XyFlex™ Area Measurement Software Instruction Manual
Contents
1. 5 2 2 Choosing the Well Orientation The WELL ORIENTATION is the type of Figure 19 Selecting the image files to analyze plate the images reflect and allows the software to find each well automatically for analysis The choices are FOUR 6 WELL PLATES or FOUR 24 WELL PLATES 5 2 3 Selecting the Resolution The SCANFLEX software allows the user to capture the image in 150 dpi 300 dpi or 600 dpi The user should record the image resolution chosen when scanning occurs The software automatically locates the center of each well in respect to the RESOLUTION selected 5 2 4 Selecting the Anchor File The ANCHOR FILE is generally the first sop ea ee pore BMP 150dpi 6well 0000hr04min jpg BMP 150dpi Swell O000hr10min jpg BMP 150dpi 6well 0000hr08min jpg image in the series This file will be the BMP 1504pi well 0000hr14min jpg image where threshold values and the Region of Interest ROI are initially defined for processing the rest of the images in the series The threshold values are the upper and lower limits of color contrast The ROI is the area in which the threshold values are applied These processes will be covered in y Well Orientation Resolution Sections 5 4 and 5 5 re spe ctively Four 6 Well Plates 150dpi Anchor File BMP 150dpi 6well 0000hr00min jpg Double click the desired anchor file name in the right window and the file name will appear in the ANCHOR FILE Figure 20 Selecting the image files to an2. Destination Directory wu x Folders i Select the primary installation directory O 3 3 3 3 0 CD Files Currently on the CD o B B kerze suppertfiles autorun ad setup N All software will be installed in the following locations To install woe into different locations click the Browse button and select another direct File and Folder Tasks 2 B Rename this Gp Move this file Copy this file B Pte this fie to the wed E mal this Directory for Flexcell Xyflex Delete this file C Program Files Flexcell Directory for National Instruments products C Program Files National Instruments Figure 1 XYFLEX CD contents Figure 2 Destination directory XYFLEX INSTRUCTION MANUAL PAGE 3 OF 19 FLEXCELL INTERNATIONAL CORP License Agreement You must accept the licenses displayed below to proceed NATIONAL INSTRUMENTS SOFTWARE LICENSE AGREEMENT INSTALLATION NOTICE THIS IS A CONTRACT BEFORE YOU DOWNLOAD THE SOFTWARE AND OR COMPLETE THE INSTALLATION PROCESS CAREFULLY READ THIS AGREEMENT BY DOWNLOADING THE SOFTWARE AND OR CLICKING THE APPLICABLE BUTTON TO COMPLETE THE INSTALLATION PROCESS YOU CONSENT TO THE TERMS OF THIS AGREEMENT AND YOU AGREE TO BE BOUND BY THIS AGREEMENT IF YOU DO NOT WISH TO BECOME PARTY TO THIS AGREEMENT AND BE BOUND BY ALL OF ITS TERMS AND CONDITIONS CLICK THE APPROPRIATE BUTTON TO
3. n EI Select Fies Select weis Mask Vet Cange Threshold Processing Modfy Wel Mark Subgroups Process Another Zoom to Fit Zoom In Zoom Out Area pixels Area Units Accept Threshold Range f 92 Quit Back to Start lt lt lt Back Next gt gt gt About Figure 28 The processed well image prior to manual editing instructions Move mouse over to expand pS Select Folder E 2222 Change Threshold Processing Modity Vel Mark Subgroups Celedet Subgroups Process Anether M EPI Zoom to Fit Zoom In Zoom Out EE Area pixels Area Units Accept Threshold Range 1 Quit Back to Start Back Next About Figure 29 Adjust the threshold and use the editing tools to modify a specific well image updated information from the MODIFY WELL screen in the selected cell Repeat this process until all the well images are acceptable and then select NEXT on the processing screen XYFLEX INSTRUCTION MANUAL PAGE 15 OF 19 FLEXCELL INTERNATIONAL CORP 7 2 Categorize the Wells into Subgroups The MARK SUBGROUPS screen allows the user to categorize the wells into particular subgroups with group names if desired Figure 30 This step is completely optional Breaking the 01 00 03 00 00 00 wells down into subgroups may 91 956 BERR Test Group A
4. 5 4 Selecting the Region of Interest The Region of Interest ROI is the selected area where the threshold is evaluated The threshold is defined as the pixel contrast coloration located between the upper and lower limits of the desired threshold 1 The ROI rectangle may be relocated and resized to locate the best fit for the ROI Figure 23 For BATs the ROI is generally located from the end of one Tissue Train nylon anchor to beginning of the opposite nylon anchor in respect to height and a width slightly wider than the widest measurement of cross section of the BAT 2 The user may advance the process of selecting the ROI for each of the individual wells 24 wells for a 6 Figure 23 Selecting the Region of Interest ROI well plate experiment and 96 wells for a 24 well plate experiment by selecting USE CURRENT ROI FOR ALL 3 The user may choose to select the ROI for each individual well by scrolling to the WELL NAME adjusting the ROI rectangle to the desired area and then selecting NEXT WELL until the process is complete Note the area defined in pixels is shown to the right of the selected WELL NAME Choose NEXT to begin the threshold range evaluation 5 5 Choosing the Threshold Range The threshold range is a determining factor in the effectiveness of the software to complete a successful mr image series area measurement te ie i evaluation Figure 24 1 The AREA UNITS should be chosen first The units a
5. TestB5 24 019 244694 2 2 15 016 simplify data extrapolation when 44957 31798 23458 24 Ha 13 572 1400 4 19 259 analyzing the processed data hes uw rwr 1 8 EE um t A3 15 016 6 0 i jTestGroupB 13 572 11 Test B6 27 796 24 971 1 Type the subgroup name in the 398 4751 848 1444 14 BEES 14 537 J B2 31 739 GROUP NAME window at the EN S top center of the screen 2 Highlight the wells in the left Test B1 11 325 6 786 Test Group C Test C6 1 327 Test C3 1 222 Test C5 0 000 Test C2 0 178 2 880 1 738 0 000 0 577 4 i 1 222 TestC4 0 000 1 990 window to be included in the Test C5 0000 Test C1 0000 0743 Test C2 0 178 222 Test Group D defined subgroup Group Mose ore Tn 10659 na wazan Test D5 18 105 25 603 lt selection in the left window may be done by using the Control and Shift buttons on the keyboard accompanied by the mouse All the highlighted wells will move to the right window by selecting INSERT This action will insert the GROUP NAME in the first available cell above the selected wells in the right window 3 To form a second subgroup repeat 5tep 1 Do not insert blank rows into the table in the right Figure 30 Create subgroups with the wells window Please note that a particular well may be placed into more than one subgroup To remove a well or group of wells from a subgroup highlight the well or wells in the righ
6. 10 9 2 3 Selecting tlie ResolutlOELun uisu uariis puri y bek kn EREA ANARAN 10 5 2 4 tn An ORO l yy 10 52 Selecting the Wells TO AT 6 11 aw Selectmp the Region of THEBEESE i u k nn h k n 12 no Choosing the Threshold K kl kc xis n k enana Rao k n A 12 O ATLE A T siseyan cay y ra M DEED k y e coves d d y NND de wek d e duke HAR n ya d dewe NNI HEU EID A And Re 14 Ju Wa OSU ay BOT rrr Wr 15 7 1 Modifying Individual Well Images eese nenne tenant tete tnt tenent tenni 15 42 Categorize the Wells THEO SUD OT OUP E EE kahn IINE 16 D O D O 17 ST Da 18 XYFLEX INSTRUCTION MANUAL PAGE 1 OF 19 FLEXCELL INTERNATIONAL CORP 1 Introduction 1 1 Use of XYFLEX Software with Tissue Train Culture Plates XYFLEX software is designed to automate the area measurement of a series of images It was initially designed to evaluate the area compaction of 3D bioartificial tissue constructs fabricated using Flexcell s Tissue Train Cultu
7. Another U Image Folder i ty A Select Image Folder Calibration File oa Calibration File Extra Functions Create Calibration File Flexcell International Corporation 437 Dimmocks Mill Road Suite 28 Hillsborough NC 27278 USA Tel 919 732 1591 Fax 919 732 5196 http Mflexcellint com Figure 16 XYFLEX start up screen Instructions Move mouse over to expand re LI LI LI LI Select Folder Select Files Select Wels Mask Wet Change Threshold Processing Mark Subgroups Calculate Subgroups Process Another Select C Xyflex BAT Images BAT Images 3 Strain Image Folder v Calibration File Load C Documents and SettingslcolinlLocal SettingslApplication DatalFlexCell_Xyflexi 5 configiTrapezoidal_BAT_Calibration_File xml v Proceed without Calibration Extra Functions Create Calibration File F e Flexcell International Corporation 437 Dimmocks Mill Road Suite 28 Hillsborough NC t Tel 919 732 1591 Fax 919 732 5196 http flexcellint com Figure 17 Image folder and calibration file selection PAGE 8 OF 19 FLEXCELL INTERNATIONAL CORP b Selecting PROCEED WITHOUT CALIBRATION will allow the user to continue the analysis using only pixels as the unit of area measurement To PROCEED WITHOUT CALIBRATION go to Section 5 2 of this manual To use area measurement units of um2 mm or cm2 choose CREATE CALIBRATION FILE This will
8. CANCEL THE INSTALLATION PROCESS DO NOT INSTALL OR USE THE SOFTWARE AND RETURN THE SOFTWARE WITHIN THIRTY SHALL BE SUBJECT TO NI S THEN CURRENT RETURN POLICY lt The software to which this National Instruments license applies is Flexcell xyflex accept the License Agreement O I do not accept the License Agreement Figure 3 NI license agreement Installation Complete The installer has finished updating your system Some of the installed products require activation Activate these products now Figure 5 Installation complete NI Activation Wizard Activating Vision Development Module 2011 Vision Run Time INSTRUMENTS Enter serial numbers For the products you want to activate You can find serial numbers on your product s Certificate of Ownership or packaging Vision Development Module 2011 Vision Run Time Figure 7 Vision activation code Start Installation Review the following summary before continuing Adding Changing NI Vision Run Time Engine 2011 Activex Support Flexcell xyflex Files Click the Next button to begin installation Click the Back button to change the installation settings Figure 4 Starting XYFLEX installation Welcome to the National Instruments Activation Wizard Complete the Activation Wizard to activate your product Activation is the process of acquiring and applying an activation code Automatically activate through a secure Internet con
9. orientation on the scanner bed The orientation is important regardless of whether the Tissue Train culture plates are utilized or if the image series analyzed is for a customized experiment XYFLEX INSTRUCTION MANUAL PAGE 6 OF 19 FLEXCELL INTERNATIONAL CORP Figure 11 The orientation and numbering of 24 well Figure 12 The orientation and numbering of 6 well culture plates on the scanner when the frame provided culture plates on the scanner when the frame provided is used for placing the culture plates on the scanner is used for placing the culture plates on the scanner The black area is the frame used to hold the plates on The black area is the frame used to hold the plates on the scanner surface the scanner surface OOOQOOO X9 9 Figure 13 orientation and numbering of 24 well Figure 14 The orientation and numbering of 6 well culture plate wells in the scanned picture The black culture plate wells in the scanned picture The black area is the frame used to hold the plates on the area is the frame used to hold the plates on the scanner surface This numbering system is used in the scanner surface This numbering system is used in the XYFLEX software for identification of wells XYFLEX software for identification of wells XYFLEX INSTRUCTION MANUAL PAGE 7 OF 19 FLEXCELL INTERNATIONAL CORP 4 3 Opening the XYFLEx Software When the XYFLEX software is completely installed the X
10. 0 85 832 72 935 PROCESSING to begin processing TE esn 677 4446 107 179 97869 79 175 the image series Figure 27 At ea 88 116 87 625 any point during the processing 778 115139 192543 105 509 106 442 the user may stop the analysis by 8402 102979 124540 selecting STOP PROCESSING e am sow upper cell in the 00 00 column wil highlight when processing is complete After Figure 27 Processing the image series processing is complete Figure 27 the user has several options for post processing see Section 7 2 The user may scroll up and down using the keyboard to highlight the series of images for a specific well Selecting STEP FORWARD and STEP BACKWARD moves the cell selection left or right across the highlighted row The red highlighted cell represents the image shown in the right window of the PROCESSING screen IMPORTANT NOTE The speed of the CPU s processor coupled with the resolution of the scanned image determines the speed the user may scroll through the images Using the mouse to select cells is more time efficient then scrolling with the keyboard arrows 3 When the processed data represents the desired results the user may choose to EXPORT TO EXCEL csv EXPORT TO TEXT txt or click NEXT to move the data into subgroups The EXPORT TO EXCEL action creates a Comma Separated Values file csv Once this csv file is saved to a designation the file
11. OM IN ZOOM OUT and ZOOM TO FIT buttons below the window to gain an improved B3 B5 B2 B4 B1 AG A3 AS A2 Ad 1 06 5 Lower Value Upper Value Threshold for All Threshold Range J 110 J 144 view of the pixilation inside the ROI 4 The user may choose USE CURRENT THRESHOLD FOR ALL to employ the LOWER VALUE and UPPER VALUE selected for the current image for all 24 wells if 6 well plates are used and all 96 a wells if 24 well plates are used une Figure 26 This selection will 128822 a cause the software to evaluate Lower Value Upper Value T Id J J for All hreshold Range 108 7 144 each well in the anchor image with the current threshold The user may choose not to use the current Figure 26 Processed well images of the anchor file only threshold for all the wells by selecting STOP UPDATE during area evaluation of the anchor image When the threshold is acceptable select NEXT to continue XYFLEX INSTRUCTION MANUAL PAGE 13 OF 19 FLEXCELL INTERNATIONAL CORP 6 XYFLEXx Processing When pre processing is complete it is time to process the image series 1 The user may double click on the well name in the left column of the window to view the image of 00 08 00 10 the chosen well with the 4377 91140 89075 90794 95 855 88 829 122 754 previously selected threshold 420 109 095 108 923 99 515 pixilated Select START 765 140643 133408 128500 r 91 04
12. XYFLEX AREA MEASUREMENT SOFTWARE INSTRUCTION MANUAL XYFLEX 3 3 0 REVISION 3 2 FLEXCELL INTERNATIONAL CORPORATION 2730 TUCKER STREET SUITE 200 BURLINGTON NC 27215 USA TEL 919 732 1591 FAX 919 732 5196 www flexcellint com FLEXCELL INTERNATIONAL CORP Table of Contents T Jii OI OL OL n yel 2 1 1 Use of XYFLEX Software with Tissue Train Culture Plates sess 2 1 2 Use of XYFLEX Software for Other Applications eese neret 2 MEE 6100910 0c e E E E 2 2 Jn talling XY ELE umane neo MU 3 S AClivati n GGG 5 ds Dd la SETUD e 6 5 ee eee ers 6 4 2 Orientation and Analysis of the Tissue Train Culture sss 6 29 STN the AYL er rrr lr err rXrrXXl rrr gw 8 92 PTO S 8 od Gre Tl TOL l sun sen Kane aye nen NN ni en Aker 9 5 2 Defining the Image File Processing Parameters eese entente nnne tenant nnne 9 9 21 Ordering the Pa Ge TIES s c nusna yan W n nen IUS Reni 9 22 2 Choosing the Well
13. YFLEX icon Figure 15 is displayed on the desktop Click on the icon to open the XYFLEX software The XYFLEX SELECT FOLDER screen Figure 16 will open 5 XYFLEX Pre Processing The XYFLEX software will begin the area measurement analysis in the pre processing stage This stage begins on the start up screen Figure 16 1 The IMAGE FOLDER window is located below the XYFLEX logo Click SELECT IMAGE FOLDER to open the directory where the images to be analyzed are located Once in the directory select CURRENT FOLDER located in the bottom right corner of the window 2 The selected directory should appear in the XYFLEX window Figure 17 next to the SELECT IMAGE FOLDER button 3 Next the user may choose whether or not to load a calibration file The calibration file is used to define a known distance in respect to the specific number of pixels in a specified area of an image to create a ratio for processing a Selecting LOAD CALIBRATION FILE will allow the user to load a pre defined calibration xml file created during a previous analysis Choose the desired xml file The file will load into the CALIBRATION FILE window Next go to Section 5 2 of this manual XYFLEX INSTRUCTION MANUAL Figure 15 XYFLEX Icon Instructions Move mouse over to expand Select Folder Select Fles Select Wels Mask Wel Change Threshold Processing Mark Subgroups Calculate Subgroups l Process
14. alyze window Figure 20 XYFLEX INSTRUCTION MANUAL PAGE 10 OF 19 FLEXCELL INTERNATIONAL CORP 5 3 Selecting the Wells to Analyze The well positions of the scanned images are in the orientation shown in Figure 21 for the 6 well and 24 well plates 05 9 D OOOO CI 0 OO GO OG XO GO Figure 21 Well positions in the scanned images for the 6 well and 24 well plates 1 The SELECT WELLS screen Figure 22 displays the default labeling of the WELLS DISPLAYED for the 6 well plate The user may alter the WELL NAME by typing a new name in the window selecting RENAME WELL The highlighted well name in the WELLS DISPLAYED column will change as a result 2 To streamline multiple groups of processed images select SAVE WELL NAMES to save the WELLS DISPLAYED column to a Text file for later recall 3 The LOAD WELL NAMES button allows the user to upload a group of Figure 22 Selecting the individual wells to analyze previously renamed wells in the WELLS DISPLAYED column 4 Selecting the CLEAR WELL NAMES allows the user to clear the renamed wells in both the WELLS DISPLAYED column and the WELLS TO USE column The names will revert back to the software default well names After the WELLS TO USE column meets the desired criteria select NEXT XYFLEX INSTRUCTION MANUAL PAGE 11 OF 19 FLEXCELL INTERNATIONAL CORP
15. ar in the left window on the SELECT FILES screen Figure 19 The images to process may be selected one at a time or grouped using the Control or Shift keys They are moved to the right window using the gt button Use the gt gt to move all the images to the right window The right window signifies the images which are going to be processed The images are processed in the same order as they appear in the window To deselect an image from being processed select an image in the right window or group them using the Control or Shift keys and press the button The moves all the images back to the left window to remove them from processing XYFLEX INSTRUCTION MANUAL PAGE 9 OF 19 FLEXCELL INTERNATIONAL CORP 2 Once the files are in the right window they may be ordered using BMP 150dpi Gwell 0000hr00min jpg SORT BY NAME or SORT BY DATE BMP 160dpi wel 0000hr02min jpg BMP 150dpi 6well 0000hr04min jpg BMP 150dpi 6well 0000hr06min jpg IMPORTANT NOTE Make sure to BMP 150dpi 6well 0000hr08min jpg BMP 150dpi6well0000hr10min jpg BMP 150dpi 6well 0000hr12min jpg pay close attention to the file names BMP 150dpi 5well0000hr 4min jpg and the times in the image descriptions so the images will be in the desired order 3 The user may also manually move image files by using the mouse to select the file in the right window and drag the file up or down 222 2222 1 Four 6 Weli Plates j 1904
16. d on the same computer but the images to be analyzed must be present on the computer with the XYFLEX software The computer with the XYFLEX software also should have the following settings turned OFF for long term image series evaluations e Auto updates e Virus scanning e Sleep mode e Automatic defragmentation The maximum number of images to be analyzed is only limited by the memory capacity of the computer Note the image resolution determines the size of each file so make sure to record the resolution of all images captured before evaluation The approximate file size in MB of the scanned images with respect to the file type resolution and quality JPEG is the only image type which uses quality are listed in the Appendix in Table 2 Always be certain that you have sufficient memory on the hard drive to contain all the images 4 2 Orientation and Analysis of the Tissue Train Culture Plates Two different plate frames one frame for four 6 well plates and one frame for four 24 well plates are provided with the SCANFLEX software package The culture plate location in the frame corresponds to the numbering of the plates and wells in the XYFLEX software If the plates are placed according to the instructions the wells will be named as shown in Figures 11 through 14 in the XYFLEX software See the Appendix for an illustration to identify the corresponding location of wells on the scanned image with reference to their physical
17. ivation flexcellint com at Flexcell International with the user codes to obtain an activation code Note Do NOT close the XYFLEX software until the activation codes have been received from Flexcell International Doing so will generate new user codes voiding the previous user codes and the activation code 4 Enter the activation code in ACTIVATION CODE 1 box and click on CONTINUE 5 If EVALUATE XYFLEX is selected you will have 30 days to use the XYFLEX software after which it will automatically deactivate Xyflex Activation Please call Flexcell at 1 919 732 1591 or email us at info flexcellint com to activate Xyflex Please have the User Code 1 and User Code 2 numbers shown below when calling or emailing Activate Xyflex have User Code 1 User Code 2 already purchased and would like to activate the product 244582117 102328670 Evaluate Xyflex ou have 30 days left in your eva Activation Code 1 Activation Code 2 Go To Website Figure 9 Options for activating XYFLEX software Figure 10 User code and activation code for XYFLEx activation XYFLEX INSTRUCTION MANUAL PAGE 5 OF 19 FLEXCELL INTERNATIONAL CORP 4 XYFLEX Software Setup 4 1 General Settings The XYFLEX software may be installed on any Windows based PC meeting or exceeding the requirements listed in Table 1 The SCANFLEX and XYFLEX software do not necessarily have to be installe
18. may be resaved or reopened and saved as a Microsoft Excel file or opened in other data analysis software programs XYFLEX INSTRUCTION MANUAL PAGE 14 OF 19 FLEXCELL INTERNATIONAL CORP 7 XYFLEX Post Processing 7 1 Modifying Individual Well Images Individual images in the processed results may more consideration if the pixilation does require not possess the desired effect For instance there may pixels pixilated white which are outside of the object being analyzed but inside of the ROI In this case highlight the cell respective to the well image and select MODIFY WELL Figure 27 1 A combination of adjusting the threshold range for a selected well image utilizing the editing tools allows the freedom of pixilation creation or deletion Figure 28 The threshold range for the selected well image may be adjusted using the LOWER VALUE and UPPER VALUE The user may also ZOOM to sections of the image to ensure a higher quality pixel selection in a particular section of the chosen image The AREA PIXELS and AREA UNITS are shown below the image window The AREA UNITS are the units chosen by the user during the CHANGE THRESHOLD screen either pixels um mm or cm 2 When the threshold in the image is satisfactory to the user select ACCEPT Figure 29 This action will navigate the user back to the PROCESSING screen with the Instructions Move mouse over to expand
19. nection O Use a Web browser email client telephone or Fax machine to acquire an activation code Apply one or more 20 character activation codes previously acquired For this computer Understanding the National Instruments Software License Agreement How can I move an activated product to a different computer Figure 6 NI activation wizard NI Activation Wizard Activating Vision Development Module 2011 Vision Run Time NATIONAL ON INSTRUMENTS Products Successfully Activated The following products have been successfully activated You may need to restart the associated products to use any new features Vision Development Module 2011 Vision Run Time If you experience any problems have any questions or have Feedback regarding product activation visit the Following Web site ni com activate If you did not register your product during the activation process visit the Following Web site ni comjregister Figure 8 Successful activation screen XYFLEX INSTRUCTION MANUAL PAGE 4 OF 19 FLEXCELL INTERNATIONAL CORP 3 1 Activation of XYFLEx Software 1 Click on the XYFLEX icon on the desktop or go to program files and open XYFLEX software 2 When XYFLEX activation screen opens up select either ACTIVATE XYFLEX or EVALUATE XYFLEX Figure 9 3 If ACTIVATION is selected two user codes will be displayed in the next screen Figure 10 Call 1 800 728 3714 or 919 732 1591 or email act
20. re System The SCANFLEX scanner and software enable the user to create scanning regimens so bioartificial tissues in 6 well or 24 well Tissue Train culture plates can be scanned periodically to determine the rate of matrix compaction by cells overtime The XYFLEX software examines area measurement of the bioartificial tissues in the image series The software creates Excel and Text files which may be used to evaluate the change in area measurement of the analyzed data 1 2 Use of XYFLEX Software for Other Applications XYFLEX software may also be used for other applications requiring area measurement The images must be captured by Flexcell s SCANFLEX software and scanner The component also should be analyzed inside of the 6 well or 24 well plate templates provided by Flexcell due to the Region of Interest ROI locations The 6 well orientation will examine up to 24 positions in a series of images The 24 well orientation will examine up to 96 positions in a series of images 2 Components e XYFLEX software V3 3 0 e User manual e Desktop computer or laptop computer not provided See Table 1 for more details Table 1 Minimum system requirements for XYFLEX Minimum system requirements USB 20 11 Microsoft Windows XP Vista Windows 7 32 Bit 95 MB Microsoft Windows XP Vista Windows 7 64 Bit 95 MB Minimum screen resolution 1024 x 768 pixels Note The above are the minimum system specifications for the compu
21. roup 135219 mio we 16a sa eam Test Group 0454 14 00 x9 00 205 Figure 32 Display of the Subgroup Averages XYFLEX INSTRUCTION MANUAL PAGE 17 OF 19 FLEXCELL INTERNATIONAL CORP 8 Appendix 4555004 Figure 33 Physical orientation of the wells in Flexcell s 24 well plates on the scanner bed A their corresponding locations in the scanned image B Zu za O Figure 34 Physical orientation of the wells in Flexcell s 6 well plates the scanner bed A and their corresponding locations in the scanned image B PAGE 18 OF 19 XYFLEX INSTRUCTION MANUAL FLEXCELL INTERNATIONAL CORP Table 2 Relationship between image file size and image parameter setting Image file type Resolution setting dpi Quality setting Image size MB SM o 80 Ta JPG JPG 300 JPG 150 XYFLEX INSTRUCTION MANUAL PAGE 19 OF 19
22. t window and press DELETE This will not delete the well altogether but simply remove it from the subgroup To clear the entire right window of all data and subgroups press CLEAR ALL XYFLEX INSTRUCTION MANUAL PAGE 16 OF 19 FLEXCELL INTERNATIONAL CORP 7 3 Calculate Subgroups The developed subgroups from the MARK SUBGROUPS screen displayed in tables on two separate tabs on the CALCULATE SUBGROUPS screen 1 The values shown in the table on the left tab Figure 31 are the calculated areas for each time point of each well for the groups of processed images from the MARK SUBGROUPS screen 2 The values shown in the table on the right tab Figure 32 are the calculated subgroup average areas per time step for each subgroup 3 The user may choose to EXPORT TO EXCEL csv or EXCEL TO TEXT txt The EXPORT EXCEL action creates a Comma Separated Values file csv Once this csv file is saved to designation the file may be resaved or reopened and saved as a Microsoft Excel file or opened in other data analysis software programs Instructions Move mouse over to expand Export to csv Export to Text Groups of Processed Images SubGroup Averages d LI A XAVE HEN EE Wm san T Bs Quit Back to Start Process Another Export to csv Export to Text Test GroupA em ux rage Mn Test G
23. take the user to the Calibration screen Figure 18 5 1 Creating a Calibration File Calibration file accuracy is vital to obtaining correct post processing results 1 Select LOAD IMAGE to open an image where a physically measured dimension is known For example a user may choose to place the highlighted green rectangle to contain the boundary of one 6 well plate in the scanned image If the user measures the 6 well plate using a set of calipers and the resulting dimensions are 92 2 mm x 136 mm the X DISTANCE window wil read 92 2 the Y DISTANCE window will read 136 and the UNITS window will display mm 2 Select SAVE and choose the directory to save the calibration file xml for processing the images The default save directory is C Documents and Settings All Users Application DataMFlexcell XYFLEXMconfig V Calibration File Name Chosen by User xml 3 Select RETURN to go back to the SELECT FOLDER screen The calibration file directory will appear in the CALIBRATION FILE window Select NEXT to proceed to the SELECT FILES screen Figure 18 Screen to create a calibration file 5 2 Defining the Image File Processing Parameters The SELECT FILES screen allows the user to arrange the files in the order which they will be processed choose the WELL ORIENTATION select the RESOLUTION of the images captured in the SCANFLEX software and choose an ANCHOR FILE 5 2 1 Ordering the Image Files 1 The images from the IMAGE FOLDER appe
24. ter to operate the scanner More memory will be needed depending on scanner frequency and image resolution see Table 2 in the Appendix for more information on image size corresponding to image parameter settings XYFLEX INSTRUCTION MANUAL PAGE 2 OF 19 FLEXCELL INTERNATIONAL CORP 3 1 2 3 4 5 6 7 8 9 Installing XYFLEX Software Insert the XYFLEX CD into the CD ROM drive of the computer The XYFLEX installation process should start automatically If not go to My Computer and double click the CD ROM drive Double click the Setup icon Figure 1 Select the destination directory to save all program files Figure 2 and click NEXT to continue Accept the National Instruments license agreement and click NEXT Figure 3 Click NEXT to begin the installation Figure 4 Click NEXT after the installation is complete to continue Figure 5 Activate the National Instruments product through a secure internet connection Figure 6 and click NEXT The NI Activation Wizard will prompt to enter the serial number for the Vision Development Module 2011 Vision Run Time Figure 7 Input the Serial Number located on the NI Vision Development Module Run Time License which is included with the equipment documentation 10 After entering the Serial Number and activating the software select FINISH Figure 8 XyF lex 3 3 0 CD lle Flexcell Xyflex Fie View Favortes Tool
25. vailable for selection in the drop down window are pixels um mm and cm2 The units chosen will be used for the remainder of the analysis xj 2 The large window below the area Lower Value T Value units lists the WELL NAME PXS 108 pixels and the AREA The WELL NAMES and the order should resemble the well names and Figure 24 Adjusting the threshold in the ROI XYFLEX INSTRUCTION MANUAL PAGE 12 OF 19 FLEXCELL INTERNATIONAL CORP order defined during the SELECT WELLS screen NOTE The first row is already filled in at the initial state as a consequence of the LOWER VALUE and UPPER VALUE of the threshold range shown at the bottom of the screen 3 Next the user should adjust the LOWER and UPPER VALUES of the threshold so the desired area to be LALO a measured is completely pixilated r with white pixels in the image Shown on the right side of the CHANGE THRESHOLD screen Figure 25 Choosing a LOWER VALUE of zero will fill the ROI with 100 white pixilation meaning 34 10096 of the area inside the ROI is selected the area measurement evaluation Choosing an UPPER VALUE of 140 will fill the ROI with 0 white pixilation meaning 0 Figure 25 Evaluating the anchor image threshold of the area inside the ROI is selected in the area measurement evaluation The LOWER and UPPER VALUES should be adjusted accordingly to obtain the desired result The user may utilize the ZO
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