Compute Stitch mode is activated the cache will be erased The stitch during focus is recommended only for small regions up
33. it will revert to brightfield 4 4 3 Stage Manager This option is useful only for inverted microscopes or for any other kind of system that can support different types of stage inserts slides well plates flasks You can choose here the layout of the stage later referred as stage configuration for the next experiment Page 41 of 195 www tissuegnostics com TissueFAXS 3 0 Application Options Current Stage Default Experiment Settings Select configuration for stage Default Scan Settings Camera Manager Contrast Manager r Identification Load Settings Name Default Misc periment Options w tiem Expe p ay Collection Figure 33 Stage Manager dialog TissueFAXS supports switching at runtime between different stage configurations At this point two base configurations are supported e Slide inserts a slide insert is usually defined by the maximum number of slides it can hold by slide dimensions and distance between two consecutive slides This type of configuration can be used on an inverted microscope for culture flasks as well e Well plate inserts a well plate insert is usually defined by the number of wells per row and column distance between two consecutive wells and the distance from the edge of the plate till the first well inserts al Ar x ie TissueFAXS is configured by default with a slide insert configuration for 4 or 8 slides r ote In order to
34. of the items from the Property Editor are not editable they appear in grey The properties of an experiment can be displayed by going to File Properties In the dialog that appears only the Comments concerning the experiment can be edited JE Experiment Properties Dema_BF Type BrightField Experiment Comment Storage Directory Ci TissuePAxs ProjectsiDemo_BF Camera Figure 169 Experiment Properties dialog 5 15 Slide Manager The Slide Manager is a feature that allows a quick and simple access to the main slide settings It can be accessed from File Slide Manager Page 119 of 195 www tissuegnostics com TissueFAXS 3 0 New Open Save Save as template Acquire Print E 7 a o Print Preview Figure 170 File Menu Slide Manager option e Experiment Editor right click on the name of the experiment and choose Slide Manager option from the contextual menu Export Regions Overviews Export FOVs Export Slides Images Export Annotations Plugins B Slide 7 Slide 8 Figure 171 Experiment Editor Slide Manager option In both cases the Slide Manager dialog will open Page 120 of 195 www tissuegnostics com TissueFAXS 3 0 3 Slide 3 4 Slide 4 5 Slide 5 6 Slide 6 7 Slide 7 8 Slide 8 K e E 5 El El Figure 172 Slide Manager dialog Here you can perform the following actions e Change the name of the
35. of the other components as well Once installed the other required components work independently of TissueFAXS and keep working unless you have uninstalled each one by one TissueFAXS is ready to use when all components are installed and there are no error messages Page 11 of 195 www tissuegnostics com Firstly turn on the hardware turn on the microscope and the UV lamp if required Secondly turn on the computer and run TissueFAXS 2 System Requirements 2 1 Hardware Requirements Minimum e Intel Pentium 3 Processor 1 GHz e 1024 MB RAM e USB Port for dongle e 100 GB available hard disk space e Single monitor system with a resolution of 1024x768 Recommended Intel Core2Duo Quad Processor 3 GHz or more 4 GB RAM Internet connection for update USB Port for dongle 1000 GB hard disk space for storing your projects Dual monitor system with a resolution of 1280x1024 or higher 2 7 2 Software Requirements Windows 2000 Windows XP Windows Vista x32 Windows Vista x64 only for brightfield experiments not compatible with PCO camera Windows 7x32 recommended Windows 7x64 only for brightfield experiments not compatible with PCO camera 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
36. of virtual slide data and analyzed results TissueFAXS enables the user to link all experimental image files with their corresponding data files into one convenient experiment folder TissueFAXS supports upright and inverted microscopes from various manufacturers including Leica Nikon and Zeiss This manual was designed for the use with Zeiss microscopes but it is transferable by a large extent to other supported microscopes 1 3 Definitions Acronyms and Abbreviations This subsection provides the definitions of all terms acronyms and abbreviations required to properly interpret this Document 1 3 1 Definitions Microscope Tool Box The MTB from Carl Zeiss a software component which gives an identical interface for all Zeiss microscopes under different operating systems Installation Kit Folder The IKF is a folder where you find a set of executable files These files are kits for installation of certain components When you install TissueFAXS you go to this folder and you find a list of executable files which you must execute one by one See Installation Overview for further information 1 3 3 Abbreviations 1 4 Conventions In this section you can find listed and explained the conventions used throughout this manual of Note Note s describes important features or instructions Y TIP Tip s highlights features or hints that can help you save time or avoid difficulties I CAUTION Caution s warns reade
37. open It contains two options Page 43 of 195 www tissuegnostics com 1 Create template based on an existing template Create template Create template based on GO Mew well plate template Figure 37 Create well plate template dialog A new window will appear Note that information about the selected template is displayed below in the drop down box our example shows a well plate insert Select template Specify template to create the configuration From A Select template Available templates Additional Parameters Details Identification Mame Well Data Distance on Distance on Y First ell offset First well Y offset Number of columns Mumber of rows Well radius 17350 75 Tu E Cama Figure 38 New stage configuration Select template dialog At this point you must choose one template from the available templates and click Next You must now specify the distance between the 0 0 point of the stage and the leftmost edge of the well plate respectively topmost edge point of the well plate actually the Stage X Origin Offset and Stage Y Origin Offset parameters that you can see in the image below The Alpha parameter represents the skew angle of the well plate insert related to the stage usually this parameter is 0 TissueFAXS 3 0 Page 44 of 195 www tissuegnostics com TissueFAXS 3 0 Performing this configuration for the well plates will make sure that the default
38. properties for each channel in the list Checked this flag indicates if the current channel is used for overlay Name the channel name Intensity the channel intensity Color the channel color e Region list for each generic slide Region Name Region Image Comments the comments referring to current region Acquired this flag indicates if current region is acquired or not Two possible values are present Yes or No Path the path for current region files Objective the objective used for acquire current region Rows the number of rows for region Columns the number of columns for region FOV s Count the number of FOVs items Patient Name the patient name Patient Reference number the individual reference number Time Lapse if acquired with time lapse Number of Runs if acquired with time lapse Time between Runs if acquired with time lapse e Regions Channels a table that contains the channels list for current region and some properties for each channel in the list Checked this flag indicates if the current channel is used for overlay Name the channel name Intensity the channel intensity Color the channel color e TMA Blocks list for each TMA slide TMA Block Name TMA Block Image Comments the comments referring to current TMA block Page 189 of 195 www tissuegnostics com TissueFAXS 3 0 Acquired this flag indicates if current
39. slide type the new name into the New Name field of the desired slide e Include the slide in acquisition by checking the corresponding checkbox in the Included in acquisition column e The Content type column informs you about the type of each slide e You can also add a comment to each slide by typing it into the Comment field of the desired slide 9 16 Slide Editor The slide editor is one of the main features of the TissueFAXS application It displays the image of the whole slide and also a panel with all the applicable functionalities Page 121 of 195 www tissuegnostics com TissueFAXS 3 0 re PELE TEE LE LE ELE LEE TE e te to i oll i A i m ft di gt 0 spr j1 E TISSUEGNOSTICS Figure 173 Slide Editor in the main window A slide preview must be acquired to use the slide editor You can perform a set of operations on a slide by right clicking on it and generating a pop up menu as shown below Page 122 of 195 www tissuegnostics com TissueFAXS 3 0 Add Region Remove Selected Items Remove Acquired Images Go To This Slide Go To This Point Acquire Slide 1 Acquire Selected Items Build Cache for Region 006 Clear Cache for Region 006 Select All Preview Label Up Label Down Show Position on Slide Export Slides Images Copy Paste Compute Stitch Figure 174 Slide editor context menu 5 16 1 Add Region In this section you will learn how to draw regions on your slide
40. the other two channels Red the amount of red in the picture Green the amount of green in the picture Blue the amount of blue in the picture A Depending on the color used in the staining different adjustments of the Red Green vf note and Blue values might become necessary Page 134 of 195 www tissuegnostics com TissueFAXS 3 0 Tissue Advanced Settings Thresholds Point Generation Automatic Apply same value for all channels Green 15 Figure 194 Advanced Settings Thresholds tab Automatic checked QGqG_Q_ Tissue Advanced Settings Thresholds Point Generation Automatic Apply same value For all channels Figure 195 Advanced Settings Thresholds tab Automatic unchecked e Point Generation Distance the initially detected region will be inflated with the specified number of pixels in order to entirely include the edges of the tissue and a small surrounding area Max Step this parameter represents the maximum distance between two generated points The smaller the distance is the more accurately the contour of the region will be represented Page 135 of 195 www tissuegnostics com TissueFAXS 3 0 Advanced Settings Tissue Point Generation Thresholds Distance gg 0 50 Max Step pr Figure 196 Advanced Setting Point Generation tab For more accurate tissue detection the following contr
41. 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 Observations 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 xml8dang en 3 The names of actual companies and products mentioned herein may be the trademarks of their respective owners 4 In order to use TissueFAXS application it is essential for the users to have sufficient knowledge of PC and Microsoft Windows operating system usa
42. the type of the slide containing the original regions to be propagated The dialog contains another option All items that will select all available slides The propagated regions will not have corresponding thumbnail images Af Note Export To find out more about this option please see Chapter 5 20 of the current manual E For certain operations acquisition propagation export you have the possibility to use vf note the multiple selection Plugins see Chapter 5 21 Compute Stitch see Chapter 4 3 2 Property Editor The properties section is located below the tree list More precisely it is a property grid that shows all the properties available for a specific project item A project item can be a slide a region a group and the experiment itself Some of the properties displayed in the properties grid are editable others are read only Here you may change create names for slides groups and regions The user can also choose to include exclude a region in the acquisition process or determine if the region is already acquired Page 117 of 195 www tissuegnostics com nuununnnnunuununnnnnnnnnnnn Acquired Acquired with time lapse Acquisition date Area Area in ROI Comment FOW matrix size Width Height FOV size Width Height Induded in acquisition Mame Patient Name Reference number Region type Storage directory Time lapse Number of runs Time lapse Time Regions Total magnification Type S
43. uses the counter will be incremented after each successful or canceled acquisition The counter will not be incremented if acquisition fails Checked at experiment creation acquisition Acquisition of ZStack TissueFAXS will not acquire Z stack If the user owns a predefined number of uses the counter will be incremented after each successful or canceled acquisition The counter will not be incremented if acquisition fails Extended focus TissueFAXS will not acquire using extended focus If the user owns a predefined number of uses the counter will be incremented after each successful or canceled acquisition The counter will not be incremented if acquisition fails Acquisition of time lapse regions TissueFAXS will not acquire snapshots but will open already acquired projects If the user owns a predefined number of uses the counter will be incremented after each successful or canceled acquisition The counter will not be incremented if acquisition fails Acquisition with stitch and compute stitching TissueFAXS will not acquire with stitch or recomputed stitch for regions Older projects will be properly displayed If the user owns a predefined number of uses the counter will be incremented each time stitch is computed manually or automatically through the acquisition Acquisition of TMA TissueFAXS will not acquire TMA Spots but will allow opening existing projects If the user owns a predefined number of uses the counter will b
44. 59 Adjust camera settings tab Press the button from the lower right corner to close the tab For more information please see Chapter 5 7 of the current manual 9 10 Drag drop panel In this panel you can drag and drop controls from the microscope panel that are used most frequently If a control is no longer needed it can be dragged and dropped on the remove button ne a When TissueFAXS is restarted the last configuration is loaded td TL Shutter RL Shutter Objectives UG Ox 25x 10x Figure 160 Drag drop panel 9 11 Camera settings for Preview and Acquisition To adjust the camera settings for Preview or Acquisition click the button for the desired channel and its current settings will appear in the camera panel After you adjust the camera settings click on the button in the camera panel to save them 9 12 Making a preview You will be ready to capture a preview image of your slide when you have set up your experiment and adjusted your camera settings for your preview objective To acquire press the Preview button 1 located within your selected slide panel To preview another slide or multiple slides click on the drop down menu of the Preview button for more preview options Page 111 of 195 www tissuegnostics com TissueFAXS 3 0 Preview selected slide Preview multiple Figure 161 Preview options panel lf Preview Multiple is chosen the following dialo
45. 9097 2609 a4 036 400 527 TissueFAXS 3 0 Page 58 of 195 www tissuegnostics com TissueFAXS 3 0 second click here cancel Figure 56 Distance calibration panel a e Draw a line between two points for which you know the distance Click in a desired point to start drawing the line then click again to determine the end of the line In our example the squares on the calibration slides are 100umX100um Use a distance as big as possible in order to improve results e Specify the distance between the two selected points the value must be in ym Press Compute button FOV Width FOV Height Pixel size and FOV size ratio will automatically be computed Press OK button in order to save the obtained values Page 59 of 195 www tissuegnostics com TissueFAXS 3 0 pecify a value for the Y lenght of the line DW Press Compute F FOY Width 554 036 FO Height 400 527 Pixel size 7 996 x 2 996 Foy size ratio Cancel Figure 57 Distance calibration panel b pate About calibration methods Only the FOV values for the current objective have been calculated recalculated this far In order to compute the FOV sizes for the rest of the objectives repeat the steps for each objective For a quick setup but less precise you can select the objective with the medium magnification between your objectives compute its FOV size and before pressing Save and Exit 775 e
46. Background threshold It is best to set this to auto You might want to try this when the spots are clearly visible on the background and the detection fails to return the best results This parameter represents a medium value between background maximum intensity and spots minimum intensity When TMA structures are adequately detected press the OK button Place Holders Page 174 of 195 www tissuegnostics com TissueFAXS 3 0 The place holders are virtual TMA spots which replace missing tissue from some positions on the matrix structure of the TMA block In the image below the place holders appear in dark green Figure 248 Place holders For a TMA spot to be considered a place holder go to the property editor and set the value Yes in the Placeholder option El Patient Mame Reference number Placeholder Region type Storage directory Time Regions Figure 249 Place holder property 6 2 Manual definition of TMA structures In order to manually define the TMA structures on a slide the slide must already have a preview image and the content type of the slide must be set to TMA 6 2 1 Definition of TMA block size Page 175 of 195 www tissuegnostics com TissueFAXS 3 0 The first step in the manual definition of TMA structures is to indicate the size of a TMA block the number of rows and columns You have two possibilities when defining the size of a TMA block e Press the left mouse button on TMA Block Size but
47. Fine Focus steps three focus measure algorithms are available e Variance this is the fastest and most robust method for finding the location of the sample It has the drawback of being easily affected by the quality of the samples e g any dirt bubbles of air in the oil if any illumination problems or any other optical artifacts This method is the default choice for Rough Focus measure because its robustness and speed e HP this method is more precise than Variance at the cost of being more time consuming It has two additional parameters X and Y which specify how much from the image is processed By decreasing these two values the speed of the HP will be increased at the cost of lowering the chance to focus on sample e g analyzing a hole of the tissue cannot provide the Z position due to lack of signal Usually a value of 50 or above should provide a good balance between speed and accuracy for objectives up to 63x For higher objectives it is recommended to have most of the image content analyzed This method is the default choice for Fine Focus measure e HP Fine this method is the slowest of all more than two times slower than HP in terms of computing time but it can provide the best focus under toughest conditions It should be used only when HP fails repeatedly on difficult samples for example on fluorescence samples with a high un uniform background This method is not recommended for Rough Focus It has an additional pa
48. Lapse right image To enable Standard acquisition mode the Time Lapse acquisition checkbox must not be ticked To enable Time Lapse Acquisition then Time Lapse acquisition checkbox must be ticked and the user should also set the values for the following parameters e Number of runs how many times the acquisition will run Time lapse the interval between runs The time lapse will be specified like this days hours minutes seconds Maximum estimated time per run This message is displayed on the right side of Time Lapse acquisition section It gives you an estimation of the time interval required for a run maximum required time estimation Time lapse acquisition Number of runs Time lapse 00 00 15 E Maximum estimated ime per run 0 0 11 Fooat Days Hours Minu tes Seconds Figure 92 Maximum estimated time per run message Page 81 of 195 www tissuegnostics com TissueFAXS 3 0 If the time interval set in the Time Lapse field is too short to acquire the item the message will be displayed in red Time lapse acquisition Number of runs Time lapse 6 00 00 03 57 A Format Days Hours Minutes Seconds Figure 93 Maximum estimated time per run low time lapse value In this case by pressing Acquire button a message box will inform you again abut the short time lapse A The time lapse is too short Do you wish to continue Figure 94 Short time lapse message box If you continue with
49. MPORTANT technical note If you have installed previous pre release versions of Visual C 2005 or Visual Studio 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 jf Note The TissueFAXS instrument you received from TissueGnostics or your local distributor has been preconfigured and you should not change anything in this software hardware configuration unless you are directed by the support team For further information about this package please visit the Microsoft homepage 2 6 TissueFAXS The TissueFAXS installation kit is the last file installed from this list of required components It consists of a single executable file Once launched it leads to a well known installing procedure You just need to run it and follow the instructions Mainly you have to choose the destination directory and that s all An important feature is that different versions of TissueFAXS may coexist on the same machine You only need to make sure that they are installed in different directories If you want to install the same version of TissueFAXS in the same directory please uninstall the existing version first instead of overwriting it This ensures that no files are in use and the new installation will be a fresh new one Also note successive installations of TissueFAXS do not require re installation
50. Mennt cccccscccsscecssceceeeceeceueeceuceceueecaueeseeseeecsaeeseusecsueesueessusessueessaeenaess 69 9 1 3 Experiment iden CO sorna prieto ooo rien roo wade vers a o elo N ADE RAE ETA A 70 Page 2 of 195 www tissuegnostics com TissueFAXS 3 0 q p o A 71 0 10 Finish EXpenment IR OA Veritas 73 5 2 FNS SON Sars sa tirita 74 9 2 1 Brightield eX OC LIMON dr eos 74 5 2 2 Fluorescence Experiment srt O ias 75 00 ACQUISIION 5 CUUINGS ssena A A Aa EE i 76 5 4 Standard acquisition vs Time Lapse acquisition ccoocncccnccocnncncncconncnnnncnnnnnnnnnonononnnnnnnnnnnnnnnnnnnnncnnnors 80 Da eje E e A EE E ee eee eee 84 AAA o 85 SAA 85 9 050 TL SMUMEF Tor transmite d NON assi aa 88 A Se 89 999 RL Shutter for rellected HON usina tipear ici 89 e 89 5 5 7 White Light Attenuator optional COMpONenNt occccccoccncccnncccccnnconcncconcnononnnononnnononnnnnnnnnnnnnnnnnnnnnnonnnononnnnnns 90 5 5 8 Fluorescence Attenuator optional component ooccccccoccnnccnoccnnononcnnonnncnncnnnnonnonnncnnonnnnnnnnnnnnnnonnnrnrnnnnrinnnnas 90 ONO CIN O tati post elite iia 90 5 95 10 Condenser Front LenS estacion 91 5 95 11 Condenser ADS nue ces cotos dll 91 BZ SIS DO NAO as olor 91 5 6 A ee ee A ere ne ee 91 5 7 o A ote Ae a Ta ac ee a a nant Soe mccansoeausenness 93 DIAN a Cameras srta dale looser le de oro orita 93 Oat z Change Default Camera msn aaa ri iii sitas 95 5 7 3 Operations with Current Camera ccoonccccnccccncononcconnnconnn
51. PolyLine Figure 204 Toolbar categories shapes Page 141 of 195 www tissuegnostics com Then add the desired area on the viewer using the mouse just like for any normal region see Chapter 5 16 1 Once created these areas cannot be modified only removed To remove the areas right click inside the desired area and then choose Remove area name Clear All Reacquire Flags Reacquire This FOV Only Reacquire Flags Go To This FOV Flag Entire Row Flag Entire Column Clear All Add Flags Clear All Remove Flags Insert Row Insert Colurnn Acquire New FOVs Transform into Defined Region Figure 205 Remove category area context menu View hide categories on regions To be able to view all tissue areas belonging to a certain category check only that particular category in Region Viewer Toolbar Subregions If you want to see all existing categories check All Categories option from Region Viewer Toolbar Subregions To hide the tissue areas belonging to a category simply uncheck that particular category in Region Viewer Toolbar Subregions Export tissue areas belonging to categories e When exporting the Region Overview you have the possibility to choose an option that also exports the categories e When exporting the FOVs images you have the possibility to choose an option that only exports the images belonging to the respective category For more details please see Chapter 5 20 of the curre
52. Print or File Print Preview 7 buttons from the main toolbar Printing is only possible for an opened experiment First a dialog appears and then items to be printed must be selected acquired items all items Please select the items from the project you wish to include in the print no Ted fotos D Draw regions on slide preview E Slide 1 19 Region 002 Region 011 19 Region 018 9 Region 019 EE slide E 9 Region 031 l bgp Region 005 L Ga Region 010 Ol side LS Region 006 oH ses GB Region 007 39 Region 030 El Slide 6 8 Slide 7 ENE slide 8 Region 020 i Ga Region 033 Show names of regions Apply illumination correction to regions images Show current date Show page number VIIA AAAI Figure 263 Print dialog choosing items to print Page 187 of 195 www tissuegnostics com TissueFAXS 3 0 The default selection for this dialog is the selected slide in the slide viewer It also offers a set of options Draw regions on slide preview the preview image may also contain region shapes Show names of regions this option is available only if the first option is selected Apply illumination correction to regions available only for Brightfield experiments the correction image will be applied to exported images if the correction image is available Show current date the print date is visible on each page of the report Show page number the page number is visib
53. a 24 4 2 Default Experiment SettingS ccoocccconcoconncconccnoncconnnnonnonannononnonnnnnnnnnnnnnnnnnnnnnnrnnnnnnnnnnnnenannennnnenaniss 28 ZW AIS e PE PERSO aston asin A E Sedan ated ening eae caw 28 EA o PA o ne O PE O eee ee ee ee 29 AS OMA 9 aaa 30 4 3 e ie Sera ere E E E E E E EEO aan Sees A EE EOE aa 32 Eos SCAN IO SI ETES T ee eee EE EA A E E eres 32 AS PP 32 Ao FO UL Ie E E o E o E E S sae naan eea 34 7s AAVA Ed FOCUS rica iia 38 o oo e E EA EEIE A E E 39 4 4 Na ARO GUNA EEPE TEISE AE E AEI AE TEE EE A SE 40 44 1 Camera Manager ascos tetas 40 44 2 Contrast ManadE lnea 40 Ad ads Man ratita 41 A o A A Pa 48 4 5 ls A o seeyietene ween cemere A 48 ESNE eg PPP e O a eee 48 4 6 calibrate Field A censcicestradseemeedte use 49 LOT Manual POCA ON pisos arcos pirita veoseel cue ated asee sececeeseacec un 50 40 2 10 8 aits iia FOV CAIDO earlier 54 AS SAIN CS IN AIO episcopado cie cica 57 4 7 o PA 61 A e e o EE e 61 AR AI SS cios coin 62 AS IMC OI IA USOS cocida 63 4 7 4 Delete A o o e 63 4 7 5 Change Password Administrator Mode ccooonncnccccccnccconcnncconcnnconononnononcnnonnnronnonnnrnnonnnrnnnnnnnnnnnnnnrnnenanens 63 4 7 6 Role Based Administration cion a ii 64 SHR New Experiment VVIZAN Cisco sdeccce aes ede ceeapacsxaiss naddvennaeenneieninedeseeuinonaine oh sees aweaisiiab nedouemndueareiensed Sresnneneyesdeseneies tede 66 Oe Vol MPO RIM OIL Ce all OM sasos E A a E E E 67 5 1 2 Browse for template experi
54. ach objective from Tools Calibrate Field of View menu Page 49 of 195 www tissuegnostics com TissueFAXS 3 0 Camera PixeLINK PL A6220 6220116 Automatic Calibration Objective 20x Air Distance Calibration Left 15071 655 Right 14405 145 Top 26042 162 Botkorn 26444250 Width 533 490 Height 402 076 Edit Speeds Mi 14138 400 um i 26243 200 pm Es 3056 150 pm Automatically compute calibration For other objectives Figure 45 Field of View Calibration control panel This dialog allows you to compute the size of the Field of View for the current objective In order to accomplish this task you have three possibilities manual calibration automatic calibration distance calibration For all FOV calibration methods it is recommended to use a slide containing a more vf Note prominent area in order to track it easier For optimal results you may use a calibration slide the calibration slide is mandatory for the distance calibration method 4 6 1 Manual FOV Calibration e Choose a reference point on the sample For a good evaluation of the reference point please make sure the whole object is in focus e Move the reference point in the upper right corner by using the software or hardware joystick Page 50 of 195 www tissuegnostics com TissueFAXS 3 0 Comite reer er oe cee Bao ic la Cl thee ES at Figure 46 Reference Point Upper Right Cor
55. ack the acquisition time could increase with as much as 30 The backlash correction should be used when the acquired images do not properly fit to each other Note If all the images have a stitch problem you should check the FOV calibration settings e Use Enhanced Camera Mode this is a feature available on few cameras that will increase data transfer speed 4 3 2 Stitching Stitching will allow the user to see high quality pictures without any inconveniences caused by stage errors camera calibration or other issues that might interfere with proper image display acquisition To enable this feature you must mark the Enable Stitch checkbox Page 32 of 195 www tissuegnostics com TissueFAXS 3 0 Acquire with overlap only For external programs Use internal stitch Overlap Settings Width Overlap Advanced Focus Speed Height Overlap Hardware Settings g pa Experiment Options Compute stitch mode Manual WARNING The sioh duning focus is recommended only for smal regions up to 100 Gelde of view because if is very dime consuming Min allowed stitch score Figure 24 Stitching dialog You can choose between internal and external stitch Acquire with overlap only For external programs The acquired images will share common parts with their neigboring images Use this option only if you want to stitch the images using third party stitching software For normal use of the TissueFAXS software this
56. adjusted in 1 10 of a second to a maximum exposure time of 50 ms The Video Mode is recommended when there is a low amount of light reaching the camera sensor This mode is recommended for Fluorescence experiments The stepsize of the exposure time can be adjusted in 1 second steps to a maximum exposure time of 10 000 ms 10 sec Mode O Async Shutter 8 Video Mode Figure 132 Camera Mode radio buttons Display Information on Camera L4 Display Information on Camera when enabled this option will display on the live image the histogram the saturation and the high background indicators Display Scale bar Ll Show scale bar when enabled this option will display the scale bar on the live image Display Gray value H Show Gray value when enabled this option will display the gray value of a pixel selected with the mouse Page 98 of 195 www tissuegnostics com TissueFAXS 3 0 Gaya Ley cu tsay Scale bar Figure 133 Information Displayed on Camera If areas appear labeled in red within a fluorescence image this means those areas are wo over saturated The settings available for both cameras are divided in two categories basic settings and advanced settings Basing settings are the most frequently used while advanced settings are rarely used and should only be changed by experienced users Usually advanced settings are only needed when the system is installed or in special conditions as described in
57. aining correction image the one you want as correction image fn The region that appears in red in this dialog is the currently opened region ote After selecting the respective image you can effectively apply the illumination correction Page 146 of 195 www tissuegnostics com TissueFAXS 3 0 Gi slide 1 Ha Group 31 49 Region 025 Region 029 Region 030 Region 033 Region 034 Region 035 Region 036 Qogojgo go Region 039 i iG Region 045 GB Region 330 CI slide 2 E slide 3 CHI slide 4 8 slide 5 698 slide 6 fEl Slide 7 698 side 8 Figure 213 Illumination Correction Select correction image e Reacquire correction image this option should be used when you wish correcting the Illumination for an already acquired region The following panel will appear Correction region size Weidth 3 Height 3 Overlap O e o 0 Please go to an empty location without tissue or other objects Lise the same settings as for acquisition Stage Control Figure 214 Acquire correction image control panel Correction region size you can specify here the width and the height expressed in FOVs of the area used to compute the correction image Generally a large area size is to be preferred but this is not mandatory Overlap the overlap parameter helps you control the size of the overlapping area shared by neighboring FOVs that form the correction r
58. ar icon looks like this The Scale bar is represented by a segment that indicates the scale of the image lt has two adjustable attributes e The color e The location list with four values Location Figure 12 Scale bar location combo box fn 0 The default values are Black for color and BottomLeft for location ole The scale bar can be found on Live Image Slide Preview Region Viewer Page 21 of 195 www tissuegnostics com TissueFAXS 3 0 One Image Viewer Exported Images Measure The Measure icon looks like this E This function is used to measure the distance between two points on the sample specified by the user by clicking on the start point then on the end point The distance and the unit of measure are displayed on the measured image Figure 13 Measured image example There are two adjustable attributes the color and the measure unit The default color is aquamarine and the default measure unit is millimeter This function is available on Live Image Slide Preview Region Viewer One Image Viewer RGB Here you can select the color used to display information about RGB or Grey value for a pixel on the camera live image Show Area in ROI If checking this option the defined regions and subregions that are going to be created will have computed the area and the area in ROI e The area refers to the total area of the region including only acq
59. are possible even without hardware For problem solving and re installation it is essential that you configure your ye microscope in terms of teaching it which hardware is provided To do so we Page 10 of 195 www tissuegnostics com TissueFAXS 3 0 recommend using the MTB configuration tool Do not forget to write the configuration into the microscope hardware when you are finished Present hardware that was not enlisted in the configuration will not be accessible The TissueFAXS instrument you received from TissueGnostics or your local distributor has been preconfigured and you should not change anything in this configuration unless you encounter technical problems 2 4 Intel Performance Primitives 5 3 Intel Integrated Performance Primitives Intel IPP is a software library for application developers providing better performances on Intel s latest microprocessors Incorporating these functions into the TissueFAXS software will ensure that the potential of your Intel based PC can be fully exploited for analysis of your samples 2 5 Microsoft Visual C 2005 Redistributables The Microsoft Visual C 2005 Redistributable Package x86 installs runtime components of Visual C Libraries required to run applications developed with Visual C on a computer that does not have Visual C 2005 installed TissueFAXS contains modules that were developed with Visual C so this Redistributables kit is required I
60. arnnnonaninnos 145 ra a lo IO eE A PAP 149 5 18 Field OF VIEW VICWON acti de 157 5 19 Field of View Viewer Overlay feature FlUOrescence occccccocccccccoccnconoccnnononcnnnnnanononnnncnnonancnnonnncnnnnnos 158 5 20 EII e A Rioncmudsveuemanusacwue dia 160 020 1 EDOL F IY SIMAO Sonnerie 160 5 20 2 Export REGION OVervieW ccccccccccseecesceceneeceeccueeceuceceueesaueesaeeceueeseueesaueesaeessaeessueesueesueessueessasessenensas 162 8 203 EDOT ONG FOV MAG Ourense sprites 167 5 20 4 Export current Slide IMage ccooocccconcoconccconcconnoconnononccnonconnnononnononnnnnnnnnonnononnrnnnnnnnnnnnnnenannrnnnnenaninos 169 3 20 EXON PIN OLAUIOMS oz ssecdccnedestcusureadepeonestcyeesued dsccestseeevec lace EEE EES EEEE EES EES EE EA EINEN EEEIEE EETA 170 5 21 Ue PGN ee 172 Page 3 of 195 www tissuegnostics com TissueFAXS 3 0 6 1 Automatic detection of TMA structures oocccocnccccnccccnccccnnnnnnnncnnonononnnnnnnnnnnnnnnnnnnnnrnnnnnnnnnnnnnnnnnnnncnnnnnnnss 173 6 2 Manual definition of TMA StrUCtUFES cooccccocnccccncoccncnononnnncononcnncnonnnonnnnnonnnonnrnnnnnnnnnnnnnnnnnnrnnnaninnnnennnnns 175 6 2 1 Definition of TMA block SIZE oocccoocccocccocccocncocncoonocononononononononononanonnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnenanenaso 175 6 2 2 Definiton of the first TMA block On Slide oocococcococncoccncocnnncononocnnnnnnonnnncnnconnnnnnnnnnnnnnnoncnnnnninnanonos 177 6 2 3 Definition of the s
61. ation Page 68 of 195 www tissuegnostics com TissueFAXS 3 0 e f you choose to create an empty experiment follow these steps Experiment Identification Experiment Folder Experiment Settings and Finish Experiment Ready e f you instead choose to start from a template or an existing experiment follow these steps Browse for template experiment Experiment Identification Experiment Folder and Finish Experiment Ready 5 1 2 Browse for template experiment In this page of the wizard you must browse for a template or an existing experiment This will conveniently copy most of the useful data including the settings for preview and acquisition channels to the new experiment J6 New Experiment Browse for template experiment You may select a template or an existing experiment Browse Description of template experiment Figure 72 Browse for template experiment After choosing the template a short description is displayed You can find below the data from the template existing experiment used for the new experiment e Experiment type e Preview objective e Preview channels e Acquisition objective e Acquisition channels e Camera e Preview schema e Label position An experiment can be saved as template by accessing the main menu File Save as Template Page 69 of 195 www tissuegnostics com TissueFAXS 3 0 Acquire Print Print Preview Slide Manager Properties Figure 73
62. ation Options Actions Technician Disabled Calibration Options Application Options Support gt Disabled Connection Details Options gt Application Options Support Test Disabled Connection Camera gt Advanced Options Application Options gt Licensing Disabled Page 65 of 195 www tissuegnostics com 5 Using TissueFAXS 9 1 New Experiment Wizard In order to acquire images in TissueFAXS it is essential to create a new experiment or open an existing one All images settings and data are saved and embedded in your experiment files and folder The only thing you can do if you don t have a current experiment opened is to preview slides or acquire single images from the camera window Opening an existing experiment Lcd To browse for an existing experiment press Open button from the toolbar or press j al Open Open from the File menu To access the recently used experiments press Open button arrow 0 from the toolbar or Open press Open arrow from the File menu J Ai O FAProjects TissueFAXS Demo_ProjectiDemo_Project aqproj F Projects TissueFAxS Demo_BFi TF 132 Br agproj View for All Users Figure 66 Recent Experiments list From this list choose the desired experiment and left click on its name to open it Currently opened experiment name appears bold on the list If you choose View for All Users option Recent Experiments dialog will open Here you ca
63. be deleted or modified These schemes are grey You can add rename or delete additional schemes from this panel Page 28 of 195 www tissuegnostics com TissueFAXS 3 0 Create new preview area on slides press Add button to create a new preview schema according to the size and the location of the sample you are currently using After giving a name to the newly created preview area you can modify its size resize using the mouse cursor on any edge of the shape or move it by using the mouse cursor to drag amp drop the area to the desired location Set the default preview area on slides by selecting the desired entry from the Preview Schema combo box e Select the label position from the Label Position section default label position is down _ The options mentioned here will only affect newly created projects Please remember vf note the label position is not stored in the preview schema Changes in this panel will affect future experiments If you want to change the preview area of a current experiment use the Preview option from the Experiment Options section in the same dialog e More detailed information concerning the Preview option can be found in the Hint section Chapter 5 1 4 number of FOVs camera used for preview preview objective 4 2 2 Reflectors Fluorescence images are usually gray scale images from black to white over a certain number of gray values the intensity of the gray values representing a cer
64. be exported Images Movies option available only if the region was acquired with time lapse Use stitch Select the file name convention Name Parts Custom Part Parts Image Format Name Preview Select the type of images you want to save Overlay the image as it appears in the One FOV Viewer Original the images for each channel as acquired Mark Crop Area it marks the contour of the shape for each FOV you can select its color Scale bar please see Chapter 4 1 2 of the current manual Apply Illumination Correction this option is enabled only for Brightfield experiments The correction image will be applied to exported images if the correction image is available Page 168 of 195 www tissuegnostics com 5 20 4 Export current Slide Image To export a slide s preview image select Export slides images from e Stage control menu Go To This Slide Go To This Point Acquire Slide 3 Label Lip Label Down e Slide editor menu Add Region Remove Selected Items Remove Acquired Images Go To This Slide Go To This Point Acquire Slide 1 Acquire Selected Items Build Cache for Region 039 Clear Cache for Region 039 Select All Preview Label Up Label Down Show Position on Slide Paste Compute Stitch Figure 241 Slide editor context menu e Experiment editor menu only on experiment item TissueFAXS 3 0 Page 169 of 195 www tissuegnostics com TissueFAXS 3 0 Export regions ov
65. blocks and spots The TMA blocks and spots can be deleted as follows e select the desired TMA blocks and spots e press the right mouse button and select Remove selected items in the menu that appears or press the Delete key Page 183 of 195 www tissuegnostics com TissueFAXS 3 0 E Preview Slide E sa hy oo Remove Acquired a de Group as TMA Block Group TMA Spots Go To This Slide Go To This Point Acquire Slide 2 Acquire Selected Items Build Cache for TMA Block 00010 401 Clear Cache for TMA Block 00010 401 Select All Preview Label Up Label Down Show Position on Slide Export Slides Images Copy Paste Compute Stitch Figure 260 Delete TMA spots 6 2 10 View acquired images for TMA spots To view the acquired image of a TMA spot double click on it 6 2 11 Grouping TMA spots When working with TMAs it might become necessary to group certain spots within a block they share similar properties they contain certain types of cells etc So TMA spots can be grouped as follows e select the desired TMA spots e press the right mouse button and select Group TA Spots in the menu that appears Page 184 of 195 www tissuegnostics com TissueFAXS 3 0 Add TMA Spot Eo Remove Selected Items Remove Acquired Images Group as TMA Block Go To This Slide Go To This Point Acquire Slide 2 Acquire Selected Items Build Cache Clear Cache Select All Preview Labe
66. can be used to operate the camera to display a live preview capture images and adjust camera settings Page 104 of 195 www tissuegnostics com TissueFAXS 3 0 PixeLINK PL 46220 6220116 Sensor saturation a d gt An i a Y Live Image Figure 150 Camera control View live preview When working with TissueFAXS you may choose to see the live image or to disable it by simply using a checkbox e To enable live preview check Live Image emmma Any time you press View button from the channel list the Live Image will be enabled If the function Live Images is disabled during acquisition the overall acquisition speed will slightly increase Page 105 of 195 www tissuegnostics com TissueFAXS 3 0 Capture images manual procedure Before manually capturing an image without using the automatic acquisition feature of the TissueFAXS software you must first choose a location and filename by pressing the following button Browse Next you are able to capture the image by pressing the Capture button If the filename you want to create already exists the TissueFAXS software will not overwrite the existing file The new filename will be incremented by a numerical extension that will generate a file name that does not already exist For example if you choose the filename C limage bmp your new image will be automatically saved as C image bmp and if the file already exists the name w
67. ckbox In the Product Improvement Program participating systems send information to TissueGnostics about how they use certain products Received data is combined to help TissueGnostics solve problems and to improve the products and their features Click the link in the lower left corner of the dialog shown above to read about this program and check agree checkbox if you want to participate If you don t check the check box the Send Error button will be disabled Page 27 of 195 www tissuegnostics com TissueFAXS 3 0 TissueGnostics Product Improvement Program Agreement 1 About TissueGnostics Product Improvement Program Focusine gt vation TissueGnostics TG js constantly involved in understanding a anta ting custo eds invarder to create ies A soy Pull i datar emeni Progg wher J i n gout hoj Figure 19 Product Improvement Program Agreement 4 2 Default Experiment Settings 4 2 1 Preview In the preview dialog you can manage the stored preview schemes The adjustable yellow area allows the user to define the default preview region on the slide s A Please specify the default preview schema Preview Schema Preview Normal Center Hint N A FOVs Camera N A Reflectors Storage Preview objective 10x Air Label Position O Label Up 2 Label Down Figure 20 Preview dialog e By default four preview schemes All Slide Large Center Normal Center Small Center are present which cannot
68. d settings feature ONLY if you are instructed by TissueGnostics personnel or authorized distributors Load settings feature is only available if logged as technician or administrator After the update is finished the application will be automatically closed Application Options Scan Settings Load Settings From Another Folder NOTE This opion wi alow you fo migrate set nas from an older version of the application MHARAING After he update is finished Ne application wi be automaticaly dosed Camera Manager Contrast Manager Stage Manager Experiment Options Figure 43 Load Settings dialog 4 5 Experiment Options 4 5 1 Preview This dialog resembles very much to the Preview dialog from the default options above The difference is that changes in this dialog affect the actual experiment Page 48 of 195 www tissuegnostics com TissueFAXS 3 0 A ERR BB Application Options Please specify the preview schema for current experiment Preview Schema Normal Center Camera N A Preview objective 1x Air Figure 44 Experiment Options Preview dialog 4 6 Calibrate Field of View The camera calibration is a basic but essential operation when using TissueFAXS as the quality of the acquired images directly depends on it We advice you paying maximum attention and perform the calibration with particular care We recommend calibrating the size of the Field of View for e
69. des in order to facilitate your work and obtain high quality results In order to define TMA blocks and spots on a slide the content type of the slide must be set to TMA rote It is not possible to set the content type of a slide if the slide contains any regions or 7 groups El General Comment Content type TMA oe Name Figure 246 Setting the content type of the slide 6 1 Automatic detection of TMA structures TissueFAXS provides automated tissue detection for TMA structures By using this feature the efforts of working with TMAs will be considerably reduced It consists in an algorithm based on contrast and uniform illumination Therefore applying the illumination correction before detection of the TMA spots might be needed You can do this by using Illumination Correction button from Slide Preview toolbar After applying the illumination correction TissueFAXS can automatically detect the TMA structures on a slide Start by pressing the Tissue Detection button from Slide Preview toolbar When the automatic detection of TMA structures is initiated the dialog below will appear In this dialog the user can restart the TMA detection using different parameters This operation can be done for the whole slide or over a specific selected area For best results the area selected for detection should contain mostly tissue The coverslip borders and other artifact structures should be avoided Page 173 of 195
70. ds to be reinstalled virus infection system files being deleted you might lose data located on the system drive Images stored as jpg will make subsequent analysis slow because jpg files are scanned by anti virus software by default You may want to consult your local system administrator on how to exclude jog files from on access scans If this is not possible we recommend storing files as tifand compressing the folders after analysis is completed Page 31 of 195 www tissuegnostics com TissueFAXS 3 0 4 3 Scan Settings 4 3 1 Scan Strategy TissueFAXS provides a type of Scan Strategy Snake that allows an optimal scanning process Application Options ay Stitching Advanced scan settings _ Use software backlash correction Note This aootes only fo XY axis of fhe siage The stage precision wil be improved but as drawback fe acquisition dime comia increase wiih as mudh as 30 Focus Advanced Focus Speed _ Use Enhanced Camera Mode This applies to Baumer cameras Hardware Settings only se Save amp Exit Cancel Figure 23 Scan Strategy dialog e Use software backlash correction This is a feature that helps avoiding certain errors that may appear when changing the moving direction of the stage caused by physical caracteristics of the stage which is the case during normal TissueFAXS operation It applies only to XY axis of the stage The stage precision will be improved but as drawb
71. e changed later if necessary only the experiment type cannot be changed Page 71 of 195 www tissuegnostics com TissueFAXS 3 0 Experiment Settings Please specify the objective the reflectors and the camera used by your experiment You can select a profile with predefined settings If you are not sure what to select you can modify these settings later Profile EC O Preview Objective Preview Settings Acquisition Objective Acquisition Reflectors Figure 75 Experiment Settings for Brightfield Experiments You may notice that for Brightfield Experiments the Transmission reflector is selected by default A check box for Transmission indicates that no reflectors are present and the microscope is set to regular transmitted light The other reflectors are present in the list but they are disabled for Brightfield Experiments In Fluorescence Experiments the panel looks like the figure below Experiment Settings Please specify the objective the reflectors and the camera used by your experiment You can select a profile with predefined settings If you are not sure what to select you can modify these settings later Profile Select Preview Objective 2 5 Ait Preview Settings Acquisition Objective 20x Air Acquisition Reflectors 0 Transmission DAPI Rhodamine FITC a Camera Figure 76 Experiment Settings for Fluorescence Experiments In this case there is a list of reflectors
72. e incremented each time a TMA spot is acquired Acquisition of well plates TissueFAXS will not acquire Well Plates but will allow opening existing projects If the user owns a predefined number of uses the counter will be incremented each time a region on a Well Plate is acquired Tissue Detection TissueFAXS 3 0 Page 192 of 195 www tissuegnostics com TissueFAXS 3 0 TissueFAXS will not allow running Tissue Detection If the user owns a predefined number of uses the counter will be incremented each time Tissue Detection is runned Illumination Correction TissueFAXS will not allow acquiring setting the illumination image If the user owns a predefined number of uses the counter will be incremented each time the correction images are applied Burst mode for Baumer during focus and acquisition TissueFAXS will not allow the use of burst mode during acquisition focus lf the user owns a predefined number of uses the counter will be incremented after each successful or canceled acquisition The counter will not be incremented if acquisition fails Correction flags TissueFAXS will not allow the use for flags If the user owns a predefined number of uses the counter will be incremented after each successful or canceled acquisition The counter will not be incremented if acquisition fails Load settings from file TissueFAXS will not allow loading settings from project If the user owns a predefined number of uses the counter w
73. e new user as the User Name the Real Name the Password Description and role Ar z All fields excepting Description are mandatory OTe Page 62 of 195 www tissuegnostics com TissueFAXS 3 0 4 7 3 Modifying Users By pressing Modify user button you can use the panel that appears to update the real name the description and the role of the user Real name Description Figure 61 Modify User dialog 4 7 4 Delete Users To delete a user select it in the User list dialog then press Delete user button You will receive a final warning before the effective deletion it looks like the image below Do you really wank bo delete the selected user Figure 62 Delete User message box 4 7 5 Change Password Administrator Mode When you are logged in as Administrator you can change other users passwords by using the dialog shown in the image below To change the password the application will first ask you to type the old password then the new password and its confirmation Page 63 of 195 www tissuegnostics com TissueFAXS 3 0 Define a new password Figure 63 Changing other user s password dialog 4 7 6 Role Based Administration In order to prevent certain untrained users to access the advanced controls of the application a role based policy exists in TissueFAXS Role based administration implies access restriction to non administrators for advanced controls of the application The ro
74. e of the microscope that will automatically adapt the contrast method to the objective and filter combination i e position of condenser opened closed shutters Page 40 of 195 www tissuegnostics com TissueFAXS 3 0 E Application Options Default Experiment Settings v Contrast Manager Default Scan Settings This setting applies for Zeiss Microscopes only Hardware Settings Default TissueFAXS will not set the contrast method of the microscope Smart Tissue FAXS wi set the contrast method according to the type of experiment E M OF TissueFAXS wil disable the Contrast Manager of the microscope See Aba Figure 31 Contrast Manager dialog The Contrast Manager has three options available in the dropdown box shown below Figure 32 Contrast Manager options e Default TissueFAXS won t try to set the contrast method of the microscope For optimal results we recommend manually setting the contrast method on the microscope e Smart TissueFAXS will set the contrast method according to the type of experiment This is the recommended setting for the Contrast Manager e Off TissueFAXS will disable the Contrast Manager of the microscope Use this option if Default and Smart options don t return desired results l CAUTION fn For a detailed view over contrast manager and contrast method please consult your gt iS microscope operating manual If the contrast method is not available for the microscope
75. e second click determines the radius of the circle Figure 178 Creating circular region e Custom with joystick This method allows you to draw the region shape by using the hardware joystick When you select this the following dialog will appear Use the joystick then push Add Point button to define your region Press Close Region button when you have done Figure 179 Custom region with joystick dialog Move the joystick to the desired points and for each of them click the Add Point button When you wish to close the region click the Close Region button Usually this function is used in conjunction with Live image using a high magnification in order to increase the precision of the region shape You can click anytime the Cancel button to reject any changes you have made 5 16 2 Modify Region All created regions can be modified resized moved as long as no images have been acquired for them e The move whole region operation can be performed in 2 steps 1 Select the region by right clicking an inner point of its shape 2 Click and hold the right mouse button in the same spot to begin the drag and drop operation Page 125 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 180 a Click an inner point of the region b Drag amp Drop operation to move the region e The resize or reshape operation Each region has special points that can be dragged in order to resize it or to change its sha
76. e similar to those of the Region Viewer The difference is that the FOV Viewer gives higher resolution images because it displays the original acquired image directly and not just a thumbnail image as with the Region Viewer The user can navigate to neighboring FOVs with the arrow buttons at the top right corner of the viewer t 4 Q The FOV displayed will be highlighted within the Region Viewer nP In the eventuality that the image does not fit the screen drag it by using the hand tool instead of scrolling up down or left right To do this hold the right mouse button down and drag the image then release the mouse button to stop Similar with the region viewer use the trackbar to see the FOV at a certain moment by moving the cursor il This option is available only if the FOV was acquired with time lapse for details please see Chapter Q Page 157 of 195 www tissuegnostics com TissueFAXS 3 0 9 19 Field of View Viewer Overlay feature Fluorescence The Overlay button in the FOV viewer button displays the same dialog for adjusting channels intensities and colors as for the Region Viewer see previous section Rhodamine MA 255 0 0 used Name Intensity Figure 226 Overlay Settings for a single FOV The difference is that the Apply button can work on multiple items as shown in the image below For current FOV For current region For whole slide For whole e
77. e steps mentioned below e From the Tools menu select Options to open the Options dialog box e Inthe Options dialog choose Hardware Settings Camera Manager Please select the default camera to be used Add new camera Frames to wait for acquisition Camera adapter magnification Contrast Manager stage Manager Load Settings Figure 124 Camera Manager dialog e Press Add new camera button The System Cameras dialog box will appear and it will display the cameras that are supported by TissueFAXS and are connected to your computer Page 93 of 195 www tissuegnostics com m Available System Cameras m P FixeLINK Cameras In the System Cameras dialog select the desired camera and press OK If your camera is not System Cameras m EJeco PixelFly Cameras m L PCO PixelFly 0 vendor PixeLINk Model Firewire Camera Release 4 Description PixeLIMK PL 4622C Color Camera PIECES 2 6 0 0 Subtype 0x2 B000001 Pu 1 0 23 0 FPGA 2 5 0 Serial Number 6220116 Firmware Version 014 00 23 00 PRA version a2 05 00 00 Mame PixeLINK PL S622 Figure 125 System Cameras dialog listed there you can press Reload to rescan for connected cameras ne Now you will be able to see the selected camera in the combo box Frames to wait This value indicates how many images the camera will skip before the image will be taken and stored If this value is too low the images may appear stretched
78. e to your local TissueGnostics distributor to be renewed e use Load license file to reload the new license file to the application Use the Licensing feature ONLY if you are instructed by TissueGnostics personnel or CAUTION authorized distributors Licensing feature is only available if logged as technician or administrator Page 19 of 195 www tissuegnostics com TissueFAXS 3 0 Renew License Save license file Measurements Load license file Remember Support NOTE In order fo renew ie icense you must save e file and send it to he olstibutor or support team After receving fhe new file vou have to load if to complete ie operation Default Experiment Settings Scan Settings Hardware Settings Experiment Options Seve AE Figure 10 Licensing dialog 4 1 3 Measurements In the Measurements dialog you can manage the display options for the scale bar and measure function Page 20 of 195 www tissuegnostics com Scale Bar Eo Color Licensing Location Remember Support Measure Color E 127 255 212 Unit of measure mm RGB Color C Show Area in ROI NOTES Be aware hat chectng is opion wil dramatically increase the acquisition Gime This option wil only analy to regions at wil be acquired after marking his check box This also anoles fo furi er adged defined regions and subregions TissueFAXS 3 0 Figure 11 Measurements dialog Scale bar The Scale b
79. eFAXS 3 0 TissueFAXS needs to calibrate your stage Please make sure that the slides are properly inserted if any Figure 4 Stage Calibration warning message If the magnification of the calibration objective is higher than 20x a warning message will appear In this situation there are two options e Press Cancel calibration then go to Tools Options Application Options Actions Calibration in order to select another objective from the dropdown list then return to calibration process from Tools Calibrate stage For more details please see Chapter 4 1 1 e Press OK to continue calibration l CAUTION If you continue the calibration as described above you should think about the risks such as damages to your objective stage or slides The selected objective For calibration has a magnification higher than 20x Press Cancel calibration in order to select a different objective From Tools gt Options gt Actions gt Calibration objective or press OK to calibrate anyway NOT RECOMENDED physical damage to the objective stage or inserted slides might occur Cancel Figure 5 Calibration objective warning message After the calibration ends TissueFAXS is ready to be used You can now create new projects open existing ones or simply adjust the microscope components and see live images from the camera as described in the following chapters Page 15 of 195 www tissuegnostics com
80. each channel In the next two sections the workflow for brightfield and fluorescence experiments will be described 5 2 1 Brightfield Experiment For a Brightfield Experiment you can select the objective used for preview To edit the camera settings for a channel click the View button e For further details see Chapter 5 7 from the current manual Page 74 of 195 www tissuegnostics com TissueFAXS 3 0 You cannot add or remove channels The default channel is Transmission and cannot ote be changed If you don t have a Transmission channel e g a missing reflector in the reflector changer you will not be able to acquire brightfield images To adjust the camera settings for the channel Transmission you must have a camera present in your project Otherwise the View button view h for each channel will not be visible i If you had made previews without any experiment and then created a new experiment CAUTION the previews will be lost f Preview Objective Preview Channels Settings for Preview Pe Tater Pts ee FE the channels for preview Acquisition O Transmission Transmission For each channel you can edit the camera settings In order to make the preview you should select at least one channel Close RL Shutter Add Remove Channels Figure 80 Preview settings panel 5 2 2 Fluorescence Experiment For a Fluorescence Experiment you can select the objective used for previe
81. econd TMA block On Slide oocooocccoccncoccncccnocccncnonnconcnonanononnnnnnnnnnnncnncnnnnnnnnnnnnos 178 6 2 4 Selection of TMA blocks ANd Spots oocccoccncocccccccncncnccncnonncnonnnnonnconnnnnnnnnnnnnnnnnrnnnnrnnnnnnnnnnnnnrncnnrnnnaninnaninns 179 02 OROSZ Ne WII DOS cates ect cast gc sc AT EE nee cneseetuedad ee ica vevetaceruieeesetteate ss 180 020 Rone a TMA DIOS Oe oe nr ere a ic 181 6 2 7 Adjust the distance between TMA Spots oocccccnccccnccoccococnoconcononcnnnncnnnnononnnnnnonannonnnnnnnnonannnnannenanconaninos 182 6 2 6 Moving TMA blocks and SDO Sustacietade ol tslp crisis lo tad aia 183 6 2 9 Deleting TMA blocks and SpofS ooccoooccoccccocccoconnononccnonocnnnnnnnnononnonanconnnnnnnnnonnnnannrnnnnnnnnnnnnenannenanennns 183 6 2 10 View acquired images for TMA Spot cooocccccnccocnccocnoconnononcononcononcnnnnononnnnnnnonannnnnnnnnnnnnannnnannonannnnaninns 184 NE O TNA DOL rA E A EEA E EEE 184 7 1 PROPIOS ico caatas 187 7 2 nn nn EE E 188 Page 4 of 195 www tissuegnostics com 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
82. ector in use is always surrounded by a black rectangle A good management of your reflectors will provide good quality acquired images and will also help you later in your image analysis Right clicking on a selected reflector will show a menu with two options Page 85 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 104 Context menu for selected reflector Edit Name the user can change the Full Name the Short Name and the Abbreviation max 4 characters for the selected reflector Also the user can write a short description for this reflector Full Name Short Name Rhodamine Abbreviation max 4 chars Description Rhodamine Cancel Figure 105 Edit details for selected reflector The user also has the possibility to choose the displayed names for the reflectors in Q TIP the application by going to Tools Options Reflectors Page 86 of 195 www tissuegnostics com Use inverted image for Transmission channel in case of fluorescence M Use white in preview overlay of fluorescence slide Please choose what name to display in application for reflectors Cel Full Name O Short Name 2 Abbreviation Figure 106 Reflector Settings dialog e Edit Color the user can set the color for the selected reflector Reflectors EOOSOEG a mim le ji tt tt Eee eee Eee eee LILI PILL LLL Figure 107 Edit Reflector Color context menu Save abe
83. ed number of uses the counter will be incremented after each export Export to Definiens TissueFAXS will deny use of plug in based on dongle If the user owns a predefined number of uses the counter will be incremented after each export Acquire raw data images TissueFAXS will not save raw data during acquisition If the user owns a predefined number of uses the counter will be incremented after each successful or canceled acquisition The counter will not be incremented if acquisition fails Measure Tool Page 193 of 195 www tissuegnostics com TissueFAXS will not allow the use of measure tool If the user owns a predefined number of uses the counter will be incremented after each use Print Module TissueFAXS will not allow the use of print module lf the user owns a predefined number of uses the counter will be incremented after each use Role based administration No settings will be hidden based on user role lf the user owns a predefined number of uses the counter will be incremented after each login Estimate and display duration No estimation will be displayed If the user owns a predefined number of uses the counter will be incremented after each successful or canceled acquisition The counter will not be incremented if acquisition fails Move the Z axis on filter change Z drive will not move when changing filters If the user owns a predefined number of uses the counter will be incremented after each succ
84. egion You can adjust the Page 147 of 195 www tissuegnostics com Af Note number of pixels that can be shared overlapped by two FOVs by using the slider Usually by increasing the value you can obtain a better correction image and thus a better illuminated region Press OK button to save the correction image or Cancel button to cancel the acquisition process Apply this correction image to the entire experiment the correction image of the current region from the region viewer will be applied to all regions of an opened experiment TissueFAXS 3 0 Each time before starting the acquisition process you will be displayed an Acquisition Information dialog where you can check the Acquire correction image option in order to obtain a correction image for the respective region TissueFAXS will use the same acquisition settings for the region and for the correction image Page 148 of 195 www tissuegnostics com TissueFAXS 3 0 You are about to perform Acquisition Please read the following summary before proceeding You have 1 region to be acquired with a total number of 36 FOVs Actions Beep after finish Time lapse acquisition _ Shut down after finish Time lapse acquisition Number of runs Time lapse 0 00 00 50 Maximum estimated ime per run O04686 FPorma Days Hours Minutes Seconds Figure 215 Acquisition Information Acquire correction image checkbox The Shading correction
85. ems Remove Acquired Images Go To The Slide Go To This Point Acquire Slide 1 Acquire Selected Items Build Cache for Region 002 Clear Cache for Region 002 Select All TissueFAXS 3 0 Set Region as Preview Area Label Up Set as Preview Focus Point Save Region as Preview Schema E Label Down Reset Focus Point Show Position on Slide Show Preview Info Export Slides Images Compute Stitch Figure 186 Preview Area context menus 5 16 5 Stage Menu By right clicking on any of the slide images you will obtain a contextual menu including more options as seen in the image below Page 129 of 195 www tissuegnostics com Go To This Slide Go To This Point Label Up Label Down Export slides images Figure 187 Stage Control context menu This contextual menu will help you better manage the physical stage and control the acquisition process Go To This Slide To make the objective lens physically move onto a particular slide you must right click on the slide and choose Go to This Slide In the main window a green box will outline the slide that is currently in observation position Go To This Point To make the objective lens physically move to a specific location on a slide right click on that point and choose Go to This Slide This is particularly useful for navigating through acquired slide previews and for determining where to draw regions of interest in the Custom with Joystick mode
86. ently used in the acquisition process when your tissue sample is compact without holes for example Floating Focus Points the focus is performed in a point that may not be the center of the block Floating Focus Points are especially used for biopsies or when your regions to be acquired are not compact e g they include holes The method identifies the location of the tissue within one block of FOVs and selects the most inner one This location may be between several FOVs and not necessarily identical to one of FOVs e Always force full focus The last focus position is ignored and the focus is always searched in the full interval This method will take more time but the obtained results will be better in case of difficult samples and environment e g samples with many tissue folds Focus Validator The Focus Validator checks and confirms that the focus search algorithm has obtained a good quality image TissueFAXS 3 0 Page 36 of 195 www tissuegnostics com TissueFAXS 3 0 It is used to confirm that the Quick Focus method has worked fine If the quality score it provides is low then a Full Focus search is initiated It is also used to confirm that the Full Focus method algorithm has obtained a sharp image If this validation fails the system will use the last known good focus position Normally the default values for the validator options should be enough to provide a good acquisition Disable focus validator it will d
87. ep 1 Page 46 of 195 www tissuegnostics com TissueFAXS 3 0 In the next steps of the wizard 2 9 simply follow the indications given for each step see figure below Move stage bo the left most border of the well plate insert Mowe stage to the top most bordes of the veel plate insert then press Mesct then press Next Move stage bo the left most border of the first row first column Mowe stage bo the right most border of the first row First column then press Mext then press Next Step 4 otep 5 Mowe stage to the left most border of the first row second column qye stage bo the bop mest border of the first row first column then press Mest then pres Next Step 6 otep More stage bo the bottom most border of the first row First column then press Next otep 6 Move stage to the top most border of the second row first column then press best Figure 42 Create well plate template Wizard Steps 2 9 Once you have created a well plate experiment and added the well plate regions you ote can control their size and position in a similar manner as described in the TMA chapter Page 47 of 195 www tissuegnostics com TissueFAXS 3 0 please see Chapter 6 After creating the well plates templates they will be available for further use in the Drop down box Pefaul 4 4 4 Load Settings The user can use this feature to load the settings from the previous version of the software l CAUTION Use the Loa
88. er rote Please ensure that you have enough rights on the selected folder Please ensure that you have enough free space on the drive where the folder is located The free space required depends on the file image format but at least 10 GB is recommended 5 1 4 Experiment Settings In this step you can set some properties of your experiment 1 Objective to be used for preview 2 Objective to be used for acquisition The default acquisition objective is set to 20x 3 Reflectors used for acquisition e In Brightfield Experiments no selection can be made here The channel Transmission is selected by default it is specific for the Brightfield Experiments e In Fluorescence Experiments you can select the desired reflectors here You can also modify the reflectors later from the Acquisition Settings tab 4 Preview area from Preview Settings button sl Camera used for acquisition A default camera is selected for each type of experiment The default camera for brightfield experiment is the PixeLINK camera and for fluorescence experiment is the PCO PixelFly camera N You can choose a preview schema from the list and select the position of the label from the slide up or down This slide label setting will remain the same for the entire experiment It can only be changed later for single slides In addition you can also select a profile with predefined settings from the dialog All settings made here can b
89. er Toolbar Subregions Show Annotations only Show Annotations should appear checked Now choose the annotation shape from the toolbar Then add the desired shape on the viewer using the mouse just like for any normal region see Chapter 5 16 1 TissueFAXS 3 0 Page 143 of 195 www tissuegnostics com TissueFAXS 3 0 To be able to see all annotations you have added simply check Show Annotation option from the Region Viewer toolbar PolyLine Measurements This feature allows drawing a line shape in order to measure a certain area Press PolyLine button and draw desired shape on the sample see Chapter 5 20 for more details Jf Annotation 01 04177 mm 4 Manage Annotations 3 i A nno El O ns e enor Annotation 01 0 21033843566400 33 2 Annotation 02 0 157318288037402 Please insert annotation content below Figure 209 Edit Annotations dialog e Locate by pressing this button the selected annotation will be located on the sample and displayed at the current size of the sample e Best Fit by pressing this button the selected annotation will be located on the sample and displayed at the current size of the region viewer e Remove removes the selected annotation e Remove all removes all existing annotations e Please insert annotation content below write desired content for the respective annotation Page 144 of 195 www tissuegnostics com TissueFAXS 3 0 Edit annotation directly on t
90. er combo box Page 90 of 195 www tissuegnostics com 5 5 10 Condenser Front Lens Condenser Front Lens Figure 118 Condenser Front Lens 5 5 11 Condenser Aperture Condenser Aperture Figure 119 Condenser Aperture control 5 5 12 Sideport Changer Sideport Changer 100 Observation Figure 120 Sideport Changer combo box 5 6 Stage Stage Components TissueFAXS 3 0 Using the control you can swing in or swing out the condenser front lens For objective lenses with magnification factors smaller than or equal to 10x and during fluorescence acquisition the front lens must always be swung out Only for objective lens magnification factors higher than 10x in transmitted light the front lens is needed for concentrating the light onto the field of view Using the slider you can open or close the aperture diaphragm SJ Note The condenser aperture determines the a contrast in the image Wrong contrast settings might impose autofocus functionality and the quality of image analysis As a good start point the condenser aperture should be close to the numerical aperture of the objective lens in use The Light Manager will store individual settings for each objective lens installed Sideport Changer control when present It is used to specify to which port observation rear port front port the light is directed Thus the user can switch between visual observation through the oculars and the digi
91. erval The current Z position should be read from TissueFAXS software in Camera window and not from any display of the microscope Larger focus intervals will make the autofocus slow and may cause the microscope to focus on objects laying on the coverslip e If you want to use the specified focus interval for all objectives check Override default Y TIP intervals with these values If you want to use the default focus interval only uncheck Override default intervals with these values Please be always very careful when making focus related changes as wrong settings CAUTION could cause damage to your sample and your objectives that when the mouse is over an item a short description of the respective item will Q For all the items described in Focus section some tool tips are available This means F TIP appear Enhanced Focus tab The options from Enhanced Focus tab will increase the speed of the focus However they are available only on certain cameras Camera Partial Scan based on width height percent only a part of the current FOV will be transferred from the camera to TissueFAXS resulting in faster focus Page 37 of 195 www tissuegnostics com TissueFAXS 3 0 Enhanced Camera Mode it will increase data transfer speed Focus Options Camera Partial Scan m Use camera partial scan Width percent Heigth Percent Enhanced Camera Mode Use Enhanced Camera Mode Applies to Baumer cameras only Fi
92. erviews Export FOVs Export slides images Export to Definiens Figure 242 Export slides images from Experiment editor No matter from where you access this option the following dialog will appear Export Slides Images Ba Please select slides Save To Folder Slide 2 lt please select gt Slide 3 Slide 4 Please select the extension for the file Ipg Slide 5 Slide 6 Include regions Slide 7 Slide 8 Scale bar Color HO 0 0 Location BotbomLeft ee Figure 243 Export this Slide Image dialog e Select the storage folder e Select the file extension e Include regions checkbox you can choose if the export image should contain or not the region shapes e Scale bar please see Chapter 4 1 2 of the current manual 5 20 5 Export Annotations To export annotations go to the region viewer toolbar choose Subregions button Export Annotations Page 170 of 195 www tissuegnostics com TissueFAXS 3 0 Show Defined Regions All Categories Manage Categories Show Annotations Manage Annotations Figure 244 Subregions contextual menu Export Annotations The following dialog will appear HEF Region 006 Figure 245 Export Annotations The first step to export is to select desired item s from the list You can also select desired items by using the buttons in the right side of the dialog e Select by area e Select by length e Select all Now press Export bu
93. es might help you in order to perform a precise analysis Add categories In order to add categories go to Region Viewer Toolbar Subregions Manage Categories A dialog will appear containing already added categories if existing where you can add new categories giving each one a name mandatory and assigning a color The list of categories is per project not per region Figure 202 Add categories dialog Page 140 of 195 www tissuegnostics com TissueFAXS 3 0 Microscope Camera Acquired Images Deol Oia 14 Export Plugins Subregions E a L f oe saa Ta Dimes C apg 1 Slide Region 004 i a Figure 203 Region Viewer example of categories You may also use the categories when you are about to export images For further details please see Chapter 5 20 of the current manual ra All existing categories are listed in the Subregions menu gt Note Remove categories In order to perform this action go to Region Viewer Toolbar Subregions Manage Categories and select the category you want to remove then press the Delete key from your keyboard Apply categories on regions To create these areas go to Region Viewer Toolbar Subregions then select only the desired category Now choose the area shape from the toolbar Microscope Camera Acquired Images Region Overlay Export Plugins al a O 7 Add
94. essful or canceled acquisition The counter will not be incremented if acquisition fails Different Attenuator settings for channels Attenuators settings will be ignored when moving from filter to filter TL Lamp settings will be ignored lf the user owns a predefined number of uses the counter will be incremented after each successful or canceled acquisition The counter will be incremented if acquisition fails If the user does not have a license for a certain feature TissueFAXS will display an informative message Your license does not allow you to use Cache module For more information please contact TissueGnostics or one of the local distributors Please find contact data by clicking on one of the following links TissueGnostics contact or authorized distributors Figure 267 Example of License Message TissueFAXS 3 0 Page 194 of 195
95. ew By default when double clicking on a region in Experiment Editor that region will open in Time Lapse mode if time lapse images have been acquired or in Standard Mode if time lapse images have not been acquired Be aware that not all operations normally available on a region are available on a set of Time Lapse images see also Chapter 5 17 Adding Removing FOVs option is not available Reacquisition of specified FOVs is not available Resize of the region is not available There is only one correction image for all Time Lapse images belonging to the same region All the categories subregions will be the same for all Time Lapse images belonging to the same region Microscope Camera Acquired Images Figure 98 Region viewer toolbar Time Lapse mode Page 83 of 195 www tissuegnostics com TissueFAXS 3 0 Assuming a region has both Standard and Time Lapse images the user can switch between Standard Time Lap these two modes by using two buttons C ee The user can choose to see the region at a certain moment by moving the cursor a E or by pressing L H and buttons The user can delete all time lapse images by pressing the Remove button An Time Lapse acquisition is not available for TMA samples see Chapter 6 ote During the Time lapse acquisition process the user can see the progress of the acquisition in the status bar of the application F lma Te a F Ban F al a
96. g will appear What type of focus for preview operation a Autofocus O Use current Focus position Do not show this dialog again Figure 162 Focus for Preview dialog Select your focus method then press OK In the next window select the slides you wish to acquire Autofocus method will use the method defined in Options Scan Settings Y ne Focus If you check Do not show this dialog again your current choice will be remembered for future use and you will not be prompted again This selection can be reset from Tools Options Application Options Remember Slide 2 Slide 3 _ Slide 4 Slide 5 Slide 6 _ Slide 7 Slide 8 L all slides Figure 163 Slides selection checkbox Page 112 of 195 www tissuegnostics com 5 13 Stopping a Preview To quit the preview there are two options you can abort the preview of the current slide in progress or the complete preview process To quit the preview of the current slide current selection simply press the Preview button 1 which has turned into a power off button during acquisition TissueFAXS will quit the current slide and move on to the next slide if available ra EY To quit the complete process click the Preview Selected Slide button from the very top panel 9 14 Experiment Editor The Experiment Editor is the drop down menu located at the left side of the application s main window lts purpose is to orga
97. ge 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 informational 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 77 June 2011 11 Document version 3 5 TissueFAXS 3 0 Page 5 of 195 www tissuegnostics com TissueFAXS 3 0 Notices about symbols and labels on product The following symbols appear on the labels of the product NnSSUE FAXS EE amp CECE ANALY ole SYSTEM TissueFAXS is a trademark of TissueGnostics GmbH TissueGnostics GmbH Taborstrasse 10 2 8 C A 1020 Vienna Austria TissueFAXS Workstation TissueFAXS is a trademark of SN TE TissueGnostics GmbH TISSUE GNOSTICS Taborstrasse 10 2 8 y A 1020 Vienna Austria C Figure 1 Product labels Serial number a WEEE symbol
98. gure 27 Enhanced Focus tab 4 3 4 Advanced Focus Application Options Choose the extended focus method for each reflector Default Experiment Settings Default Default Default Default Hardware Settings Experiment Options _ Use Extended Focus C Save Z Stack Save At Figure 28 Advanced Focus dialog TissueFAXS software is shipped with one default extended focus method Default that works for all reflectors Z Stack Z Stack represents a set of images that are acquired above and below the optimal focus position By combining these images a final FOV image will result containing a higher level of details than any of the initial Z Stack Page 38 of 195 www tissuegnostics com TissueFAXS 3 0 Check this option only if you intend to use some external software to fuse the images Be aware that enabling this option will require more space on the hard drive You can choose to use the Z stack for each individual reflector by marking the corresponding checkbox from the Save Z Stack column To be able to use the Z stack for individual channels you must previously check the vf Note Save Z Stack option below the reflectors section Use Extended Focus For each reflector present in the system you can choose the extended focus method used to combine the Z Stack Check this option only when objects are present on multiple focus plans If you don t want to apply the extended focus to one or
99. gure 50 Generic tissue sample The point in the center is the reference object If using a calibration slide place the center of the calibration slide on the live image like in the figure below Page 55 of 195 www tissuegnostics com TissueFAXS 3 0 Stage Control Ls 4 PixeLINK PL 4622C 6220116 Sensor saturation Bl Live Image Figure 51 Calibration slide centered In order to bring up the red cross marking the center of the calibration slide on the live image use the key combination Alt C Do not use images with repeating patterns For instance slides with fluorescence beads might contain many similar objects and auto calibration will fail or return wrong results Ensure the speed of the stage is low e Press Automatic Calibration button The stage will be moved and the software will determine the following The correct flip settings You will be prompted only if the current flip settings are wrong Page 56 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 52 Message warning that the current flip settings on camera are wrong The angle that is formed when the camera is misaligned You will need to manually rotate the camera and rerun the calibration to recheck this The dimensions of the FOV FESUITS Computed Foy size 534 025x406 178 Pixel size 2 996x2 954 FOV size ratio 1 5140 Image size ratio 1 3333 Angle 0 0217290940453051 Do ou wish
100. he for Region 006 Remove Acquired Images Propagate Region 006 to Slide Plugins Export Regions Overviews Compute Stitch Export Annotations Figure 230 Export FOVs Images Option b If you want to export more regions or FOVs simultaneously you can select multiple Y TIP items from the Experiment Editor tree You can also select Experiment item from Experiment Editor tree to export all the regions or FOVs In both cases the following dialog will appear Page 160 of 195 www tissuegnostics com Save to folder lt please select gt Save images Save movie Oniy i he regi n amp acquired with time Janse In case of images acquired with overlap Use stitch Options File name convention Parts Separator Image Format pg Mame Preview Experiment Region FieldOfwiew Filter jpg Please select what type of images you want to save Al 1 BESS eee pr A KAKARA Please select if you want to save images for unselected FOVs Export only FOVs belonging to categories es Mark crop area Color Scale bar Color E o 0 0 e Location Apply Illumination Correction Note Oniy if the correction image is available Figure 231 Export FOVs Images dialog TissueFAXS allows you configure the export as you desire by choosing exactly the contents that is of interest to you In the dialog displayed above you can adjust the following settings e Select the storage f
101. he sample An annotation can also be edited directly on the image viewer it can be renamed its content can be changed or it can be removed To edit an annotation you must use the little black framed square on the contour of the annotation If pressed this little square will display an edit box where you can operate the following actions Figure 210 Edit annotation directly on the sample e Edit the name of the annotation write the desired name in the upper left corner of the box then press Enter key to save the changes e Write a comment in the space below the name you can also erase the existing comments all the changes concerning the notes are automatically saved e Delete the annotation by pressing Es button e Close the edit box by pressing E button Export Annotations please see Chapter 5 20 5 5 17 4 Illumination Correction Shading Correction Occasionally on the acquired images some shades may appear They can be caused by imperfections of any component of the lightpath specks impurities on the camera objective TissueFAXS allows you fixing such shading problems by using the Illumination Correction function In this chapter you will find how to use this feature a You can access the Illumination Correction menu by pressing the Illumination Correction This operation is available only for brightfield experiments button mn from the Region Viewer control To apply illumination correction a c
102. he storage folder for your acquired images e FOV Group Name which acts as a naming template for files to be acquired e Separators inside the filename the character used as a separator should not be used as part of the filename provided by the user e Counter is used to avoid duplicate filenames when pressing the Acquire button multiple times e Channel usually it is the reflector name e Format of the graphics file note Filename Preview section contains the file or the list of files obtained after the o acquisition process At any time you can press the button to verify the validity of the filename in the specified location e g if the name is valid if you have specified an existing folder if you have enough rights etc Intensity Profile An Intensity Profile represents a set of camera related settings that you can save for further use in order make available different combinations of settings for the acquisition process Page 107 of 195 www tissuegnostics com TissueFAXS 3 0 Saves Load Delete Figure 154 Profile Options menu e Save the current camera settings can be saved in order to make them available for further use You must specify a name and a short description for each profile A default name is already generated It contains the camera name and type the objective magnification and the reflector selected on microscope you can also add your own information to the already existing name
103. hence TissueFAXS will build the cache after the acquisition and the stitching are done if stitching option is enabled lf the stitching or the cache is performed at the end of the acquisition process a progress bar will warn the user about the status of the action The progress bar can be found in the status bar of the application If stitching is set to Manual no cache is built Build Cache started for Region 066 Figure 8 Build cache after acquisition when stitch option is enabled Progress bar e When opening a region if Tools Options Actions Use default cache action for regions without cache is checked and Build Cache is set for Default Cache actions lf Use default cache action for regions without cache is not checked TissueFAXS will ask every time when opening a region without cache if you wish to build it jf Note e Using the region contextual menu Build Cache for Region Page 18 of 195 www tissuegnostics com TissueFAXS 3 0 Create Group Acquire Region 001 Remove Remove Regions Clear Cache for Region 001 Remove Acquired Images Propagate Region 001 to Slide Export Plugins Compute Stitch Figure 9 Build cache feature in region contextual menu 4 1 2 Licensing In the Licensing dialog the user will renew the license for TissueFAXS by following these steps e use Save license file button in order to save the license file on your hard drive e send this fil
104. hutter for reflected light RL Shutter is used to block or unblock the light coming from RL lamp This way the light maintains the same intensity compared to on off cycle and the lifetime of the lamp is increased RL Shutter RL Shutter is closed Click the RL shutter icon to open the RL shutter Figure 112 RL Shutter control a RL Shutter RL Shutter is opened O Figure 113 RL Shutter control b 5 5 6 Fluo Arc A Fluo arc is a device used to dim the fluorescent lamp It should be run at 100 or it will flicker and give bad results Page 89 of 195 www tissuegnostics com TissueFAXS 3 0 Fluo Arc control when present Figure 114 Fluo Arc control 5 5 7 White Light Attenuator optional component Adjusts the transmission light intensity using some White Light Attenuator MD 12 filters SA E Figure 115 White Light Attenuator control 5 5 8 Fluorescence Attenuator optional component Fluorescence Attenuator ND 100 Adjusts the reflected light intensity using some filters Figure 116 Fluorescence Attenuator control 5 5 9 Condenser Condenser The Condenser combo box allows you to select the desired contrast method F Note In fluorescence it is recommended to move the front lens out of the lightpath of the condenser wf Note a Phase Contrast PH and Differential Interference Contrast DIC are optional components Bright Field Figure 117 Condens
105. ill be C image 001 bmp ff Note Acquire Single FOV manual procedure Acquiring a FOV means capturing one image in case of multi color fluorescence one image per reflector Reflectors are to be found in the channels list in the Acquisition settings for the current experiment It is necessary to have a current experiment created in order to acquire a single FOV In addition be sure that the camera settings for all reflectors have been properly adjusted before acquiring an image Live Image Browse Capture Acquire Intensity Profile Save A a oe Figure 151 Acquire FOV panel Acquire Clicking on the right side of the Acquire button will pop up the following menu Intensity Profile Acquire Current Foy Advanced Options Figure 152 Acquire FOV menu Acquire Pressing the Acquire button is the same as choosing Acquire Current FOV from the pop up menu Page 106 of 195 www tissuegnostics com TissueFAXS 3 0 le Advanced Options For Acquiring Single FO Save To Folder please select gt Foy Mame FOY Mame Sep Counter E Format File Name Preview Demo BF 001 jpq Figure 153 Advanced Options for Acquire FOV You also have the possibility to adjust the Advanced Options The first time you attempt to acquire you will be prompted to go to the window shown above where you will be asked to provide the following information e T
106. ill be incremented after each load Load settings from profile TissueFAXS will not allow loading settings from profile If the user owns a predefined number of uses the counter will be incremented after each load Camera profiles TissueFAXS will not allow loading settings from profile If the user owns a predefined number of uses the counter will be incremented after each load Build cache for acquired regions If license is not present TissueFAXS will not display compute the cache and it will use thumbnails only If the user owns a predefined number of uses the counter will be incremented after each build Export FOV images TissueFAXS will deny export if license is not present lf the user owns a predefined number of uses the counter will be incremented after each region export Export Region Overviews TissueFAXS will deny export if license is not present lf the user owns a predefined number of uses the counter will be incremented after each region export Export movie for time lapse TissueFAXS will deny export if license is not present lf the user owns a predefined number of uses the counter will be incremented after each region export Export of XML files TissueFAXS will deny use of plug in based on dongle If the user owns a predefined number of uses the counter will be incremented after each export Import regions from XML files TissueFAXS will deny use of plug in based on dongle If the user owns a predefin
107. ill be prompted to manually focus the image when necessary Current position no type of focus will be performed and the stage will maintain for all FOVs the same position during acquisition Autofocus when using this type of focus TissueFAXS will automatically detect the best focus position based on the parameters you can find described below The Autofocus method consists of two steps In step one using a fast method TissueFAXS tries to determine a short interval where the focus is best In step two using a high precision method specified by the Fine Focus Measure parameter TissueFAXS determines the best focus position from the fine focus interval determined in step one Generally in order to determine the fine focus interval TissueFAXS uses the Rough Focus Measure and scans the entire interval specified in the Focus interval section This approach is called Full Focus However in order to improve the speed of the focus if a previous focus position was found TissueFAXS will directly use the Fine Focus on a small interval around the last focus position This approach is called Quick Focus An If the Quick Focus method fails a Full Focus will be automatically performed ote ji Page 35 of 195 www tissuegnostics com The following parameters are for autofocus only and have no effect on the Current position or Manual Focus Rough Focus Measure and Fine Focus Measure For both Rough Focus and
108. images acquisition dialog will appear see above the Reacquire correction image paragraph If you frequently acquire correction image you can set this option as default from Tools Y TIP Options Application Options Remember 5 17 5 Flags In TissueFAXS the flag option is used in the reacquisition of the FOVs when the initial acquisition process has generated unclear or unsatisfying results By using the flags you can modify the shape of your region by adding or removing FOVs Region Overlay Export Plugins a a 1 O O 7 Add PolyLine Subregions Page 149 of 195 www tissuegnostics com Figure 216 Region Viewer Toolbar Reacquire Flags In order to view and manage the flags you must first ensure the Set Flag button I from region viewer toolbar is pressed 4 To add flags you have two options e Directly left clicking on the FOV you choose for focus e By pressing Set Reacquire Flag from the contextual menu that appears when right clicking on the FOV chosen for focus For both adding flags methods the selected FOV will be set as a focus point and all its neighboring FOVs will be selected for reacquisition as well The size of the neighboring area is the size specified by Tools Options Scan Settings Focus Focus Strategy Neighbors To remove flags you have three options e Directly left clicking on the FOV e By pressing Clear Reacquire Flag from the contextual
109. ing thumbnail This gives the user a visual overview of all regions of interest in the experiment that can be acquired later Page 114 of 195 www tissuegnostics com TissueFAXS 3 0 Acquired Acquired with time lapse No Acquisition date 4 16 2010 2 17 PM Area 7 7912 mm Area in ROI 0 0000 mm E Comment write a comment FOY matrix size 6 6 Width 6 Height 6 El FOY size 937 907 402 342 Width 537 907 Height 402 542 Induded in acquisition Yes Name Region 002 El Patient Name write patient s name a TuT irene Bae a y sal a ee Te Figure 165 Thumbnail for regions Grouping Regions This is one important feature of the Experiment Editor allowing regions to be grouped together This is particularly useful when you wish to distinguish multiple regions of interest that exist on the same slide For example you may have two patients tissue samples on the same slide and want to organize your regions images into a group for each patient To create a group right click on the Slide in the list and select Create Group Once created the group can be renamed and regions can be dragged and dropped into the respective group Page 115 of 195 www tissuegnostics com TissueFAXS 3 0 Acquire Slide 1 Remove Remove Regions Build Cache for Slide 1 Clear Cache for Slide 1 Remove Acquired Images AS Slide 2 E Slide 3 E Slide 4 Export E Slide 5 Plugins Propagate Slide 1 to Slide Compute Stitch
110. ions defined within the Region Viewer having the same properties as the regions from the Slide Preview You can see them in the experiment editor and they can be acquired just like normal regions The advantage of the subregions is to ensure higher flexibility and more detailed analysis on a more detailed area of tissue To add subregions go to Region Viewer Toolbar Subregions Show Defined Regions only Show Defined Regions should appear checked TissueFAXS 3 0 Page 139 of 195 www tissuegnostics com TissueFAXS 3 0 All Categories Tumor Normal Adjacent Tissue Manage Categories Manage Categories Figure 201 Add Subregions context menu Now choose the subregion shape from the toolbar O O 7 Subregions 7 Then add the desired subregion on the viewer using the mouse just like for any normal region see Chapter 5 16 1 To be able to see all subregions you have added simply check Show Defined Regions option from the Region Viewer Toolbar 5 17 2 Categories Sometimes you may want to emphasize certain small areas on your region that could contain high interest information for your research These areas can be exported for analysis or they can be used as a highlight tool For instance a tissue may contain both tumor areas and normal adjacent tissue non tumor areas For each type of area e g tumor non tumor you may create a category in order to highlight that particular area on the image Using categori
111. isable any focus validation and as a consequence any images returned by the focus algorithm will be accepted as being in focus lt should be used only as a last solution when the sample has a very low contrast Maximum allowed score increases the tolerance of the focus validator Setting low values means that only very good images will be accepted as being in focus Setting high values means accepting lower quality images e g blurred Basically this parameter should be increased when using samples of a lower contrast Fidelity of details pixels size of relevant details If you set it to minimum 3 then finest details are searched for and will make a difference in the validator score lf you set to a higher value then only rough elements will count in computing the score However this depends on the images you are processing Most of the time the default value should provide good results The Load default settings button restores the default options only for the focus validator settings Focus Interval In this dialog you can specify the z boundaries of the rough focus search You can adjust your focus search interval to match extra thick or thin coverslips or culture plates To determine the proper focus interval we recommend manually focusing with the objective of interest You can then check the current Z position in the camera window and add 250 um above and below this position for the upper and lower Z positions of your int
112. king the respective point Add Region Remove Selected Items Remove Acquired Images Go To This Slide Go To This Point Acquire Slide 1 Acquire Selected Items Build Cache for Region 039 Clear Cache for Region 039 Select All Preview Area bel up Label Down Reset Focus Point Show Position on Slide Show Preview Info Export Slides Images Copy Paste Compute Stitch Page 131 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 189 Set as preview focus point context menu e Reset focus point use this option to reset the focus point to the default position center of preview area Add Region Remove Selected Items Remove Acquired Images Go To This Slide Go To This Point Acquire Slide 1 Acquire Selected tems Build Cache for Region 039 Clear Cache for Region 039 Select All Preview Area Label Up Set as Preview Focus Point abel Donn Reset Foes Pointe Show Position on Slide Show Preview Info Export Slides Images Copy Paste Compute Stitch Figure 190 Reset focus point context menu You can reset the focus point for the current slide or for all slides Rid The focus point is reset for Current slide O All slides Do not show this dialog again Figure 191 Reset focus point dialog Page 132 of 195 www tissuegnostics com 5 16 6 Export this slide image To export a slide s preview image select Export this slide image from the me
113. l calibration for other objectives i select Automatically Compute At any time you can press Cancel if you don t want to save the changes Page 60 of 195 www tissuegnostics com TissueFAXS 3 0 Calibration Camera PixeLINK PL 4622c 6220116 Automatic Calibration Objective 20x Air 213233 675 FOV 213708 526 39496 131 Righ Bokttorn 39597 2769 Width 904 036 Height 400 527 Edit Speeds ma 213501 100 um Ws 39696 700 um El 645 975 um O promaticay compute calibration For other objectives i Save amp Exit DP cancel Figure 58 Field of View Calibration control panel final operations 4 7 Users In order to use TissueFAXS you must be authenticated by owning a username and a password 4 7 1 View Users To manage the users you must be logged in as Administrator In TissueFAXS there is only one Administrator and a set of users who may be managed in the application when the Administrator is logged in From the main menu choose Admin then Users and a panel will appear Page 61 of 195 www tissuegnostics com TissueFAXS 3 0 Real name Administrator Description Figure 59 Users dialog 4 7 2 Adding Users Click on the Add user button and you will get the following panel User Name Password SY SY Re type password Description Role User Figure 60 Adding Users dialog Here you can fill necessary information concerning th
114. l Up Label Down Show Position on Slide Export Slides Images Copy Paste Compute Stitch Figure 261 Grouping TMA spots a Time Lapse acquisition mode is not available for TMA samples Therefore the vf note Acquisition Information dialog that appears before acquiring TMAs will display a Time Lapse acquisition option disabled and a message that warns the user about this fact see image below Page 185 of 195 TissueFAXS 3 0 www tissuegnostics com You are about to perform Acquisition Please read the following summary before proceeding You have 24 regions to be acquired with a total number of 144 FOWs arning You have selected TMA Spots to acquire The time lapse acquisition fe A samples is not supported Actions _ Shut down after finish _ Acquire correction image Time lapse acquisition Maximum estimated dime per nun 0 4 35 Figure 262 Acquisition Information dialog acquiring TMAs Page 186 of 195 www tissuegnostics com TissueFAXS 3 0 7 Print Printing your TissueFAXS project will prove to be very useful at some point especially for the fact that the software will not simply print your project but you will be able to decide which information the report has to contain and how the data will be displayed 7 1 Print Options You have two options to access the Print option and the Print Preview option e By pressing Print Print Preview e By accessing File
115. lained in the following tutorials Ata glance TissueFAXS provides e Fully automated slide scanning e Autofocus up to 63x Oil objective e Multi channel acquisition for fluorescent imaging e High resolution image overview by TissueStitching e Freely selectable areas of interest e Channel overlay and data storage Features of TissueFAXS automated image acquisition e Slide readout e Pre scanning to identify tissue sections smears and biopsies e Multi slide management and stage movement e Self adjusting color and monochrome CCDs e Automated objective lens and filter block handling e High speed and high precision autofocus e TissueStitching for overviews of entire sections e Time Lapse acquisition e Channel overlay and data storage TissueFAXS rapidly yields high resolution images of tissue sections for subsequent analysis in HistoQuest or TissueQuest the cell analysis software for brightfield and fluorescent images respectively The process of image acquisition to data analysis and quantification are streamlined with the TissueGnostics software combination Quick and easy data analysis in HistoQuest and TissueQuest is accomplished with these key features e Powerful algorithms for image processing e Automated identification of cells e Analysis of staining intensities e FACS style dot plot operations e Backward connection from dot to cell Page 7 of 195 www tissuegnostics com TissueFAXS 3 0 e Storage
116. le on each page of the report 7 2 Print Items If you choose the Print option a dialog will appear that permits to choose the printer If you choose the Print Preview option you will be able to see a preview report Print date 04 June 2009 16 05 41 Ay i TISSU E oO ECAC A MOSTICS o Fk O U E Demo _BF TissueFAXS Report File Hame Demo BF aqproj Experiment Type Brightfield Experiment Description Product Version Location C TissueFAXS Projects Demo BF Preview Objective EC Plan Neofluar 7 5x 0 0 75 M27 2 5 Air Acquisition Objective EC Plan Neofluar 70 0 50 M27 20x Air Camera PixeLINK PL A622C 6220116 Figure 264 Preview report example first page The preview report contains the following e Experiment Name File Name e Experiment Type e Experiment Description e Product Version the TissueFAXS version used to print this info Page 188 of 195 www tissuegnostics com TissueFAXS 3 0 e Location the location of the experiment e Preview Objective the objective lens used for the preview operation e Acquisition Objective the objective lens used for acquisition e Camera the camera used for this experiment e Each Slide selected in the list Slide Name Slide Image Content Type Comments the comments referring to this slide Objective the objective lens used for preview e Slide Preview Channels a table that contains the channel list for the current slide and some
117. les can be assigned in two ways e When creating a new user choose one of the three available options from the Role dropdown box User Name Demo User Real Name i Password Re type password Description Role Administrator Figure 64 New User assigning role When modifying a user choose one of the three available options from the Role dropdown box Page 64 of 195 www tissuegnostics com TissueFAXS 3 0 Real name Demo User Description User Technician Administrator Figure 65 Modify User assigning role The following user roles are available 1 User e This role cannot modify wide system settings like FOV calibration or scan strategies 2 Technician e This role can modify wide system settings like FOV calibration e Inherits all the rights of the User role 3 Administrator e This role can modify the list of users and their roles e Inherits all the rights of the Technician role List of settings and the minimum access level If using the Role Based Administration feature some settings will be available only for Administrator or Technician Please see table below for more details Access Level access Options Default Experiment Settings Technician Disabled Storage Raw Data Options gt Scan Settings Scan Strategy Disabled Options Scan Settings gt Speed Disabled Options Application Options Actions Technician Disabled Move To a Safe distance Options Applic
118. les having many bright areas sensor saturation Max allowed value Figure 145 Sensor saturation Gamma adjusts the contrast of the image You can use this to highlight the differences between the light and dark areas or in order to minimize these differences Always force on off at startup Figure 146 Gamma control Page 102 of 195 www tissuegnostics com TissueFAXS 3 0 5 8 Camera Panel The Camera Panel one of the essential tools in TissueFAXS contains the stage control the camera controls and a panel where controls for the microscope can be dragged This panel containing controls for the microscope proves to be very useful as it allows you to have ready at hand exactly the controls you need at the moment If you want to remove the controls from the camera window drag them into the dustbin above You will be able to drag them again from the microscope panel if needed later 5 bis Con Erte A a 2 a AAA y fia i ge T ETRO un EM A POELE FLAMADO 220l ii n a a named t Te Shon tirir har Sensor Sara bon Bs lor aage Sapte Aspa v Intereey Profle I Save X Figure 147 Camera Panel 5 8 1 Stage control This panel helps you to accurately control the movement of the microscope s stage Stage Control Al 2 o400 400 um Y 526858 400 um zi TE Mo Figure 148 Joystick control Stage control components e Joystick control The four a
119. lide Please choose what name to display in application for reflectors gt Full Name 0 Short Name Abbreviation Figure 21 Reflectors dialog e Use inverted image for Transmission channel in case of fluorescence experiment choose how the transmission image will appear in the overlay image the normal image or the inverted image of the Transmission channel which means a fluorescence like appearance e Use white in preview overlay of fluorescence slide for fluorescence experiments if only one channel is present in the preview settings the default color for that channel is white If the preview is made on a nuclear marker like DAPI and is displayed in dark blue the resulting preview image will be difficult to see against the black background White however gives a stronger contrast e Finally you can choose how the name of the reflector will be displayed in the application Full name Short name or Abbreviation see also Chapter 5 5 2 4 2 3 Storage In the storage section you can define various data for the file to be stored like the file names convention the file types and the default locations Page 30 of 195 www tissuegnostics com 1 Options Application Options Scan Settings Hardware Settings Experiment Options Default Experiment Settings Es EJ Ls File Mame Convention Mame Parts Parts Separator Image Format Name Preview Experiment Region FieldOfView Filter jpa
120. ls the smallest value for which the High background is green and the highest value for the upper parameter for which the Saturation indicator is green Page 96 of 195 www tissuegnostics com Lower Upper Sensitivity Threshold Lower J 00 Advanced Options Figure 128 Lower Upper parameters controls You can automatically adjust these parameters by pressing the Auto button By choosing Advanced Options the following dialog will appear JE Parameters Detect Lower Upper Limit Parameters Undersaturation Percent 0 00 2 00 Detect High Background Parameter UnderSaturation value Saturation Percent 0 00 a DO Detect Saturation Parameter Saturation Value Figure 129 Options for detect Lower Upper Limit Parameters control panel The advanced options control how TissueFAXS detects the presence of oversaturation and undersaturation lack of signal in the camera image UnderSaturation Percent If the number of pixels with intensity value higher than the under saturation limit is over this percent of the total number of pixel then the image is considered undersaturated Saturation Percent If the number of pixels with intensity value higher than the over saturation limit is over this percent of the total number of pixel then the image is considered oversaturated UnderSaturation Value The maximum acceptable intensity value for witch a pixel is considered undersa
121. ly for Brightfield experiments The correction image will be applied to exported images if the correction image is available p Restrictions regarding the images to be exported wf Note For all export formats excluding tiff with multiple tiles the image size must not exceed 80000000 pixels For tiff with multiple tiles the uncompressed size must not exceed 2000 Mb Export Overviews from Categories To export categories first check Export only overviews from categories You should also select the types of categories to be exported by pressing the button The following dialog will appear listing the available categories Page 165 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 236 Export subregions Select categories In the lower part of the Export Region Overview dialog you will now see a section including all the overviews that will be exported For each overview the name and the image size is displayed Page 166 of 195 www tissuegnostics com TissueFAXS 3 0 Save to folder lt please select gt Save images Custom part fo Name preview Experiment Region Subregion jpg eden Save movie Upton available only for regions acquired with time lapse Options Options for tiff with multiple tile 20 so 100 O Export regions overviews Export only overviews from categories Enable option O I want to stitch thumbnails I want to specify a custom size for images t
122. menu that appears when right clicking e By pressing Clear All Reacquire Flags from the contextual menu that appears when right clicking and then choosing one of the three available options For Current Region For Whole Slide For Entire Experiment Set Reacquire Flag Clear Reacquire Flag For Current Region Reacquire This FOV Only For Whole Slide Reacquire Flags For Entire Experiment Go To This FOV Flag Entire Row Flag Entire Column Clear All Add Flags Clear All Remove Flags Insert Row Insert Column Acquire New FOVs Remove Subregion Figure 217 Clear All Reacquire Flags context menu TissueFAXS 3 0 r For the first two remove flags options you may notice that by clicking on the focus rote flag all neighboring flags will be removed including the focus flag itself However by clicking on a neighboring flag non focus flag only this flag will disappear Reacquire FOVs Page 150 of 195 www tissuegnostics com TissueFAXS 3 0 e If you choose the Reacquire This FOV Only option only the current selected FOV will be acquired e f you choose the Reacquire Flags option you have three choices For Current Region For Whole Slide For Entire Experiment et Reacquire Flag Clear Reacquire Flag Clear All Reacquire Flags Reacquire This FOV Only Go To This FOV For Whole Slide Flag Entire Row For Entire Experiment Flag Entire Column Clear All Add Flags Clear All Remove Flags Insert
123. meter to correct the orientation of the image if the left right or top down of the camera are different that those of the stage This parameter is generally adjusted when the system is installed The FOV calibration feature is able to automatically set this parameter to its correct value Figure 142 Flip Image control Page 101 of 195 www tissuegnostics com TissueFAXS 3 0 Rotate Image this parameter is used to rotate the captured image Use this when the camera is placed in a 90 180 270 degrees position in relation to the stage Rotate Image None 90 Diso 270 Figure 143 Rotate Image radio buttons Gain artificially adjusts the intensity of pixels by software multiplication of pixel values Figure 144 Gain control Sensor saturation This set of parameters has no effect on the final images This sensor is used only to warn the user of possibly over saturated pictures that can later affect the illumination correction process The two parameters are Max allowed values if you have a very bright image that is considered oversaturated the sensor turns red you can increase this value in order to make the sensor accept it Sensor saturation area if the number of bright pixels with a value above maximum allowed is lower than Sensor saturation area the image is considered to be fine otherwise it is considered oversaturated Generally you might want to increase this value when working with samp
124. more reflectors choose None option from the reflector s dropdown list Figure 29 Reflectors Extended Focus dropdown list fn To be able to use the extended focus for individual channels you must previously F Note check the Use Extended Focus option below the reflectors section Using any of the two options Use Extended Focus Save Z Stack will significantly increase the acquisition time l CAUTION 4 3 5 Speed In the speed dialog box you can adjust the speed of stage movement during acquisition These settings do not affect the speed settings of your joystick Generally all speed values can be set to maximum However if you are working with well plates or cell cultures in general you may want to reduce these values in order to avoid troubling the content The Acceleration parameter describes how fast the stage will try to reach the defined speed Page 39 of 195 www tissuegnostics com TissueFAXS 3 0 Magnification xv speed XV Acceleration ZSpeed ZAccdleraton e scan Strategy Stitching Focus Advanced Focus Save AB Figure 30 Acquisition Speed dialog 4 4 Hardware Settings 4 4 1 Camera Manager This option allows you to add and remove a camera from the list of the cameras connected to the computer It also helps you setting the current camera For more details please see Chapter 5 7 of the current manual 4 4 2 Contrast Manager The Contrast Manager is an automated featur
125. n enabled this option will display the scale bar on the live image Display RGB value Show RGB value when enabled this option will display the RGB value on the live image Advanced Settings In order to access the advanced mode right click in the area where the camera parameters are Ad d Mod The following menu will appear dieta The Advanced tab will appear when checked Frame rate the maximum number of images captured per second Page 100 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 137 Frame Rate control Pixel Addressing this parameter is used in order to decrease the size of the captured image and decrease the time needed for the capture operation This will increase overall acquisition speed but the image quality will be affected reduced Pixel Addressing Decimate Mone Figure 138 Pixel Addressing combo box Color Temperature this parameter is similar to white balance making the picture look colder or warmer Color Temperature K Figure 139 Color Temperature control Pixel Format the format used when transferring images from the camera only experts in imaging should change the settings Fixel Format BAYERO GRAB Figure 140 Pixel Format combo box Resolution this is the size in pixels of the captured images Resolution 1600 x 1200 Figure 141 Resolution combo box Flip this parameter is used to flip the captured image You can use this para
126. n see the previously opened experiments by other users see Chapter 4 7 6 and you can open any experiment by double clicking on it You can delete items from the Recent Experiments dialog by selecting desired item s ad pressing Delete button from your keyboard You can also use the contextual menu that appears when right clicking on any item in the list Open Remove selected Remove All Remove All User Lists Figure 67 Recent Experiments contextual menu TissueFAXS 3 0 Page 66 of 195 www tissuegnostics com TissueFAXS 3 0 Demo User F Frojects TissueFAXS Demo _Project Demo Project agproj M FProjects TissueFAXS Demo _BF TF 132 BF agproj Figure 68 Recent Experiments dialog 5 1 1 Experiment Creation Its easy to create a new experiment as TissueFAXS will guide you through a 5 step user friendly wizard e To begin creating a new experiment follow the steps below Go to File New menu Page 67 of 195 www tissuegnostics com TissueFAXS 3 0 Save as template Acquire Print Print Preview Slide Manager Properties h a Close Exit Figure 69 New Experiment menu Or go directly in the toolbar Figure 70 New Experiment toolbar At this point you have two possibilities to create a new experiment Experiment Creation How you want to create the new experiment New empty experiment From existing template experiment Figure 71 Experiment Cre
127. ner e Press Top then Right Page 51 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 47 Manual calibration a e Using the joystick move the reference point from the upper right corner to the lower left corner Page 52 of 195 www tissuegnostics com TissueFAXS 3 0 u J T Pt Ld i if q Pi AAA A Mal Y lea had Pe Le oe tomare T pl red jale ve Cn hom cala ba Sree PGE at se ii y Figure 48 Reference Point Lower Left Corner e Press Bottom then Left At this point the Width and the Height values will automatically be computed e To save the computed values press Save and Exit Page 53 of 195 www tissuegnostics com TissueFAXS 3 0 Camera PixeLINK PL 4622C 6220116 Objective 20x Air Left 212945 555 Right 213477 045 Top 39707 S Bokktorn 40109 438 i width 533 490 y Height ge 402 076 pl 39506 400 um Figure 49 Manual calibration b If you already know the FOV size you can introduce the Width and Height values by Y TIP yourself in their corresponding fields 4 6 2 Automatic FOV Calibration e Choose a reference point on the sample For a good evaluation of the reference point please make sure the whole object is in focus Page 54 of 195 www tissuegnostics com TissueFAXS 3 0 Stage Control Fi x 70707900 pm 7 m y 2434300 pm 2 427 018 ym Fi
128. nize your slides regions of interest and acquired images into one complete list Regions of interests are displayed as thumbnails images in this list see the image below TissueFAXS 3 0 Page 113 of 195 www tissuegnostics com MC m jm Demo_Project Acquired Yes Acquired with time lapse No Acquisition date 4 16 2010 2 17 PM Area 7 7912 mm2 Area in ROI 0 0000 mm Comment write a comment El FOV matrix size 6 6 Width 6 Height 6 El FOV size 537 907 402 342 Width 937 907 Height 402 342 Induded in acquisition Yes Name Region 002 El Patient Name write patient s name Reference number write reference number Region type Rectangular Storage directory F Projects TissueFAXS Demo El Time lapse Time lapse 00 30 00 Time Regions Time Regions Total magnification 20 Type Region E stitch Stitch computed Mo Support stitch None as Slide 1 Number of runs 239 Region 002 5 Region 006 TissueFAXS 3 0 Figure 164 Experiment editor Experiment with slides and regions From top to bottom the editor consists of three main parts a tree like representation of the experiment a property grid and a description area Experiment tree like representation In TissueFAXS you can draw a region on your preview image with the rectangle circle or custom draw tool as described in Chapter 5 16 1 The Experiment Editor will automatically display each new region in the tree list along with its correspond
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130. nt manual Transforming regions from categories into subregions for reacquisition If the user needs to reacquire the tissue area from a category it is possible to transform it into a Defined Region and then perform reacquisition In the region viewer right click on the border of a category and from the contextual menu choose Transform into Defined Region option The category will transform into a Defined Region it will disappear from the region viewer and it will be visible in the experiment editor so it can be reacquired TissueFAXS 3 0 Page 142 of 195 www tissuegnostics com Demo category 1 0 0815 mm2 ay 4 gt j dl il gt p a Right Click MA Set Reacquire Flag Clear Reacquire Flag Clear All Reacquire Flags Reacquire This FOV Only Reacquire Flags Go To This FOV Flag Entire Row Flag Entire Column Clear All Add Flags Clear All Remove Flags Insert Row Insert Column Acquire New FOVs Remove Demo category 1 Figure 206 Transform region from a category into a subregion for reacquisition 5 17 3 Annotations Subregions Press the Subregions button in order to access the Subregions contextual menu The following options will be available Show Defined Regions All Categories Demo 1 Demo 2 Manage Categories Show Annotations Manage Annotations Export Annotations Figure 207 Annotations contextual menu Show Annotations To add annotations go to Region View
131. nu You will be prompted to choose the destination directory and the encoding type for the image For more details concerning this feature please see Chapter 5 20 4 of the current manual 5 16 7 Automatic tissue detection After the preview is done TissueFAXS is able to auto detect the tissue regions on the slide This is a great tool provided by TissueFAXS that can help you save time and improve the accuracy of the tissue detection In order to detect generic tissue samples please make sure that the Content Type property of the slide is set to Generic Then press Detect Tissue button from the slide editor toolbar The following dialog will appear Detection Parameters Mmm Titus Ares Repons Debected Reg ITISSUEGNOSTICS Figure 192 Tissue Detection panel By default the detection is run on the entire preview image If you want to refine the results you can run the detection on a smaller area TissueFAXS 3 0 Page 133 of 195 www tissuegnostics com TissueFAXS 3 0 e select the desired area by drawing it on the displayed image e click on Run Selection If you want to cancel selection press the Clear Selection button Detection Parameters In order to refine the results of the automatic detection TissueFAXS provides a set of parameters you can adjust according to your needs in order to obtain the desired results e Minimum Tissue Area the smallest area a tissue region can have All the regions with
132. o see the live image or not e Capture an image with the current camera the main purpose when using the camera e Adjust the settings for the current camera in order to obtain the desired results in preview or image acquisition you are able to constantly adjust your camera settings 5 7 4 Remove Camera You can remove the camera you are using any time you need To perform this operation follow the steps described below e From the Tools menu select Options to open the Options dialog box e Inthe Options dialog choose Hardware Settings Camera Manager e Select the camera you want to remove from the combo box and press Remove camera button Q If the camera is not present in the combo box its removal is no more necessary TIP 5 7 5 Settings for PCO PixelFly Camera The settings available for both cameras are divided in two categories basic settings and advanced settings Basic settings are the most frequently used while advanced settings are rarely used usually when the system is installed or in special conditions as described in sections below Changes in the Advanced Settings menu should only be done by informed users CAUTION understanding the relevance of these controls Changing these parameters in an unqualified way may cause TissueFAXS to malfunction Basic Settings Exposure Time controls the amount of time available to the sensor for collecting light to form an image In Assync Shutter Mode
133. o stitch 2 so 100 Mark crop area Color C 255 255 255 Scale bar Select desired Color MW O 0 0 categories Location BottomLeft Include categories oe pee subregions to C Apply Illumination Correction Only if the correction image is available del A X ineme estes ee 04 Eta a m Region 006 Demo 1 ERE 1742 EZ 1368 1668 N Region 006 Demo 2 Width 3484 Height 26 18 27363336 27363336 m Region 006 Demo 3 Width 1742 Height 3927 20522502 20522502 Figure 237 Export Region Overview export subregions 5 20 3 Export One FOV Image You can access this option by pressing the Export button from the One Image Viewer toolbar a Q Ka Overlay port gt Le O be 3 Figure 238 One Image Viewer toolbar The following dialog will appear Page 167 of 195 www tissuegnostics com TissueFAXS 3 0 Save to folder lt please select gt Files Save images Save movie Onik i the region is acquired wif ime pose In case of images acquired with overlap Use stitch Options File name convention Name Parts Parts Separator Image Format jpg Name Preview Experiment Region FieldOfView Filter jpa Please select what type of images you want to save Overlay Mark crop area Color MW Black Scale bar Color E o 0 0 Location BottomLeft Figure 239 Export single FOV Image dialog Select the storage folder Select files to
134. older e Select files to be exported Images Movies option available only if the region was acquired with time lapse Use stitch e Select the file name convention Name Parts Custom Part Parts Image Format Name Preview TissueFAXS 3 0 Page 161 of 195 www tissuegnostics com TissueFAXS 3 0 e Select the type of images you want to save Overlay The FOV images will be composed from all the channels as currently specified in region viewer Original The FOV images will be exported separately for each channel as they were acquired e Select what images will be saved All Flags Only and Flags with Neighbors these options are available only if you have at least one flag set e Invert selection Only the FOVs not marked with flag or neighbors of the flagged FOVs applies if Flags with Neighbors is selected are exported e Export only FOVs belonging to categories in this case only the FOVs that belong to the selected categories are exported by pressing the browse button les Select Categories Esl Figure 232 Export FOVs Categories included In the selection list only those categories will be listed that contain tissue areas on a Not b i gt cdas their overview image e Mark Crop Area it marks the contour of the region for FOVs near the region border you can also select its color e Scale bar please see Chapter 4 1 2 of the current manual e Apply Illumination Correcti
135. ols are available e Zoom in and Zoom out buttons allow zooming the image Another option would be to type the desired zoom value in the zoom editor e View Original Size displays original size of the image e View Best Fit displays the entire region 5 16 8 Slide Overlay Located in the toolbar the Slide Overlay button iy will allow you to view your preview image for each reflector channel in fluorescence experiments Check the desired reflector in the panel shown below in order to obtain the desired preview iea are Ter E gt Ayo EEE 0 0 255 Rhodamine MN 255 0 0 Co E Figure 197 Slide Overlay Dialog 5 16 9 Custom Selection Custom Selection button AD allows the user to freely draw a selection over the sample in order to select the desired group of ROIs This feature helps avoiding multiple clicks for one by one selection of the desired items Page 136 of 195 www tissuegnostics com TissueFAXS 3 0 Los Region 002 2 Region 003 ad Region 004 d L3 2 Region 006 23 Region 007 3 Region 008 Alia Region 009 E Slide 2 J Slide 3 E Slide 4 E Slide 6 Figure 198 Custom Selection 9 17 Region Viewer A region is an array of FOVs disposed in a matrix structure Each FOV has its own position within the matrix which represents the number of rows and columns The FOV Matrix Size property of the region represents the number of rows
136. ombination from the keyboard e Paste Region this operation can be done by choosing the Paste option from the region contextual menu or by using the Ctrl V combination from the keyboard Q Itis possible to select multiple regions to be copied by using the mouse selection TIP Page 127 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 184 Multiple selection of regions to be copied 5 16 4 Preview Area A region can be set as a preview area for future preview operations in the current experiment Qe vy Set Region as Preview Area Save Region as Preview Schema Add as Rectangular Region TISSUEGNOSTICS Figure 185 Set new preview area context menu Page 128 of 195 www tissuegnostics com If the preview area chosen in the experiment setup needs to be adjusted a rectangle can be drawn on the slide clicking the left mouse button to accept a drawn rectangle Right clicking within the selection will allow doing the following e Set region as preview area set current region as preview area for current experiment This will affect all subsequent slides in this experiment e Save region as preview schema saves current region as preview area for future use and set it as preview area for current experiment You can give a name to this preview scheme and store it to use it again in future experiments e Add as rectangle region creates a region that will be acquired later Add Region Remove Selected It
137. on this option is enabled only for Brightfield experiments The correction image will be applied to exported images if the correction image is available 5 20 2 Export Region Overview You can access this option from e Region viewer gt Export Region Overview e Experiment Editor Region Context Menu Page 162 of 195 www tissuegnostics com If you want to export more region overviews simultaneously you can select multiple Y TIP items from the Experiment Editor tree The following dialog will appear Save to folder lt please select gt Files Save images Custom part Name preview Experiment Region Subregion jpg Extension ipg Save movie pion available only for regions acquired with Gime lapsej Options Options for tiff with multiple tile idth 256 Height 256 _ v None y O Export regions overviews Export only overviews from categories 3 1 want to stitch thumbnails I want to specify a custom size for images to stitch 2 100 Mark crop area Color C 255 255 255 Scale bar Color HO 0 0 Location BottomLeft Include categories Categories 20 L Apply Illumination Correction Oniy if the correction image is available Region name sid Image size pixels Uncompressed size Mb Estimated size Mb p Region 006 Width 8710 Height 6545 Figure 233 Export Region Overview dialog Some information is displayed for each region name size uncomp
138. orrection image is required mandatory Page 145 of 195 www tissuegnostics com TissueFAXS 3 0 The correction image is an image computed in order to store information about the shades in the light path By applying this image to a certain region the shades will be eliminated and the images will be uniformly illuminated La Illumination Correction Select Correction Image Reacquire Correction Image Apply this Correction Image to Entire Experiment Figure 211 Illumination Correction menu The Illumination Correction menu contains the following items e Illumination correction choose this option to automatically apply the correction image to your region If there is no correction image available you will receive a message box that offers you three possibilities Compute a correction image using the already acquired images this is the solution when a correction image is not available and reacquiring the correction image with the same settings hardware as the region is not possible anymore Acquire a correction image this is equivalent to Reacquire correction image from the Illumination correction menu Cancel the whole process of illumination correction TissueFAX5 The region has no correction image What do you wank to do Figure 212 Compute correction image message box e Select correction image by choosing this option you will be displayed a dialog where you can choose from the listed regions cont
139. ot be visible You can select more than one channel for the preview operation because each slide can be scanned multiple times once for each channel in the list that will yield the final overlay image For each channel you may adjust the camera settings light intensity exposure time colors etc by pressing the View button next to each reflector in the list Make your adjustments in the new window that appears and press Save When all your channel settings are saved you are ready to acquire your preview image It might be an option to make the preview for fluorescent experiments in darkfield illumination 5 3 Acquisition Settings Acquisition settings can be accessed by clicking on the Acquisition tab in the upper left corner of the main TissueFAXS window If the Acquisition tab is not visible you can make it visible by pressing the Settings button from the application s toolbar iPr ea Acquisition Objective eerie 2 Ai se Figure 83 Acquisition tab Acquisition settings are similar to the preview settings except for the fact that they act Y TIP when acquiring regions The list of objectives which may be used for acquisition is not limited as for the preview An important difference is that you can select the channel for auto focus in the acquisition workflow Page 76 of 195 www tissuegnostics com TissueFAXS 3 0 Acquisition Objective Acquisition Channels Preview e a o 20 Ar
140. ounting e number of positive and negative cele and for quanticaton of staining intensities TissuelAXSnlus is used for the siandardizaton of issue analysis in cambinatoa User Name Administrator Password Copyright 2006 2011 TissueGnostics GmbH All rights reserved Figure 2 Login Panel Page 13 of 195 www tissuegnostics com TissueFAXS 3 0 3 2 Splash Screen The Splash Screen appears while TissueFAXS loads and establishes communication with the hardware components microscope stage cameras TISSUE GNOSTICS HEDICAL e BIOTECH BALUTIOAW ES Connecting to microscope Figure 3 Splash Screen 3 3 Device Calibration To start an image acquisition you must choose to calibrate the stage when starting the application You may choose to skip this step if you simply want to view previously acquired images in TissueFAXS If you later decide to perform acquisition you will be prompted to calibrate the stage at that time Stage calibration is important for acquiring high quality stitched images as well as the protection of the hardware components from physical damage During the calibration the application finds the hardware limits with extreme precision You will see the objectives and stage moving automatically as the system measures the coordinates of the stage in relation to each objective in nanometers To initiate the calibration procedure press Continue Page 14 of 195 www tissuegnostics com Tissu
141. pe depending on the region type as described below For all the regions these points can be seen when the mouse hovers above the Y ne region For Custom Regions only the currently selected point can be moved it appears marked by a little black cross like in the image below so you have to modify all desired points one by one in order to reshape the region Figure 181 a Resize move point custom region b Drag amp Drop operation to move the point For Rectangular Regions the entire rectangle is resized when you move the selected point it appears marked by a little black cross like in the image below Page 126 of 195 www tissuegnostics com TissueFAXS 3 0 Figure 182 a Resize move point rectangular region b Drag amp Drop operation to resize the rectangle Likewise Circular Regions are resized by moving the selected point it appears marked by a little black cross like in the image below Figure 183 a Resize move point circular region b Drag amp Drop operation to resize the circle e Remove Region right click on the region and choose Remove Region e Remove acquired images this option will erase all the acquired images and associated files from the storage area Notice the fact that this option is available only for acquired regions 5 16 3 Copy and Paste region e Copy Region this operation can be done by choosing the Copy option from the region contextual menu or by using the Ctrl C c
142. rameter if compared to HP the Resolution Its default value is 25 and is related to the size of the relevant objects in the image Setting this value lower translates into higher focus sensitivity also for small objects Focus Strategy Neighbors This parameter can be adjusted according to the flatness of your tissue section as the focus directly depends on the subsidence of the tissue If the tissue section would have been perfectly flat a single focus for its entire surface would have been enough But more often each tissue section has its own relief and the focus must be performed repeatedly The higher the magnification will be more the relief will influence the focus The Focus Strategy Neighbors helps you save time when performing the focus by avoiding unnecessary focusing The regions to be acquired will be split in blocks of Fields of View The Focus Strategy Neighbors parameter defines how many FOVs will share the same focus estimation In the actual example 3x3 is selected in which case TissueFAXS will focus once for every block of 3X3 fields of view The location of the focus point within the block is determined based on the Focus Points parameter After focusing in one point TissueFAXS will scan all neighboring FOVs using the same focus position e Focus Points Fixed Focus Points the focus is performed on the FOV from the center of the block determined by the Focus Strategy Neighbors Fixed Focus Point is the most frequ
143. regions created by TissueFAXS will match the actual well plates on the stage Additional Parameters select template Aditional Parameters E Additional Parameters Instance parameters Alpha Stage X OriginOFFset Stage Y OriginC FFset Cancel Figure 39 New stage configuration Additional Parameters dialog 7 In case of slide inserts the distance is computed between the 0 0 point of the stage ote and the first slide on the insert Once you re done you can select the newly created configuration using the previously described steps 2 Create new well plate template By choosing New well plate template Elli al eoliano retire eit from the Create template dialog you will be guided by a wizard to setup TissueFAXS for using a well plate insert Page 45 of 195 www tissuegnostics com TissueFAXS 3 0 Welcome This wizard will help you to setup TissueFAss for using a well plate Insert To continue press Met Figure 40 Create well plate template Wizard Step 1 here you must choose the name and the type of the template to be created For the well plate type some options are already available by default but you can also create a new type by choosing Custom option from the dropdown menu Specify generic parameters E Welcome E Step 1 Shep 2 Shep 3 Step 4 Step 5 Step 6 Step Step E Step 9 Results Figure 41 Create well plate template Wizard St
144. ressed size estimated size If one of the regions is too large the respective region will be highlighted in red TissueFAXS allows configuring the export as desired by choosing exactly the contents of interests In the dialog displayed above the user can adjust the following settings e Select the storage folder e Select files to be exported Images Movies option available only if the region was acquired with time lapse TissueFAXS 3 0 Page 163 of 195 www tissuegnostics com TissueFAXS 3 0 e Enter Custom Part type desired custom part for the name of the exported item part consists of items from the File Name Convention see Chapter 4 2 3 The custom fn The file name to be exported consists of a default part and a custom part The default tid ote part is defined by the user e Select the file extension choose exported item format also available tiff with multiple tiles in order to export bigger images Files Save images Custom part Mame preview Experiment Region jpa Extension Save movie Options for tiff wit png tiff with multiple tiles Figure 234 Export Region Overview Extensions If choosing as extension the tiff with multiple tiles the items from the Options for tiff with multiple files will be enabled Tile size the dimension of an image tile composing the tiff there are three predefined options Compression there are two options None and jpeg
145. rrows of the Joystick control can be used to move the stage on the X and Y axis The adjacent Up and Down buttons control the Z axis and are used to focus on the current field of view Page 103 of 195 www tissuegnostics com TissueFAXS 3 0 e Autofocus button _ E by clicking this button the microscope will autofocus on the current field of view e Edit Speeds Edit Speeds this button allows you to adjust the speed at which the stage is moving on X Y and Z axis when using the hardware joystick the joystick control or when the following commands are used Go to this point Go to this slide and Move one FOV After you have adjusted the speeds click the Done button to save them If you want to cancel just press Esc or click elsewhere outside the popup To adjust the stage speed during acquisition go to Tools Options Scan Settings Speed Stage T al gt RN Axis Speed J a Ls Z Axis Speed J Figure 149 Editing speeds for joystick and Z axis e Load gag this button moves the stage down to the load position so you can add or remove slides e Work Work this button moves the stage up to work position fn These stage controls work only with motorized focusing drive yf Note If you are working with a TissueFAXS i system which uses an inverted microscope the objective revolver will be moved down and up rather than the stage 5 9 Camera Control The camera control
146. rs about possible damage to equipment or to data or about potential problems in the outcome of what is intended Italics are used in order to highlight a title and also new or unfamiliar terms usually at their first appearance in the text Page 8 of 195 www tissuegnostics com TissueFAXS 3 0 Bold are used in order to highlight important terms or to mark the names of the features GUI elements throughout the text of this manual the arrow indicates a menu choice For example choose File Save as template means you have to choose Save as template from the File menu Page 9 of 195 www tissuegnostics com TissueFAXS 3 0 2 Installing TissueFAXS 2 1 Installation Overview Where do find the installation kits for the required components Usually the TissueFAXS installation kit is provided with a set of other installation kits for the rest of the components which can be found in the same folder Most of the components are also found on official websites You need an internet connection in order to download those components In order to make the installation procedure easier you are provided with a folder where you find all required kits Install them in any order you want but make sure TissueFAXS is the last one to be installed If you are not sure how to start try this order NET Framework 2 0 Runtime with Service Pack 2 Microsoft Visual C 2005 Redistributables Intel Performance Primitives 5 2 MTB RDK 1 6
147. s to increase the size of the TMA spots e use CTRL keys to decrease the size of the TMA spots Page 180 of 195 www tissuegnostics com TissueFAXS 3 0 ar Pre view g Mi es t Sl Figure 257 Resize the TMA spots 6 2 6 Rotate a TMA block The TMA blocks can be rotated as follows e place the mouse cursor next to the lower right corner of the TMA block e press the left mouse button on the blue arc that appears next to the lower right corner of the TMA block and rotate the TMA block using the mouse e release the left mouse button when finish Page 181 of 195 www tissuegnostics com TissueFAXS 3 0 Drevje Do Figure 258 Rotation of TMA blocks 6 2 7 Adjust the distance between TMA spots The distance between TMA spots can be adjusted as follows e select the desired TMA spots e increase the distance on the X axis using CTRL keys e decrease the distance on the X axis using CTRL lt keys e increase the distance on the Y axis using CTRL keys e decrease the distance on the Y axis using CTRL keys Page 182 of 195 www tissuegnostics com TissueFAXS 3 0 le Preview Slide 8 SENO Figure 259 Increase Decrease the distance between TMA spots 6 2 8 Moving TMA blocks and spots The TMA blocks and spots can be moved as below e select the desired TMA blocks and spots e move the selected objects using the mouse or gt arrow keys 6 2 9 Deleting TMA
148. sections below Basic Settings Exposure Time The amount of time available to the sensor for collecting light to form an image Increase this parameter when the light intensity is low Page 99 of 195 www tissuegnostics com TissueFAXS 3 0 Exposure Time ms Figure 134 Exposure Time control White Balance this parameter adjusts the intensity of the Red Green and Blue colors in order to render a neutral color correctly white is usually used as a reference You can press Auto to autobalance the colors When the transmitted light is not white you can use this parameter in order to correct the hue of the light in order to obtain the reference white light white Balance Red Green Blue 0 00 400 00 Figure 135 White Balance control Saturation represents the purity or strength of a color due to absence of black white or gray Adjusting color saturation will affect how intense the tones of the color are in your image Use this in order to enhance colors on tissues with faded colors The default value for Saturation is 150 If the user wants to set the default value for this parameter he must press the Default button Saturation 5 Figure 136 Saturation control Display Information on Camera Le Display Information on Camera when enabled this option will display on the live image the histogram the saturation and the high background indicators Display Scale bar E Show scale bar whe
149. software and the CPU will affect performance and might affect the results of the analysis in an unpredictable way TissueFAXS 3 0 Page 12 of 195 www tissuegnostics com TissueFAXS 3 0 3 Starting TissueFAXS 3 1 Login Dialog After starting the application a login panel will appear In order to login to TissueFAXS you must enter a username into the User Name field and type the proper password into the Password field Press Agree and Login to be authentified By pressing this button you explicitly agree to the TissueGnostics Disclaimer 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 when the Administrator is logged on for more details please see Chapter 4 7 TISSUE AY on SNOSTICS MEDICAL E BIOTECH BOLUTION amp ISSUE CE IM ATTENTION CONSULT OPERATING INSTRUCTIONS FOR USE Disclaimer rar is a miroscopebased cell analysis sysiem for cells in aoyoats parafin a Ss and E TMAS Jt consists of ihe softwere modules TissuelAXS TissueQuvest 2 HistoQuest and is used for acquisition of images in the Averescence and briahitield mode for c
150. ss that will be set as sender The email address of Tissue Gnostics support The available SMTP server port to be used when sending the error report Contact your network administrator for details Use Submit Command check this in order to access an external executable file that will send the files to a server Auto Save Report check this option in order to save the log files on the hard drive no matter if the report is sent or not Test Connection is used to see if the connection to SMTP server works Submit Report will help you send a mail containing your possible questions or problems regarding TissueFAXS Beside the information you type in the form the email automatically includes the log files the running processes the configuration files of the application To effectively send the mail press the Send Report button Page 25 of 195 www tissuegnostics com TissueFAXS 3 0 Submit report Report description Report details Read Product Improvement Program Terms Figure 17 Submit Report dialog Generate exception is used to simulate an error in order to test if sending emails with the specified settings works Page 26 of 195 www tissuegnostics com TissueFAXS encountered an error GI Error description Generated test exception Error detail Application TissueFAXS Version Loaded Modules C Program Files TissueGnostics TissueFAxs C Windows SYSTEM32 ntall
151. tain marker staining intensity In order to acquire a marker a corresponding reflector filter block must be used When using multiple markers in order to get a better overview for the entire region TissueFAXS allows you overlaying images acquired with different reflectors You can choose for each reflector color channel the optimal color Look Up Table that will represent the corresponding marker in the final image Later on we will use the following terms A Region Overlay is the image created by superimposing images of the same region acquired with different reflectors A channel is the image of a region acquired with a certain reflector The user has also the option to change the names of the reflectors by going to Tools Options Reflectors The colors of the reflectors will be used as default for new experiments only and after acquisition each region can have its own color configuration You can edit these default colors according to your needs in the dialog below see also Chapter 5 5 2 Page 29 of 195 www tissuegnostics com TissueFAXS 3 0 Here you can assign colors for each reflector present in the list EC Transmission C 255 255 255 MN O 0 255 Storage MM 0 255 0 OO 255 255 255 Scan Settings MN 255 0 0 Hardware Settings ER C 255 255 255 Experiment Options Use inverted image for Transmission channel in case of fluorescence fe Use white in preview overlay of fluorescence s
152. tal live image through a camera The stage is the hardware component of the microscope that allows viewing different inserts and their contents tissue cell cultures etc by moving on the X Y axes TissueFAXS provides a virtual image of the stage with its existing inserts Page 91 of 195 www tissuegnostics com TissueFAXS 3 0 Microscope Acquired Images nr m o i it ic A TM es AH i em poe ea er i i a rg cea foe i i rin a A 7 A a E 7 g E d i E ings ite Li pai i e a E a e e j z ans Pr j j A j pai i i MNMESUECAOS TIOS TMESUZEMOSTIDZ MNMESUECMOSTIOS TMESUIEMOSTIOEZ TIESGUECADOSTIEOS NESVECHOSsTICsS NS SuUcECYNOSTIcCsS Figure 127 Stage Control The currently selected slide can be recognized by a surrounding red frame Figure 122 Slide selected by the user The slide currently placed under the objective lens can be recognized by a surrounding green frame AA a ea thy ir E Figure 123 Active Slide on the microscope For more information please see Chapter 5 15 of the current manual Zh Note Page 92 of 195 www tissuegnostics com TissueFAXS 3 0 5 7 Cameras TissueFAXS supports the following cameras e PixeLINK cameras select this type of camera for Brightfield imaging e PCO PixelFly cameras select this type of camera for Fluorescent imaging 5 7 1 Adding Cameras To add a supported camera follow th
153. that may be individually selected For both types of experiments you can adjust the preview area size by clicking on the Preview eindin button The preview area or yellow box can be manually adjusted with the mouse or you may select a pre defined schema from the drop down menu Page 72 of 195 www tissuegnostics com APETECE ENEE Please specify the preview schema for your experiment Preview Schema 6x11 FOWs Camera PixeLINK PL 46220 6220116 Preview objective 2 53 dir Label Position 2 Label Up C Label Down Figure 77 Preview Area Settings panel You can give a name to your Preview Schema and save it for future projects This is useful when you have multiple slides which frequently have the region of interest tissue in the same specific location Defining your preview schema over your region of interest will save time otherwise the microscope will scan the entire slide in order to preview it The Hint area displays the preview schema size in terms of number of fields of view FOVs Additionally the type of camera and objective used for preview is shown In the Label Position area you can specify how your slide is oriented on the stage This is not a definitive setting For instance even if your slides have the labels oriented down you can change this later in the project for individual slides or for multiple slides To do this later just right click on the slide s and change its label position 5 1 5
154. the acquisition process despite the short time lapse the acquisition process will start and a warning message will be displayed in the status bar located in the lower part of the application Time lapse acquisition overlaps Applying Focus Strategy Time Lapse Acquisition Run 2 from 5 Figure 95 Acquiring with short time lapse warning from status bar For every region acquired with short time lapse this information will be displayed in the Region properties section from the Experiment Editor see Chapter 5 14 El General Acquired Yes D Acquired with time lapse Yes Acquisition date 5 4 2011 1 42 PM Area 12 0415 mms Area in ROI 9 4261 mm Comment Time lapse acquisition overlapped FOV matrix size 10 10 iman 444 300053 334 5455 Mioa ll n F FUE at ae Figure 96 Experiment editor region acquired with short time lapse If you frequently use Time Lapse acquisition you can set this option as default from Y np Tools Options Application Options Remember along with the desired parameters values Page 82 of 195 www tissuegnostics com TissueFAXS 3 0 Actions L Do not show preview focus dialog again Licensing Measurements L Do not show reset focus point for preview dialog again L Do not show slide renaming dialog again Number of runs Time lapse 0 00 30 00 E u Fomai Days Hours Minutes Seconds Figure 97 Remember dialog Time lapse acquisition section How to vi
155. the stage to the safe distance For the rest of the objectives you may customize this behavior in the dialog shown below Usually you will prefer to enable this behavior for any longer objective or for objectives that come very close to the slide for focus For inverted microscopes the best action is to enable it for all objectives Page 17 of 195 www tissuegnostics com TissueFAXS 3 0 Calibration This is a safety setting and you should access it after installing TissueFAXS in order to choose a safe objective for calibration This choice means that the application is helping you avoid possible damages of the objectives stage or slides caused by an inappropriate objective being used during the calibration procedure If an objective with a magnification higher than 20x will be selected for calibration every time before performing calibration TissueFAXS will send you a warning message please see Chapter 3 3 Cache The image cache consists of scale down acquired images and it is used to speed up the display of big regions and decrease the memory requirements for this operation If no cache is built you will be able to see only low resolution images 7 Other operations like export and analysis are not affected in any way by vf Note the presence or lack of the cache The cache can be built e During acquisition if Tools Options Actions Build cache during acquisition is checked This will increase acquisition time
156. titch Stitch computed Support stitch General Yes No 4 16 2010 2 17 PM 7 7912 mm 0 0000 mm2 write a comment m 6 6 RR d 537 907 402 342 537 907 402 342 Yes Region 002 write patient s name write reference number Rectangular F Projects TissueFAXS Demo 0 00 30 00 Time Regions 20 Region No None Figure 168 Region properties section In the example above the region has the following properties e The item is acquired e The item is not acquired with time lapse e Area total area of the region including only acquired fields of view e Area in ROI area for the cropped shape including only acquired fields of view e The acquisition date e The comment indicates sample related data e The item consists of a virtual matrix containing 6x6 Fields of View e The FOV size Width and Height e The item is included in acquisition e The item name e Data concerning the patient TissueFAXS 3 0 Page 118 of 195 www tissuegnostics com TissueFAXS 3 0 e The type shape of the region is rectangular e The storage directory is shown here e Time lapse number of runs and time lapse are shown here e The total magnification e The type of selected item is Region e Stitch re By selecting more items at the same time multiple selection all the common properties shared by these items can be simultaneously modified in order to have the same value Some
157. tive magnification and the reflectors selected for acquisition you can also add your own information to the name Experiment Profile Info Please type the name and the description for the experiment profile Marne PCO PixelFly 0 20x DAPI FITC Cy 5 Description Camera Objective Preview reflectors Acquisition reflectors Cancel Figure 87 Save Profile dialog Page 78 of 195 www tissuegnostics com TissueFAXS 3 0 e Load an existing profile can be loaded for current experiment You must select any profile you want from the existing list Description Preview reflectors Acquisition reflectors Figure 88 Load Profile dialog e Delete if an existing intensity profile is no longer needed it can be deleted by selecting it and then pressing the Delete button Page 79 of 195 www tissuegnostics com TissueFAXS 3 0 Description Camera Objective Preview reflectors Acquisition reflectors Figure 89 Delete Profile dialog Estimation of acquisition duration TissueFAXS can estimate how long the acquisition process will take This estimation is pretty rough at the first fields of view but after at least two focus operations its accuracy will increase This feature is useful in case of unattended acquisitions and it is available in the bar from the lower left corner of the application Type Region El Stitch Stitch computed No Support stitch Internal 2
158. to 100 FOVs CAUTION because it is very time consuming Min allowed stitch score decrease this value only if you are acquiring regions with a large empty area In any other cases you should let it to the default value 4 3 3 Focus In this section you can adjust all the focus relevant parameters organized in two tabs Focus Options and Enhanced Focus Page 34 of 195 www tissuegnostics com TissueFAXS 3 0 Focus Options tab Options Application Options Focus Options Enhanced Focus Default Experiment Settings Focus Search Algorithm Autofocus Rough Focus Measure Variance li Fine Focus Measure Stitching Focus Focus Strategy Neighbors Width 3 Height 3 Advanced Focus eae Focus Points Fixed Focus Points Speed _ Always force full focus WOTE Use this in difficult environments Hardware Settings Focus validator Maximum allowed score aa Experiment Options 0 0 1 0 Accept shaper images Accept blurred images High fidelity Low fidelity _ Disable focus validator Load default settings Full Focus Interval Current Z Position 0 00000 um _ Override default intervals with these values Upper Z Position umi Lower Z Position 700 um WARNING The recommended focus interval should be af least 400um ANOTE If hected al objectives wil use ese vales Figure 26 Focus Options tab Focus Search Algorithm Manual focus when using this type of focus the user w
159. to accept the computed values Figure 53 Output of the Automatic FOV calibration slides than calibration slides you can run this procedure on different areas to ensure Q For best results set the speed of the stage at a minimum value When using other TIP a os the correct value is computed p If the image is flipped the algorithm will detect this and will set the image to the right rote position e To save the computed values press Save and Exit 4 6 3 Distance Calibration e Insert the calibration slide in an empty insert position e Choose the objective for which you want to perform the calibration e Enable the live image focus and center the calibration slide on the live image e Access the calibration function from Tools Tools menu choose Calibrate Field of View option Page 57 of 195 www tissuegnostics com Calibrate Stage Options Skin Run Algorithms Figure 54 Tools menu e The Field of View Calibration control panel will appear Camera PixeLINK PL 4622c 6220116 Automatic Calibration Objective 20x Air Top Right Top FOV Bottom Width Edit Speeds ma 13501 100 um Yi 39696 700 um Es 645 975 um _ Automatically compute calibration For other objectives Figure 55 Field of View Calibration control panel e Now press Distance Calibration button O and the Distance Calibration panel will appear EA 213708 526 39496 131 3
160. ton and drag the mouse down and right while holding the mouse button until the desired size of the TMA block is defined E Preview Slide 8 OBOE E Figure 250 Definition of the TMA block size e Using predefined size for the TMA blocks by pressing TMA size button A dialog will appear where the desired number of rows and columns can be set Page 176 of 195 www tissuegnostics com TissueFAXS 3 0 TMA Block Size Please specify the number of rows and columns for TMA blocks that will be created Fors Columns Figure 251 TMA block size dialog 6 2 2 Definiton of the first TMA block on slide The first TMA block on a slide is defined by indicating the diagonal between the most upper left and the most lower right TMA spots of the TMA block To indicate this diagonal follow the steps below e hold the SHIFT key e press the left mouse button in the center of the upper left TMA spot of the TMA block e holding the left mouse button drag a diagonal to the center of the lower right TMA spot in the TMA block e when you finish drawing the diagonal release the left mouse button and then release the SHIFT key This TMA block will become the template for all the other TMA blocks having the same size the same number of rows and columns Page 177 of 195 www tissuegnostics com TissueFAXS 3 0 YE Preview Slide 8 aJe ae Figure 252 Manual definition of the first TMA block on the slide 6
161. tton and choose in the browser that opens the location folder and specify the name for the csv file to be exported Finally press Save button to finish the export The exported csv file will contain the following data e Experiment name e Slide name e Region name e Annotation name e Area Length Page 171 of 195 www tissuegnostics com TissueFAXS 3 0 e Unit measure e Description if any 9 21 Use Plugins A plugin is a software program that extends another program s functionality without the necessity of reinstalling the main program For TissueFAXS the following plugins are provided by default Plugins for Brightfield experiments e Launch HistoQuest for entire experiment from Analysis Launch HistoQuest e Launch HistoQuest for the current region displayed in region viewer from Plugins Analysis Launch HistoQuest in region viewer In both cases above all version of HistoQuest starting with HistoQuest 1 0 2 0210 will automatically import corresponding data from TissueFAXS Plugins for Fluorescence experiments e Launch TissueQuest from Analysis Launch TissueQuest e Launch TissueQuest from Plugins Analysis Launch TissueQuest in region viewer In both cases above TissueQuest will start Page 172 of 195 www tissuegnostics com TissueFAXS 3 0 6 TMA Support Although the TMAs can be treated and acquired as regular regions TissueFAXS offers enhanced support for this type of samples sli
162. turated Saturation Value The minimum acceptable intensity value for which a pixel is considered oversaturated TissueFAXS 3 0 Page 97 of 195 www tissuegnostics com TissueFAXS 3 0 Advanced Settings In order to access the advanced mode right click in the area where the camera parameters are Ad d Mod The following menu will appear E Advanced Mode The Advanced tab will appear if checked Pixel Addressing this parameter is used in order to decrease the size of the captured image and decrease the time needed for the capture operation This will increase overall acquisition speed but the image quality will be affected reduced Pixel Addressing None Figure 130 Pixel Addressing combo box Flip this parameter is used to flip the captured image You can use this parameter to correct the orientation of the image if the left right or top down of the camera are different that those of the stage This parameter is generally adjusted when the system is installed The FOV calibration feature is able to automatically set this parameter to its correct value Do not change this setting unless you encounter failure in stitching the overview images vertical Figure 131 Flip for the Image checkbox Camera Mode You can choose Async Shutter or Video Mode The Async Shutter is recommended when there is a high amount of light reaching the camera sensor like in Brightfield The stepsize of the exposure time can be
163. uired fields of view e The area in ROI refers to the area for the cropped shape including only acquired fields of view Page 22 of 195 www tissuegnostics com TissueFAXS 3 0 E The Area and the Area in ROI measurements are also displayed in the Experiment J Note editor see Chapter 5 14 Region 011 Area 1 3379 mm Figure 14 Measurements Area and Area in ROI fn Checking this option will dramatically increase the acquisition time ote a This option will only apply to regions that will be acquired after marking this checkbox This also applies to further added defined regions and subregions Page 23 of 195 www tissuegnostics com TissueFAXS 3 0 4 1 4 Remember Licensing L Do not show preview focus dialog again Measurements L Do not show reset focus point for preview dialog again support Do not show slide renaming dialog again Default Experiment Settings ai Scan Settings Time lapse acquisition Hardware _ Hardware Settings vw Experiment Options a El Fomai Days Hours Minutes Seconds Figure 15 Remember dialog This dialog can be used to specify default values for some of the most commonly used options of the application e Uncheck Do not show preview focus dialog again if you want that dialog to be displayed before the preview operation otherwise check this option e Uncheck Do not show reset focus point for preview dialog again if you want that
164. w the channels used for preview and the camera settings for each channel To add or remove channels press the Add Remove Channels button Add Remove Channels In the dropdown list that appears the already selected channels will appear checked Preview Objective Preview Channels i Preview 5 E a Pe o o o 7 Acquisition List with available channels Add Remove Channels button Check desired DAPI A channels Oeys Microscope Camera if Acquired Images i AN Rhodamine Peeters rR ADRES E E sidez HST slides Fr fH Slide 4 s HiB Slide 5 slides i Slide 7 y A aa aw st gp de pio tA A taal a ES Figure 81 Preview Settings panel Channel list Page 75 of 195 www tissuegnostics com TissueFAXS 3 0 Preview Objective Preview Channels fname Rec A A A Preview Acquisition gt Darr DAPI m Rhodamine Rhodamine FITC w Transmission Transmission Add Remove Channels Figure 82 Preview Settings panel Channels inserted To edit the camera settings for a channel click the View button next to it For further details see Chapter 5 7 of the current manual You can change the names of the channels by clicking on each in the settings field 2 To adjust the camera settings for any channel you must have a camera present in va MOTE your project Otherwise the View button vi for each channel will n
165. www tissuegnostics com TissueFAXS 3 0 TissueFAXS 3 0 User Manual Page 1 of 195 www tissuegnostics com TissueFAXS 3 0 Table of Contents 1 1 e ES O Lm0 S UR 7 1 2 GANS e or a ie td tee haters etc asec te o A AA 7 1 3 Definitions Acronyms and ADbreviatiONS cooocccocccoccconncocnconnnncncocnnonnnonnnonnnnnnnnnnonnnonanonanonnronanonaninaninons 8 ToL BOIU a A o OO oe ee eee eee 8 Es nn ee ee A ee eee eee eae 8 LI ADOOS sees a nce accu ti otrora ras 8 1 4 CONVE Said 8 2 1 Ec LAO PQ o 10 2 2 iNET Framework 2 RUIN ee 10 2 0 NTE RODK Wi AAA A PP e sess ate ee sega a ba asec E 10 2 4 intel Pertormance PrMIIVOS 9 3 esmero a in 11 2 5 Microsoft Visual C 2005 Redistributables ooccoocccoocnococnoconccccncconnoconnonanconanonnnnonnnnnnnnonannonaninos 11 2 6 A eo O OE E oriacenn S 11 2 7 VOLS REGUN GIONS eden cada aA EIE EEEE E EEDE 12 2 11 Hardware TREGUINGIMCIINS smc rior rra a E Eai 12 ZT 2 SOM Wale TR OG UI CTO INS e parra cti 12 3 1 A ea as caeeraatenntcdan sean aces annnuane neat uaee aaa dae slaw cate gaat se saat E 13 3 2 o A er een nee een ETA I ee eens en eee ee te ee ener 14 3 0 DVICO CANOU Renee eee en ere eee ee er ee ee ee ee 14 4 1 ADPICANON OPINAS cairo een eee ee ee 16 ETA ONIN Sa E E E nese aden denen altuna AAE E A EA 16 A A AP ee ee EE AA ee 19 AS MO ASSIM esisin a EEE Ea EE E EA EE EE E E ia 20 E SS o non EE E o o ee AE E R 24 E O O eeen E EE E E NEE OE E E E O EE A EE i
166. xperiment Figure 227 Apply overlay menu Page 158 of 195 www tissuegnostics com Demo Region 0041 FOV 00021 a 80 Fit A Overlays Export tr 4 m Used Name Intensity Color Alexa 488 Gi O 255 0 Alexa 647 100 MN Wyo 255 wf Cy3 100 Em Web System 1 we Bled Choose desired color d Pre F Apply For current FOW For current region For whole slide For whole experiment E Demo Region 004 FOV 00021 A ls E Fit 1 Overlay Export 4 Used Name Intensity Color Alexa 488 GE O 255 0 DAPI 100 ET 0 255 255 eii P if A Maa Cy3 100 Custom Web System L Figure 228 FOV Overlay Example of adjusting color for one channel TissueFAXS 3 0 Page 159 of 195 www tissuegnostics com TissueFAXS 3 0 The user can also adjust the intensity for each channel by typing the desired value in the corresponding field ao 5 20 Export TissueFAXS allows exporting acquired images as a complete region overlay or image by image In certain cases csv files can also be exported 5 20 1 Export FOVs Images You can access this option in two ways Region viewer Export Fields of View Region Overview Figure 229 Export FOVs Images Option a e Experiment Editor Region Context Menu Create Group Acquire Region 006 Remove Remove Regions Build Cache for Region 006 Clear Cac
167. y instead of making new settings for the camera For more information about intensity profiles please see Chapter 5 9 Additionally TissueFAXS stores TL lamp intensity value for Transmission and attenuator settings value for Fluorescence channels per channel Choose an existing intensity profile Use this button to view or edit camera settings Acquisition Channels PixeLINK PL A622C 6220116_20x_Transmission_Demo2 tl Figure 85 Camera Settings Experiment Profile Settings for Acquisition Here you can select the objective and the channels for acquisition For each channel you can edit the ke the acquisition at least one channel Page 77 of 195 www tissuegnostics com TissueFAXS 3 0 An Experiment Profile represents a set of settings camera acquisition objective preview channels acquisition channels that you can save for further use in order to make available different combinations of settings for the preview and acquisition To access Experiment Profile options go to acquisition settings tab and press Experiment Profile button Acquisition Objective 20 Air Preview Figure 86 Profile Options menu e Save the current preview and acquisition settings can be saved in order to make them available for other experiments You must specify a name and a short description for this profile A default name is already generated It contains the camera name and type the objec

TissueFAXS 3.0 - TissueGnostics

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