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User Manual 2011A

1. 1 13 x 0 75 mm 0 84 mre Analysis Filter Settings v F Object Counting Analysis Filter Manual Calaedinn v bal Save Filter i Post Filter Summary Object Properties for Current Image Area pr Eccentricity Mean intensity Summed intensity Total Count 225 Avg Mean Intensity 60 4 min 200 E min C min C min Avg Area ur 283 1 Avg Summed Intensity 7817 3 C max C max C max C max Figure 9 13 The Analysis Filter Settings Task Pane Adjust settings to exclude Objects with Minimum Area of less than 200um The exclusion of these Objects is reflected not just in the image but also In the Post Filter Summary in the Vessel View window The values in the Post Filter Summary will change to reflect changes in the Filter Settings In Figure 9 13 the Total Count reads 225 Prior to setting the minimum Object Area to 200um the Total Count was 338 The Metric data under the Graph Export tab When Filter Settings are adjusted they are initially applied ONLY to the currently selected image This is because a certain degree of processing is required to apply the new settings to the image The time required to reprocess a single image to reflect updated Filter Settings is quite short The time required to reprocess an entire Analysis Job is more substantial Thus in the interest of time Filter Settings are initially applied to one image at a time in order to facilitate ease of manipulation This is the ca
2. 10 1596SWTCN Scratch Wound 2s LLL Is F Now Search Label test 96 Figure 8 112 Search Label for test 96 options Start Date Time End Date Time Label Cell Type Figure 8 113 Search Label for test 96 See Figure 8 113 By enclosing the terms in quotation marks the search looks for all scan labels that include the terms test AND 96 together in that order for the same vessel There were no results using this search string 161 Page if SEN ENC A UN i B E i DAt Finally search Label t t See Figure 8 114 Search By For k t When was the vessel scanned Refine Search By fs Label 6 Vessels Shown Columns Cell Type End Date Time s12 2008 12 1 Test aii weekend Test A10 weekend Test A10 Weekend Test CHO weekend Test 410 weekend Test A10 Start Dake Time S 12 2008 11 4 HT 10080 00 1 7 2008 2 32 PM 1712008 2 00 PM LH 20DS 6 23 PM 1i7iz008 2 00 PMI 1 4 2008 5 38 PM 1 4 2006 6 00 PM 1 4 2008 5 38 PM i 1 4 2008 6 00 PM 1 4 2008 6 23 PM 1 4 2008 6 23 PM Weekend Test Figure 8 114 Search Label for t t Refine Search By Search results that were obtained using the Search By function can be filtered once more using the Refine Search By box The Refine Search function performs a follow up search on the results of the coarse Search By function Note that the Refine Search f
3. Customization Dialog Export Dialog Help Figure 8 161 Undo Zoom 8 10 6 Microplate Graphing The Microplate Graphing selection provides another option for graphing all experiments conducted in a microplate format Cc Microplate Graph eS loll lej uxa File Edit View Experiment 1 Object Count per mm over 1 day Unfiltered No filter parameters set PEA E AEA 66 44 A Sa F F p p P L p Er P g 5 5 gt eS A e E LA A SAS A AS AA A SRR RBA KRH NAK BAA RAL KLKRRRRAI AKARARAR CRAL ARK NI ARRA SKRIK SAR AAA A EEEEEEPEO M 66 A p r ale Ta L w p SSSA MALAAAML Ax id ad a adhd ad ad af a ad aL a Figure 8 162 Microplate Graph 199 Page Using the Microplate Graphing function users can easily graph all wells individually in high or low density microplate Unless selected otherwise the Microplate Graphing color scheme directly mirrors the information in the Plate Map editor Furthermore Microplate graphing can be customized using the Menu options These options include e Editing Titles Subtitles Axis Range Control Show Hide x and y axis labels Logarithmic y axis scaling S M L marker sizes In addition users can export images of the Microplate Graph by selecting one of 4 different file tyoes PNG TIFF JPEG or BMP and selecting one of 5 different predet
4. ESSEN BIOSCIENCE N Two Phillips Pan Head Screws D Figure 2 3 Removal of the Drawer Brace 12 Page N 3 Installation of the IncuCyte Hardware 3 1 Positioning the Controller It is important to put the controller into position before placing the microscope into the incubator The controller should be placed on a level surface within 3 feet of the incubator s cable port The top of the incubator often works well Do not however connect any of the controller cabling at this point 3 2 Placing the Microscope into the Incubator IMPORTANT The IncuCyte EX microscope is NOT designed for use inside a cell culture incubator It is not compatible with the size temperature or humidity of an incubator environment NEVER place the IncuCyte EX into a cell culture incubator When choosing a shelf for the microscope keep in mind that flasks and plates are placed into the IncuCyte cell drawer from above For incubators that are at table height a shelf installed near the bottom of the incubator is usually most convenient Approximately 8 inches of height will be used by the instrument In most incubators this usually allows room for at least two shelves to be installed above the microscope Remove all shelves above the targeted shelf before setting the microscope into the incubator Feed the two cables from the microscope out through the cable access port on the back or side of the incubator The cables can usuall
5. January 09 Wed 10 Thu H 11 Fri H 12 Sat H 13 Sun E 14 Mon H 15 Tue 16 Wed 17 Thu 3 18 Fri VASE 10 30 00 AM 00900000000 OLOLOLOLOLOLOLOLOLO OOO0000006 Tot totototo DOG ci 00000009 0000 enin 3 14 1 Confluence w13 Coming 100mm 8D 100mm 25 6 Confluence w13 14 6 Confluence v13 Image Mean Focus Position Exposure Time Autofocus Sharpness Statistic Mean E rag Time Range January S 17 Thu Start Time 10 30 PM 18 Fri 19 Sat 20 Sun 21 Mon 1 30 AM 4 30 AM 7130 AM 10 30 AM 4 30 AM 7 30 AM a 10 30 4M ar 1 30 PM 5 ensasi cestessis oa dos man S80 0888 888088 fa TLETT a 4 30 PM k C CCLETCTLECT sOes8 dm sa Ose Region All Wells y H 7 30 PM ie e ti ase t T r o 10 30 PM sosss Saslees ee k LETT 19 Sat 20 Sun H affitti etti etti tE z z Group al Coming 384 New BD 384 74 a oe 9 28 5 Confluence w13 ps 17 2 Confluence w13 Figure 8 129 Ready to Graph Press the Graph button A graph will now appear in a new window Figure 8 130 The graph will be assigned a default title in this case All Mean vs Time The same graph can be obtained by using the Vessel View graphing option e Look at the graph in Figure 8 130 Because Region All Sectors Group All was selected only a single trace appears that represents the mean of all sectors in the dish In Figure 8 1
6. Dilute wells and the direction in which the replicates run If for example a dilution series runs from left to right and the User wishes the replicates to be arranged horizontally a box will appear allowing the User to specify how many wells exist at each concentration singlets duplicates triplicates efc Once the dilution series is specified click OK and the compound will appear on the plate map in the specified series Cells Add a cell type in the same manner as a compound Figure 8 93 Once a region of the plate is selected click Add ___ to open up a new window Here specify passage number and seeding density and click OK to add the cell type to the plate map Growth Conditions Once a growth condition is specified in the list simply click Add ___ to add it to the plate Because the Growth Conditions category is used for conditions where no concentration or dilution series is specified clicking Add __ will add the condition directly to the plate with no intervening options Adding Multiple Well Items To sequentially add multiple items to a grouping of wells i e cells growth conditions compounds select the plate map icon second to the right on the top menu that reads Keep Selection After Adding to Wells Normally after adding an item to a well or group of wells that grouping will automatically be deselected With the Keep Selection icon activated the sel
7. Fluorescent images can ONLY be viewed and manipulated using the Vessel View window See Figure 9 1 Therefore this window plays a more important role in fluorescence imaging relative to non fluorescence imaging Vessel View 5 19 PM on 10 30 2008 File Utilities View fir30001 admin aue g SYSE 2008 October 30 Thu seo ooo ene ane oea nen 5 19PM S28 S08 SES O88 135 13 5 19 PM orien 3 19 PM B88 17 7 Contuence ui4 11 19 PM 31 Fri H November A B2 Imagelof9 1 Image Properties Graph Export Fluorescence v Minimum Intensity 5 6 3 Maximum Intensity 104 3 J Auto Scale Always As y Phase Contrast I um 400 1 908 1 52 mm 2 89 mv Analysis Filter Settings Figure 9 1 The Fluorescence Vessel View Window 201 Page N 9 2 Viewing Fluorescent Images in the IncuCyte FLR 9 2 1 Fluorescence scanning generates both an HD phase contrast and fluorescence image at each scan pattern location Open the Vessel View window and select the Image tab The Image tab in the FLR Vessel View Window provides the User with a selection of viewing options Itis divided into two Channels A and B Use the corresponding pull down menus to make your selections See Figure 9 1 You can select either Phase Contrast or Fluorescence for each channel In Figure 9 1 Channel A is set to Fluorescence and Channel B is set to Phase Contrast The two channels are independent and the settings for
8. Follow these steps to perform the fluorescence calibration Place a microslide tray in the front position of the IncuCyte Fluorescence calibration requires use of the front tray position and the remaining positions can be occupied with trays at the time of calibration Load a calibration slide with supplied dye Use 40ul dye per slide Place the pipet tip at one end of the slide window and expel the liquid dye The liquid will wick into the slide window After all the dye has been expelled use the pipet to transfer a small amount of dye to the other end of the window to be sure it is completely filled Avoid getting any dry spots in the window as this will interfere with proper calibration Place the calibration slide in the FAR LEFT CUTOUT POSITION of the microslide tray The orientation of the slide within the cutout position is not important However it is ESSENTIAL that the calibration slide is the ONLY slide in the microslide tray Verify that the rest of the tray is empty Select the Tests tab under the Administer IncuCyte tasks bar Then select the Fluorescence Calibration test Press the Run button A new window will open to confirm that the IncuCyte is properly configured to run the test Press the Yes button to start the calibration See Figure 6 10 The calibration procedure will begin Fluorescence Calibration requires 15 minutes Calibration status will be indicated at the top of the IncuCyte software window an
9. For recommendations on how to reduce background fluorescence see the Essen BioScience application note on this topic The Refinement Pull Down Menu The Refinement option allows the User to adjust the degree of Refinement or separation between adjacent Objects 209 Page Refinement Edge Split Edge Split values have a range of 1 00 to 1 00 Based on the Edge Split value the software makes an attempt to split or separate Objects that are close together Positive values will cause more Objects to be split Alternatively negative values will result in fewer Objects being split Refinement None No attempt will be made to separate adjacent Objects Preview Using Current Image Button Pressing the Preview button allows the User to run an analysis on a single currently selected image in order to preview evaluate the Analysis Parameters The Preview function will be more fully discussed in Section 9 3 4 page 211 Launch Analysis Button Pressing this button will launch an Analysis Job for all selected images based upon the selected Analysis Parameters See Section 9 3 5 page 215 for more information Save Analysis Parameters Button The User may define Analysis Parameters that will be applicable to multiple experiments Rather than having to manually reset these same parameters each time an analysis is launched parameter settings can be saved named and reopened at a later time Use the Save Analysis Parameters button to
10. Scan overlap as a result of the longer initial scan The increased scan length required for 96 well plates must be taken into account when setting up the scan interval s It is possible that even though all subsequent scans will be fine the longer first scan could overlap with the beginning of the second scan If this is the case then the second scan will be skipped Scanning will start again at the third scheduled scan time If such an overlap will occur a warning window will appear indicating that the second scan will be skipped The second scan can be set to start after a somewhat longer interval with subsequent scans occurring at the regular shorter interval But if the scanning interval is offset in this way it is important to remove the very first Scan Bar from the Timeline after it is completed This is because after the IncuCyte has finished the scans for the day it will come back and start the scans set for the beginning of the next day If the offset Scan Bar is not removed the IncuCyte will include it in the next day s scan thus disrupting the new interval and possibly causing another overlap 79 Page N 12am 2am 12am 2am y I I 4am 6am Sam 10am 12pm 2pm 4om 6pm Spm 10pm 12am Figure 8 29 Scan overlap When scanning starts for the next day the offset initial scan time will still be on the Timeline causing an overlap Figure 8 29 This overlap would prevent the scan from being set However if
11. 1 Include Vesse Label in trace names Figure 8 122 The Graphing Window from the View Completed Scans Screen 170 Page UN Time Plot ESSEN BIOSCIENCE The overall Graphing features available in the Vessel View window are the same as those available using the View Completed Scans Graphing Box but there are some additional options specific to the Vessel View Custom Region The Region pull down menu in the Vessel View window offers a Custom Region option See Figure 8 123 When Custom Region is selected the vessel color will change from light orange to green thus alerting the User that this is a special Image Mean Confluence v1 5 Focus Position Exposure Time Autofocus Sharpness hw 10 00 PM G 24 Sat 25 Sun 26 Mon 27 Tue Histogram 00000000 Ls i s Mts t s ts Wt it Ht 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 Region Custom Region Label Group enter a custom label All E Include Vessel Label in trace names Figure 8 123 Custom Region Selected graphing Mode When Custom Region is selected the User can select deselect a subset of individual wells with the mouse as desired Normally when a graph is generated it is automatically assigned a Label based upon the selected Region and Statistic Because in this case the Region is variable it is poss
12. 47 Page A ESSEN BIOSCIENCE Select the Options button to expand the temporal criteria of the search Search for vessel using term of interest Start Date Time Label Cell Type 1 25 2008 5 32PM HO Movie Plate multiple 1 10 2008 12 4 HD Flask Test HO Flask Test cs HO Flask Test HD Flask Test HD Flask Test 1 9 2008 9 11 AM HD Flask Test seep HO Flask Test lin 12 10 HD Flask Test Time Tree displays Data can be scans ONLY forthe Selected vessel graphed exported etc as from View selected vessel and Time Completed Scans Screen Figure 7 8 Find Scanned Vessels Screen 7 3 1 Find Scanned Vessels EX As mentioned in View Completed Scans the Find Scanned Vessels Screen is the default screen that opens when the EX software is launched and it is the jumping off point for viewing scan results Users of the Standard Model IncuCyte and the IncuCyte HD frequently set scan schedules involving a small number of vessels that are scanned many times In contrast the IncuCyte EX is specifically designed to scan an extraordinarily large number of vessels each of which is scanned infrequently Therefore it is generally easier for the User to locate their particular vessel of interest via the Find Scanned Vessels screen See Figure 7 9 For more information see Section 8 9 page 164 48 Page ESSEN BIOSCIENCE IncuCyte Ses o File View Scan P
13. Essen BioScience manufactures a special 24 and 96 well plate type called the Essen ImageLock plate These plates facilitate very precise repeated imaging within each well Under standard imaging conditions slight variations can occur in the location of imaging from one scan to the next resulting in small jumps between frames When very precise repeated imaging is required for instance when generating a movie use the Essen ImageLock plate By selecting an ImageLock plate and running it in ImageLock mode the highest fidelity movie will be generated If an ImageLock plate is selected as the vessel type under the Physical Layout tab the Scan Type box directly beneath will include a Phase Contrast ImageLock option Figure 7 4 While the default setting will be Phase Contrast ImageLock an ImageLock plate can be run in either Phase Contrast ImageLock or Phase Contrast mode 42 Page Middle Tray Tay Type Vessel Type 96 well Essen magelock ss Scan Pate New Select Al Select None Figure 7 4 Running in ImageLock Mode If an ImageLock plate is run in the standard Phase Contrast mode then locking will not occur and the results will be comparable to those of a standard non lmageLock plate NOTE It is recommended that ImageLock plates ONLY be run in Phase Contrast ImageLock mode For information on obtaining the Essen ImageLock plate contact Essen BioScienc
14. IncuCyte Users Manual 2011A Essen BioScience Inc 300 West Morgan Road Ann Arbor Michigan 48108 USA 1 734 769 1600 www EssenBioScience com User Manual Written by Elizabeth Leslie Edited by Thom Nelson Jason Wantuck and Dan Appledorn 1 Page N Table of Contents INCUCYTE USERS MANUAL cscccccoccocceccecceccsscesccsccecceccsccsccsscsscsccccsccssceccsscssccacesceccecescccecescsacesceacense TABLE OF CONTENTS acciri aE Eea a aeaa aaaea aaia WARRANT ona E E E 6 1 1 LIMITATION OF WARRANTY cces overarergacecanaecinenesnicee rane rasiusseeauconteadseaoteadeeatenaneercammecanteaaeueeans 6 1 2 EXCEUSIVE REMEDIES careia rano ena i T EE E E EE E E 6 1 3 WARNINGS AND DISCLAIMERS siacacaciusepecssniceaiieasisanedaaspeiuneaces sennacunectunuipunede uneseanseavereaseoqeeanes 6 GETTING TO KNOW YOUR INCUCYTE eeeeessssscecccosssscceccsssssececosssssccecosssssseeooo 7 2 1 TIN FRODUC LION aesir EN eaeee ripeness ees ates 7 2 1 1 Ineu ve MOGEIS eissira ise E SEES EE ERT EE EAs E 7 2 1 2 TPCT USE ieseni esien ENEE EEEE EE EEEE E 7 213 OY TS OT ye NIA Qian tree entre eee mere RPE eee ee mre ne ore nner eon er ore mene eran meer es 7 2 1 4 The IncuCyte HD Imaging Module iicicccccccccceseeeesetesteeteeeesestesscssssssssssssssssssssssssseesssseaas 7 ZA De ACC ye IIe sia wince cre ses E I E E E EA 8 2 1 6 Special Model IncuCyte Instructions ccccccccccccccccccccssesseeesesseeeeeeeeeeeeeeaaaaaaassssseseeeeeeeees 8 22 SAPE Wb AA
15. Restoring archives is only recommended under 2 circumstances e Searching Vessels archives cannot be searched e Running Analysis Jobs See Manipulation of Archived Data FLR page 117 To restore an archive browse to the archive location and then select the scans to be restored Archive Vessels When scans are archived as described in Section 8 6 7 page 110 all vessels on the screen will be archived together into a single archive file However it is 114 Page also possible to archive one or more individual vessels while not archiving others from the same screen There are 3 ways to archive vessels e Select Archive Scans from the View Completed Scans Scan pull down menu Figure 8 68 e Right click on the desired vessel in the View Completed Scans screen e Access Archive Current Vessel from the Vessel View Utilities pull down menu or in the Vessel View Tasks Pane Unlike entire scan archives it is NOT possible to append to a vessel archive NOTE The Scans tab is only available at the Administrator level however individual vessels can be archived at the User and Guest levels The remaining functions available under the Scans pull down menu will be discussed in Section 8 7 9 page 152 Preferences Help Search Chrl ShiFk 5 Show Log Cbrl ShiFE L First Scan Chrl ShiFt F Previous Scan Ctrl ShiFE P View Vessel Ctrl ShiFt GraphiExport Ctrl ShiFk Archive vessel Ctrl Shift 4 Delete Vessel Ctrl
16. item and press the Properties button below the list of items 5 Copy down the settings found under the General tab and other tabs Some of these setting will be modified to create the 2 device network and you will want to remember the current settings when it comes time to reset the port to its regular configuration 6 On the General tab select the Use the following IP address Radio Button 7 Type in the following IP address 192 168 128 1 Figure 4 1 page 18 8 Type in the following Subnet mask 255 255 255 0 9 Press OK to exit the property sheet 10 Press OK again to effect the change to the connection 11 Follow the steps in Section 4 1 2 page 18 for verifying the presence of the controller on the network 12 Follow the steps in Section 4 1 1 page 16 for verifying the presence of the controller on the network Recall that the ultimate goal is to configure the controller s primary Ethernet port with a static IP address selected for your corporate network Once the 2 device network has been established and verified you must connect to the controller s desktop in order to configure the primary port Follow the steps in Section 5 4 page 23 Additional information on creating User accounts can be found in Section 8 6 3 page 95 Once on the desktop the controllers primary Ethernet port can be configured with a static IP address in the same way that the PC s primary port was config
17. 354429 354461 354516 354596 354607 354640 354649 354650 354651 354657 354670 354689 356407 356461 356516 356519 356640 356649 356650 356651 356689 356690 356692 356693 356698 356700 3300 3340 3372 3474 3585 3595 3596 3598 3599 3603 3610 3628 3665 3666 3667 3841 3842 3843 3903 3904 3917 3997 4379 655087 655088 655090 655098 655160 655162 655180 655182 3860 096 3861 096 MGB101 1 1 LG MGB096 1 2 LG L MGB096 1 2 LG CC L MGB096 1 2 LG PDL L MGB096 1 2 LG FN L MGB096 2 2 LG MGB096 2 2 LG L MGB096 2 2 LG CC L Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate White Wall Glass Bottom MatriPlate 0 17mm with Lid Microplate White Wall Glass Bottom MatriPlate 0 17mm Collagen with Lid Microplate Plate Name Tray Type BD BioCoat Poly D Lysine 96 well Microplates Black Wall Clear Bottom Optilux BD BioCoat Poly D Lysi Whi l il Corning CellBIND 96 Well Clear Flat Bottom Black Polystyrene Microplate with Lid Std Microplate CellStar 96W Plate PS ST TC FLT BOT BLK uCLR Lid 127 8 86 15 MM TC W LID CellStar 96W Plate PS ST TC FLT BOT WHT uCLR Lid 127 8 86 15 MM TC W LID PS F Bottom chimney well Crystal Clear Sterile Lid Condensation Rings PS F Bottom chimney well Crysta Clear Lid Condensation Rings 10 per Bag Microplate Microplate Microplate Microplate _ Microplate Microplate Microplate _
18. 5 oO 8 3 40 Time Hours Drag and Drop Marwal Alignment Auto Alignment is Apply Clear jse the Apply button to align the plots Use the Clear button to align the plots by ther ana Andy _ cea m first point Figure 8 150 Alignment set at 15 Graph All Mean vs Time Fie Yiew Edit Average Smooth Growth Over Time Flask1 Flask2 Confluence v1 0 Percent Drag and Drop Manual Alignment Auto Alignment po Apply Clear Use the Apply button to align the aa een Clear button to align the plots by their Figure 8 151 Alignment set at 50 Manual Alignment Deletion This function enables manual alignment or deletion of each individual trace on a graph When the Manual Alignment tab is selected the graph will initially be aligned at the value set in the Auto Alignment box While the Auto Alignment function is used to align all traces on a graph to the same point Manual 192 Page Alignment facilitates the alignment of individual traces upon a single graph at different points In Figure 8 152 the graph consists of 3 traces all aligned at 23 confluence But the starting confluences differ between the traces Align all three traces to the same starting confluence using the Manual Slider Select each trace to be manipulated using the Trace Selection Box to the right of the Slider In Figure 8 152 Well 1 is selected The selected trace can be pulled to the left and right using the slide
19. A Layout can be saved in the Schedule Upcoming Scans screen by clicking on File at the very top left of the screen Create the configuration to be saved Select Save Layout As from the File pull down menu This will bring up the Save Layout window Figure 8 36 Save Layout EXAMPLE 2 Enter the 86 Page N name of the new Layout Save the Layout to add it to the Current Layouts box To open load an existing Layout select Load Layout from the File pull down screen Highlight the desired Layout from the Load Layout window lt Save Layout Current Layouts Example Layout Mame Example 2 Save Layout Figure 8 36 Save Layout EXAMPLE 2 An existing Layout can be deleted using the File pull down menu 8 6 Administer IncuCyte The functions available on the Administer IncuCyte screen will vary according to the Permission Level of the User this topic is further discussed in Section 5 3 2 page 21 The Administer IncuCyte screen is only available to Administrators and Users not to Guests Administrators have access to all administrative functions but Users have access to only a subset of functions The Administer IncuCyte screen automatically opens to the Device tab Figure 8 37 shows the screen available to Administrators and Figure 8 38 shows that available to Users The Administrator screen includes tabs for Device Accounts Tests Update Scans and Logs and all features within each tab are active At the
20. Controller Non Condensing Non Condensing Non Condensing Non Condensing Operating 0 C to 42 C 0 C to 42 C 0 C to 42 C 0 C to 33 C ananta 5 to 95 RH 5 to 95 RH 5 to 95 RH 5 to 90 RH Microscope Non Condensing Non Condensing Non Condensing Non Condensing fo ya He Wy lr J ae pe bag 9 5cm x 44cm x 52cm 8 8 x 17 7 x 18 3 a7s X17 25 KS 9 5cm x 44cm x 52cm 8 8 x 17 7 x 18 3 Sy By a Yee eit hoe 9 5cm x 44cm x 52cm 9 9 x 17 7 x 18 3 ST Woe hPa 9 5cm x 44cm x 52cm 8 8 x 17 7 x 18 3 Other image formats are available via export functions in the software Other wavelengths are available by special order Please contact your sales representative Notes Choice of 10X or 20X objective is only an option on the IncuCyte FLR IncuCyte FLR is only available with HD optics 243 Page 13 IncuCyte Catalogue Catalogue Item Number IncuCyte Microscope System Includes Onsite Installation and Training 4362 Essen IncuCyte Controller Unit 4364 Essen IncuCyte Base Software 5025 0106 IncuCyte Calibration Tray Customer must choose 6 additional IncuCyte Trays prior to purchase IncuCyte HD Microscope System Includes Onsite Installation and Training 4362 Essen IncuCyte Controller Unit 4364 Essen IncuCyte Base Software 5025 0106 IncuCyte Calibration Tray Customer must choose 6 additional IncuCyte Trays prior to purchase IncuCyte
21. FLR Microscope System Includes Onsite Installation and Training 4368 Essen IncuCyte FLR Controller Unit 4376 IncuCyte FLR Gantry 4364 Essen IncuCyte Base Software including Fluorescent Object Counting Software 5025 0106 IncuCyte Calibration Tray 1221 0275 IncuCyte Microslide and Calibration Tray Customer must choose 6 additional IncuCyte Trays prior to purchase and must choose between the following 2 lens options 10x Objective 5050 0109 00A or 20x Objective 5050 0110 00A IncuCyte EX Microscope System for integration with TAP Includes Training 4387 Essen IncuCyte EX Controller Unit 4364 Essen IncuCyte Base Software Requires integration by TAP IncuCyte EX HD Microscope System for integration with TAP Includes Training 4387 Essen IncuCyte EX Controller Unit 4364 Essen IncuCyte Base Software Requires integration by TAP CellPlayer 96 Well Cell Migration Invasion Assay Kit Includes 9500 4400 96 well Cell Migration Software Application Module The WoundMaker 96 Tool 5025 0191 WoundMaker 96 Well Rinse Boat Assembly 4379 Fifteen 96 well ImageLock Plates and 5025 0116 IncuCyte Micro Plate Tray Certificate of Analysis 1500 0078 A00 Two 2 Biocision BCS 147 96F CoolBox System Includes CoolBox Microplate System with CoolSink 96F 1500 0079 A00 One 1 Biocision BCS 133 M30 CoolBox System Includes CoolBox 30 System with CoolRack M30 and 1500 0080 A00 On
22. It is important to remember that aside from being synchronized the controller time settings should also be correct Synchronizing the controller to an incorrect time setting can lead to unexpected scan results Therefore if a disparity between the IncuCyte controller and the local computer becomes apparent the accuracy of the local computer settings should be confirmed prior to adjusting the controller settings This is especially important if a single IncuCyte controller is being accessed by multiple individual computers 90 Page NOTE Synchronization is ONLY required if a prompt is issued by the software In this case a prompt will appear following login Figure 8 40 and Figure 8 41 lf you are prompted to synchronize it is important to confirm that your personal computer has the right time and the correct settings Every time the IncuCyte software is launched it automatically compares the Time Zone and Daylights Savings Time settings with the controller s settings There is also a comparison between the controller time and the local PC time If these settings or values are not in agreement a window will be launched to this effect NOTE We strongly recommend that only individuals at the Administrator permission level implement changes to the controller time settings Synchronization is ONLY permitted when the device status is idle For instance synchronization cannot occur during a scan It is important to clo
23. Nunc EasyFlask Vent Close Cap Nunc EasyFlask Filter Cap 75cm Flask with Vent Cap 75cm Flask with Filter Cap a es 430725 Rectangular Canted Neck Cell Culture Flask with Phenolic Style Cap a Brand Catalog s Flask Name Tra Collagen 150 cm2 Flask with vented cap Poly D Lysine 150 cm2 Flask with plug seal Poly D Lysine 150 cm2 Flask with vented cap Collagen 150 cm Flask with plug seal cap ibronectin 150 cm2 Flask with plug seal cap BD Falcon O0OmL Canted Neck Standard TC Vented Cap OOmL Canted Neck Standard TC Plug seal Cap ollagen 150 cm2 Flask with vented cap Poly D Lysine 150 cm2 Flask with plug seal cap Poly D Lysine 150 cm2 Flask with vented cap Collagen 150 cm2 Flask with vented cap 3291 CellBIND 150cm Rectangular Canted Neck Cell Culture Flask with Vent Cap E 430823 150cm Rectangular Canted Neck Cell Culture Flask with Plug Seal Cap g 430824 150cm Rectangular Canted Neck Cell Culture Flask with Phenolic Style Cap e On 0n On O G1 O1 On O O O1 01 Ly lt alo 430825 150cm Rectangular Canted Neck Cell Culture Flask with Vent Cap TPP TP90150 690ml Cap TP90151 oO 90mL Filter Cap Brand Catalog s Flask Name Tray Type anani 3150 Traditional Straight Neck Cell Culture Flask with Phenolic Style Cap g 31451 Traditional Straight Neck Cell Culture Flask with Vent Cap 175cm Flasks Brand Catalog s Flask Name Tray Type BD Falcon 353118 750ml Straight Neck Standard
24. Only the Object Counting Task Bar is open In the left image Adaptive Segmentation and Edge Split Refinement have been selected In the right image Fixed Threshold Segmentation and no Refinement have been selected Angiogenesis New Analysis Optimize preview the parameters for Angiogenesis Analysis and launch a new analysis See Section Error Reference source not found Error Reference source not found page Error Bookmark not defined NOTE The Angiogenesis Task Bar will ONLY be available if the User has purchased the Angiogenesis Module 207 Page N 9 3 3 Analysis Tools Available in the IncuCyte FLR Tasks Pane All analyses must be launched from the Vessel View window Tasks Pane There are typically two steps to running an Analysis Job Use the Preview Current Image button to optimize Analysis Parameters Additional information and analysis strategies are provided in a Technical Note that can be downloaded from the Essen BioScience web page Launch a new Job Analysis Open the Object Counting Task Bar See Figure 9 4 Opening this Task Bar allows the User to adjust image Analysis Parameters to preview and launch Analysis Jobs and to save Analysis Parameters There are three pull down menus available related to setting Analysis Parameters Parameters Abbreviated in the Tasks pane as Params Segmentation Refinement There are three buttons available related to previewing launching jobs and saving Parameter
25. The calibration procedure requires up to 1 hour and it utilizes a special calibration tray that is shipped out with the unit The calibration tray is the same size and is inserted in the same fashion as the regular trays See Section 6 1 3 page 27 for proper insertion of trays into the IncuCyte Each of the three tray positions front middle and rear must be calibrated individually CALIBRATION TRAY Figure 6 5 The Calibration Tray 29 Page LN A ESSEN BIOSCIENCE What follows is an overview of the calibration procedure A more detailed explanation of the procedure can be found in Section 8 6 5 page 97 For now See Figure 6 6 IncuCyte far x mm File View Scan Preferences Help fIr30000 admin Ready To Scan Search Scans By Label for GO Task List Device Accounts Tests update scans Logs A View Completed Scans Motion Cakbration A Find Scanned Vessels Tray Position Front Test __ Calibrate and Confirm Run j r Confirm Oni Schedule Upcoming Scans 7 5 V Quick Test Sa Administer IncuCyte 5 Optics Tests ViewAfStoric tests and start new optics tests Go al 84 well Plate Calibratiop Selecta tray a front middle or Tray Position Front Run rear rs Calibration Select Calibrate Run a fluorescence calibration i Run and Confirm and Quick Test Calibration results will appear in the Logs tab L
26. This subsequent filtration of the data is performed using the Analysis Filter Settings in the Analysis Filter Settings Pane The Analysis Filter Settings Pane can only be opened during Preview Mode or after an Analysis Job has been opened Analysis Filter Settings will be more fully discussed in Section 9 3 6 page 222 Unapplied Analysis Preview Mode When Analysis Preview Mode is first opened the image will reflect the analysis results of whatever Segmentation and Refinement Parameter settings were present at the time the preview was launched However it s possible that after some initial evaluation the User decides to adjust these settings Changing any of the Segmentation or Refinement settings will shift the User into Unapplied Analysis Preview Mode The Banner at the top of the Vessel View window will change from beige to yellow and the text will read Unapplied Analysis Preview This Banner change is a warning to the User that Segmentation and or Refinement Parameters have been changed but they have not yet been applied to the image To apply the newly selected parameters press the Update Analysis Preview button The Image and graphical data will be updated to reflect the new settings and the User will be returned to Analysis Preview Mode 9 3 5 Launch New Analysis Once the appropriate Segmentation and Refinement Parameters have been selected it s time to launch an Analysis Job A new analysis can be launched from Analysis Pre
27. be two options for image export Export all Images Export Image __ of Total Single Time Image Export Select Single Time instead of Time Range See Figure 8 100 Then use the arrow buttons to scroll to the appropriate time or use the mouse to select a time Export Time4rea ange El 22 ttun Single Time reer Single Time 2 08 PM c08 PM 5 05 PM 11 08 Ph E 23 Wed Figure 8 100 Select Single Time for Image Export Select the sectors or wells for image export Remember only the selected images for the highlighted time point will be exported In Figure 8 98 only Image 1 in well A1 has been selected If multiple images were obtained per well or sector select which images to export If the images are exported in bulk then the same image will be exported for every selected well or sector In Figure 8 98 three images were obtained per well so it is possible to export a maximum of 3 images per well However in this same figure only one image image 1 has been selected for export Next use the browse button to select the target file location Select the Sequence Type Recall that images can be exported as movies or images In this case only images are desired so highlight Set of Individual Images in the Sequence Types drop down menu The movie options will still be 147 Page available in this menu but if you try to export single images as a movie you will get an error message Choose Image Type e Phas
28. sterile 160 box Microplate Perkin Elmer 6007440 CellCarrier 384 384 well microplate black clear bottom Collagen sterile 32 box Microplate UO UO OO UO UO NIN A I UN J BR i Dinisis D WiW venues O 10 O O 6007441 CellCarrier 384 384 well microplate black clear bottom Collagen sterile 3 box 6007450 CellCarrier 384 384 well microplate black clear bottom Poly D Lysine sterile 32 box Microplate 6007451 CellCarrier 384 384 well microplate black clear bottom Poly D Lysine sterile 3 box Microplate scar 152041 384 Well Optical Bottom Plates Black Clear Collagen Microplate vV 3 3 3 176751 ____ 384 Well Optical Bottom Plates Black ClearCC3_ Microplate V 176752 ___ 384 Well Optical Bottom Plates White ClearCC3_ Microplate vV W 311503 White Tissue Culture Treated 384 well Krystal Microplate with lid Microplate v 312003 Black Tissue Culture Treated 384 well Krystal Microplate with lid Microplate vV Porvair Sciences 312030 Black Tissue Culture Treated 384 well Krystal Microplate with lid Microplate 312503 Black Tissue Culture Treated 384 well Krystal Microplate with lid 311003 hite Tissue Culture Treated 384 well Krystal Microplate with lid Microplate aa 313030 White Tissue Culture Treated 384 well Krystal Microplate with lid Brand Catalog s Dish Name Tray Type BD Falcon 353001 35 x 10mm Easy Grip Cell Culture Dish tissue culture treated polystyrene Cor
29. to scan in the Scan on Demand mode is 20 longer than the estimated time in the 24 Hour Repeating scheduler This additional time is required so that the user has time to replace the Scan on Demand vessel with the 24 Hour Repeating vessel scanning using the Scan on Demand Scheduler is initiated by clicking the red scan button For additional information please refer to Section 8 5 9 Page 83 41 Page IMPORTANT 1 Remember the scanning process causes the IncuCyte to become warm In order to prevent overheating it is recommended that total scan time not exceed 45 minutes Although the Scan on Demand function can be used during cooling times and between scan times it is recommended that you do not scan too often 2 Scans of vessels in the Scan on Demand mode cannot be linked together using the Unique ID following a scan The Unique ID must be entered prior to scanning Furthermore scans of vessels in the Scan on Demand mode cannot be linked to vessels in the 24 Hour Repeating scheduler 3 All changes to the 24 Hour Repeating schedule must be Applied or Reloaded in order to access the Scan on Demand mode 4 Users MUST replace the 24 Hour Repeating vessels into the IncuCyte following a Scan on Demand Failure to do so can result in several problems including failed skipped scans or insertions of incorrect Scan on Demand vessel scans into the 24 Hour Repeating scheduled vessel scan sequence 7 1 5 ImageLock Mode
30. 02 29 08 13 55 15 hayam day ed betreneeeg te ho i gt San of 119 9 counts cbtaned wath a 2 20 meat exponse i Pent Gif fom mean per regor L Anos Tet i 25 25 191 0 Bigtres Contrast dee ttt A cesta Tors soot mgs sent to Quantitative results of selected Optics Test Figure 8 57 The Optics Test Window Initiate a new test by pressing the Run Optics Test button A window will appear to indicate that the front position of the IncuCyte must be empty for the test to function appropriately If this position is empty then press Proceed If not press Cancel remove any tray in the front position and then start again A new window Optics Test Started will appear indicating that the optics test has been initiated The Optics Test window will become inactive while the test is running When the test is complete the Optics Test window will become active once again and the time and date of the most recently completed test will appear above those of previous tests The Optics Test Started window will remain open until it is closed by pressing OK 105 Page Exporting Results Three options are available to export the optics test results 1 Export Image 2 Export Test 3 Export all Tests An Essen BioScience employee will indicate which test s need to be exported Pressing any of the Export buttons will open a Save As window to select a location for saving the exported file Save the file to the appropriate
31. 158 Results of the Rate of Change transformation The top graph is the original in this case a scratch wound experiment The bottom graph shows the rate of chage at each data point 197 Page ESSEN BIOSCIENCE Zooming the Graph Zoom Once a graph has been created it is possible to Zoom into any particular area for closer inspection To Zoom left click the mouse and then highlight the portion of the graph to be expanded See Figure 8 159 Release the mouse to see the Zoomed portion of the graph See Figure 8 160 File wew Edt Growth Over Time Flask 30 Time Hours 37 33 Hours 40 44 Drag and Drop Manual Alignmant Auto Aligimant Graph on Graph v Right click and hold on this graph and drag onto another to create a graph overlay lt Graph Average Graph File View Edit Growth Over Time Flask 1 pN 5 a og A Se SEI SD OD Pe ST a a P tot Confluence v1 3 Percent Figure 8 160 Zoomed Portion of the Graph Undo Zoom 198 Page To reverse the zooming e Right click on the expanded graph A menu will appear Select Undo Zoom from the menu and the graph will return to its full size format e Type the character Z on the keyboard Viewing Style F Border Style d Font Size w Show Legend Numeric Precision Plotting Method Data Shadows Grid Options Include Data Labels l in Data Points Undo Zoom r Fr F F Maximize
32. 194 Vessel View 44 49 131 154 157 166 178 195 Vessel View FLR Bannet ccc cceeeceeeeeee 210 vessels 25 28 29 35 36 60 64 67 73 86 114 124 127 235 237 I PIS O E EE O EE T T 149 M an A einnsean eE Naa 6 WSIS ceeuceeneee 36 52 53 62 68 180 191 194 Windows 2000 00 ccecceecccescceeccesccesceesseeeess 20 Windows Vista cc eeceecceccecccceeceecees 16 20 23 Windows XP 008 16 17 18 19 20 21 23 ZOOI sargia 135 142 198 199 213 259 Page
33. 22 97 Delete Vessels cccccceccceesceccessceeeceeeseeeesens 116 Device Status oo cee cceccceecceecceescceesceeseeeesens 107 Device TaD iscncesnttncicecovesceosere ctseuadebeaaneunnssanewsioaars 90 PH oa esscsa capstone NN 16 1S a AEE E E 25 36 62 125 B PE E A A 62 Drag and Drop52 53 54 55 156 158 183 184 185 186 189 Dragging Graphs eeeesseessesseee 52 181 183 Drawer Brace Removal ccccccccesessssseeeeeees 1 FCCONUICIVY siinse oniinn 224 Edge Spiers E e 207 210 Error Dafo errauskin aa da 54 127 UT O E 16 17 18 19 23 Existing VESSEL scisicensrtascesevinsesnqetebarnaecaetaieredoncerbe 73 Export 53 54 94 106 120 133 136 141 142 143 144 145 146 147 148 149 150 154 172 173 174 175 176 177 Fiber COlOL en a a A 214 Fier GOR e 214 225 Filter Settings 212 215 222 224 225 226 227 228 229 230 231 232 233 234 TEE niga sendedos sans ets bonede ceinecia desanteuscenecsetepeaes 13 Fixed Threshold ce ee eeeeceeeceeceeeeees 207 209 flask 25 29 36 60 62 65 70 125 127 134 181 190 235 237 238 I aes ete sais ae est endo eae A dente ee eee 62 fluorescence calibration kit e eee 33 103 Fluoresence Intensity Values 000008 203 Foreground Intensity cccccessseeeeeeeees 209 E e C E A A E E 65 Graph49 154 166 177 178 179 180 186 187 194 195 198 AND E E A E E E A E AE 7 HOD Enabl
34. A24 0 182 0 17 B1f 0 0799 0 186 C1 0 0799 0 205 D1 0 0799 0 21 E1 0 0799 0 161 Fi 0 0799 0 165 Gi 0 0799 0 158 H1 0 0799 0 148 I1 0 0799 0 155 J1 0 0799 0 192 K1 0 0799 0 185 L1 0 0799 0 186 Mi 0 0799 0 0747 Ni 0 0799 0 18 01 0 0799 0 333 Pil 0799 0 171 Wel Offsets for Cutout 1 Al 0 226 0 0993 A2 0 242 0 0993 A3 0 252 0 0993 A4 0 23 0 0993 AS 0 249 0 0993 Ab 0 218 0 0993 A7 0 243 0 0993 AB 0 242 0 0993 A9 0 294 0 0993 A10 0 278 0 0993 ALI 0 27 0 0993 A12 0 286 0 0993 13 0 291 0 0993 A14 0 272 0 0993 A15 0 294 0 0993 A16 0 322 0 0993 A17 0 273 0 0993 A18 0 269 0 0993 A19 0 303 0 0993 A20 0 346 0 0993 A21 0 338 0 0993 A22 0 334 0 0993 A23 0 327 0 0993 A24 0 382 0993 Bi 0 226 0 166 C1 0 226 0 165 Dif 0 226 0 133 1 0 226 0 15 Fi 0 226 0 125 G1 0 226 0 141 H1 0 226 0 101 I1 0 226 0 159 J1 0 226 0 169 K1 0 226 0 168 Lif 0 226 0 154 Mi 0 226 0 0517 N1 0 226 175 O1 0 226 0 307 Pi 0 226 0 148 gt gt End 08 17 10 12 41 02 17 2010 12 36 55 PM gt gt 384 wel Plate Calibration completed successfuly lt lt gt gt 384 wel Plate Calbraton of the Middle Tray Start 08 17 10 12 34 50 Wel Offsets for Cutout 0 Al 0 246 0 399 A2 0 345 0 399 A3 0 329 0 399 A4T 0 292 0 399 AS 0 266 0 399 A6 0 283 0 399 A7 0 222 0 399 AB 0 256 0 399 A9 0 237 0 399 ALO 0 271 0 399 A11 0 281 0 39
35. Additionally if it is desired to rename an archive be sure to rename the iaf file and NOT the folder If the folder is renamed the data will no longer be accessible IMPORTANT NEVER rename an archive folder The iaf file can be renamed but NOT the folder If the archive folder is renamed then the archived data will no longer be accessible 119 Page 8 6 8 Logs Tab All IncuCyte activities are monitored and stored and they can be accessed using the Logs tab of the Administer IncuCyte screen Figure 8 73 These logs can be consulted to investigate a variety of IncuCyte related issues They can also be used by Essen BioScience towards the diagnosis of problems Each of the logs can be exported by first selecting a log and then clicking on the Export button at the bottom left of the screen Only the log that is currently selected will be exported Therefore multiple logs must be exported individually Exporting logs may be required in the event of a problem as Essen BioScience may request a copy of the log s via email to facilitate analysis Device Accounts Tests Update Scans Logs Log Types Entry Types Views Filter al v ial v crd Log DateTime Entry Source b IncuCyte firmware 20081 0 88 started successfully Device 2 29 2008 2 33 PM 99 Camera Found and successfully initialized in CyclePo Device 2 29 2008 2 33 PM 99 Cycling power to camera Device 2 29 2008 2 32 PM IncuCyte firmware v20081
36. B13 Image 1 of 4 eae 1 3 3 4 2008 12 06 PM 4Mar_66 My Label 3 4 2008 12 05 PM 4Mar_65 My Label 3 4 2008 12 04 PM 4Mar_64 My Label GraphjExport 3 4 2008 12 03 PM 4Mar_63 My Label 4Mar_ 62 My Label Figure 8 116 Find Scanned Vessels Screen in the EX When the IncuCyte EX software is launched it opens by default to the Find Scanned Vessels Screen See Figure 8 116 In most respects the Find scanned Vessels screen in the EX functions as previously described in Section 8 8 Find Scanned Vessels starting on page 158 However there are some important differences Because the EX identifies vessels by barcode the Search function is expanded to include the Bar Code parameter See Figure 8 116 Thus the default searchable parameters available in the Search By box are Bar Code Label Cell Type Passage In the EX the parameters available in the Search By pull down menu also include the available Custom Vessel Fields see Section 8 5 6 page 75 for more information on Custom Vessel Fields In Figure 8 117 the first Custom Vessel Field has been assigned the name Seeding Date and Seeding Date is now available in the Search By pull down 164 Page N ESSEN BIOSCIENCE Pm t m Pe a Seeding Date 46 5 Contl uence wi we Start Date Time Bar Code Label Seeding Date CORN DCP 345252 Test Flask 9 March 2008 Sf1 1 2008 1 48
37. Choosing View Vessel will open the Vessel View window for whichever vessel is currently selected Vessel View is fully discussed in Section 8 7 3 Vessel View Overview page 131 Graph Export Graph Export data associated with the selected vessel See Section 8 10 Preparing to Graph page 165 and Section 8 10 4 Export Metrics page 172 Archive Vessel Archive an individual vessel using this option See Section 8 6 7 page 114 Restore from Archive Under some conditions scans that were previously archived can be restored from their archive location back onto the IncuCyte controller After scans have been restored they appear on the controller as they did prior to archiving and they do not need to be opened separately by browsing to an archive This function is available with some limitations 154 Page The Restore Archive function is only available on IncuCytes equipped with a 2009A or later software release Archived scans can only be restored if they were performed and archived using an IncuCyte equipped with the 2009A or later software release The archive that is to be restored must be derived from the same controller from which was originally archived Restoring archives is only recommended until 2 circumstances Searching Vessels archives cannot be searched Running Analysis Jobs See Manipulation of Archived Data FLR 117 To restore an archive browse to the archive location and then select the scans to be r
38. Like the Time Zone DST settings must also be in agreement between the local computer and the IncuCyte controller If this is not the case a message prompt will appear after login but prior to launching the software As in the case for synchronization of Time Zones the exact verbiage of the message will depend on the User s permission level In Figure 8 44 the local computer is not set to DST but the IncuCyte is In order to synchronize the local and IncuCyte controller settings press the Synchronization button Set Device Time Local Time 2 29 2008 10 07 58 AM Eastern Standard Time Daylight Savings 1210104 time 2 29 2008 10 07 54 AM Eastern Standard Time Daylight Savings Figure 8 44 Daylight Savings not Synchronized 93 Page N As in the case for Time Zone synchronization the connection will be briefly terminated and can be reestablished after about one minute Synchronizing the Time If the difference between the local computer time and the IncuCyte time is too great a message will appear after logging in but prior to the actual launch of the software Figure 8 45 Time Synchronization Required The Time on controller 1010104 needs to be synchronized Please use Administer Incucyte gt Device Synchronize as soon as possible Figure 8 45 Time not Synchronized Either proceed to the Administer IncuCyte task bar yourself or locate an administrator to synchronize the time Press
39. Microplate Microplate Microplate _ 252 Page HD gt N 96 Well Plates ga ESSEN bue b Lome 3IOSCI at ENH N f Brand Catalog s Plate Name Tray Type HD a ViewPlate 96 96 well Microplate White Clear Bottom Sterile TC treated with Lid 50 box 6005182 _ ViewPlate 96 96 well Microplate Black Clear Bottom Sterile TC treated with Lid 50 box Perkin Elmer ViewPlate 96 96 well Microplate White Clear Bottom Sterile TC treated with Lid 3 box 6005264 ViewPlate 96 96 well Microplate Black Clear Bottom Sterile TC treated with Lid 3 box 96 Well Microplate Nunc Edge Nunclon treated Sterile without lid 96 Well Microplate Nunc Edge Nunclon treated Sterile with lid Nunc 96 Well Microplate Nunc Edge Non treated Nonsterile with lid is 92096 ___ 96 Well Plate All Clear Sterile with Lid individually wrapped S Microplate V Brand Catalog s Plate Name Tray Type HD 02o naor Y Oaoa ll S D eel D QD NIC PIL C 353961 ____ 384 well Microplates tissue culture treated flat bottom withlid Microplate v BD Falcon 353280___ 384 well Microplates black clear BD Optilux TC treated sterile wid S Microplate v 353284 384 well Microplates black clear BD Optilux TC treated sterile wid Microplate vV _ 353963 384 well Microplates white clear BD Optilux TC treated flat bottom with lid 354660 Poly D Lysine 384 well Microplates whi
40. PM CCFF2345235H2 Test Flask 9 March 2008 3 11 2008 1 44 PM BCFY2345235H2 Test Flask 9 March 2008 3 11 2008 1 39 PM ACF 234525H2 Test Flask 9 March 2008 Seeding Date 9 March 2008 lt Not Currently Sek gt gt Saal Figure 8 117 Adding Custom Vessel Fields in the EX menu A Search was performed using Search By Seeding Date For March and the results are displayed in Figure 8 117 Note that an additional column has been created to display the results of the Seeding Date Search However even ce Seeding Date co VESSEIS DNT Bar Code A CCFF234523HZ Test Flask 9 March 2008 Passage End Seeding Date 9 March 2008 Figure 8 118 Columns Pull Down Menu Remains the Same EX though and an extra column exists to display the results of the Seeding Date search note that the Seeding Date column is NOT available in the Columns pull down menu See Figure 8 118 Additional Custom Vessel Parameters will only be displayed as columns in the results section if they were actively searched 8 10 Preparing to Graph The IncuCyte graphing feature can be accessed in one of 4 ways 165 Page gt re o s b K YY e Selecting the Graph Export button in the Selected Vessel box in the Completed Scans screen This will open the Graphing Box e Selecting the Graph Export tab in the Vessel View window See Graph Export Tab page 136 e Choosing
41. Required to save movies NOTE It is possible you will get a message titled Windows Media player 10 Stating that This version of Windows Media Technologies is incompatible with this version of windows This message can be disregarded Just click Cancel The incompatibility message communicates that the machine already has a newer version of Windows Media Codecs for movie generation It will be followed by a message entitled Windows Media Player Codecs Setup stating that The Codec package for the 7 Player has been installed successfully 5 If prompted install the Windows Media Encoder Required to save movies 6 Close the installer when completed 5 2 IncuCyte Control Software Installation using Windows Vista and Windows 7 1 Review the license agreement To continue installation you must Agree 2 Choose a destination folder and Install 3 If prompted install the NET Framework 3 5 20 Page N 4 If prompted install the Windows Media Encoder Required to save movies 5 Close the installer when completed 5 3 Connecting 5 3 1 Setting the Date and Time The IncuCyte controller settings will be synchronized with the local computer at the time of installation It is unlikely that any further synchronization will be required after that point However if for some reason synchronization is required at a later date the IncuCyte software will issue a prompt to this effect The pr
42. Seeding Date 9 March 2008 Custom Scan Fields are properties that do change from one scan to the next An example would be the ACH automated cell handler Scan ID Scan Job 564 See Figure 8 23 A single custom Vessel Seeding Date and a single custom Scan Job 564 have been assigned The remaining 4 custom fields remain empty Not Currently Set for both the Vessel and Scan fields 75 Page A D ESSEN BIOSCIENCE _ 45 6 Confluence v1 2 ae EEE Pr ie ee Jan ane ESE uaa nape Peete Menes Voss scan LE Image Properties Metrics vessel g March 2008 ACH Scan ID lt Not C thy Set lt lt Wok Currently Sek gt SS ee lt lt Wok Currently Sek gt gt lt Not Currently Seto gt y Doo lt lt Not Currently Sets gt ee Graph Export Figure 8 23 The EX Vessel and Scan Tabs 8 5 7 Setting Scan Times Click on the Timeline to add a Scan Time 12am 2am 8am 10am 12pm 2pm 4pm 6pm Figure 8 24 Timeline with no scan times set and a current time of 3 40pm noted by a dotted blue line 76 Page Setting a Scan Start Time To select a scan start time left click on the Timeline A gray Scan Bar will appear on the Timeline at that site Figure 8 25 The exact scan start time can be visualized by positioning the mouse over the Scan Bar The start time and estimated scan length will appear in a yellow box y I i 12am 2am 4am
43. Settings have been Pinned to a job they will automatically be selected when a job is opened and the Pin to Job button will be inactive Pinning is a time saving measure Because the settings have been pinned all images in the Analysis Job have already been post processed to include the specific filter preferences Therefore the data can be graphed and exported more rapidly Although any saved Filter Settings can be applied to any completed Analysis Job only one set of Filter Settings can be Pinned to any one Analysis Job at any given time How To Pin To Pin to a Job select and open a completed Analysis Job Then select the desired Filter Settings and press the Pin to Job button Only saved filters can be pinned and filters can only be pinned to jobs that are currently open How to Unpin To Unpin a saved Filter Setting from an Analysis Job you can Pin a different saved Filter Setting instead Alternatively select the Unfiltered option from the Filter Settings pull down menu and then Pin the Unfiltered selection to the job Now no filters will be applied to that job 229 Page Delete or Rename Analysis Filter Settings Use the Manage Analysis Filters option under the Analysis Filter pull down menu to delete or rename previously saved Filter Settings Recall that when an Analysis Job is launched ALL saved Analysis Filter Settings are automatically applied to that job for User convenience Therefore deleting a
44. Shift D Jump To Start Ctrl Shifk J Change Start Cbrl ShiFE C Figure 8 68 The Scans Pull Down Menu selecting Archive Vessel will open the Archive Vessel window Figure 8 69 Select the Browse button to choose the archive destination Then select Archive Vessel to initiate the archiving process To see the size of the archive select Estimate Archive Size 115 Page N ESSEN BIOSCIENCE As in the case for archiving scans use the Shift key to select a series of scans for archiving or use the Ctrl key to select a collection of scans for archiving Archive Vessel ee Vessel Information Label Tyler test Cell Type Location Middle Tray Right Position Scans to Archive Browse to a Destination Destination Estimate Archive Size Archive Vessel Cancel Figure 8 69 Archive Vessel Window Delete Vessels Individual vessels can be deleted without having to delete an entire scan The Delete Vessel function can be accessed by e Selecting the Delete Vessel in the Scans pull down menu Figure 8 68 e Right clicking on the selected vessel in the View Completed Scans screen See Tasks Available by Right Clicking on a Selected Vessel page 131 Once a vessel is deleted it can only be restored from a vessel archive made with 2009A or later software Additional Archive Notes Appending to an archive When multiple archives are generated they can be either created in new files or added to ex
45. TC Vent Cap w barcode 354478 Collagen 175 cm Flask with plug seal cap 248 Page F 175cm Flasks Brand Catalog s Flask Name Tra BD Falcon e Corning 182cm Flasks On On O1 O1 G1 O G1 O1 O O O On O O1 O O Ly lt O1 lt Brand Catalog s Flask Name Tray Type 660160 CellStar T182 Flask 550mL TC ST LoPro CN Plug Cap 550 ML 182 CM STERILE 660175 CellStar T182 Flask 550mL TC ST LoPro CN Filter Cap 550 ML 182 CM2 Greiner 185cm Flasks lt lt Brand Catalog s Flask Name Tray Type ee 144881 SoLo Flasks Nunclon A Polystyrene Sterile Angled Neck Vent Cap ey 144903 SoLo Flasks Nunclon A Polystyrene Sterile Angled Neck Filter Cap Ooo 6 O Brand Catalog s Flask Name Tray Type a 7 emi 7 500cm Tripleflask a ok a e O O O O OJO Brand Catalog s Flask Name Tray Type 132867 Nunc TripleFlasks Nunclon A Straigh Neck Vent close Cap 132913 Nunc TripleFlasks Nunclon A Straigh Filter Cap 132920 Nunc TripleFlasks Nunclon A Straigh Filter Cap 84 cm Autoflask Brand Catalog s Flask Name Tray Type 779160 AutoFlask Polystyrene TC Treated for Adherent Cell Culture Microplate 249 Page N 92 6cm Roboflask Brand Catalog s Flask Name Tray Type 3059 Corning RoboFlask Cell Culture Vessel for Manual Use TCT with Bar Code No Septu Microplate 3067 CellBIND Surface RoboFlask
46. User even if the program is closed and then opened again at a later date If the Auto Align button is checked then whenever graph overlays are generated by dragging they will be aligned at this set value When multiple traces are included on a single graph in Calendar Mode their relative positions are constrained by the absolute times and dates of each point within the graph However when Calendar Mode is no longer checked and the traces are displayed merely as confluence relative to hours of growth individual traces can be aligned with each other according to a specific selected percent confluence Under what circumstances is such an alignment useful Take for example a situation where growth curves obtained from two separate flasks are to be compared Unfortunately however the initial seating densities of the flasks were not the same When the graphs are overlaid the resulting graph appears as in Figure 8 147 189 Page Graph Sector C3 Mean vs Time Fie View Edt Averages Smooth Growth Over Time Flask Flask2 100 Doe daii F a ae eed w Je 30 40 Tirne Hours 71 53 Hours 0 20 Drag and Drop Manual Alignmert Auto Alignment 14 Apply Clax Use the Apply button to align the eae Geer button to align the plots by ther Figure 8 147 Plot Overlay with Auto Alignment set to 14 In order to facilitate a more accurate comparison reset the Auto Alignment value to one that is common to both graphs for exam
47. Vent Cap Corning 430168 Rectangular Canted Neck Cell Culture Flask with Plug Seal Cap ol Rectangular Canted Neck Cell Culture Flask with Phenolic Style Cap Rectangular Canted Neck Cell Culture Flask with Vent Cap 50ml Canted Neck Standard TC Plug seal Cap 25cm Tissue Culture Flask Nunc EasYFlasks Polystyrene Sterile Angled Neck Filter Cap Poly D Lysine Nunc EasYFlasks Polystyrene Sterile Angled Neck Filter Cap Collagen Nine Nunc EasYFlasks Polystyrene Sterile Angled Neck Vented Cap 156367 Nunc EasYFlasks Polystyrene Sterile Filter Cap Nunclon a Canted Neck Standard TC Vented Filter Cap 163371 unclon Canted Neck Standard TC Plug seal Cap 90025 Oml Canted Neck Standard TC lt lt VENT gt gt Cap 90026 75cm Flasks Brand Catalog s Flask Name Tray Type 14 358023 250ml Straight Neck Standard TC Phenolic Cap ey 3581383 250ml Canted Neck Nontreated Plug seal Cap 353134 250mI Canted Neck Standard TC Phenolic Cap 3581385 250ml Canted Neck Standard TC Plug seal Cap 250ml Canted Neck Standard TC Vented Cap BD Falcon Collagen 75 cm Flask with plug seal cap Collagen 75 cm Flask with vented cap Gelatin 75 cm2 Flask with vented cap Fibronectin 75 cm2 Flask with plug seal cap Collagen IV 75 cm Flask with plug seal cap Poly D Lysine 75 cm Flask with plug seal cap Poly D Lysine 75 cm2 Flask with vented cap Gelatin 75 cm2 Flask with plug seal cap Collagen 75 cm Flask with plug
48. Window ssssccsccccccccccccceeeneneeseeeeesseeeeeeeeeeeeeeaeaaaaaeasasseeeeeeeeeeeeeqaaagas 156 FINDS CANNED Vs SCG iiie ris oine E EE EOE OE 158 E A ET E A eee T T E O T AT eee 159 Tesee ed Vese DON aa E EA 163 PIND SCANNED VESSELS EX rotieren i EE EE 164 PREPARING TO GRAPH osiin a e inc e E i 165 Prepare to Graph from the View Completed SCANS SCre N cccccccccsesveeeseeceeecnaasesescees 167 Prepare to Graph From the Vessel View Window 111sssssseecceeceeceeccceeaaeessessssseeseeeeess 170 PoPa MO G5 10 61 5 ae Grete Cen TE Ree aCe eee een wy Oren E EEE 172 POV CS ao Pas crass scasaaceacisecgut tess ey eds sc tonto tao sae ee A 72 E LE I PE EEE EA TEE E TE E EE E E TE E 177 Microplate Graph E spessa E OE R R 199 THE INCUCYIE M SELMTE Tareis E 200 INCUCY TEL ELR caii aaa a 201 THE INCUCYTE FLR VESSEL VIEW WINDOW ssessssseseeeeeeseseererererererereeererererererees 201 VIEWING FLUORESCENT IMAGES IN THE INCUCYTE FLR oeer 202 Fluorescence SCUINGS sixcscesonscanisactssnannancevasacensiucasavdsesacbuedauaiedevenaveoannsaetdevessanvnrasssacdeceos 202 Phase Contrast Settings sccesosssssaveurssuaspasetsnssodeonasaeeesieraeseduastehdesasashosuasaneeduasadeusuaneeaddvaseds 203 Te A S de ooreet cansicacanacteoveussocaaaearacdianarsednawerbudsauaitcasaasesdeeacahiceuasaraceuaeacscouaeeedeocet 204 INCUCYTE FLR SPECIFIC SOFTWARE APPLICATIONS sssssssseseresesereeererererererereeee 204 The IncuGyie FLR Tasks Pane s sa
49. a specific vessel can also be generated by left double clicking with the mouse on the selected vessel displayed within the tray This will open a new window entitled Vessel View which displays the same details described above Alternatively select the Vessel View button at the bottom of the Selected Vessel box See Section 8 7 3 page 131 for more information on the Vessel View window 44 Page N ESSEN BIOSCIENCE Grab any border of the Time and Use the Search box to IncuCyte screen with date of the the mouse and drag to selected Overall dish re size Scan Status Scan confluence displayed here Confluence for selected sector highlighted in blue search all completed scans according to various Criteria The Time Tree lists dates and times of all a completed heds isas naaa scans Select fy atime to view e E the scan for 25 a ae that time Complete 1 Scans eens ote Cs 09 Sot 10 Sun 11 Mom 12 Tue E 13 Wed 14 Thy 15 Fri 16 Sat 17 Sun 18 Mom 19 Tue 20 Wed 22 Fri 23 Sat 24 Sun 25 Mon 5 26 Tue 12 00 AM 3 00 AM Highlighted scan is displayed Image of the Use the Image pull down menu selected Sector to select which Sector image to is displayed view In this case four images under the were obtained per sector so Ve
50. ability to scan in the Scan on Demand mode Simplistically this means that the IncuCyte can be used to scan individual vessels at times when the IncuCyte is not scanning This includes both periods in between regularly scheduled scans in the 24 Hour Repeating scheduler as well as in designated Cooling Times To access the Scan on Demand scheduler click on the vertical tab within the Schedule Upcoming Scans screen labeled Scan on Demand However all changes to the 24 Hour Repeating Schedule must either be applied to the IncuCyte by clicking the red Apply button or reloaded to an unchanged state by clicking the Reload button before the Scan on Demand scheduler can be accessed If changes are not applied or reloaded the Scan on Demand tab will be unavailable 00000000 COOCOO COG OOOO000 OOOOQOOOO QOOOQOOOO 00000000 0000000 9900 00 00000000 O OOOOO Figure 8 31 The Scan on Demand Scheduler 83 Page To insert a vessel in the Scan on Demand mode the Tray Type must be selected followed by the Vessel Type This can be accomplished in exactly the same manner as it is in the 24 Hour Repeating mode Because only one vessel can be scanned at a time in the Scan on Demand mode the user also has to select the Tray Position Front Middle Rear in addition to the Cutout Position Right or Left in the example in Figure 8 31 All three tray positions and either cutout position can be used The same is true for all of
51. added to the network and you are done If not you should attempt to find the controller on the network by continuing to the next step 16 Page N 3 At the top of the window there is a text area that reads Network gt Left click in an empty area to the right of this text and type the following ICXXXXX followed by the Enter key lf the controller is found on the network you will be prompted for a User name and password Press the Cancel button to exit If the controller is not found then a Cannot Find message will be displayed after a brief search period 4 1 2 Verifying the IncuCyte Controller Network Connection using Windows XP 1 2 3 Open My Computer from the PC s Windows Start menu Under the heading Other Places on the left side of the window open My Network Places Under the heading Network Tasks on the left side of the window click once on View workgroup computers Look for the computer named ICXXXXxX recall that XXXXX is the 5 digit controller serial number If you find it in the window then the controller was successfully added to the network and you are done If not you should attempt to find the controller on the network by continuing to the next step Verify that the Address toolbar is displayed at the top of the window Under the View menu time at the top of the window verify that the Toolbars gt Address Bar
52. additional options are available when exporting data e Include Experiment Details in the Header e Fill Holes in the Data with the Following Characters e Include Error Measurements e Break Data Into Individual Images Include Experiment Details in the Header Choose this box to include export of the following additional details along with the data the selected Metric usually confluence cell type passage number and any notes If multiple files are exported the additional details will appear in every file Fill Holes in the Data with the Following Characters It is possible to set a scan pattern that does not include all the possible wells or sectors in the vessel If the data are exported using the Layout option Show each Scan as its Own Table the table will have empty spaces where sectors or wells were omitted from the scan pattern See Figure 8 126 Include Error Measurements 175 Page Use this option to include either standard error SE or standard deviation SD for each measurement Note that SE and SD are not available for individual images but are calculated for the selected grouping all rows columns none When None is selected error measurements are available only if there are multiple images per sector Break Data Into Individual Images When there are multiple images per sector you have the option to break data down into measurements from each image Time Stamp 1 17 2008 16 30 Elapsed o hours 1 2 3 4 A
53. at the bottom right of the Movie Image Set Export window See Figure 8 101 Click the Movie Designer button and a new window appears that is virtually identical to the Export Designer shown in Figure 8 97 of the Export Current Image Single Image section As discussed 149 Page in that section the designer is used to interactively crop and scale movie frames and to add a legend or timestamp to each image if desired If a Timestamp is added then a timestamp that displays the corresponding time days hours and minutes will appear on each frame of the movie If Show Legend is selected a scale bar and field of view dimensions will appear in the exported image or movie as they do in Vessel View In addition to these customization options there is also a frames per second parameter that can be adjusted from a minimum of 1 to a maximum of 30 frames per second When the mouse is placed over this control a thumbnail preview of the movie at the selected frame rate is shown as an aid Name prefix Example enter a filename prefix Movie Designer Crop Scale Legend etc aa Figure 8 101 Detail of the Movie Designer option in Movie Image Set Export As mentioned when discussing the Export Design window perhaps the most important tool found in the Export Designer is launched via the Output Preview button in the lower left corner of the Export Designer We strongly encourage you to preview your customizatio
54. er mm E Object Count per Image g Object Confluence hi Label enter a custom label Include analysis filter details Group All X Unfiltered No filter parameters set F Include Vessel Label in trace names Figure 9 16 Graphing Data from Processed Jobs To generate a Time Plot choose Time Range and then select the desired Start and End Times Select images for graphing and export as described for non fluorescent data Section 8 10 2 170 The fluorescence Graph Export window also lists any Filter Settings that are currently applied to the data In Figure 9 16 only one Filter Setting has been applied Area gt 50um To include the Filter Details with the graph or exported data select the Include Filter Details check box located above the Graph button The Metrics available for the Object Counting Application are as follows Average Object Area The average area for all Objects within an image Average Object Mean Intensity The average Mean Intensity for all Objects within an image Average Object Summed Intensity The average Summed Intensity for all Objects within an image 231 Page Object Count per Image The total number of Objects within an image Object Count per mm The total number of Objects within an image divided by the image area in mm Object Count per well The average Object count per mm times the area of the well in mm Object Summed Intensity per mm The total Summed Intensi
55. establish new User accounts click on the Administer IncuCyte task bar in the upper left corner of the main window The available Administrator functions are organized within a series of tabs Select the Accounts tab to access password and accounts information The Create User function is self explanatory The Change Password menu allows Users to set and change their own passwords This function is available to Users at all permission levels The Create User and Delete User functions however are only available to Users with the Administrator permission level New Users must have a unique User name Passwords are case sensitive and new Users should be encouraged to change their password the first time they log on When creating a new User it is important to select an appropriate Permission Level Users with an Administrator Permission Level have all of the functions of the IncuCyte control program available to them A User with a User Permission Level is able to change his her password view device information configure the device to collect data and perform all post collection analysis view plot annotate etc The Guest Permission level is one step below the User level in that guests cannot configure the device to collect data Additional information regarding this topic can be found in Section 8 6 3 page 95 lt is important to note that there are some differences between the permissible functions of the Standard Model Inc
56. file or e mail In these cases the graph can be dragged as either the raw data or the image To drag the image into Word select Drag and Drop Image to Word or Outlook To drag the graph image into EXCEL PowerPoint or email select Drag and Drop Image to Other Document To drag the raw data from the graph select Drag and Drop 183 Page N Raw Data to Document If the graph is dragged as Raw Data into EXCEL the data will appear neatly within the spreadsheet cells See Figure 8 137 If the raw data are dragged into Word they will appear within columns Growth Over Time Date Time Elapsed Column 1 Column 2 Coumni Column E Column3 Y Columnd ColumnS ColumnG 4 5 2006 15 45 0 5 116157 5 62277 4 5 2006 18 45 3 5 873641 6 483157 4 5 2006 21 45 6 6 748467 7 517222 4 6 2006 0 45 9 7 93768 8 310006 4 6 2006 3 45 12 9 286036 9 822143 4 6 2006 6 45 15 10 79348 11 6282 i 4 6 2006 9 45 18 12 91238 13 6381 5 4 6 2006 12 45 21 15 41242 15 66134 z 4 6 2006 15 45 24 18 40523 19 5973 4 6 2006 18 45 27 22 04792 23 05909 2 4 6 2006 21 45 30 26 11257 28 860244 8 4 7 2006 0 45 33 30 45502 34 0718 4 7 2006 3 45 36 35 27699 39 10634 4 7 2006 6 45 39 40 49081 44 93753 4 7 2006 9 45 4 46 00566 50 74551 4 7 2006 12 45 45 51 61444 56 49158 4 7 2006 18 45 51 63 54694 69 3348 gar Figure 8 137 Graph data dragged into EXCEL as Raw Data left and as Image right The format in which the data will be dragged and dropped into the
57. file s and the corresponding installation instructions The three different update options are described below Update Database Your IncuCyte will arrive equipped with the most recently available database of compatible cell culture vessel types see Section 6 1 Selecting and Placing Trays and Vessels into the IncuCyte page 25 and a Selection of trays to accommodate them However as new vessel types are created and as customer demand requires additional vessel types and trays will be added to the database Any such updates can be uploaded into your IncuCyte using the Update Database button Load License New applications for the IncuCyte are being developed as an ongoing process Some specific applications will require additional software modules in combination with the standard software package To upload these new software applications select the Load License button IMPORTANT Before loading a new license or upgrading an IncuCyte it is ESSENTIAL to remove any vessels from the Schedule Upcoming Scans screen and then press Apply it s ok if the vessels trays are still physically located in the IncuCyte If any vessels are present even if no scans are scheduled when the new license is loaded the software will experience a serious problem Therefore completely clear the Schedule Upcoming Scans screen before loading a new license Update Device As new versions of the IncuCyte software become available
58. graph for Flask 2 has been dragged onto the graph for Flask 1 The resulting graph is displayed in Figure 8 136 The new graph still contains the title from the original Growth Curve for Flask 1 however it now contains two traces labeled as Flask 1 and Flask 2 182 Page Graph Col1 Mean ys Time File Vi Edit Smooth Flask 1 Flask1 Flask 2 If only a single trace for example a single well from a 12 well plate needs to be dragged then it the desired well must be graphed alone and dragged individually Alternatively if the entire graph is dragged and dropped individual traces can be deleted See Section 8 10 5 page 192 To drag and drop graphs onto each other within the IncuCyte software be sure that the Graph on Graph selection is chosen from the Drag and Drop pull down menu If a graph overlay is attempted with the wrong Drag and Drop selection a window will appear indicating that the overlay cannot occur There are 3 commonly made errors that can prevent creating a graph overlay from occurring If you are not able to drag your graph be sure that e The correct Drag and Drop option is selected e The right mouse button is selected and not the left e You are not trying to drag the same graph onto itself Dragging Graphs from the IncuCyte Software into Different Software It is also possible to drag a graph created within the IncuCyte software directly into a Word document EXCEL file PowerPoint
59. in graphing and exporting data from an experiment Once defined Regions appear in the Graph Export window See Graph Export Tab page 136 in the Regions drop down list and allow graphing exporting of a custom grouping of wells Any replicates defined within the region are automatically detected and can be grouped by selecting Replicates in the Graph Export Group drop down list For example the User can create a region such that an entire dilution series along with negative and positive controls appears on the same graph without the need to drag and drop individual plots onto one graph In addition the labels on individual wells or groups of wells replicates will automatically appear in the figure legend of the graph To create a Region on a plate map simply select the desired area of the plate in the Plate Map Editor and click on Save Selection as Region under the Regions tab on the left side of the window Figure 8 94 Creating a custom Region Enter a name for the region and hit OK to save the region D Wgl e a agla l Click and drag to select areas all i z Le Click to toggle individual well selection Use the tabs below to populate the selected wells Right click a selection to edit well contents A SSP 1000 SSP 300 seen ee To create a region select an area onthe plate _ nM nM ie map and click this button i DMSO DMSO Pi 0 2 0 2 SSP x Del
60. increase the rate of processing by joining an ongoing Analysis Job See Figure 9 8 The Open and Manage Jobs window is displayed as it will appear under two different circumstances On the left a completed job Paclitaxel has been selected At the bottom of the window the analysis type is indicated Object Counting Analysis as well as the User admin who launched the analysis The time and date at which the job was completed are also indicated 218 Page N Analysis Jobs Open and Manage Analysis Jobs Open and Manage Al Hame Hame Paclitaxel Paclitaxel Hydosyurea A Example Hidoxyures AY Example Object Counting Analysis admin Ubject Counting Analysis admin Completed at 12 2 2006 9 56 AM Analysis Job Incomplete 25 se Figure 9 8 Join an Analysis The buttons at the bottom of the window give the User the options of opening the job viewing the job details or deleting the job On the right a job Example has been selected that is currently being processed The incomplete status of this job is indicated by the yellow construction icon to the left of the job name The analysis type and User are listed at the bottom of the window However there is no date of completion available since processing is ongoing Instead the window indicates that the Analysis Job is incomplete Additionally the button that read Open on the left now reads Join If the computer parti
61. is built to interface with an automated cell handling system There are some features of the IncuCyte software and function that are unique to the IncuCyte EX instrument These special features of the IncuCyte EX will appear with an orange background and an icon to the left as demonstrated in this paragraph 2 1 4 The IncuCyte HD Imaging Module An IncuCyte equipped with the HD Imaging Module utilizes a newly developed unique imaging technique that generates extremely high quality high definition images of cells This system introduces a new generation of imaging technology All cell culture vessels previously compatible with the Standard Model IncuCyte are also compatible with the IncuCyte HD However the innovative features of HD imaging also facilitate production of exceptionally high quality images of cells in 96 and 384 well plates HD images of 96 well plates are superior to those obtained with the Standard Model IncuCyte and 384 well plates cannot be imaged in the absence of HD Some aspects of the IncuCyte software are uniquely applicable to HD enabled units Aspects of the IncuCyte software that are only applicable to the HD enabled IncuCyte will appear with a blue background and an icon to the left as demonstrated in this paragraph 7 Page FLR N 2 1 5 The IncuCyte FLR The IncuCyte FLR is an HD enabled instrument that is also equipped with fluorescence scanning capabilities When ves
62. issued since the original trays were released Updated versions include trays 5 7 and 9 The original trays 5 7 and 9 incorporated the use of Spacer Disks in order to accommodate a wider variety of vessels The old style versions of these trays are no longer being sold however some Users might retain these trays from their original purchase The Trays 5 7 and 9 listed under the Tray Type Find search Figure 8 6 represent the new Tray Type versions Information regarding the older versions of these trays can be accessed by clicking on Help Unavailable Trays at the top of the IncuCyte software screen 62 Page A ESSEN BIOSCIENCE Triple Wide Trays Tray Types 10 and 11 are triple wide trays and a single triple wide tray occupies the entire IncuCyte drawer Therefore only one of these trays can be used at any given time See Figure 8 7 ola x Preferences Help ic10094 admin Next Scan 21 minutes 3 00 PM Search Scans By Label gt for GO Ta P rar z wy View Completed Scans File View SA Find Scanned Vessels aca Physical Layout Properties Front Tray Me Administer IncuCyte 24 Hour Repeating Tray Type Vessa Tipe Scan Type Scan Pattem a 4 New Scan on Demand unavailable U enson esea Figure 8 7 Tray Type 11 Because this is a triple wide tray the IncuCyte can only accommodate one tray ata time Because the triple wide tray wi
63. lt is VERY IMPORTANT to always check the New box when a new vessel is scanned Otherwise all the scans from the previous vessel will be included with the data from the new vessel Figure 7 1 If you forget to check the New box see Section 8 7 9 Change Start page 155 Failure to check the New box appropriately will also interfere with searches under the Find Scanned Vessels task bar Ee ee ee et ee eee ae Data from Expenment 1 Data from Experiment 2 Figure 7 1 The New box was not checked at the beginning of scans for a new vessel Experiment 1 Therefore when the flask is graphed the graph for the previous vessel is connected to the graph for the new vessel Experiment 2 Setting Scans in the IncuCyte FLR Two scan Types are available in the IncuCyte FLR Phase Contrast or Fluorescence amp Phase Contrast If Fluorescence amp Phase Contrast is selected the vessel will be scanned both fluorescently and in HD mode e Fast Fluorescence Allows for faster scanning and analysis with no loss in sensitivity See Section 8 5 2 Setting Vessel Properties FLR e Standard lf Phase Contrast is selected the vessel will only be scanned in HD mode 3 Page e ESSEN BIOSCIENCE 7 1 3 Set Scan Time s Select Tray Type Vessel Type Scan Type and Scan Pattern The New box The Properties box The Fast Fluorescence Option IncuCyte FLR only The Apply button Red indicates an unsave
64. movie click the Slideshow button below the time tree in Vessel View to scroll through time points of the selected image without exiting Vessel View While in Slideshow mode you can switch to any image in the vessel by clicking on its location at the top of the window Also switch to the Graph Export tab to view changes in any selected metric over time To change the speed at which IncuCyte cycles through images click on the Preferences Menu at the top of the main IncuCyte screen Movie Export To export movies follow the steps indicated below Select Time Range in the drop down menu adjacent to the Time Tree in the Movie Image Set Window Choose movie start and end times The movie will include all times including and between the selected start and end times Choose the wells or sectors and the desired image numbers as described for image export It is possible to simultaneously export movies for all images from all wells sectors at the same time Choose a target folder select the Sequence Type select Image Type Current or Phase Contrast Original settings see Export Images Overview pg 142 Select a Name Prefix As discussed in Export Image Set Multiple Images page 145 the Example box will be automatically populated If movie Sequence Type Windows Media Video MWV9 or Windows Video AVI is selected a button with the text Movie Designer Crop Scale Legend etc appears above the Export button
65. of a scan containing the selected search terms and occurring during the prescribed time span Each line in the results section represents a single vessel The Start Date Time and the End Date Time for each vessel is indicated along with the vessel label cell type and experiment type Selecting highlighting one of the vessels will display the vessel in the Selected Vessel box to the left of the search results All available scan times dates for that vessel will be displayed in the Time Tree The Vessel Box will automatically display the image for the very first scan of the selected vessel Scroll through the Time Tree to view scan results as in the View Completed Scans screen Displayed Columns The number of columns that are displayed in the Results area can be adjusted Click the Columns pull down menu above the search results to view display options By default the Start Date Time Label Experiment Type and Cell Type will be displayed The remaining two parameters End Date Time and Passage can be displayed if they are selected under the Columns pull down menu Alternatively the columns for any of the currently selected parameters can be unselected Remember that when a Search is performed only columns that are selected for display can be searched So if you want to search by Cell Type be sure that the Cell Type column is being displayed BEFORE starting the search When all 5 columns are selected use the slide bar at the bottom of the
66. of each scanning session are indicated as Scan Started and Scan Complete Self Test start and end times are also included Some additional physical manipulations of the instrument will also be recorded including drawer opening and closing and the pushing of the manual Stop button at the front panel Although it may initially seem trivial drawer opening and closing can be highly relevant under certain circumstances Each time the drawer is opened and closed it can cause a slight displacement of the images to be scanned For example if large image displacements are observed within a movie consult the Device log to confirm that the drawer was not opened between scans Client This log records all IncuCyte manipulations by Users The Client log indicates the User name time date and a brief description of each action Use the Client log as a resource for tracking all User related IncuCyte activities Diagnostic This log records the details and results of all diagnostic tests performed on the instrument These include the Calibration and or Confirm Tests Optics Tests and others Maintenance When type Maintenance is selected the Add Maintenance Log Entry button at the bottom of the screen will become active Maintenance Log entries should ONLY be made by Essen BioScience employees during service calls Users should NOT make any entries into the Maintenance log IMPORTANT Users should NOT make any entries into the Maintenance Log
67. of items used by the connection Select the Internet Protocol Version 4 TCP IPv4 item and press the Properties button below the list of items Copy down the settings found under the General tab and other tabs Some of these setting will be modified to create the 2 device network and you will want to remember the current settings when it comes time to reset the port to its regular configuration On the General tab select the Use the following IP address Radio Button Type in the following IP address 192 168 128 1 Figure 4 1 page 18 18 Page N 9 Type in the following Subnet mask 255 255 255 0 10 Press OK to exit the property sheet 11 Press OK again to effect the change to the connection 12 Follow the steps in Section 4 1 1 page 16 for verifying the presence of the controller on the network 4 2 2 Verifying the Direct Network Connection using Windows XP Open My Computer from the PC s Windows Start menu 2 Under the heading See also on the left side of the window open Network and Dial up Connections 3 Identify the network connection used in the 2 device network It will most likely be labeled Local Area Connection but could have a different name Right click the connection and select Properties from the menu 4 Under the General tab find the box with a list of items used by the connection Select the Internet Protocol TCP IP
68. or on a sturdy bench top Start removing materials from the top as shown in the exploded diagram Figure 2 1 The accessory kit can be removed first through the opening in the top foam piece The kit contains all of the trays for holding flasks and plates that were ordered with your system 9 Page Accessory Kit IncuCyte Shipping Bolt Microscope Figure 2 1 IncuCyte Microscope Packaging The microscope weighs approximately 33 pounds so it is recommended that one person hold the box while a second carefully removes the microscope from the bottom foam piece and places it on a stable work surface The unit should then be removed from its plastic bag not shown in Figure 2 1 and inspected for any obvious signs of external damage If the unit appears in good condition use a wrench to remove the shipping bolt before further inspection and use After removing the bolt the drawer on the unit should open and close smoothly Please inspect the drawer for any signs of damage The controller should be removed from its box and bag in a similar fashion see Figure 2 2 The controller box will contain the controller and under most circumstances it will also include a power cord appropriate for your local utilities However while Essen provides power cords compatible with most utilities there will be instances in which Essen does not stock the compatible cord In these rare circumstances the purchaser will have to provide their own c
69. presents 3 additional graph related options File Use the File Menu to open save or print a graph or to close the window Figure 8 156 The File Pull Down Menu lt Graph All Wells Mean Edit view Open Graph Chrl o Save Graph Cbtri S Prine Close Graph Alt F4 oe ee Figure 8 156 The File Pull Down Menu NOTE If a graph was created in IncuCyte and then dragged and saved into another program such as Word then the graph can be opened directly through that program It will however be a static image that cannot be manipulated Edit 195 Page The Edit Menu provides the option to edit the graph title and labels associated with traces on the graph View Use the View Menu to select different error measurements standard error or standard deviation change the y axis to a log scale or view filters from an analysis Tools The Tools Menu provides options to edit how the data are viewed on the graph Smooth Lines Sometimes if the data are particularly noisy the Smooth feature might be more useful than Outlier Removal When Smooth is selected a new window will appear to facilitate selection of the smoothing size i e the number of points over which smoothing will occur If 3 data points are selected then each point on the graph will be averaged using its two neighboring points Figure 8 157 NOTE It is important to remember that smoothing is a tool for generating cleaner cur
70. scans as a timeline and a list Change Scan Order To change the order in which IncuCyte scans vessels either 1 click and drag a vessel on the timeline into the desired position or 2 use the Move Before and Move After buttons to rearrange the scan order Add a Cooling Time To add a cooling idle period between two vessels the Auto cool option is recommended Auto cool will find the most efficient vessel schedule possible with the current scan duration and interval If a specific scan order or timing is preferred click the Auto Cool Settings button Use the Auto Cool Settings window to adjust how Auto Cool will rearrange the current vessel schedule To minimize the number of scan groups and or the length of the entire scan period check the first box to allow Auto Cool to rearrange the order in which IncuCyte scans the vessels Check the second box to ensure all scan groups groups of vessels scanned without interruption begin at a set interval on the half hour quarter hour etc To manually insert cooling times click the Add Cooling Time button and drag the cooling time block to the desired location on the timeline To change the length of the cooling time click the appropriate Cooling Minutes box in the list below the timeline and type in the cooling time in minutes Cooling times appear as blue intervals on the timeline in the Schedule Upcoming Scans screen See Figure 8 25 Timeline with one scan bar starting at 12 p
71. section Entry Types For each Log Type three log categories are available through the pull down menu All 121 Page Choosing All displays all history affiliated with the selected Log Type whether or not errors were associated with those events History All IncuCyte activities that did not involve errors will be displayed for the selected Log Type Errors Any IncuCyte related errors associated with the selected Log Type will be displayed under this category Figure 8 74 Log Types Entry Types Device wt Log 2 29 2008 2 33 23 PM 99 Camera Found and successfully initialized in CyclePower4ndRetry 2 29 2008 2 33 12 PM 99 Cycling power bo camera 2272008 11 59 24 AM 77 No images were acquired during the scan zjar 2008 11 36 33 AM 11 Scan aborted 227 2008 11 36 33 AM 34 MQ Job Pause IMS motion job killed 2272008 11 36 33 AM 18 Scan aborted because stop button was pressed 2 20 2008 1 04 02 PM 11 Scan aborted IP 0 2008 1 04 02 PM 61 VQ Job Autofocus Video job killed zlep 2008 1 04 02 PM 18 Scan aborted because stop button was pressed Entries To Display 1000 w Figure 8 74 Device Errors The Errors log will be used primarily by Essen BioScience in the event of a problem If an error appears to have occurred such as a scheduled scan does not take place consult the Device Errors log to see if an error has been recorded Errors will be recorded with an error description a numerical code an
72. target software will be determined by the format in which the graph was created for example sorted by column vs row SUMMARY 1 To drag one plot onto another within the IncuCyte graphing software select Drag and Drop as Graph on Graph 2 To drag the graph image into MS Word or MS Outlook select Drag and Drop as Image to Word or Outlook 3 To drag and drop the image into other software programs select Drag and Drop as Image to Other Document 4 To drag and drop other raw data select Drag and Drop Raw Data to Document Preferences Outlier Removal and Drag and Drop Auto Alignment Select Preferences from the top of the main IncuCyte screen to open the Preferences Outlier Removal Window Two features of the Preferences window will be discussed here See Figure 8 138 Outlier Removal This function facilitates the increasingly stringent removal of outlying vessel data Selecting Outlier Removal will reduce the standard deviation of the Metrics The outlying data are selected automatically by the software according to an algorithm points cannot 184 Page be excluded manually If no outlier removal is desired select None under the pull down menu however standard is the default setting and it is the setting recommended for general use NOTE Increasing the stringency of Outlier Removal will not necessarily improve the appearance of a curve To improve the appearance of a noisy curv
73. that it is an Existing Vessel Figure 8 21 The New box is active while the Label Cell Type Passage and Notes boxes are inactive and unchecked To change the properties of an Existing Vessel and start a new scan series check the New box This will activate the previously inactive windows Any previously existing text within these windows can be edited or deleted Every time new 73 Page N ESSEN BIOSCIENCE material is added to the Properties Section the new User name date and time will appear automatically in the Notes box abel MCF7 kill curve Plate Map Seeded at 5000 cells well Plate was seeded 24 hrs before scan time per well A1 5 B1 5 Figure 8 21 An existing vessel in the Physical Layout left and Properties right tabs of the Schedule Upcoming Scans screen Label Name of the vessel Cell Type Cell type used in the vessel Passage Passage number of the cells can be selected by scrolling up and down or a number can be typed directly into the passage box Plate Map Use this button to open the Plate Map Editor and create a plate map for the vessel microplates only Once a plate map has been created and applied to a vessel a small plate map icon will appear next to the plate map button Hovering over this small icon will provide additional information for the applied plate map Notes Any extra information desired In the Vie
74. that no scans are scheduled to occur 8 4 Task Bars The IncuCyte Task Bars allow the User to select the main screen display or User interface There are four interface options Each of these options will be further discussed on an individual basis View Completed Scans This is the screen currently visible in Figure 8 4 It shows the time and date of the selected scan in bold at the top and the results of the scan below faa File View Scan Preferences Help 1710094 admin ia Next Scan 2 hours 36 minutes 5 08 PM Search Scans By Label Task List Er Find Scanned Vessels E Schedule Upcoming Scans a m Sa Administer IncuCyte H ji s ih oe i ia coma a 38 4 Confluence wi3 1 18 2008 7 30 00 PM Selected Vessel Middle Tray Left Position AULE pie l 7 a Ei j f TE gi a a eje a i b eoeoees Be 1 1 ia i 12 Oe le Ts k F ar L ia i 8e8080 D0000 eee ma 7 G od 29000800080 1 Pe ite hier ati be ver a coming a6 r B 26 3 Confluence v13 SiG atmo om H i ms ILLI I D e660 a fe amp TEEL CEOE Ek Ba aa a a a a eee Bitja aie ial alae b OOOOGS F Completed Scans a January 09 Wed 10 Thu 11 Fri 12 Sat 13 Sun 14 Mon 15 Tue E 16 Wed 17 Thu 15 Fri 19 Sat 20 is 3 Coming 100m0 47 9 Confluence w13 49 5 Confluence via L Mew B
75. they can be uploaded using the Update IncuCyte button 8 6 7 Scans Tab and Archiving for Data Management NOTE The Scans tab is ONLY available at the Administrator level however it is possible to archive individual vessels at the User and Guest Levels See Archive Vessels page 114 Device Status When scans are initially carried out the data are stored on the IncuCyte controller While the controller can hold a large amount of data it will gradually fill up as more and more scans are performed The amount of free space remaining on the controller hard drive is displayed at the bottom left of the Schedule Upcoming Scans screen in the Device Status box Figure 8 59 displays the Device Status box which indicates that 445 43GB 97 of free space 107 Page N remain on the controller hard drive In order to free up space on the controller hard drive scan data can be removed from the controller to a new destination through a process called archiving 4 08 PH Mext Scan 4 15 PM Set By Admin Set Ot 2 24 2009 3 11 PM Status Ready To Scan Free Space 445 435 GB 97 Database 17 MB 100 Free Figure 8 59 The Device Status Box IncuCyte FLR Temperature History As a quality control measure and diagnostic tool IncuCyte FLR is equipped with a temperature sensor and graphing tool for viewing the temperature history inside an IncuCyte A current temperature reading for the instrument is available at the
76. vessels and or from different days i te ca pee itii Figure 7 15 Creating an average graph 7 4 3 Export of Graph Data Data can be exported by selecting the Export button underneath the graph settings Export parameters can be adjusted according to the desired Layout Destination and Other options Further reading on this function is recommended See Section 8 10 4 Export Metrics page 172 When generating the selected Metrics the stringency of outlier removal can be adjusted by selecting the Preferences option at the top of the screen Select from None no outlier removal Standard or Aggressive Whenever a graph is created it will conform to the currently selected degree of stringency None is the IncuCyte default setting and it is recommended See Section 8 10 5 Preferences Outlier Removal and Drag and Drop Auto Alignment page 184 for additional information on outlier removal Outlier removal applies to all Metrics 54 Page Preferences Cutler Removal Standard Aggressive Figure 7 16 The Preferences Window The Preferences window contains some additional options that relate to dragging and dropping graphs and images See Sections 8 10 5 Preferences Outlier Removal and Drag and Drop Auto Alignment page 184 for more information on these options Note that increasing the stringency of outlier removal does not necessarily result in a smoother graph To reduce noise in the graph see Section 8 10 5 re
77. 0 87 started successfully Device 2 29 2008 2 16 PM IncuCyte firmware 20081 0 87 started successfully Device 2 29 2008 2 07 PM Scan complete Device 2 29 2008 2 00 PM Scan started Device 2 29 2008 1 55 PM Optics test report O Test run from 02 29 08 13 55 0 Diagnostic 2 29 2008 1 54 PM Optics test report O Test run from 02 29 08 13 54 0 Diagnostic 2 29 2008 1 53 PM Optics test report Test run from 02 29 08 13 53 0 Diagnostic gt Figure 8 73 The Logs Tab Any IncuCyte activities will be automatically updated into the Logs Section as they occur However if the Logs screen is opened up directly after an activity has occurred or if the activity occurs while the Logs screen is already open the change will not appear among the Log entries To ensure that the most up to date information is being displayed click on the Refresh button at the bottom right portion of the screen The Logs tab consists of three components e Log Types e Entry Types e Views 120 Page The Filter box is a search function that filters out all Log entries that do not match the criteria entered into the Filter box Log Types Five types of logs can be viewed using the Log Types pull down menu All The All log contains the combined log entries from device client diagnostic and maintenance Device The Device log records all device activities irrespective of the User The start and stop times dates
78. 20090619 E e 20090619 Angiogene 7 10 20 20090626 Ang Co cul TC 77 6 200 4 Standard Je Standard Standard Standard Standard LA Standard 7 2 20 7 2200 17 2 20 7 2 200 7 2200 6 26 20 7 2 200 7 6 200 7A G 200 v 6 200 7 6 200 7 8 200 pe 24 well 24wel 24 wel 24 well 24 nell omm pe Ejim View Analysis Job Figure 9 10 The Analysis Jobs window _ Analysis Job Details Press the Details button to see the Analysis Parameters and other information associated with the job Add or remove any relevant Notes The nature of any errors that occurred during processing will be displayed here The Details button is active whenever a completed or ongoing job is selected in the Tasks Pane Close an Analysis Job Close the job by clicking the red X at the far left of the green banner closing the Vessel View window opening a new job or launching a new preview or a new Analysis Job Delete an Analysis Job To delete an Analysis Job select it in the Tasks Pane and then press the Delete button Keep in mind that processing Analysis Jobs can be time consuming and that deleted jobs cannot be restored So be cautious when selecting a job for deletion Analysis Jobs and Archived Scans When scans vessels are archived any Analysis Jobs that were affi
79. 31 a flask has been graphed In this example the Group None option was checked so one trace appears for each sector When multiple wells traces appear on a single graph each one is listed at the top of the window Figure 8 131 The wells sectors will be assigned default titles indicating the vessel label and the well A1 A2 A3 etc 178 Page gpm gt F p F how a Senn Renee iN ee Ge See Bee pe BIOSCIENCE N Graph All Sectors Mean vs Time DER File Edit view All Sectors Mean vs Time BD All gt i D o D a a lt gt o o D 3 Z f Oo 18 Fri Jan 2008 Calendar Mode Drag Drop and Alignment Settings pm Drag and Drop Manual Alignment Auto Alignment Right click and hold on this graph and drag onto another to create a graph overlay Figure 8 130 Dish Mean Because Region All Sectors Group All was selected a single trace appears on the graph Graph All Sectors Mean vs Time File Edit View All Sectors Mean vs Time Corning 25 B1 Corning 25 D1 Corning 25 F1 Corning 25 C2 x Corning 25 E2 Corning 25 G2 Corning 25 B3 O Corning 25 D3 A Corning 25 F3 VY Corning25 C4 Coring 25 E4 Corning 25 G4 Corning 25 B5 Corning 25 D5 Corning 25 F5 Corning 25 C6 Corning 25 E6 Corning 25 D7 Corning 25 F7 Corning 25 C8 Corning 25 E8 Corning 25 D9 Corning 25 F9 Corning 25 E10 gt a o o D a
80. 4 26 B 9 14 C 4 59 D 4 03 E 9 21 F 3 08 G 3 85 H 4 04 2 0 J 4 48 K 7 02 L 8 63 Mi 4 87 N fod O 7 29 P 6 5 Figure 8 126 Export of a 384 well plate without filling the Holes Rather than leaving these spaces empty it is possible to select characters to fill the spaces In Figure 8 127 the word BLANK has been selected for the empty spaces Other Options Include experiment details in header Fill holes in the data with the following characters BLANK optional Figure 8 127 Fill Holes with the Word BLANK 176 Page Export the same data set and the results will now be displayed as shown in Figure 8 128 Time Stamp 1 17 2008 16 30 Elapsed Mo OD G H J K L M N O p Figure 8 128 Empty spaces are filled with the word BLANK 8 10 5 Create a Graph Create a graph From the View Completed Scans screen with the graphing window open choose the graphing Metric Confluence the Start and End times the Region and Group See Figure 8 129 The graphing window is open to the right Start and End times have been selected The Metric Confluence is highlighted and the Statistic Mean Region All Wells and Group All have been selected 177 Page N ESSEN BIOSCIENCE IncuCyte File View Scan Preferences Help 1 10094 momo v Next Scan 2 hours 44 minutes 8 08 PM Search Scans By Label Task List Ma Administer IncuCyte Completed Scans
81. 6am 8am 10am 12pm 2pm 4pm 6pm Spm 10pm 12am Figure 8 25 Timeline with one scan bar starting at 12 pm The scanned vessels are separated by a cooling period The Scan Bar can be moved up and down the Timeline by left clicking and dragging it Fine adjustments in the start time can be made by right clicking on the Scan Bar A pull down menu will appear as in Figure 8 26 Selecting 1 Min 5 Min 1 Min or 5 Min will reset the Scan Bar by the corresponding lengths of time A FHI Jam 4pm 5 min 1 Min 1 Min 5 Min Delete All Set Interval npty Heta Figure 8 26 Scan Bar Pull Down Menu This same pull down menu can be used to delete the selected Scan Bar or delete all scans on the timeline To set multiple scan times simply set Scan Bars at all desired times Setting Scan Intervals Frequently it will be desirable to set up a series of scans to occur at a regular interval Rather than setting each start time individually a fixed scanning interval can be selected by right clicking on the timeline and selecting Set Interval Figure 8 26 This will open the Set Timeline Scan Intervals window shown in Figure 8 28 If an interval is desired that is not present as a selection within this menu then the interval must be set manually by setting a Scan Bar at each selected start time as described in Setting a Scan Start Time 7 Page Set Timeline Scan Intervals Starting 2 00 00 PM 2 Add Scans Emso F
82. 8 140 Drag and Drop Manual 4lignment Auto Alignment gt A Apply dear Use the Apply button to align the plots Use the Clear button to align the plots by their Apply First point Figure 8 140 Set the value at which Auto Alignment occurs Whatever alignment value is set under this tab will apply to all future graph overlays To change the value select a new value and then press Apply See Section Auto Alignment under Other Graph Features page 189 for more information NOTES Outlier removal applies to ALL Metrics See Section 8 7 10 page 156 for a discussion of the remaining Preferences Window feature 186 Page Other Graph Features Error Bars When multiple wells or sectors are combined to generate a single trace Error Bars can be added that represent the standard deviation between the wells or sectors Error Bars can be added in one of two ways e Select View Error Bars from the top of the main IncuCyte screen When Error Bars are selected from the main screen then all graphs generated subsequent to that time will automatically be assigned Error Bars Deselecting the Error Bars will eliminate all Error Bars from subsequent graphs e Select View Error Bars from the top of the graph window When a graph window is open the Error Bars option on the main screen will be inactive As with the main View menu all subsequent graphs will be affected by this selection Error Bars are ONLY availab
83. 84 well Plate calibration use 100 Page Catalogue Number BD Biosciences Black 353962 write ao 312003 Porvair 311003 Figure 8 53 384 Well Plates Approved for HD Calibration Matrix We recommend that customers select the same plate type for calibration that they also plan to use most frequently for scanning If the desired plate type is not among our approved list of candidates then select the plate type that seems closest to your particular plate of interest Clear walled plates CANNOT be used for 384 well Plate calibration so in that case select the approved plate from the same manufacturer It is helpful to pre warm plates prior to using them for calibration If room temperature plates are placed directly into the warm incubator condensation can form on the bottom of the plates and interfere with the calibration results ONLY clear bottom COMPLETELY EMPTY opaque walled 384 well plates from the approved list can be used for the 384 well Plate calibration procedure Follow these steps to for the 384 well Plate calibration procedure Figure 8 54 Put two 2 empty 384 well plates into a microplate tray both spaces in the microplate tray must be occupied select the appropriate tray position in the software all three tray positions need to be calibrated Press Run The calibration for each tray position takes only a few minutes Repeat for all three tray positions 101 Page Device Accou
84. 9 A12 0 301 0 399 A13 0 255 399 AL4 0 253 0 399 A15 0 252 0 399 Al6 0 25 0 399 A17 0 281 0 399 Al8 0 274 0 399 AL9 0 293 0 399 A20 0 339 0 399 A21 0 311 0 399 A22 0 324 0 399 A23 0 335 0 399 A24 0 321 0 399 B1 0 246 0 391 1 0 246 0 443 D1 0 246 0 412 E1 0 246 0 488 F1 0 246 0 486 Gi 0 246 0 497 H1 0 246 0 483 I1 0 246 0 482 J1 0 246 0 516 K1 0 246 0 562 L1 0 246 0 596 M1 0 246 0 648 N1 0 246 0 679 01 0 246 0 624 1 0 246 0 682 Wel Offsets for Cutout 1 Al 0 344 0 333 A2 0 431 0 333 A3 0 388 0 333 A4 0 384 0 333 AS 0 329 0 333 Ao 0 333 0 333 A7 0 29 0 333 AB 0 304 0 333 AS 0 314 0 333 A10 0 318 0 333 All 0 356 0 333 A12 0 375 0 333 A12 0 37 333 Al4 0 327 0 333 A15 0 329 0 333 A16 0 336 0 333 A17 0 356 0 333 A18 0 33 0 333 Ai9 0 382 0 333 A20 0 416 0 333 A21 0 421 0 333 A22 0 437 0 333 A23 0 471 0 333 A24 0 482 0 333 B1 0 344 0 35 1 0 344 0 312 D1 0 344 0 353 E1 0 344 0 342 Fi 0 344 0 418 G1 0 344 0 387 H1 0 344 0 373 11 0 344 0 384 11 0 344 0 424 K1 0 344 0 428 L1 0 344 0 451 M1 0 344 0 497 N1 0 344 0 528 1 0 344 0 473 1 0 344 0 477 gt gt End 08 17 10 12 36 55 2 49 PM Next Scan 5 00 PM Set By admin Set Af 8 17 2010 1 36 Status Ready To Scan Free Space 67 61 GB 7 Database 591 MB 85 A AAOAAAN Figure 11 1 The IncuCyte Logs can be saved t
85. 9 Both traces start at 0 time When the two disparate graphs were initially overlaid see Figure 8 147 at Auto Alignment setting 14 the composite graph displayed both traces one on top of the other Even though an alignment value was set 14 no actual alignment was possible because the traces had no points in common i e the trace for Flask 2 has no readings at 14 confluence If an alignment value is set that is outside the range of one or more traces then the program cannot generate an accurate alignment To set a new Auto Alignment value scroll through the values in the alignment box or simply type in anew value Then press Reset or hit Enter on the keyboard Another useful application of the Auto Alignment function is the comparison of multiple traces at different stages of growth In Figure 8 150 growth between two flasks plated under different circumstances is compared When the traces are aligned at 15 Figure 8 150 there is an obvious disparity in growth between the two samples However when the traces are aligned to 50 cells from both samples grow at a similar rate Figure 8 151 Apparently the cells in Flask 1 191 Page ESSEN BIOSCIENCE T grow more rapidly at low confluence but cells from both flasks grow at a similar rate once confluence reaches 50 Graph All Mean vs Time Fie Yew Edt Average Smooth Growth Over Time Flask1 Flask 2 8 3 8 es z 5 o p a o gt D
86. An ongoing analysis can be exited by closing all the open Processor Windows Exiting an Analysis Job will suspend participation of the local PC in an ongoing analysis Remember however that even though your PC is no longer involved in processing the controller will continue to process the Analysis Job on its own lf the analysis is still incomplete at a later time because the controller hasn t completed it yet it can be rejoined by any PC by selecting the job and pressing the Join button once again Closing the IncuCyte software will NOT automatically exit an ongoing Analysis Job Even after the software has been closed the Processor Windows will remain open and the PC will continue processing the data If the Processor Window s are closed by the User the controller will continue processing the data lt is possible to have multiple incomplete Analysis Jobs listed in the Open and Manage Jobs window that are not being actively processed by any PC In this case the controller will continue to process the jobs in the order in which they were added Once the controller has completed the first previously incomplete Analysis Job it will move on to the next until all pending jobs are fully processed In Summary Analysis Jobs can be exited by Closing all the Processor Windows Logging off of your computer Shutting down your computer Termination of an Ongoing Analysis Job The ONLY way to fully terminate an ongoing Analysis Job includin
87. Apply button or reloaded to an unchanged state by clicking the Reload button before the Scan on Demand scheduler can be accessed If changes are not applied or reloaded the Scan on Demand tab will be unavailable 24 Hour Repeating Time bar Scan on Demand Time bar 12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm 12am Tray Type Microplates x Select Tray Type Tray Position Front Vessel Type Scan Type and Cutout Position Left x Scan Pattern Vessel Type Efail Comings X Scan Type Ruorescence amp Phase Contrast v Fast Fluorescence i Load button ican Pattem Sample Pattem X v NY s Unique ID Load 3 Label Ceol Type Unique ID and Passage 1 label boxes Noe The Properties boxes IncuCyte tray is c i 12345 67 8 8 oN 2 ADUVUIIOIYOY IO 9 F ONR ASSA ce a JO E OO QEeG a 6 C F HOQOOQO O O OO OO OO OOGE 6 O OO OO O O OOOE Scan on Demand tab SAT AADAC Clear all properties e Edit Scan Patterns Vessel Scheduling The Scan button Red indicates changes made to SOD and ready to scan Figure 7 3 Scan on Demand Screen Within the Scan on Demand scheduler the 24 Hour Repeating scan schedule including all of the time bars will be faded to the background but will be clearly visible See Figure 7 3 The current time will be indicated as in the 24 Hour Repeating scheduler but will be
88. C COMMIS A A e ea teseceaetrasneaanesiansatesetiaatysau adducts ehantenasctecaniadeeauaaaieaaseiaeececnsaaeeaee 95 SCOTA G d OMT CECC ete MCR ERCE eC CTER Er 97 DOSES EEE EAN E E EEEN A E O EA E O E A 97 TO GUE AEN AE E E E E E E E E E E 106 8 6 7 8 6 8 8 7 1 amp 2 8 7 3 8 7 4 8 7 5 8 7 6 SAI 8 7 8 8 7 9 8 7 10 8 8 6 8 1 8 8 2 8 9 8 10 8 10 1 8 10 2 8 10 3 8 10 4 8 10 5 8 10 6 8 11 9 1 9 2 O24 9 2 2 9 2 3 9 3 9 3 1 O32 9 5 3 9 3 4 93 3 9 3 6 YAT 9 3 8 9 3 9 10 11 11 1 11 2 12 13 14 15 Scans Tab and Archiving for Data Management o ooonnnnnnnennnsesonnnnnssssseenensssseeereesse 107 LOS O E E E RN 120 VIEW COMPLETED SCANS isssadearsacssetemnonreanonsstetoonondoanensssusionvaseannnnstedheasaceanpasneeahenoertanvaies 124 T a E E AE E E E 124 OELE OU VOSSO eocena reens r E a E E 127 VESSEL View OV EIVICW ss cecenscncacasacaceutaanasesesesichesaanosacdiensanudsaxnsedevadactuesewaseeseuavesasanasieouecbes 131 WES SCL VIEW Fe LAI EEEE EE E OEA E EE E 136 TNC Fide Map EAUOT arrere iias e E E EE O 136 Export Images ANd MOVIES iccccccccccssvccccccccccecneesceecceeeeeesseeeeeeeeeaaeeeseeeeeeeeaaaseseeeeeeaaaaaseeeees 141 Export Images and Movies FLR ccccccccccssseescccceeeenneseseeeeeeeee ae eeseeeeeeeeaaaeeeeeeeeeeaaaaaeeeees 150 AIO E E E E S E E 152 The Scans Pull Down MON sccccccccccsssseeccccccceccneessecceeeeaeeeeeeeceeeeeaaeeeeeeeeeesaaaeseeeeeeeeaas 152 The Preferences
89. CP Based Networks 4 1 1 Turn off power to the unit and connect the controller s primary Ethernet port to the network with a standard Ethernet cable Power on the controller and wait a few minutes before verifying the controller is networked correctly lf connected correctly the controller will appear as a computer on the Microsoft Windows Network as a member of the Workgroup group of computers Assume that the controller is assigned the name ICXXXXX in the factory where XXXXX is the 5 digit serial number of the controller This serial number is found on a label on the front panel of the controller behind the hinged front plate To verify that the ICXXXXX controller has been successfully added to the network a Windows based PC hereby referred to as the PC on the same network is required These verification methods are discussed separately for Windows Vista and Windows XP PC s Verifying the IncuCyte Controller Network Connection using Windows Vista or Windows 7 1 Under the Windows Start menu select Network Vista or Computer Windows 7 a Windows 7 only On the left side of the window there is a list of destinations Favorites Libraries etc Look for the Network destination and select this by left clicking 2 Look for the computer named ICXXXXX recall that XXXXX is the 5 digit controller serial number If you find it in the window then the controller was successfully
90. Cell Culture Vessel for Automation Septum Cap Microplate 3068 CellBIND Surface RoboFlask Cell Culture Vessel for Automation Septum Cap Microplate coming 3069 Corning RoboFlask Cell Culture Vessel for Automation TCT with Bar Code Septum Microplate 3070 Corning RoboFlask Cell Culture Vessel for Automation TCT with Bar Code Septum C4 Microplate 3071 Corning RoboFlask Cell Culture Vessel for Manual Use TCT with Bar Code No Septu Microplate 6 Well Plates Brand Catalog s Plate Name Tray Type y yp Pk Microplate T Brand Catalog s Plate Name Tray Type 2 well Flat Bottom Standard Tissue Culture Treated Microplate 2 well Flat Bottom Standard Tissue Culture Treated Microplate oly D Lysine 12 well Multiwell Plates Microplate SE e Collagen 12 well Multiwell Plates Microplate Fibronectin 12 well Multiwell Plates Microplate Laminin 12 well Multiwell Plates Microplate 354503 Matrigel Matrix 12 well Multiwell Plates Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate 356470 Poly D Lysine 12 well Multiwell Plates 3336 Corning CellBIND 12 Well Microplate Flat Bottom Standard Clear Plate with Lid Corning 3512 Costar 12 Well TC Treated Microplates Standard Clear Plate 3513 Costar 12 Well TC Treated Microplates Standard Clear Plate 150628 12 Well Plates Nunclon A Sterile Polystyrene TPP 92012 12 Well TPP Individually wrapped 92412 2 Well TPP 4 p
91. D 20090612 expt UEA 7 1 200 20090619 Angiogene 7717200 20090619 Angiogene 7717200 20090619 Angiogene Cell Type TCS HU Scan Type EER Standard Standard TCS HU Standard s Standard j Standard Seared 6 26 20 6 26 20 6 1 3 20 6 1 9 20 6 19 20 6 26 20 6 26 20 7 2 20 772 200 7 2 200 EM 24 well 24well 24wel 2b wel aa E Row A 25 per well ft a 7 2 200 a 7 2 200 772 200 772 200 Row c 25 per well Row A 25 per well Row C 25 per well really 7 2 200 7 2 200 20090619 Angiogene 7 2 200 20090619 Angiogene TCS F 2003061 9 Angiogene vz 20090619 Angiogene Standard Standard Standard e Standard 6 1 oe 20 6 1 9 20 6 19 20 6 19 20 LEZ 200 Z 2 3 200 172 200 7 2 20 A 24 wel 24 well 24w Zamal 7 2 20 77 6 200 iZ 6 200 7 6 200 iZ 6 200 v 1 O 20 20090702 full plate analysis 200900706 20090706 whole plate analy m 20090706 whole plate 16 ATA 20090706 whole plate analy 20090710 VEGF GFP infec z 7 2 20 20090619 Angiogene _ 7 6 200 20090619 Angiogene 7 6 200 20090619 Angiogene 776 200
92. Displays Metrics Display Metrics and access Graph Export functions See Section 8 10 Preparing to Graph page 172 and Section 8 10 4 Export Metrics page 172 These same functions can also be accessed using the Vessel View utilities pull down menu See Figure 8 95 p Vessel and Scan Tabs EX EX Two additional tabs Vessel and Scan are available on the IncuCyte EX See Section 8 5 6 page 75 for more information on these tabs 8 7 4 Vessel View FLR FLR The Vessel View window for fluorescently scanned vessels contains expanded options and functionalities relative to vessels scanned non fluorescently See Section 9 IncuCyte FLR page 201 for information regarding viewing and manipulation fluorescently scanned images 8 7 5 The Plate Map Editor To assist in experimental design and data analysis the IncuCyte software includes a Plate Map Editor that allows the User to custom design a plate map for any microplate based experiment The User may design and save a plate map at any point before or during an experiment or after the experiment has been completed Access to the Plate Map Editor is available from four locations e Main IncuCyte window Select Plate Map in the File menu at the top of the window 136 Page gt P Pas Ca D wW an w ia wo Dus ae S I Bm ta z l o From this location the User may save a plate map to a file but not to an individual vessel e Schedule Upcoming Scans Properti
93. Image Format JPEG TIFF JPEG TIFF JPEG PNG TIFF RAW JPEG TIFF Exported Movie Format WMV AVI Metamorph WMV AVI Metamorph WMV AVI Metamorph WMV AVI Metamorph High Definition HD i No Yes Yes Optional Optics Phase 400 Kbytes 150 Kbytes Image Size typical 150 Kbytes 400 Kbytes Fluorescence 1 1 Mbytes with HD 400 Kbytes Fluorescence co as J n a n a 450 490 nm 500 530 nm n a Excitation Emission 500 Gbyte Hard Disk 500 Gbyte Hard Disk gt 900 Gbyte Hard Disk gt 900 Gbyte Hard Disk Internal Image Storage 2 Gbyte RAM 2 Gbyte RAM 2 Gbyte RAM 2 Gbyte RAM Ethernet 10 100 Mbps Ethernet 10 100 Mbps Ethernet 10 100 Mbps Ethernet 10 100 Mbps Network Connection RJ45 Connector RJ45 Connector RJ45 Connector RJ45 Connector 100 240 VAC 100 240 VAC 100 240 VAC 100 240 VAC 47 63Hz 47 63Hz 47 63Hz 47 63Hz Power 2 0 Amps max 120V 2 0 Amps max 120V 2 0 Amps max 120V 2 0 Amps max 120V 1 0 Amps max 230V 1 0 Amps max 230V 1 0 Amps max 230V 1 0 Amps max 230V Controller Dimensions H x W x D Microscope Dimensions H x Wx D 224mm x 450mm x 465mm 224mm x 450mm x 465mm 251mm x 450mm x 465mm 224mm x 450mm x 465mm 30 Ibs 30 Ibs 33 Ibs 33 Ibs troll ight Controller Weigh 13 6 kg 13 6 kg 15 0 kg 15 0 kg n 32 5 Ibs 32 5 Ibs 36 Ibs 37 Ibs Microscope Weight p E 14 8 kg 14 8 kg 16 3 kg 16 8 kg 0 C to 33 C 0 C to 33 C 0 C to 33 C 0 C to 33 C Operatin P E 5 to 90 RH 5 to 90 RH 5 to 90 RH 5 to 90 RH Environmental
94. NCE C Export Current Image Image type ji Phase Contrast using current display settings JPEG X Filename os Display Options Scaling 1 00 kl Final Size 956x624 Include Timestamp Include Legend Click and drag the red guides to choose the area to be exported Figure 8 97 The Export Current Image window with Export Designer open Export Image Set Multiple Images It is possible to export all or a subset of all the images obtained for a particular vessel in bulk format by selecting the Utility Export Movie or Image Set See Figure 8 98 This window displays the selected Vessel and its corresponding Time Tree The window is divided into two main Sections Export Time Area Range and Save Location and Options Note that one or more possible images from each well or sector can be selected for export The image number in the check box refers to which image will be exported It does NOT refer to the total number of images that will be exported Thus if only the checkbox for image 3 is selected then only image 3 will be exported for each selected well sector and or time point That is only ONE 145 Page ESSEN ON BIOSCIENCE image will be exported per sector or well and that image will always be image number 3 To export all three images for each well sector the checkboxes for all three image numbers must be selected Cc Movie Image Set Export Export Time Area
95. NOT be performed Scans are selected for deletion using the Time Tree in the same way as they are selected for archiving see Section 8 6 7 Scans Tab page 110 113 Page N After scans have been selected simply press the Delete Scans button A window will appear to confirm that scans should be deleted 2 Choose a Time Day Month or Year Selecting a scan time will delete that scan Selecting a day will delete all scans in that day Selecting a month will delete all scans in that month etc 2005 A May 09 Mon 5 15 PM 3 15 PM 11 15PM 10 Tue 2 15 4M 5 15 AM 6 15 AM 11 15 4M 2 15 PM 3 30 PM 6 30 PM 9 30 PM 11 Wed 12 30 AM v A wv Delete Scans Figure 8 67 Delete Scans Restore from Archive Under some conditions scans that were previously archived can be restored from their archive location back onto the IncuCyte controller After scans have been restored they appear on the controller as they did prior to archiving and they do not need to be opened separately by browsing to an archive This function is available with some limitations e The Restore Archive Function is only available on IncuCytes equipped with a 2009A or later software release e Archived scans can only be restored if they were performed and archived using an IncuCyte equipped with the 2009A or later software release e The archive must originate from the same controller to which it is being restored
96. O S84 3 Image 1 of 4 23 5 Confluence vi Graph Export Figure 8 4 View Completed Scans Screen 59 Page Find Scanned Vessels Search for individual scanned vessels Schedule Upcoming Scans Use this screen to schedule scans EX Note that the Schedule Upcoming Scans Task Bar is NOT available for the EX IncuCyte EX Administer IncuCyte This screen facilitates access to the administrative features of the IncuCyte including instrument logs calibration and changing the password and User account management 8 5 Scheduling Scans Many of the functions covered in this Section will not be relevant to the IncuCyte EX User The two functions that ARE relevant regard setting Scan Patterns and setting vessel Properties Each of these sections is discussed for general IncuCyte use followed directly by an additional section related to the specifics as they pertain to the EX We strongly recommend that Users read both the general information regarding Scan Patterns and vessel Properties and then read the special sections specific to the EX See the following sections Section 8 5 3 Setting Scan Patterns page 67 section 8 5 4 Setting Scan Patterns EX page 70 Section 8 5 5 Setting Vessel Properties page 73 The microscope can heat up during excessive scanning Therefore we recommend keeping scan times under 45 minutes and duty cycles under 50 As an example 40 minute long scans scheduled every t
97. Range 4 06 PM Time Range x Start Time Images Save Location and Options Target folder Q incuCyte INCUCYTE ARCHIVE DATA Anc Name prefix TEST Sequence type Windows Media Video high quality WMV9 compression i Example TEST_A1_1 wmv Image type Phase Contrast using current display settings v i Movie Designer Crop Scale Legend etc Export 1 file Cancel Figure 8 98 Movie Image Set Export Window Select Wells Sectors for Image Export One or more wells sectors can be selected for image export Select one or more wells sectors by clicking on them individually with the mouse Select a region of the vessel by clicking and dragging with the mouse Deselect one or more individual wells sectors by holding the Alt key and then clicking with the mouse Deselect a region of the vessel by holding the Alt key and then clicking and dragging with the mouse Right click on a well sector to activate additional options for selection deselection See Figure 8 99 146 Page Select this Column Select this Row Select All Deselect this Column Deselect this Row Deselect All Figure 8 99 Right click for More Options It is possible to individually select up to 9 images per well sector for export if the scan pattern includes 9 images or less However it is also possible to set a scan pattern that includes more than 9 images per well sector In this case there will
98. Removal and Graph Drag and Drop Auto Alignment see Preferences Outlier Removal and Drag and Drop Auto Alignment page 184 For information on dragging and dropping graphs in general see Dragging Graphs page 181 For more information on slideshow viewing see Slideshow Mode page 141 156 Page N ESSEN BIOSCIENCE _ Preferences Outlier Removal None Image Drag and Drop To Microsoft Word Outlook or Publisher To Microsoft PowerPoint Excel or WordPad Graph Drag and Drop Auto Align at Y Value when Drag and Drop occurs Vessel Viewing 1 5 Seconds before loading next image in slide show mode Figure 8 106 The Preferences Window A variety of images can be dragged and dropped from the IncuCyte software into other programs The following images can be dragged and dropped from the following screens Vessel View Window e The Selected Vessel image e The large image e The Metrics View Completed Scans Screen e Any tray e The selected vessel in the Selected Vessel box e The Metrics and image in the Selected Vessel box Find Scanned Vessels Screen e The selected vessel in the Selected Vessel box e The Metrics and image in the Selected Vessel box 157 Page ESSEN N BIOSCIENCE Select the appropriate option in the Preferences Window based on the intended destination of the image Drag and Drop See Figure 8 107 Whichever option is selected will apply to all drag and drops u
99. Std Dev lof4 2of4 3of4 4of4 Min Figure 8 120 The Graph Export Tab in the Vessel View Window 8 10 1 Prepare to Graph from the View Completed Scans Screen The default Metric is Confluence It is also the most commonly used Metric The remaining Metrics Image Mean Focus Position Exposure Time and Autofocus Sharpness are diagnostic tools used to confirm that the software is functioning within the specified parameters These Metrics will not be relevant to the User under normal circumstances NOTE Metrics other than Confluence are only available at the Administrator permission level Select Statistic Image Mean Median When multiple images are available within each well then selecting the Mean vs Median will select the image Mean or image Median respectively for graphing If a single well is graphed then each point on the graph will represent the Mean or Median for all images in that well at that time 167 Page N Trace Mean Median When multiple wells are represented by each point on the graph for instance if all wells within a column are combined to generate a graph of that column then the Median or Mean refers to the combination of the individual wells Summary 1 If each point on a graph represents a single well or sector then selecting the Mean or Median refers to the Mean or Median of all the images within that well or sector This is the case when the data are graphed as a single well or
100. User level however only the Device Accounts and Logs tabs are present Additionally some of the features within these tabs are inactivated 8 7 Page N ESSEN BIOSCIENCE Set Device Time 12 11 2009 2 48 59 PM Eastern Standard Time Daylight Savings 12 11 2009 2 49 46 PM Eastern Standard Time Daylight Savings Device System Control Power off the device Restart the device Export scan diagnostics Export application events Export setup files Export database Export device test logs Figure 8 37 Administer IncuCyte at the Administrator Permission Level 88 Page Esessssssssssssssesed Set Device Time Local Time 12 11 2009 2 49 58 PM Eastern Standard Time Daylight Savings ic 10036 time 12 11 2009 2 50 40 PM Eastern Standard Time Daylight Savings Synchronize Device System Control Power off the device Shutdown Restart the device Restart Export Export scan diagnostics Diagnostics Export application events Events Export setup files Setup Files Export database Database Export device test logs Tests Figure 8 38 Administer IncuCyte at the User Permission Level 8 6 1 Administer IncuCyte EX On the IncuCyte EX the Administer IncuCyte tab is ONLY available to Users at the Administrator permission level Figure 8 37 The complex nature of the scan schedules and the robotic comp
101. XCLUSIVE REMEDIES ESSEN BIOSCIENCE INC SHALL NOT BE LIABLE FOR ANY DIRECT INDIRECT SPECIAL INCIDENTAL OR CONSEQUENTIAL DAMAGES WHETHER BASED ON CONTRACT TORT OR ANY OTHER LEGAL THEORY 1 3 Warnings and Disclaimers Doing any of the following may void your warranty WARNING DO NOT RUN THE STERILIZATION CYCLE ON AN INCUBATOR WITH THE INCUCYTE GANTRY INSIDE WARNING DO NOT TURN OFF THE INCUCYTE AND LEAVE THE GANTRY INSIDE THE INCUBATOR CONDENSATION BUILDUP CAN DAMAGE THE ELECTRONICS Essen BioScience is not responsible for data loss due to hardware failure 6 Page N 2 Getting to know your IncuCyte 2 1 Introduction 2 1 1 IncuCyte Models Thank you for choosing the IncuCyte from Essen BioScience There are currently 5 models of the IncuCyte available e The Standard Model IncuCyte e The IncuCyte EX e The IncuCyte HD e The IncuCyte EX HD e The IncuCyte FLR HD 2 1 2 Intended Use The IncuCyte is intended to facilitate live cell monitoring via customized imaging protocols It is intended for experimental purposes only Please read carefully through the operating instructions in this manual to ensure that you can properly and safely utilize all features of your new instrument 2 1 3 The IncuCyte EX Unlike the Standard Model IncuCyte the IncuCyte EX microscope is NOT designed to operate within a cell culture incubator The EX is a special model instrument that
102. age Wessel 12 Wwell BD Falcon well BO Falcon well Corning 12 well BD Falcon 12 Wwell Corning 24 well BD Falcon 24 well Corning 4 well Essen ImageLock 46 Wwell BD Falcon 48 well Corning 96 well BO Falcon Clear Wall 96 Well BD Falcon Optilux g6 well Corning 96 well Greiner RoboFlask Figure 8 18 Select the Vessel Type for the new Scan Pattern in the EX A single Sample Pattern will be available for each Vessel Type by default However additional Scan Patterns can be created as required The process by which new Scan Patterns are created is basically the same as for the Standard Model IncuCyte Section 8 5 3 page 67 Existing Scan Patterns can be edited by highlighting the desired Scan Pattern and then selecting a new pattern The estimated scan length for each Scan Pattern is indicated in the Estimated Time to Scan window In Figure 8 19 the estimated scan length for the selected Scan Pattern is 1 minute and 20 seconds IMPORTANT Changing the Scan Pattern also affects the scan length of the target vessel It is important to consider that significantly increasing the scan length of your own vessel could negatively impact the schedules of other unrelated vessels Images per Well Estimated Time To Scan 00 01 20 Figure 8 19 Estimated Time to Scan Press the Refresh button to insure that you are viewing the most up to date set of scan patterns for the selected vessel It is recommended to always p
103. aphs can be viewed with or without error bars and the error bars will represent the inter sector Mean or Median as selected in the Statistic drop down menu All All the sectors within the flask will be combined into a single trace None any of the sectors will be grouped No error bars are available for graphs of single sectors Region Single Sector If the Region Single Sector is selected a new drop down menu will appear Choose the desired sector for graphing Error bars are not available for graphs of a single sector 8 10 2 Prepare to Graph From the Vessel View Window Time Plot Press the Graph Export button in the Graph Export tab of the Vessel View window to open a new graphing window See Figure 8 122 In this figure a single well A1 has been selected Note that it is possible to independently choose both the well letter and number because this is a high density plate Time Range bd Image Mean Eoo pm Confluence v1 5 i 6 00 PM Focus Position i 10 00 PM Exposure Time 24 Sat k Autofocus Sharpness 25 Sun 23 82 3 8 15 3 8 8 00 PM Start Time on oo oo on ae an oe oo an IA s s F 26 Mon 27 Tue z i 12 00 AM i 2 00 AM 4 00 AM 6 00 AM Statistic Histogram 48 scans 8 00 AM Mean 10 00 AM i 12 00 PM tommogwsa gt e s 3 0 C Region Single Well A
104. ard Model IncuCyte except that the Tray Position cannot be selected because the EX contains only a single tray View the calibration results in the Logs tab under Log Type Diagnostic Device Accounts Scan Patterns Tests Update Scans Logs Motion Calibration Tray Position Front Test Calibrate and Confirm Confirm Only J Quick Test Figure 6 9 Calibrate Device EX FLR 6 5 Calibration FLR Because all IncuCyte FLR s are HD enabled instruments they must undergo standard tray calibration as well as 384 well calibration Additionally the fluorescent IncuCyte requires a special fluorescence calibration procedure 32 Page See the Essen BioScience IncuCyte FLR Technical Note entitled Using the IncuCyte Calibration Kit for more information A calibration kit will be provided with the purchase of the IncuCyte FLR and the instrument will be calibrated at the time of installation It is currently recommended that the instrument be recalibrated approximately twice a year however if the User suspects a drift in the calibration signal then the system can re calibrated at any time The fluorescence calibration kit includes liquid sufficient for multiple calibrations as well as three calibration slides These calibration slides are disposable and should be used only once New calibration kits can be purchased from Essen BioScience See Section 13 IncuCyte Catalog page 244
105. ard fluorescence images which means that the very finest spatial features that can be detected in standard images are observed with less detail in an FF image For assays where high throughput is preferred over high content the advantages of FF far outweigh the resolution afforded by a standard fluorescence scan One example is a nucleus counting assay where the FF resolution is more than adequate to detect cell nuclei 66 Page N 8 5 3 Setting Scan Patterns Overview The scan pattern determines the pattern s and location s of images that will be obtained within a vessel After the vessel type has been selected the Scan Pattern menu box becomes activated for that particular vessel The Scan Pattern menu box will only be active when a vessel is highlighted and the Scan Pattern selected will apply only to that one vessel If two or more vessels are highlighted simultaneously either using the SHIFT Ctrl keys or by choosing the Select All button then the selected Scan Pattern will be applied to all highlighted vessels The Scan Pattern is indicated within each vessel as a pattern of green dots A default Sample Pattern will be available for each vessel type when the IncuCyte arrives Initially this will be the only scan pattern available However new scan patterns can be created by the User by selecting a vessel and then clicking on the Edit Scan Patterns button located at the bottom left corner of the Schedule Upcoming Scans screen t
106. ate 99 Page If the calibration was performed recently the results will appear at the top of the log window However if the object is to locate calibration results from some point in the distant past they will likely be preceded at the top by other more recent events The number of entries displayed at any given point can be adjusted at the bottom of the Logs tab using the Entries to Display pull down menu Try increasing the number of displayed entries if you are unable to locate your results within the current log screen Entries To Display Figure 8 52 Select the number of entries to be displayed in the Log window 384 Well Plate Calibration HD only The standard calibration procedure previously described in Section 6 2 is adequate for scanning all vessels in the IncuCyte HD with the important exception of 384 well plates Because scanning of 384 well plates requires such a high degree of precision it also requires a special calibration procedure Again all three IncuCyte tray positions front middle rear must be individually calibrated for 384 well plates In order to calibrate the IncuCyte for 384 well plate scanning complete the standard calibration procedure as described in Section 6 2 The additional 384 well Plate Calibration requires a single microplate tray and two 2 EMPTY clear bottom opaque walled 384 well plates The following is a list of 384 well plates that have been tested and approved for 3
107. atically be applied to all images for all scan times included in the Analysis Job The Manage Analysis Filter Settings option will become available after at least one Filter Setting has been saved Use this selection to rename or delete Filter Settings New Filter Settings can only be selected when the Analysis Filter pull down menu is set to Manual Selection Adjusting any of the Filter Metrics when saved Filter Settings are open will cause the pull down menu to revert to Manual Selection Use the Save Filter button to save selected Filter Settings 9 3 8 Filter Settings Once an image has been processed either via the Preview function or as part of a complete Analysis Job all Objects will have Object Property values These values can be visualized by holding the mouse over an Object in the Object Segmentation Mask Object Properties Definitions Area Total area of an Object um Eccentricity A measure of how round compact the Object is The values for Eccentricity range from 0 to 1 with a circle having a value of 1 224 Page Mean Intensity The average fluorescent intensity within an Object Summed Intensity The sum of the intensities of the pixels in the Object Pixel 594 603 Channel 4 Pixel Intensity 107 0 AU Channel B Area 360 6 um Mean Intensity 66 6 AL Summed Intensity 11162 AU Eccentricity 0 63 Figure 9 12 View the Processed Values Associated with an Object See Figure 9 12 The co
108. ating new pattern s press Exit to close the Scan Pattern Manager window The new EXAMPLE pattern can be Renamed or Deleted The Sample Pattern cannot The newly created Scan Pattern can now be selected under the Physical Layout tab using the Scan Pattern pull down window See Figure 8 16 It is possible to set scan patterns that generate an extremely large number of images per well For instance up to 121 images are available per well of a 6 well plate There may be times when a large number of images are required However more images means longer scan lengths and more data to store Therefore be judicious about selecting scan patterns and use the minimum number of images that will provide representative data Rear Tray Tray Type Microplates a Vessel Type 6 well Coming 6 Scan Type Phase Contrast a Figure 8 16 The EXAMPLE Scan Pattern is now available in the Scan Pattern pull down menu A Scan Pattern created for a specific vessel type will be available only for that particular vessel type For example if a scan pattern is created for a Corning 75cm flask it will NOT be available for a Becton Dickinson 75cm flask even though both are 75cm flasks A scan pattern CANNOT be deleted if it is currently selected in the pull down menu and displayed in the tray or if it is saved as a Layout Saving Layouts will be discussed further in Section 8 5 13 page 86 8 5 4 Setting Scan Patterns EX To create scan
109. bam am 10am 12pm 2pm dpm 6pm opm 10pm 12am Figure 8 33 Scan on Demand time bar illustrating scan time overlaps with 24 Hour Repeating Schedule Clearly additional scheduling rules apply to the Scan on Demand scheduler in order to protect regularly scheduled scans from being skipped These rules include e The Scan on Demand Time bar is typically yellow If the Scan on Demand Time bar is Red as in Figure 8 33 this means that the Scan on Demand scan time overlaps with a scan time in the 24 Hour Repeating Schedule Clicking the Red scan button will result in an error e The estimated time indicated by the width of the yellow bar to scan in the Scan on Demand mode is 20 longer than the estimated time in the 24 Hour Repeated scheduler This additional 84 Page time is required so that the user has time to replace the Scan on Demand vessel with the 24 Hour Repeated vessel scanning using the Scan on Demand Scheduler is initiated by clicking the red Scan button Figure 8 31 For additional information descriptions warnings and important notes regarding Scan on Demand please read Section 7 1 4 Page 40 8 5 10 ImageLock Mode Essen BioScience manufactures a special 24 and 96 well plate type called the Essen ImageLock plate These plates facilitate very precise repeated imaging within each well Under standard imaging conditions slight variations can occur in the location of imaging from one scan to the next resulting in smal
110. be eliminated from the graph Select the Manual Alignment tab use the pull down menu to select the trace for Well 6 and then click the Delete button There are 2 circumstances under which traces from a graph CANNOT be deleted These are e fthe graph contains only a single trace e f all the wells or sectors from a vessel are graphed simultaneously then the trace for the very first well or sector cannot be deleted If all wells from a microplate are graphed simultaneously this will be the trace for Well A1 The above two deletion rules also hold true for the target graphs onto which plot overlays are created Drag and Drop Manual Alignment auto Alignment 5 ws CEO e Figure 8 154 Trace Selection Box and Manual Alignment Slider Selecting an Individual Point on the Trace 194 Page N Use the mouse to select an individual point on a trace Hold the mouse over the point and the time and date of the scan represented by that point will be displayed See Figure 8 155 Corning 100mm Sector C3 Figure 8 155 Hold the mouse over a specific point on the graph The vessel label and the well sector Connections will be displayed in a yellow box next to the point Double left click on a selected point and it will open the Vessel View window for that scan The Vessel View window cannot be opened by double clicking on the points in an Average Graph The Graph Pull Down Menu Selecting the graph window pull down menu
111. bottom of the Device Status box Figure 8 60 To access your instrument s temperature history click on the graph box next to the temperature reading to bring up the temperature history window Figure 8 61 Here scan periods are shown in red while idle periods are blue At the bottom right of the screen choose to view the last 24 hours one week or 30 days As with the standard graphing window click and drag over an area within the graph to zoom in on that area The IncuCyte FLR temperature sensor makes periodic measurements of the ambient temperature inside the instrument Although this sensor is very accurate it only approximates the internal temperature of the device due as it is in a fixed location and does not move around with the microscope Nonetheless the sensor is very useful for measuring the heating and cooling trends in the device over long periods of time By plotting the temperature over time one can not only track the rise and fall of the temperature as the device scans but also as the heat that is both lost from the incubator by opening the door and injected into it by the incubator heating system The temperature sensor therefore is a useful tool for verifying the target temperature setting of the incubator itself as well as for tracking the influence of IncuCyte scans on the transient temperature inside the device See Scan Length for recommendations on how to minimize the influence of the IncuCyte on incubator tem
112. button A new window will open allowing the User to adjust the Min Max Values of the x axis as well as the Bin Count The software will assign recommended settings based 232 Page N ESSEN BIOSCIENCE upon the data but it is possible to substitute Custom values as desired After Bin Settings have been selected press the Graph button Pressing the Graph button directly will generate a Histogram even if Bin settings are not viewed adjusted first The Histogram will simply use the settings assigned by the software Increasing the number of Bins results in finer resolution of the distribution across the selected Metric See Figure 9 17 In this example a histogram has been generated that displays the distribution of Object Area in Well A1 Note that it is possible to Drag and Drop both the Histogram Data and Image Because the Include Filter Details box was checked the Filter Settings is also displayed on the graph Histogram Well A1 2008 10 30 17 19 Object Area File Edit View Well A1 2008 10 30 17 19 Object Area Histogram 4500 objects total 164 3 objects per mm Area gt 50 pm 5 200 150 100 iin o mm See _ 100 00 160 00 220 00 26000 340 m 400 00 460 00 520 00 580 00 640 00 Drag Drop Settings v Raw Data to Document v Right click and hold on this graph and drag into another application as a text data table Raw Data to Document Image to Word or Outlook Image to Other Document Figu
113. cccoscsterssarisesceasidecasasstarcoseddecasasetuaceasetesasansecebedeeecsts 205 Task Bars Available in the IncuCyte FLR Tasks Pane cccccccssseseeseeeeeeseeetessessstesesseans 205 Analysis Tools Available in the IncuCyte FLR Tasks PAne cccccccccccccccceceeeeceeeeeeeeeees 208 Preview Using Current Image Analysis Preview Mode 1ccccccccccseveeeseceeeeceanesssecees 211 Launch New Analysis ccccccccccccssessscecccecccasusseeceeccaaanssseeececsaaanaseeeeeesaaauseeeeeessuaaaaeeesees 215 Open and Manage Analysis JODS nnnnenononnnnnnnnnnnesooennnnsssseeennnnsssseerensssssseeressssseerressss 222 The Object Counting Analysis Filter Pull Down Menu u ccccccccccseseeecccceceeeeneseseseeeeeens 224 OP SONI a E TE dvd wares otiedudes tosses Sones 224 Graph Export Data from Fluorescent Scans Unprocessed VeSSe1S ccccccsseecceeeeeeeees 230 GETTING OPTIMUM PERFORMANCE FROM YOUR INCUCYTE 235 TROUBLESHOOTING sar ciecs hss aes sds cte cs state o aN a aop a aa aa aoa LaNa 236 IMAGES AND WHAT THEY MEAN o ii ascssecnaosceusetenanecevicacnsssnnsedeaenaeansaseamiagan onan eneeeeennreees 236 CONTACTING TECHNICAL SUPPOR T vccncsvnssipasinnsdsvntvnsienyassbad ssinenbnssinasansd a idide 241 SPECIFICA TIONS sis sesasseactissccvcesasssveisuassrseuasiaxctsisccncerscherstunneeaseassisieieseressersienniavs 243 INCUCYTE LCATALOGUT ssori anaa EN aana 244 ORDERING CONTAC DP sect ssicsvsaiasiesstsnssetsasnsssscivcinssceteciecsscitctcassse
114. cd caesioenteanedtnaaieaoenesanidd caeeioe taanenteeaies 27 Placing Trays into the IncuCyte eeeeeennnnnnnnnennenrerrerrrrrrrrrrrrrrrrrrrrrrrrrrrerrrerrererererrerens 27 Placing Trays into the IncuCyte EX eeeeeeeeeeseeeseeesesererreerrrrrrerrrrrrerrrrrerrrrrrrererrrererrrens 28 Placing Vessels into the InCuCyte ccccccccccscssssccecceeeececcccecceeeccecceeceeeeeeeeeeeeeeeeeeeeeeees 28 TENC ALBRA TION oia EE 29 CALIBRATION OF THE INCUC YTE M FID uirri iaioa kio atone stansaness il CALIBTATON E A rii E E abe seamaanseabiantseianeetioeess 32 CALIBRATION PLE oiiire rE a AE E A E 32 OVERVIEW OF CELL CULTURE MONITORING eessssssssssssssssssessssssssssssssseseseee 35 SCH DOLINO OC CANS sereoo eE T A 35 The Scan Active Light and Stop Button cccccccccccssssccccccccccaeesseececeeecaaeessecceeeeaaaaseseeeeeeaas 35 Physical Layout Select Tray Type Vessel Type Scan Pattern and Scan Type 36 ECON L E S oaa a EE EE EAEE 38 CO OD CI aae E E E E E E E EEEE 40 imace ig A MOR O eseria EE E EAE E E S 42 imaceLoc kM Mode PGR apa pastries EEEE EAE 43 ME COVETED SCAN anian poigstneta eo sions vnawteosoesiawtennelenaatvntons sass bonseusouiacennteadesoneeaus 43 View Completed SCONS TX araroa a E EEEE 46 PIND SCANNED VESSEL orminn a 47 FiS canned Vessels TA ei E A A E E ar 48 GRATIN E ae E O E E E E A a 49 Crea OTA E EE EA E E Terr nar eereemrr Te 49 Creating Average GLADNS wo cccccccscsecccccccccnccnssseccceccacaassseeecceeaaus
115. cipating in the analysis has a multi core processor then the Analysis Job can be joined by that same computer Press the Join button and a second Processor Window will open If the computer participating in the processing has a multiple core processor then multiple Processor Windows can be open at any given time one for each core in order to expedite the rate of processing Although it is still possible to continue joining and open more Processor Windows we recommend against opening more windows than the total number of cores Opening an excessive number of Processor Windows can actually decrease the rate of processing Ongoing Analysis Jobs can also be joined by other computers that have the IncuCyte FLR software To join the analysis on a separate computer launch the IncuCyte software locate the vessel open the Vessel View window select the ongoing analysis and then press the Join button 219 Page While joining an ongoing Analysis Job will generally increase the rate at which processing occurs at some point excessive joining will result in diminishing returns because the IncuCyte controller will be overwhelmed Therefore it is important to monitor the progress of job processing If multiple computers are working on a single job and the rate of processing decreases then reduce the amount of joined processing This same principle applies to excessive joining on a single computer Exiting and Rejoining an Analysis Job
116. create a new Scan Pattern click the New button in the manager window A window will appear requesting a name for the new pattern Enter a name and click OK Now the desired vessel will appear with no scan pattern The only active selections will be the Clear and Cancel buttons and the Images per Well pull down menu Figure 8 12 2 Scan Pattern Manager for 1C10104 Pattern Editing Press Save when Finished editing Hold down the Alt Key to remove g to select and deselect wells Click and dra ps of wells Images per well I v Clear Cancel Figure 8 12 Making a new Scan Pattern Select the number of images to be scanned per well or sector Then left click on the empty well s sector s to which the pattern will be applied in the vessel image above the Pattern Editing Box The scan pattern will appear within the selected area To remove the pattern from a well sector left click on it The desired number of images will appear in all wells sectors that have been selected Choosing a different image number will change the image number in all wells already containing a pattern NOTE It is NOT possible to have different scan patterns between different wells sectors of a single vessel Right click over a well to enter remove a scan pattern from multiple wells or sectors at once Figure 8 13 68 Page Select this Column Select this Row Select All Deselect this Column Deselect this Row Deselect All f Fig
117. croplate Graphing Currently histograms are only available with the IncuCyte FLR Object Counting Application and Microplate Graphing is only available when Graph Export is selected via the Vessel View Menu under Utilities or through the Graph Export tab in the Vessel View screen 51 Page Dragging Graphs Graphs can be dragged to either Create a Plot Overlay within the IncuCyte software or Drag the entire plot image or raw data into a different software program such as EXCEL MS Word PowerPoint or Outlook To Drag and Drop a graph choose the Drag and Drop tab at the bottom of the graph window Then use the pull down menu to select the appropriate Drag and Drop option Drag and Drop Manual Alignment Raw Data to Document Wr Graph on Graph Raw Data bo Document Image to Word or Outlook Image to Other Document Figure 7 12 The Drag and Drop Pull Down Menu Plot Overlay For example in a multiwell plate graph two wells individually Then right click on the graph for the first well and drag it onto the plot for the second well Now both traces will appear on a single plot Use the Delete button under the Manual Alignment tab to delete a trace that has been dragged but is subsequently not desired see Figure 7 13 To create a Plot Overlay the Graph on Graph option must be selected Figure 7 13 Use the mouse to drag the trace from one graph to the next to create a plot overlay Alternative
118. d Figure 8 66 Archiving Results Note that the archiving process copies the selected scan data from the controller to its new location Therefore after the process is completed these data still remain on the controller hard drive Generally it is a good idea to delete these data from the controller following archiving in order to free space on the hard drive If the controller hard drive is full new scans CANNOT be performed The Archiving Complete window conveniently presents the opportunity for deleting the previously archived scans However ONLY these scans can be deleted at this point To delete other scans see the Delete Scans Section that follows If deletion is not desired at this time simply click Done and the entire process will be complete with no scan deletion However if the archived scans should be deleted click on Delete Scans These Scans will be PERMANENTLY deleted Once scans have been deleted from the controller they CANNOT be retrieved Therefore consider carefully before deleting any scans Note Multiple archives CANNOT be saved to the same folder A folder must be created for each individual archive Delete Scans The second option under the Task pull down menu is Delete Scans Figure 8 67 This task can be used to delete scan data from the controller hard drive Unlike the Archive Scans option selected scans will NOT be copied prior to deletion If the controller hard drive is full new scans CAN
119. d Time l 7 30 PM columns rows or sectors In this 1 18 2008 10 30 PM example a single column liis 19 Sat 1 30 AM selected Choose whether or not to group Region Single Column w eee asi conn Group All Graph the data g lt Export the data inthe desired format Figure 7 10 The Graphing Box 50 Page ESSEN BIOSCIENCE Select Edit to edit graph titles and labels orto d Smooth or Average Graphs a ar _ m a4 D o x All Wells Mean vs Time Plate Conflu Choose View to toggie Error Bars on and off Use the File menu to Open save Print or Close a Graoh 5 BO i a h DH 30 AD Well Mean we Tone Costermization i q Gammal Pit S ocx Fort Coke Site T Hn Ties dl A an we 40 ti Sub Tie E Enie Spia E 3 MaBr e O 30 Shado K aj Fgh Coke bebe gaa rn i Mhe paad Ford Som i Lape lt a Apr 2007 Drag Drap and Abgnment Settings Drag and Drop Manual Alignment Auto Alignment Graph on Graph Right click an Right click or left double click an the graph background to make modifications to the graph format Figure 7 11 Graph Example Note that the Graphing Box looks somewhat different when accessed via the Vessel View as opposed to the View Completed Scans screen In the Vessel View the Graphing Box contains a space for a Histogram and an option for Mi
120. d change wrea ENESENN MOOQOOOQOOOOO 000000000000 OG OOG 00000000000 000000000000 000000000000 G 000000000000 Fill in Vessel Properties IncuCyte tray NIN P to the schedule Figure 7 2 Schedule Upcoming Scans Screen First select Physical Layout then the Properties and last set Scan Times using the Timeline Look at the Timeline at the top of the IncuCyte screen with the vertical 24 Hour Repeating tab selected The current time will be displayed as a dotted blue line 2 20pm in Figure 7 2 Left click with the mouse on the Timeline to set a scan start time This will generate a gray Scan Bar Hold the mouse over the Scan Bar to display the exact scan start time and scan length Right click on the bar to adjust the start times by small increments Left click and drag the bar to move the start time by large increments To set multiple scans create multiple scan bars individually or use the right click Scan Bar menu to select a Scan Interval Scan Bars will appear for each scan time Use the right click Scan Bar menu to delete a single scan or all scans To delete a scan you may also left click and drag the Scan Bar off the timeline The Timeline represents a 24 hour Repeating time period Once all the scans for the first 24 hour period have been completed the IncuCyte will automatically 38 Page start over for the next 24 hour period This
121. d in the status box at the bottom left 33 Page Me ESSEN BIOSCIENCE Calil bration Ready e Fluorescence Calibration uses a Microslides tray populated with a single Essen Instruments Fluorescence Calibration Slide in the far left Please verify that The Microslides tray is loaded in the front position A loaded Essen Instruments Fluorescence Calibration Slide is placed in the far left All other cutout positions in the tray are empty Are you ready to start the calibration Figure 6 10 Fluorescence Calibration FLR Calibration Results When calibration is complete the results can be viewed under the Logs tab Log Type Diagnostic The log will state whether or not the calibration passed If the calibration failed the log will include recommendations to avoid potential issues the next time through 34 Page N 7 Overview of Cell Culture Monitoring The following is a general overview of cell culture monitoring A more detailed explanation of all of this material can be found in Section 8 page 56 Software Reference 7 1 Scheduling Scans 7 1 1 So finally everything is ready to go You have selected the trays and vessels that you want and arranged them within the IncuCyte drawers You have checked that all vessels are clean fit snugly and lay flat within their trays Now it s time to use the IncuCyte software to schedule the desired scans within the 24 hour Repeating Schedule To get started select
122. d the corresponding time and date Examples of errors are displayed in Figure 8 74 In the event of a Device Error the best approach is to restart the device and try again If the error is persistent and cannot be eliminated consult Essen BioScience for advice In the event of a Calibration Error check that the Calibration Tray is in the correct location and properly seated and then retry the calibration procedure If the error persists consult Essen BioScience for assistance 122 Page Views The log contents can be viewed as a Text or Grid format When the log is viewed in the Grid format The Source of the entry is also displayed e g Diagnostic Device Client Figure 8 75 Use the Filter box to eliminate log entries that do not contain your word of interest Figure 8 76 Note that only the information within the Entry Section can be searched the logs CANNOT be filtered by Date by Source or by the IsError column Log entries can be filtered in either the Grid or Text format Log Types Entry Types VIEWS Filter A Log Date Time Entry Source 7 29 2008 5 08 Scan started Device 7 29 2008 2 16 Scan complete Device 7 29 2008 2 10 No vessel was discovered in the right position of t Device 7 29 2008 2 08 Scan started Device 7f29 2008 1 51 Selftest ended Device 7 29 2008 1 36 Self test started Device 7 26 2008 11 1 Scan complete Device 7 26 2008 11 1 No vessel was di
123. d timestamp in pixels are fixed and will thus appear differently depending on scaling Experiment with the Scaling option to see how this affects both the timestamp and legend on the final image Perhaps the most important tool found in the Export Designer is launched via the Output Preview button in the lower left corner of the Export Designer We strongly encourage you to preview your customization settings prior to export 143 Page The preview allows you to view the image at the exported size as well as in a full screen mode that fills your monitor Once you are satisfied with the customization click OK to exit the Export Designer To export with the customization settings established in the Export Designer make sure that the radio button next to the Customize with image designer button is selected prior to clicking the Export button One final note Figure 8 97 shows a selected image type of Phase Contrast using current display settings but other image types like Phase Contrast Original for example can also be customized prior to export After the Image Type has been selected browse to the file destination and select the Export button Remember that the current image can also be dragged and dropped from the Vessel View window by grabbing it and dragging with the mouse Dragged and dropped images will always be saved in the format of a JPEG file 144 Page ESSEN BIOSCIE
124. d with that vessel will be displayed in the Time Tree the preview still only applies to the currently selected image Click on the red X at far right end of the yellow banner Open an Analysis Job Change a Parameter Setting Changing a Parameter Setting will launch Unapplied Preview Mode See Unapplied Analysis Preview Mode page 215 Graphing and Export in Analysis Preview Mode In Preview Mode single time export of the selected image is available and in some cases histograms can be generated Navigate to the Graph Export tab and view the selections in the Metric pull down menu The Metric pull down menu will now include new options associated with processed data for example Object Count per Well The data corresponding to the Metric for this single image will be displayed in the Graph Export tab Select the Graph Export button to open the Graph Export Window The Time Tree in this window will include a single date for the one image that was previewed Because only one image from one time point can be previewed it is NOT possible to create a time plot in Preview Mode However it is possible to export the data associated with this single image and in some cases it is possible to generate a histogram See Graph Export Histograms of Processed Jobs page 232 for information on graphing histograms Note that the Graph Export window includes a list of any filters that have been applied to the image Use the Include Filter Details ch
125. discontinuous data sets are desired then each data set must be exported individually 172 Page Show each scan as a single row in one large table columns Al A2 B1 B2 i Destination Clipboard All scans in one file Folder File Prefix Preview enter a file prefix Other Options Include experiment details in header Fill holes in the data with the following characters optional Include Figure 8 124 The Export Metrics Window Note that in Figure 8 124 some of the options are inactive Which regions of the Export Metrics window are active are determined by selections made within the Export Metrics window as well as those Region and Grouping settings made prior to opening the Export Metrics window For example under the Layout heading Show Each Scan as its Own Table can only be activated if the Group option was set to None during the Graph Export Setup see Figure 8 119 and Figure 8 120 The Export Metrics window Is divided into 3 main sections e Layout e Destination e Other Options Each of these sections will be discussed individually 173 Page Layout Use this section to select the overall Layout of the exported data The data for each scan time can be displayed within a single long row or displayed as its own table Show Each Scan as a Single Row In this case each scan time will be listed to the far left and the data for each well will be displayed as a single row to t
126. e Matrigel Matrix 100 mm Culture Dishes for Hepatocytes _ _ o o O 9 Gelatin 100 mm Culture Dishes _ __ _ ooo o 9 O Collagen 100 mm Culture Dishes __ oS o 9S Poly D Lysine 100 mm Culture Dishes _ __ _ ooo S 9 O Gelatin 100 mm Culture Dishes _ __ oS o 9 Corning 100mm Ultra Low Attachment Culture Dish __ o o O O 9 O Corning CellIBIND Surface 100mm Culture Dish _ __ _ _ _ Ooo 9 O Corning 100mm TC Treated Culture Dish o 9 O Corning 100mm TC Treated Culture Dishes in 6 pack Carriers o 9 CellStar Dish PS 100x20mm 58cm TC ST Vented __ _ o o O Oo 9 O CellCoat Dish PS 100x20mm 58em wPDL ST __ O S 9 CellCoat Dish PS 100x20mm 58cm WCOLL ST ____ O Oo 9 Dishes Nunclon A Polystyrene o o S 9 Dishes Nunclon A Polystyrene o S oo 9 O Dishes Nunclon A Polystyrene o S S 9 Dish Name Tray Type Culture Area 148cm Dia x H 150 dia x 25mmH CellStar Dish PS 145x20mm 141cm ST TC Vented 145 0 20 MM Brand BD Falcon 93150 Catalog s 354629 9500 4380 154534 Slide Name Tray Type BD BioCoat Poly D Lysine 2 well CultureSlides FLR Calibration Slide Nunc Lab Tek II Chamber Slide System 8 Well Configuration 256 Page N 16 Index e EEEIEE A OESE EAEE 117 119 NET Framework J J ersero 20 96 well plates cccccccccccssssssseeeeeees 29 39 79 A B SUG Cfo sinccesaasunsalonsnncedsedoascavectesdscesens 202 204 A B Slider Bar cccccccceeccescccecceescesceesceee
127. e 1 Biocision BCS 106 CoolSink 96F for 96 well flat bottom plates Extracellular Matrix used in the Invasion Assay must be ordered separately CellPlayer Angiogenesis Software Application Module 4411 IncuCyte Fluorescence Calibration Standards Kit 4380 4363 43381 4376 4371 4384 4443 1221 0239 1221 0240 1221 0241 1221 0242 IncuCyte Tray 7 Tray which holds up to one T225 tissue culture flask 1221 0243 IncuCyte Tray 9 Tray which holds up to two 100mm Petri Dish tissue culture dishes 1221 0244 IncuCyte Tray 10 Tray which holds up to two T225 Large plate tissue culture flasks or 1221 0245 large plates IncuCyte Tray 11 Tray which holds up to three Large Petri tissue culture flasks 1221 0246 IncuCyte Tray 12 Tray which holds up to three T25 tissue culture flasks 1221 0247 1221 0248 IncuCyte Tray 14 Tray which holds up to two T75 tissue culture flasks 1221 0249 244 Page IncuCyte Tray 15 Tray which holds up to two T75 tissue culture flasks 1221 0250 IncuCyte Tray 17 Tray which holds up to two T75 tissue culture flasks 1221 0340 IncuCyte Tray 18 Assembly Tray which holds 3 ibidi slides and contains features to HRP 5025 0169 restrain ibidi apparatus tubing IncuCyte Tray 19 Tray which holds up to four 60mm dish tissue culture dishes 1221 0347 IncuCyte Tray 20 Tray which holds up to four T25 tissue culture flasks 1221 0348 IncuCyte Tra
128. e Sales information is included at the end of this manual Section 13 page 244 7 1 6 ImageLock Mode FLR With an FLR instrument simply select Fluorescence amp Phase Contrast ImageLock from the Scan Type pull down menu displayed in Figure 7 4 7 2 View Completed Scans After scans have been generated select the View Completed Scans task bar to view the results See Figure 7 5 The times and dates for all completed scans are displayed in the Time Tree Select a specific time to display the scan for that time The time and date of the scan currently displayed is shown at the top of the screen 02 26 08 6 00am for Figure 7 5 Highlight a specific vessel for a closer look by selecting the vessel with the mouse left click once The selected vessel will be highlighted in yellow and the details of the vessel will be displayed in the Selected Vessel box to the right of the trays These details include e Image All images obtained during the scan can be viewed under the Image tab e Properties Selecting the Properties tab will display all the Properties entered when the scan was set including any Notes Additional Comments can be added at this time 43 Page e Metrics Selecting the Metrics tab displays the quantitative results calculated from each image of each well or sector of the selected vessel These values will be used to generate any graphical representations of the data 7 2 1 Vessel View A closer look at
129. e try using the Smooth Feature page 197 Graph Drag and Drop Auto Alignment When graphs are dragged to create a plot overlay they can be set to automatically align at a Preferences Cutler Removal i Aggressive To Microsoft Word Outlook or Publisher To Microsoft PowerPoint Excel or WordPad Graph Drag and Drop Suto Align at Value when Drag and Dop occurs Figure 8 138 The Preferences Window specific point on the Y axis Select the Drag and Drop Auto Align at Y Value when Drag and Drop occurs check box in the Preferences Window Figure 8 138 Now look at Figure 8 139 In the left graph Graph Drag and Drop Auto Align was NOT checked In the right graph the Auto Align box WAS checked and Auto Alignment was set to a Y value of 22 185 Page F r re ee DT pa a F J Coico bariw DT hipa Te Py See 2 deere Set fe Eee pE deere Se Overlay without Drag and Drop Alignment Overlay with Drag and Drop Alignment Fink oa Fei Fit oa Fai F eb i a Figure 8 139 Auto Alignment during Drag and Drop The Drag and Drop Alignment selection Yes or No chosen via the Preferences window will apply to all graph overlays but the numerical value at which alignment occurs must be set using the graph window See Figure 8 140 Select the Auto Alignment tab at the bottom of the graph window and choose the value at which alignment will occur The default value is 22 as displayed in Figure
130. e 8 88 Tasks Pane Right or left click on the far left border of the Vessel View window to open the Tasks Pane The Utilities visible within the Tasks Pane can also be accessed using the Utilities pull down window at the top of the Vessel View window Most of these utilities will be described elsewhere e View Large Current Image Increase the current image to full screen size e Export Current Image See Section 8 7 4 page 136 e Export Movie or Image Set See Section 8 7 4 page 136 e Archive Current Vessel See Section 8 6 7 Archive Vessels page 114 1383 Page Time Tree To the left of the selected vessel there will be a box displaying scan times The blue bar above the box displays the instrument number IC 10094 in Figure 8 89 and the User name MeMe in Figure 8 89 or the folder to which data have been archived to be discussed later By selecting a time the corresponding image will be displayed The Vessel View Time Tree displays ONLY the scans that occurred for the selected vessel 110094 Meme 2006 January 17 Thu 4 30 PM T o0 PM 10 30 PM B 18 Fri 1 30 AM 4 30 AM iad AM 10 30 AM 1 30 PM 4 30 PM Trag PM 10 30 PM Figure 8 89 Time Tree User MeMe Instrument IC10094 Selected time 10 30pm Selected Vessel The selected flask dish or microplate is displayed to the right of the Time Tree Use the Selected Vessel to view the images in different sectors wells by selecting the
131. e Contrast using Current Display Settings JPEG e Phase Contrast Original JPEG e Phase Contrast Original 8 bit TIFF Enter the desired name into the Name Prefix box The prefix will be automatically assigned additional time and area information that will appear in the Example box below Select Export The Export button will display the total number of images files to be exported Time Range Image Export Time Range Image Export is similar to Single Time Image Export and a review of the Single Image export instructions will be useful Select Time Range instead of Single Time in the drop down menu adjacent to the Time Tree Use the mouse or the arrow buttons to select the scan times for export All images including and between the selected times will be exported It is NOT possible to select non consecutive times for export Select the desired wells sectors and images Use the Browse button to choose the destination file Choose Sequence Type Set of Individual Images Select the desired Image Type Enter a Name Prefix Click the Export button Export Image Set Multiple Images with Customization Following the same setup guidelines discussed in the previous section Export Image Set Multiple Images it is possible to customize the set of exported images by customizing the image cropping and scaling and or adding legend and timestamp information to each image To accomplish this the Sequence Type must be set to Image se
132. e Upcoming scans task bar is absent This is because scans in the EX must be scheduled through the automated cell handling system The Selected Vessel Box contains two extra tabs Vessel and Scan in addition to Image Properties and Metrics Also the vessel barcode is displayed directly below the image of the Vessel In Figure 7 7 the vessel barcode is CCF7234523HZ 46 Page Selected Vessel COPY s4o25H2 12345 67 6 9 101112 1314 15 16 45 9 Confluence v1 2 zem rmnm m fs Image Properties Metrics Yessel Scan Seeding Date g March 2008 lt Not Currently Set gt EE lt Not Currently Set gt SS lt lt Mok Currently Sek gt po lt lt Nok Currently Sebo gt Figure 7 7 The Selected Vessel Box in EX The properties within the Vessel and Scan tabs can be viewed locally but they can only be populated remotely by the automated cell handling device Other features of the View Completed Scans Screen are unchanged except that you can search vessels by Barcode For more information on this subject see Section 8 9 page 164 7 3 Find Scanned Vessels Select the Find Scanned Vessels task bar to open the corresponding screen Use this screen to search for previously scanned vessels Note that in the Find Scanned Vessels screen the Time Tree ONLY displays scan times for the selected vessel In contrast the View Completed Scans screen displays ALL scans that have occurred on the instrument
133. e appropriate voltage for your local power Also ensure the local line voltage falls within the range printed on the IncuCyte Controller e NEVER open the IncuCyte controller It does not contain any parts that need to be maintained repaired or changed by the User e Ifthe IncuCyte is not used in the manner as described in this manual provided protections may be impaired Your IncuCyte is of rugged construction but it is still a precision instrument If you treat it with the appropriate care it will reward you with many years of trouble free operation Should you have problems that require service please contact Essen BioScience Inc 300 West Morgan Road Ann Arbor Michigan 48108 USA tel 1 734 769 1600 8 Page fax 1 734 769 7295 sales essenbio com www essenbioscience com 2 3 Unpacking and Checking the IncuCyte The IncuCyte System is shipped in two boxes one containing the automated microscope and the other containing the controller NOTE The IncuCyte consists of 2 components the controller and the microscope The controller remains outside the cell culture incubator while the microscope is placed inside the incubator and holds the cells for monitoring These two components are shipped in separate boxes Remember the IncuCyte EX microscope is NOT designed for use in a cell culture incubator To unpack the microscope start by setting the box with the correct side up on the floor
134. e center of each well Figure 8 35 Scan Pattern Manager for 1C10104 Tray Microplates v Vessel 24 well Essen ImageLock v Available Patterns Sample Pattern i or system operation and may not be edited Figure 8 35 Setting Scan Patterns in ImageLock Plates In the Sample Pattern there are three images per well and all three images are arranged in a straight line along the center of each well 8 5 11 ImageLock Mode FLR It is possible to perform fluorescence scans in ImageLock mode If you have an FLR instrument select Fluorescence amp Phase Contrast ImageLock from the Type pull down menu 8 5 12 Reload Pressing the Reload button at the bottom of the Schedule Upcoming Scans screen will reset all settings physical layout properties scan times etc back to the way they were prior to the last time the Apply button had been selected NOTE When the Schedule Upcoming Scans window is open but idle you are not actively scheduling a scan the window will refresh every two minutes This way if someone applies a schedule change from a different PC the revised schedule will automatically appear on your screen FLR 8 5 13 Layouts Sometimes the same Layout i e vessel type s scan pattern s etc will be used on a regular basis It would be convenient to save this Layout so that the vessels and scan patterns do not have to be set up every single time the configuration is required
135. e in Fred s password once and then confirm Use the Permission Level pull down menu to select Fred s Permission Level User Select the Create User button A window will appear indicating that a new User has been created 96 Page Current Users Create New User UserID Fred Password started Repeat Password ea dasa Permission Level User Delete User Figure 8 48 New User Created NOTE New Users and password changes are effective immediately on creation Delete User Highlight a User name from the Current Users and press Delete User NOTE Unless the Administrator level functions are required it is recommended that Users log in at the User level because the IncuCyte response time will be faster at this level If administrator level manipulations are required log out and then log back on at the Admin level 8 6 4 Scan Patterns This tab is ONLY available on the IncuCyte EX To set Scan Patterns on the EX see Section 8 5 4 page 70 8 6 5 Tests NOTE The Tests tab is ONLY available at the Administrator level The Tests tab includes several different tests related to proper IncuCyte function However the only test that will be relevant to the User under normal circumstances is the Calibration Device test Motion Calibration Before the IncuCyte can be used for the first time the physical locations of the trays must be calibrated within the instrument A special Calibra
136. e test again If the test fails repeatedly then contact Essen BioScience The Results of the Calibration and or Confirm tests can be viewed under the Logs tab Select Logs and then use the pull down menu under Log Type to select Diagnostic The calibration results will also be visible under the Log Type category All The results will be displayed in the large window below If no results are visible press the Refresh button at the bottom of the screen to update the log The results will be displayed as coordinate values These values will not be important to the User under normal circumstances At the end of the coordinate tables the Log will indicate whether the test Passed or Failed Figure 8 51 Device Accounts Tests Update Scans Logs Log Types Entry Types VIEWS Filter l gt gt End O1f08 08 17 08 19 1 6 2008 5 00 06 PM gt gt gt Rear Tray Passed Cal Confirm lt lt lt gt gt Shark 01 08 08 16 43 12 Rear Cal Coord sys info x off 13 37 mm Figure 8 51 View the calibration results under the Logs tab Log type Diagnostic All three tray positions must be individually calibrated When one tray position has completed the test s move the Calibration Tray to the next position and repeat When all positions have been calibrated the instrument will be ready to use If the results of the calibration are not displayed press the Refresh button at the bottom of the screen to bring all the logs up to d
137. each channel can be independently adjusted However the images from the two channels can be overlayed using the A B Slider Bar to form a single composite image See The A B Slider page 204 Fluorescence Settings The Fluorescence settings allow the User to adjust the DISPLAY characteristics of fluorescence components within an image Adjusting the display settings will affect which components of an image will be displayed as fluorescent and also the brightness of those fluorescent images relative to each other It will NOT affect the absolute calibrated fluorescence values Calibrated fluorescence values are measured in terms of pixel brightness using Arbitrary Units AU s Figure 9 2 shows the case of Channel A and B both set to Fluorescence The mouse over Pixel Intensity for fluorescence image NEVER CHANGES The Pixel report will change based upon whether or not an image has been processed Pixel 326 279 Pixel Intensity 106 4 AU Figure 9 2 View Pixel Intensity and Coordinates Selecting a Minimum and Maximum Intensity basically clips the brightness at which fluorescent components will be displayed In Figure 9 1 the Maximum Intensity is set to 104 3 Nonetheless it is possible that the image will still contain areas with calibrated pixel values greater than 104 3 However these areas will be assigned the SAME degree of brightness in the display as an area with a pixel value equal to 104 3 Thus all areas
138. eckbox to apply the Filter Settings to any graphed or exported data Uncheck the box to graph export the data with no filter settings applied See Section 9 3 8 Filter Settings page 224 for information on Filter Settings It is important to remember that in Analysis Preview Mode all analysis settings are applied ONLY to the currently selected image Selecting a different image or the same image at a different scan time will cause you to exit Preview Mode 212 Page In Figure 9 6 the Object Segmentation Mask is being applied to a cell line which expresses GPF in the nucleus so each Object represents a nucleus If the goal is to launch an analysis that will count all the nuclei in the image then the Segmentation and Refinement Parameters should be adjusted such that the Segmentation Mask identifies the fluorescent nuclei visible in the fluorescence image as accurately as possible both in terms of size and number The degree to which the two images overlap can be evaluated using the A B Slider Bar In Figure 9 6 Channel A is set to Fluorescence and Channel B is set to Object Segmentation Mask Because the Slider Bar is pulled all the way to the right the image displays 100 Segmentation Mask However if the Slider Bar is pulled to the left the image blend will shift to include more of the Fluorescence Image Use this image overlay function to evaluate how accurately the Segmentation Mask is identifying the fluorescent nuclei in the
139. ected grouping will remain so after addition of a well item Deleting Editing Well Items from Lists Once the three lists are populated the User may edit or delete items from the lists Editing an item will open the same window as when adding an item to a list only the fields in the window will already contain the information for the selected item Edit the desired fields Short Name Description Color and hit OK to save the changes To delete an item from a list simply click on the item hit Delete and OK Note that this action does not delete any instances of the item already added to the plate map Editing Well Contents Once a well or group of wells are populated the User can edit its contents and appearance To edit well contents first select the desired wells by left clicking over them then right click within the selected region to bring up the editing window From here the User can create a custom description of the selected 1389 Page N ESSEN BIOSCIENCE well s delete items and change the color scheme of the well s Alternatively use the Undo and Redo buttons in the menu bar at the top left of the window if a mistake is made while editing a well s contents In addition text size font style can be changed using the font button in the menu bar Creating and Using Regions Regions on a plate map allow the User to define a specific set of wells e g a dilution series which aids
140. ed Note that the IncuCyte EX automatically opens to the Find Scanned Vessels screen The View Completed Scans screen is still accessible in the EX but it is not the default setting 56 Page Me ESSEN BIOSCIENCE File view Scan Preferences Help 1210094 adminy 7 Next Scan 2 hours 36 minutes 5 08 PM Search Scans By Label Task List Selected Vessel Middle Tray Left Position ro imik Ly an A mj 16 we J sila z i i ae r q E k iei im See iss ey aye a E k faan DOCE e 00000 oe E 1 CERI FE OII IOL He Mi rey eclelia 9666606 5 a eeee00 im i is im dih E TIILI SEEE 80000 oo D T T tei i T a ie 3 woon GL L EE SPOSSSSCSCCSCCS 00000000G Hm ime a a a a Boo ete nr Coming 96 38 4 Confluence w13 w EDE k a as epiki ejim oe COE hoe oe oe oe oe a ad Coa Ta mn ie ih oon i ee A i Completed Scans E January 09 ted fe 10 iTho 11 Fri 1 Sat 13 Sun 14 Mon 15 Tue 16 Wed 17 Thu E 26 1 Confluence v13 l 18 Fri _ Image Properties Metrics CUA ce A H G G E G Q a fpiscecese see cee ses coe i REHEREHEREHRHH 10 30 PM f PEHA RHENE C3 Image 1 of 4 o E tl 19 Saath gt Coa are me oe Oe Ceee eee CeCe Hl 20 Sun sel it Coming 384 O New BD 384 i T 4 5 Conflue
141. ed censier aN 8 22 201 Histogram ccceeesseeseeeeeeeeeees 51 171 232 233 TAS EE E 43 127 130 167 mage LOCK ocsi 42 43 73 85 86 ImageLock Mode FLR cc ccceeeeeeeeeeeees 86 25 7 Page N IncuCyte EX 7 13 22 27 28 36 46 48 49 56 60 71 75 89 90 97 102 106 129 134 136 156 164 MnicU yte TAD wssasssssceansesaciesnscossasaasassensonds 7 39 AS AE EEE EOE EEES 257 TG CSG EE 147 149 IP 17 18 19 23 DO ECE 0G ssayarceicesgs a cenneesee EA 223 Job Processor WindOW ccccceesceecccesceeeseeeees 218 Join an Anal ySis cccccccccccsssssssssseeeeeeeeees 218 219 NOM c A 219 IPE oa 142 144 Tomp O SCAM essi i 155 Me ETE EEE E A AEE IT 73 Launch Analysis Button cccccceeeeeeeeeees 210 DSA OU eec 39 61 70 73 86 87 MAY OG sates ts ers ha ecera soda usitigaweboet ie 70 86 Load VNC CNS asics cas ea tin ee a 107 Manipulation of Archived Data 114 117 155 Manual Adjustment cccscceeeeeeeeeeeeeees 209 Manual Alignment cceeeeeee 53 192 194 Maximum Intensity cccccseeeeeeeees 202 203 Mean Intensity siicvccsccatasccacencteseeissnsssveecnes 2254 231 Measurement Mode cccceeceeceeeees 135 136 WISTS as daccacsicaxorsnvescectnaveces 44 81 130 136 166 INCL OT AUE 3 soci sierapbedeeueascvtecnenweseestataarenassenies 62 67 Microplate Graph c seeeeeeees 168 199 200 Microplate Graphi
142. ed under the Logs tab Log Type Diagnostic The log will state whether or not the calibration passed If the calibration failed the log will include recommendations to avoid failure the next time through Optics Test The Optics Test is a trouble shooting function that would be requested by an Essen BioScience employee It tests the quality of the camera image In order to run the Optics Test the front position of the IncuCyte drawer MUST be COMPLETELY empty no tray Running the Optics Test To run an optics test follow the steps indicated below Press the Go button in the Optics Test Section This will open the Optics Test window Figure 8 57 The dates and times of previously run optics tests will appear in the top left portion of the window The image result for the highlighted selected test will be displayed to the right of the dates and times and the written results for the highlighted test will be displayed below the image If no optics tests have been performed on the instrument the Current Test image will contain the words No Image Found 104 Page BIOSCIENCE N ESSEN Most recently completed test appears at the top of the list highlighted in blue Dates and times of In this case it is also the previous Optics Tests currently selected test Select a date to view Run Opt Tet the results for that test Image of selected Optics Test test report Test run from C2 29 08 125500 to
143. ed vessel to the IncuCyte microscope and then select Generic within the software A generic vessel image will appear in the tray The IncuCyte will attempt to obtain the best images possible from the generic vessel however because the IncuCyte is programmed to optimize imaging according to the measurements of each specific vessel type it is possible that the images obtained through the Generic option might be of a slightly lower quality Selecting Scan Type For some vessels or with the IncuCyte FLR the User may choose a specific scan type e Phase Contrast ImageLock Mode Essen ImageLock plates can be scanned in IlmageLock Mode See Section 8 5 10 page 85 e Fluorescence amp Phase Contrast Mode Vessels can be scanned in Fluorescence and Phase Contrast Mode with the IncuCyte FLR See Section 8 5 2 page 66 e Fluorescence and Phase Contrast ImageLock Mode ImageLock plates can be scanned in Fluorescence amp Phase Contrast ImageLock Mode with the IncuCyte FLR See Section 8 5 11 lmageLock Mode FLR page 86 65 Page N FLR 8 5 2 Setting Vessel Properties FLR With the IncuCyte FLR the default scan type is Fluorescence amp Phase Contrast The User may also select to scan a vessel in Phase Contrast mode only e Fluorescence amp Phase Contrast If Fluorescence amp Phase Contrast is selected the vessel will be scanned both fluorescently and in HD mode Fast Fluorescence allows
144. ees 202 AC EE E E 8 11 13 ACCOUNTS ccccesccscovscecccecescescansceseecs 21 22 95 Ada pPUVE dressi inna a 209 Adaptive Threshold sssssseeeceeeeeeeeeees 209 Administer cecceeceeceeeees 22 60 87 95 120 Alignment Wi1ndOWS ssssssseeeeeeeeees 27 28 Always Autoscale cccssssssssssseeeeeeeeeeeeeeees 203 Analysis Filter Settings 212 214 215 Analysis Filter Settings Pane 212 214 215 Analysis Job 205 208 210 211 215 218 Analysis Job Errors ssssseeeeeeeeeeeeeeeees 221 Analysis JODS cccccccceeseee 205 206 207 208 Analysis Parameters ssssceeeeeeeeeeeeeees 208 Analysis Preview Mode 211 212 214 215 ANGIOGENESIS ccceeeeeeeeeeeeeeeeeeeeeees 152 204 207 Arbitrary Units cccccssssssessseseeeeeeeeeeeeeeaas 202 archive 57 109 111 114 115 116 117 118 119 Archive Scans 006 109 110 111 112 113 Archive Vessel ccccccecceecessessevsees 115 154 Archive Vessels uiers noii 114 Are DIVINE sess soscgcnspesaiedndanidntesnateeatirs 107 112 113 Ve stces seventies 18 19 62 145 198 224 225 226 Auto Alignment e 189 190 191 192 Auto Alignment ccccccccssssssesseseeeeeeeeees 52 191 Average graph se ncusteecansateacgneuapsseanseessenasteetonnsdetie 53 Average Lines Together c0000008 53 196 ANT psclo
145. eetannictacdsoninteesscacamnnencosnen 36 73 74 password 5 17 22 23 60 95 96 97 117 Permission Level cecceceeeceeceeceeees 22 87 96 PAN EL a PENENT OE E 229 plate 16 25 52 62 73 79 183 194 237 238 Plate Map 74 136 137 140 141 171 200 PNG OVC AY onc itersncugapecnon ae teaglinsep hlaneravecennpe tebe 52 Preferences 54 55 156 158 184 185 186 189 Preview 175 208 210 211 212 214 215 222 224 226 Properties 18 19 36 39 43 73 129 136 VUE Ke CSU san sos AEE AEN T 30 99 Radio BU OM cccdicccsbecenesds pevecaestecvandvdevsessecdee 18 19 Rate of Change ixccceriunciselesncsiniventiacwceieien 196 197 Refinement 207 208 209 210 211 213 215 217 218 Renea enan i 31 99 120 BR OAC EE nee ne soso A A A 72 86 Restant IDC VC Ce araea ENE 94 Restore from Archive c ceeeeeeeeee 114 154 Save Analysis Parameters Button 210 scan 35 36 38 39 42 43 49 59 67 68 69 70 76 77 79 80 81 86 111 112 113 121 122 124 125 127 154 195 235 Sean BAP eee ee Ta 38 77 79 Scan on Demand 40 41 42 83 84 85 Scan PUC erenn i 36 67 68 70 BS AE ete cugeaeoeie 152 Sector Shading sesonieccasvnccentiavivad svalenzearnbes 125 126 e E eS 53 68 127 180 194 Segmentation 205 207 208 209 Segmentation Mask 205 212 213 214 215 Seguente Types 147 148 149 SHOW LOT
146. en navigate to the destination software Then choose Paste The data should appear in the previously selected format 174 Page Export Data to One or More Files The data can be exported to a single file by selecting the radio button for All Scans in One File For example suppose a 96 well plate was scanned at 10 different time points If the data are exported to a single file when the file is opened it will contain the data for all 10 scan times Alternatively the data from each time point each scan can be saved to a different file In this case select Each Scan in a Separate File NOTE The option of saving Each Scan in a Separate File will only be available if the radio button for Show each Scan as its Own Table is selected in the Layout Section of the Export Metrics window Continuing with the same example as above a 96 well plate that was scanned at 10 time points will generate 10 individual files and each file will contain the data for a single time point To save scans to separate files browse to a destination file and then enter a file prefix The Preview box will be populated automatically as the File Prefix is entered Press the Export button Whether the data are exported in a single or multi file format the file s will be saved in text format with a txt extension Open the data as a text file or open the file in an alternate software program and then save to that program as desired Other Options Two
147. ence image to appear overlain on the Phase Contrast image With the slider all the way toward the Phase Contrast channel any areas of the Fluorescence image brighter than the Maximum Intensity setting will remain in the image 9 3 IncuCyte FLR Specific Software Applications There are some image analysis applications that are unique to the IncuCyte FLR and they can only be accessed through the FLR Vessel View window These applications are The Object Counting Application The Angiogenesis Application The Object Counting Application is a standard component of the IncuCyte FLR software However the Angiogenesis Application is an independent software module that must be purchased separately An overview of both applications will be provided in this manual The circumstances under which these two applications would be applied are very different however both of them operate from the same basic platform In this regard they share many common elements An overview of these common elements will be provided first followed by some basic definitions specific to each application For more in depth information please refer to the corresponding application notes or contact Essen BioScience for assistance Some important definitions are included below 204 Page Analysis Job Quantitative image analysis using an FLR software image analysis application Segmentation A fundamental basis for FLR image analysis is the separatio
148. er bag lt lt 250 Page p 24 Well Plates Brand Catalog s Plate Name Tray Type Microplate Microplate Microplate Microplate Collagen 24 well Multiwell Plates Microplate Fibronectin 24 well Multiwell Plates Microplate Microplate I Taleo Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Essen 4365 24 well Essen ImageLock Plate Microplate Microplate Greiner Microplate Microplate ma Soaic 92024 24 Well TPP Individually wrapped Microplate 92424 24 Well TPP 4 per bag 48 Well Plates Corning 3524 Costar 24 Well TC Treated Microplates Standard Clear Plate Microplate lt Brand Catalog s Plate Name Tray Type 353078 48 well Flat Bottom Standard Tissue Culture Treated 353230 48well Flat Bottom Standard Tissue Culture Treated BD Falcon 96 Well Plates Brand Catalog s Plate Name Tray Type HD 353075 TCT 96 well Plate All Clear Microplate V 353220 __ Black Wall Clear Bottom Optilux Microplate V 353872 _ TCT96 well Plate All Clear Primaria Microplate V BD Falcon 353916 TCT 96 well Plate All Clear Microplate V 353986 _ TCT 96 well Plate All Clear Ready Stack Microplate V 353947 White Walll Clear Bottom Optilux Microplate V 251 Page D 96 Well Plates Brand BD Falcon Corning Essen Greiner Iwaki Matrical Catalog s 354410
149. er customization of the export image is available by using the Customize with image designer tool This button launches a new window that allows the user to customize the cropping and zoom level as well as to add a legend and timestamp if desired With the legend option a scale bar and field of view dimensions will appear in the exported image as they do in the Vessel View Figure 8 97 shows a Vessel View including both the Export Current Image and Export Designer windows Open Export Designer window by clicking the Customize with image designer button in the Export Current Image window Here the full size image is cropped as it appears in the Vessel View window whether viewed full size or zZoomed in The cropping boundaries can also be changed in the current window by clicking on and dragging the red boundary lines within the preview window Similarly the cropped rectangle can be moved by clicking and dragging within the current boundaries The final size of the cropped image in pixels is displayed below the Scaling control Use this control to adjust the size and resolution of the final image As the scaling increases the final size increases and vice versa This allows you to customize the final image size which can be very convenient when exporting data for presentation formats By increasing the scaling you can digitally zoom in ona small cropped area NOTE The sizes of both the scale bar an
150. ermined file sizes Print and Print Preview and Drag and Drop functionality have also been added to the Microplate Graphing option 8 11 The IncuCyte Self Test The IncuCyte will perform a device Self Test every 5 hours if it is not in regular use Therefore if insufficient scanning is taking place or if scans are scheduled at an interval of greater than 5 hours Self Tests will occur If a Self Test is ongoing the status of the test will be displayed in red at the top left of the main window and the green Scan Active light on the front panel of the IncuCyte panel will be lit It is important to note that the Self Test is completely normal behavior and it does NOT indicate that there is any problem with IncuCyte function While the Self Test cannot be disabled it can be terminated by either e Starting a scan during the Self Test or e Pressing the red Stop button on the front panel of the IncuCyte lt IncuCyte File 5 Preferences Help 1210094 admin Rs Status Self Test 76 8 163 Device Self Test 200 Page N FLR 9 IncuCyte FLR The IncuCyte FLR is an HD enabled instrument that is also equipped with fluorescence scanning capabilities When vessels are scanned in Fluorescence amp Phase Contrast mode both an HD and fluorescence image will be generated Alternatively the IncuCyte FLR can also scan images in HD mode alone i e non fluorescently 9 1 The IncuCyte FLR Vessel View Window
151. erties Metrics B1 Image 1 of 1 lt H Graph Export Figure 8 84 Flask area is divided into Sectors The selected well or sector will also display a window indicating the corresponding Metric in this case confluence Note Sectors are only used in flasks and dishes NOT in microplates OOODQODOQOQOOO OOQOOOQOOOOO L 01010101010101010101010 Image Properties Metrics E9 Image 1 of 1 eB Graph Expart Vessel View Figure 8 85 Microplate area is divided into Wells 128 Page Properties Tab The Properties tab displays the properties associated with the selected vessel Properties include Scan Type This will usually be standard See Section 8 5 5 page 73 Label Cell Type Passage Number Vessel Type User Name This refers to the user who set the scan Add Notes Add any relevant Notes Notes This is an inactive window that displays Notes that have been added in the Note that have been previously added cannot be edited Some vessel Properties can be edited These include the vessel Label Cell Types and Passage number New Notes can be added but previously added Notes cannot be edited To edit properties enter the new information and press the Update button at the bottom of the window Properties Tab EX The vessel Properties can be viewed but not populated or updated through the IncuCyte EX software These boxes are remotely populated by the automated cell handler The vessel Type Labe
152. es tab Vessel View Properties tab e The User may open the Plate Map Editor independently of the main IncuCyte software by browsing for the IncuCyte GUI program group via the Windows Start menu in the lower left of a Windows desktop With the Plate Map Editor open the default screen is set to a blank 96 well plate layout Figure 8 92 The Plate Map Editor To change the plate layout click on the New Plate Map blank sheet icon on the menu bar at the top left of the window The Plate Map Editor supports plate layouts from 6 well to 384 well formats To load a plate map already saved to a file click on the Open folder icon on the menu bar and browse to the file Undo Redo 2 d New Save Open f ef bel D i se the tabs below to populat Right click a selection to edit well contents Compounds Cells Growth Conditions Regions l User Selected Wells Compounds used in this plate Well Item Definitions New X Edit X Delete Select all wells that contain this compound Well Item Options Add Compound fo Plate gt Plate Map Notes Plate Map Design Area Export Plate Map Figure 8 92 The Plate Map Editor Selecting Wells To add well items cells compounds efc to a well or grouping of wells the desired area must first be selected on the plate layout Left click on a well or click and drag over a group of wells to select a specific area of the plate in which t
153. estored Delete Vessel Delete the selected vessel See Delete Vessels page 116 Jump to Start Select Jump to Start to jump to the very first scan of the selected vessel This is a very useful option Change Start What if you forget to check the New box when you introduce a new vessel into the scan schedule Use the Change Start option to reset the first scan time 12 15 AM 1 45 AM 3 15 4M 4 45 AM 6 15 AM 7 45 AM 3 15 4M 10 45 AM 12 15 PM 1 45 PM 3 15 PM 4 50 PM 6 06 PM 13 Frit 11 37 AM 11 39 4M 11 45 AM 1 45 PM 1 51 PM 6 00 PM 14 Sak 15 Sun 12 00 AM 6 00 AM 10 47 AM 6 00 PM 1 Select Start and End Time Select a Time A006 Select Stark Time January 13 Fri Select New Vessel Start lx Starting At 1 13 2006 1 51 00 PM 1713 2008 1 51 00 PH 1413 2006 1 45 00 PM 14 Sat 15 Sun 16 Mon 12 00 AM 6 00 AM C Select End Time 10 47 OM 1 16 2006 10 59 AM 11 51 00 4M 1473 2006 6 00 00 PM 1774 2006 12 00 00 4M 1 14 2006 6 00 00 AM 1 14 2006 6 00 00 PM 1 15 2006 12 00 00 AM Figure 8 104 Changing the Start Time Completed Scans are visible to the far left 155 Page To change the start time of a vessel follow these steps Select the desired vessel in the View Completed Scans screen Then select Change Start from the Scans pull down menu The Select New Vessel Start window will open This window will list all scan times for t
154. ete Figure 8 94 Creating a custom Region To create a graph of or export data from a saved Region go to the Graph Export window from the View Completed Scans screen or the Vessel View window and 140 Page find the Region in the Regions drop down list Regions created from the Plate Map Editor will be highlighted in blue Select Replicates from the Group drop down list to view each group of replicates e g a specific drug concentration as its own plot on the graph or to arrange exported data into replicates Printing a Plate Map At any time during the creation of a plate map the User may print a hard copy of the map to put in a lab notebook or to use while in the lab for example The Print and Print Preview buttons are both found in the menu bar at the top left of the Plate Map Editor In addition to the plate map the hard copy will also include any notes written in the Plate Map Notes section below the Well Item lists in the Plate Map Editor Exporting an image of the Plate Map In addition to simple print functions Users can also export Plate Map images using the Export Plate Map button at the bottom left hand corner of the editor This brings up a selection menu where the user can select from 4 different file formats PNG TIFF JPEG BMP and 5 different predetermined sizes 8 7 6 Export Images and Movies Images can be exported either individually or in bulk Movies can be genera
155. f grey the higher the Metric value The View Completed Scans screen with active Sector Shading is displayed in Figure 8 82 IncuCyte Bf File View Scan Preferences Help ic10094 admin i Ready To Scan Search Scans By Label sv for TUE tt 1 15 2008 8 30 00 PM orita cans o coming 25 ea Selected Vessel F 32 9 Confluence w13 i tal Find Scanned Vessels 2 Z an ac A O l ia Tii iaw G Schedule Upcoming Scans a Administer IncuCyte _ _ 86 6 Confluence v13 Completed Scans January 09 Wed fH 10 Thu gt Se Image i i E 11 Fri gt gt z ge Properties Il Metrics H 12 Sat z g es 13 Sun 14 Mon 15 Tue 2 30 4M s 5 30 4M _ Coming 75 8 30 AM 73 6 Confluence v13 11 30 4M 2 30 PM 5 30 PM 11 30 PM E 16 Wed SA ENN ai Ze W 17 Thu B2 Image 1 of 4 1 F 19 Sat 20 Sun v tH 18 Fri 50 E o o M ii HARAN Figure 8 82 View Completed Scans Screen with Sector Shading selected Because the selected Metric is confluence darker shading represents higher confluence 126 Page e Error Bars are discussed in Section 8 10 5 page 187 e Condensed Metrics are discussed in Section 8 7 2 page 131 lf not all available spaces in the tray were occupied then the spaces will contain no images of a vessel and they will read Empty or Deferred Figure 8 83 e Empty No vessel is set to scan in this positio
156. followed by the yellow Scan on Demand Time bar Similar to the 24 Hour Repeating schedule the width of the time bar will 40 Page correlate to the amount of time required to scan the vessel inserted into the Scan on Demand Tray Scan on Demand allows users to scan only one vessel in one tray position at a time The Tray Type Tray Position Cutout Position Vessel Type and Scan Type must be entered into the scheduler prior to scanning In addition to a Label users can also supply a Unique ID to each vessel This Unique ID can either be generated manually by typing or pasting information into the Unique ID field or a Unique ID correlating to the current year day time will be assigned to the Scan on Demand vessel This information can be used to link subsequent scans together However it is important to note that if the user fails to type in or select using the load button the correct Unique ID scans will not be linked together and cannot be accomplished post scanning Clearly additional scheduling rules apply to the Scan on Demand scheduler in order to protect regularly scheduled scans from being skipped These rules include e The Scan on Demand Time bar is typically yellow If the Scan on Demand Time bar is Red this means that the Scan on Demand scan time overlaps with a scan time in the 24 Hour Repeating Scheduler Clicking the Red scan button will result in an error e The estimated time indicated by the width of the yellow bar
157. for archived data Double Click on a Row To Select the Scan Search Search By w for Oo eS Time of Scan Label Cell Type Passage Type User Name Tray Position Cutout Position Vessel Type When was the vessel scanned Don t Remember Within Last Week Past Month Past Year Between Figure 8 103 The Search Window 153 Page Select the Options button to narrow the time frame of the search If you know your scan occurred very recently select the radio button for Within Last Week If you don t recall when the scan took place select Don t Remember Or if you Know the scans occurred within a specific time frame use the Between option to narrow your search to within specific dates You CANNOT search within a single day Finally the search function is not case sensitive Show Log Selecting Show Log will bring up a new window displaying the Device log entries for that day ONLY First Scan The First Scan option brings up the View Completed Scans screen for the very first scan recorded in the IncuCyte Previous Scan This selection brings up the View Completed Scans screen for the scan immediately previous to the one currently being displayed Next Scan Selecting Next Scan brings up the View Completed Scans screen for the scan immediately following the one being currently displayed End Scan End Scan brings up the very last scan recorded in the IncuCyte View Vessel
158. for faster imaging and analysis by creating smaller images with no loss in sensitivity and is recommended for most applications In the Properties tab select the Fast Fluorescence checkbox See below for more information e Phase Contrast lf Phase Contrast is selected the vessel will only be scanned in HD mode Fast Fluorescence Users are encouraged to use the Fast Fluorescence FF acquisition mode because it provides some very important performance advantages Perhaps the most important attribute of FF is the fact that the overall acquisition time is approximately 50 faster This leads to smaller temperature rises for the same amount of vessel coverage Another important advantage to using FF is the fact that the amount of time a specimen is illuminated in fluorescence is reduced by almost a factor of three For specimens and assays sensitive to photobleaching this is a huge reduction over a standard fluorescence experiment Finally FF images are nearly four times smaller than standard resulting in faster image load times exports and archives Processing of fluorescence analysis jobs is also nearly four times faster than standard fluorescence analyses And because FF images are smaller the IncuCyte controller disk usage is also reduced dramatically In order to maintain the same sensitivity as standard fluorescence images the tradeoff that FF makes is in the area of image resolution FF images have less Spatial resolution than stand
159. g Open Ended Jobs is to select the job in the Open and Manage Jobs window and then press the Delete button See Figure 9 8 All processing will cease and the Analysis Job will no longer appear in the window If any Processing Windows were open at the time the analysis was terminated they will indicate that the job was completed Close these leftover windows 220 Page Exiting vs Terminating Ongoing Analysis Jobs Exit an ongoing Analysis Job by closing any open Processor Windows Exiting an ongoing analysis will suspend processing by the PC however processing will continue on the IncuCyte controller The Job will remain in the Manage Jobs Window with a construction icon indicating that the Job is still incomplete Terminate an ongoing Analysis Job by pressing the Delete button below the Open and Manage Jobs Task Bar Pressing the Delete button will terminate all processing on all PCs and also on the controller The Analysis Job will be removed from the Manage Jobs window Analysis Job Errors lf an Analysis Job was completed with errors then the name of Job will appear under the Analysis Jobs Open and Manage Task bar with an icon indicating that error s occurred during the processing Information regarding the nature of the processing errors can be viewed by pressing the Details button at the bottom of the window See Figure 9 9 Jobs that were completed with errors can still be opened but keep in mind that the job conta
160. g a complete Analysis Job can require a significant amount of time Additionally once an Analysis Job has been completed it is NOT possible to adjust any of the Segmentation or Refinement settings Therefore it is best to use the Preview function to optimize all Segmentation and Refinement Parameters prior to running an analysis of a full length experiment IMPORTANT It is NOT possible to adjust the Segmentation or Refinement Parameters of a completed Analysis Job To launch a preview select a representative image and then press the Preview Current Image button See Figure 9 6 page 214 Pressing the Preview button puts you in Analysis Preview Mode Note that several things will happen immediately upon pressing the Preview button The Preview Current Image button is renamed to Update Analysis Preview 211 Page The Banner at the top of the Vessel View window changes from blue to beige and it reads Analysis Preview The Analysis Filter Settings Pane at the bottom of the Vessel View Window is activated Object Segmentation Mask becomes one of the options in the A and B Channel pull down menus Holding the mouse over Objects within the image has the potential to display additional information that was not available in unprocessed images See Object Properties Definitions page 224 There are 4 ways to exit Preview mode Navigate to a different image or to the same image at a different scan time Even though all scans associate
161. garding the Smooth feature 7 4 4 Saving and Printing Graphs Graphs can be saved or printed using the File pull down menu at the top of the plot If a graph has been created and saved within the IncuCyte software it can ONLY be opened again through the IncuCyte software using this menu selection If the graph file is selected by another method for example just locating the file on the hard drive and clicking on it it will not open If however a graph is created and then saved into another software program for example MS Word it can be opened directly through that program In this case however it will be a static image that cannot be manipulated 55 Page e ESSEN BIOSCIENCE 8 Software Reference The software automatically opens to the View Completed Scans window under the Task List bar The first time the software is opened the screen will be empty Figure 8 1 However once scans have been performed the software will open to show the very last scan that was completed D0 Fie View Scan Preferences Help ic10094 admin x Ready To Scan Search Scans By Label gt for GO This device does not contain any scans Task List tA Find Scanned Vessels G Schedule Upcoming Scans a Administer IncuCyte Completed Scans Image Properties Metrics 41 Image 1 of 4 ee ep Graph Export Vessel View Figure 8 1 View Completed Scans Screen with no scans complet
162. gs 228 Page Saving Analysis Filter Settings The User may define Filter Settings that will be applicable to multiple experiments To save Filter Settings press the Save Filter button and give the settings a name The saved settings will now be available under the Object Counting Analysis Filter pull down menu Saved Filter Settings can be deleted or renamed using the Manage Analysis Filters option also available under the Object Counting Analysis Filter pull down menu Users are encouraged to save all Analysis Filter Settings that they anticipate applying on a regular basis Every time a new Analysis Job is launched any saved Analysis Filter Settings will automatically be processed with the new job Obviously all possible saved Filter Settings will not automatically be applied when the job is opened however if any saved Filter Settings are subsequently selected in the Object Counting Analysis Filter pull down menu then all images included in the job will already have been post processed with the selected Filter Settings Thus application of the settings will require no additional processing and the entire data set can be more easily and rapidly manipulated Export and graphing functions will also be expedited Pin to Job When saved Analysis Filter Settings are opened the Save Filter button becomes the Pin to Job button Pinning expresses a preference for a particular Filter Setting for a particular Analysis Job If Filter
163. he Edit Scan Patterns button is visible in Figure 8 5 This will bring up the Scan Pattern Manager window Figure 8 11 The Edit Scan Patterns button can also be selected when no vessels are highlighted but in that case the Scan Pattern Manager window will contain a random vessel that is not necessarily the one desired New Tray Types and Vessels can be selected from within the Scan Pattern Manager window Scan Pattern Manager for IC10104 v i 6 well Corning v Available Patterns Sample Pattern Pattern Editing This pattern is required for system operation and may not be edited Images per well Figure 8 11 Scan Pattern Manager Window Create a New Scan Pattern The Scan Pattern Manager window is shown in Figure 8 11 A large view of the selected vessel will be displayed with the scan pattern indicated as green dots The selected tray is indicated in the Tray menu box and the selected vessel name is displayed in the Vessel menu box The manager window will automatically open to show any vessel that was previously highlighted in the tray However a different vessel can also be selected using the Tray and Vessel pull down menus 67 Page The first time the manager window is opened it will display the Sample pattern The Sample pattern CANNOT be deleted or renamed In this case it is the only pattern available so the Rename Delete and Clear Pattern buttons are inactive as in Figure 8 11 To
164. he Time Zones have not yet been synchronized It is VERY IMPORTANT to proceed with the Time Zone synchronization regardless Set Device Time Local Time 2 28 2008 3 28 13 PM Central Standard Time Daylight Savings ICi l 4 time 2i28i2008 3 28 17 PM Eastern Standard Time Daylight Savings Figure 8 42 Time Zones not Synchronized IMPORTANT Even if the local computer and device times appear to be in agreement prior to Time Zone synchronization it is still ESSENTIAL to proceed with the synchronization process Press the Synchronization button to align the disparate time zones A new window will appear Figure 8 43 Hit OK to proceed with the synchronization 92 Page Synchronize Time Zone You are about to synchronize the controller Time Zone The connection will be closed You can reconnect in about 1 minute You Will be prompted when reconnecting iF you need Synchronize time Figure 8 43 Synchronize Time Zone The connection will be terminated and the device will synchronize the Time Zone You will be able to reconnect in about one minute and the IncuCyte will be properly synchronized with the local computer Daylight Savings Time Please note that the Daylights Savings Time DST check box in the IncuCyte software is never active DST cannot be adjusted through the IncuCyte software It must be selected on your local computer and the IncuCyte settings can be adjusted accordingly
165. he controller operating system as little as possible is the best way to guarantee stability of the software that controls the IncuCyte The easiest method for accessing the controller desktop is through the Windows Remote Desktop Connection tool Windows XP Users can access this utility through the Windows Start menu under the Programs gt Accessories gt Communications menu Users of Windows Vista will find the utility in the All Programs Accessories menu For other Windows operating systems this tool is available as a free download from Microsoft at the following URL http Awww microsoft com windowsxp downloads tools rdclientdl mspx Run the Remote Desktop Connection software and type in the name of the IncuCyte controller in the Computer edit box If connecting for the very first time the name used will be the factory set name Make sure that you have connected and verified that the controller is on the network see Sections 4 1 1 page 16 and 4 1 2 page 17 Press the Connect button and wait for a Windows login prompt It may take as long as 15 seconds before the prompt appears Log into the controller with a User name Administrator and the Essen provided password The controller operating system should contain the necessary components required to rename the computer configure the primary Ethernet port install security software etc Please consult with your IT department for answers to que
166. he right In Figure 8 125 the data are displayed as a single row In this figure Region All Wells Group None were selected prior to opening the Export Metrics window Note that wells from which no data were obtained are omitted Experiment Corning 364 Date Time Elapsed Corning 384 A1l Corning 384 E1 1 17 2008 16 30 4 28 9 21 1 17 2008 19 30 3 03 10 4 1 17 2008 22 30 6 5 7 12 6 1 18 2008 1 30 8 18 15 3 Figure 8 125 Data as a Single Row Show each Scan as its own Table Alternatively the data can be displayed as a single table one table for each scan time This option will only be available if Group None was selected in the Graph Export setup prior to pressing the Export button If each scan is displayed as its own table the columns labels can be displayed or not by checking the corresponding check box If data are exported in the form of a table then wells or sectors from which no data were obtained because they were skipped in the scan pattern will appear as blank spaces See the section about Other Options page 175 for more information on this topic Destination Choose a destination for the exported data There are two possible destinations for exported data e Export to Clipboard e Export to one or more files Export to Clipboard Choose export to Clipboard to export the data directly to a new location such as EXCEL PowerPoint or MS Word Simply select export to Clipboard press the Export button and th
167. he selected vessel plus those of the same vessel type in that same position immediately before Look at Figure 8 104 A new start time is being set for that vessel The current first scan time start time for that vessel is January 13th 1 51pm This is the first time listed in the graphing Time Tree the Select Start and End Time window to the far right and it is highlighted in the Select New Vessel Start window to the immediate left However scans were obtained for the same vessel type immediately before 1 51pm e g 1 45pm These scans are visible in the Completed Scans Time Tree to the left and also in the Select New Vessel Start pull down menu To change the first start time to 1 45pm use the mouse to select 1 45pm in the Select New Vessel Start window Select OK The start time for that vessel will be changed to 1 45pm Open the graphing Time Tree for the vessel The first time listed will be 1 45pm Change Start EX The Change Start function is NOT available for the IncuCyte EX 8 7 10 The Preferences Window Select Preferences from the top of the View Completed Scans screen lt IncuCyte Fie view Scan Preferences Help 10094 radmin 7 Next Scan 1 ho Task List i View Completed Scans Figure 8 105 Select Preferences from the top of the screen next to Help Selecting preferences will open the Preferences Window Only the Image Drag and Drop option will be discussed here For information on Outlier
168. hemselves If there are any questions about this filter please contact Essen BioScience for more information Figure 3 2 IncuCyte Front Panel The Power Supply Filter is located behind the front panel on the side indicated by the arrow 15 Page N 4 Establishing Network Connections The IncuCyte controller houses an embedded control computer that must be connected to your network in the same way that ordinary PC s running Microsoft Windows are connected We recommend that you consult with your IT department during the network connection configuration and verification processes A standard Ethernet cable is used to connect the controller to a network hub or switch The unit is equipped with two network ports The leftmost port is used as the primary network interface to connect the IncuCyte controller to the network The jack on the right is an auxiliary port for servicing the controller See Figure 3 1 for details The controller is configured at the factory for automatic assignment of its IP address on a network using DHCP Dynamic Host Configuration Protocol Follow the procedures described in Section 4 1 DHCP Based Networks to connect to this type of network If the network is not configured to assign IP addresses dynamically then a static IP address must be assigned to the controller See Section 4 2 page 17 Static IP Based Networks to reconfigure the IncuCyte controller for a fixed IP network 4 1 DH
169. hree hours results in a duty cycle of 22 2 40 minutes 180 minutes Under these conditions any local temperature rises will typically be less than 1 degree C and relatively short lived The IncuCyte is capable of collecting nearly 1500 images in 40 minutes so another good reason to avoid such over scanning is to limit disk usage and processing time This will keep hard drives from filling up unnecessarily and keep the system more responsive We have found that between 0 5 and 1 image per square centimeter is usually sufficient to characterize the growth of cells in most tissue culture vessels For example 48 images will usually characterize a T75 flask very well With good technique that results in uniform seeding even fewer images can be used Also with some slower growing cell types scanning every 4 or even 6 hours may be sufficient After connecting to your IncuCyte press the Schedule Upcoming Scans task bar The first time this screen is opened it will be empty Figure 8 5 60 Page IncuCyte File View Scan ic10094 admin IEA pi View Completed Scans Find Scanned Vessels Schedule Upcoming Scans Preferences Help v Status Processing 77 12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm 12am Physical Layout Properties Rear Tray A Administer IncuCyte Tray Type None x Vessel Type None x m be Scan Type Scan Pattem Ne
170. ible for the User to assign a custom Label If the Label box is not populated the default Label will simply read Custom Region accompanied by the Statistic To save a custom region for later use click the Save button to the right of the Label box The custom region will then be saved under the Region pull down menu for the current vessel type To delete a saved custom region select it from the Region pull down menu and click the X next to the Region box The selected region will then appear as a Custom Region giving the option to edit it or permanently delete it by choosing a different saved region Regions from a Plate Map lf a plate map is associated with a vessel microplates only any Region created in the Plate Map Editor will be available for graphing To create a plate map or assign regions see Section 8 7 5 page 136 Histograms Currently tt only possible to create Histograms when graphing Object Counting data and the Histogram option is disabled in Figure 8 123 See Graph Export 171 Page N Histograms of Processed Jobs page 232 for information on graphing histograms However it is possible that the Histogram function will be expanded in the future 8 10 3 Prepare to Graph FLR It is ONLY possible to graph fluorescent Metrics from the Vessel View window Non fluorescent Metrics e g confluence can be graphed using the Graphing Box in the View Completed Scans screen FLR Some additional Metrics are avai
171. ibrate the tray in the designated position and follow up with a test to confirm that the calibration is correct If the calibration tray is absent or was not placed into the appropriate tray the process will abort Confirm Only Quick Test Figure 8 50 Motion Calibration with Calibrate and Confirm and Quick Test selected for the Front Tray Position Confirm Only If the instrument has been previously calibrated but there is some question as to whether the calibration is still accurate calibration can be confirmed using Confirm Only This single test requires less time than the Calibration and Confirm tests together and it is useful for obtaining a rapid update on calibration status 98 Page N e Quick Test In most cases the Quick Test will be used for both the Calibrate and Confirm or just the Confirm tests The Quick Test is not a separate kind of test but refers to the length detail with which the Calibrate Confirm tests are performed The Quick Test check box should be checked along with the selected test type Figure 8 50 If the Quick Test box is not checked the Calibration Confirm tests will require significantly more time If the Quick Test fails repeating the test with the Quick Test check box unchecked will not increase the chances that the test will succeed However it will provide more information that can be used to trouble shoot the source of the problem Generally if the Quick Test fails simply try running th
172. iliary controller port A 2 device network is established between a PC and the controller via either a Direct Connection using a crossover Ethernet cable or b connection through a hub or switch using standard cables With both devices powered down connect the PC and the controller in one of the two configurations shown using the auxiliary network interface on controller see After connecting power up the PC and the controller The key to establishing a simple 2 device network lies with the correct configuration of the PC s Ethernet port Like the auxiliary port on the controller the PC Ethernet port must be configured with a static IP address The steps for establishing and verifying this network are explained in Section 4 2 1 for Windows 4 2 1 Verifying the Direct Network Connection using Windows Vista or Windows 7 1 2 3 Open the Control Panel under the Windows Start menu Select Network and Internet then Network Sharing Center at the top of the page In the left pane of the window select Manage network connections under the Tasks List Vista or Change adapter setting Windows 7 in the upper portion of the pane Identify the network connection used in the 2 device network It will most likely be labeled Local Area Connection but could have a different name Right click the connection and select Properties from the menu In the new window you will find a box with a list
173. ill abort the scan that is ongoing at that time However if scanning is terminated in this way the IncuCyte will start scanning again at the next scheduled scan time The ONLY way to terminate all future scans is by deleting all the Scan Bars and then selecting Apply IMPORTANT 1 The scanning process causes the IncuCyte to become warm In order to prevent overheating it is recommended that total scan time not exceed 45 minutes 2 Scan times CANNOT overlap If attempting to set scan times that overlap the IncuCyte will generate an error message 3 The Apply button MUST be selected before new parameters will be saved and scanning will be initiated Further reading on this topic is recommended See Section 8 5 7 page 76 39 Page A 7 1 4 ESSEN BIOSCIENCE Scan on Demand In addition to the 24 Hour Repeating scan schedule users also have the ability to scan in the Scan on Demand mode Simplistically this means that the IncuCyte can be used to scan individual vessels at times when the IncuCyte is not scanning This includes both periods in between regularly scheduled scans in the 24 Hour Repeating scheduler as well as in designated Cooling Times To access the Scan on Demand scheduler click on the vertical tab within the Schedule Upcoming Scans screen labeled Scan on Demand However all changes to the 24 Hour Repeating Schedule must either be applied to the IncuCyte by clicking the red
174. image For a closer inspection use the Zoom slider To determine the best Analysis Parameters that will mostly accurately reflect all images within the experiment it is best to preview multiple images that represent different experimental conditions and or different time points 213 Page Z Vessel View 5 19 PM on 10 30 2008 Tasks File Utilities view Utilities Analysis Preview FLR30001 admin x 5 2008 October Export movie or image set 30 Thu Archive current vessel TTT 11 19 PM 31 Fri Analysis Jobs Open and Manage November view large current image Export current image Object Counting New Analysis Image Properties Graph Export Params Manual Selection Fluorescence v Segmentation Adaptive v1 0 Minimum Intensity 61 C Sackom aa Maximum Intensity 945 v Foreground Intensity Manual Adjustment ii Auto Scale Always Refinement Edge Split Ka f FB Edge Sensitivity n Object Segmentation Mask v 7 fe Update analysis preview Use Filter Color LJ Launch new analysis E Show Object Centers ad Save analysis parameters Angiogenesis New Analysis E vV ee e oe 7 gt i A Pee Show Legend i i eS 400 o gt o 1 90 x 1 5 mm 2 90 rge ah Analysis Filter Settings Figure 9 6 The Vessel View Window in Preview Mode The Banner at the top of the Vessel View Window is beige and it reads Analysis Preview The Analy
175. ime Tree has been processed according to the selected Segmentation and Refinement Parameters Therefore the User is free to jump between different images and different scan times and in all cases the images will be fully processed e The Vessel Image at the top of the Vessel View window will only indicate a scan pattern for those wells sectors that were included in the analysis Wells sectors that were not included in the analysis cannot be selected e Time plots can be created and data for all images and time points can be exported e The Open and Delete buttons become inactive It is not possible to delete a job that is currently open View Analysis Jobs To view a history of all analysis jobs run on a specific IncuCyte click on the Scan drop down menu at the top of the main user interface window and choose View Analysis Jobs A new window will appear showing all analysis jobs and related information date created date completed label cell type etc about each job Figure 9 10 Double click on a job to bring up that analysis 222 Page ESSEN BIOSCIENCE N _ Analysis Jobs mat hater t WA atic r Name Cre Created 20090630 A1 A2 analysis 20090630 analysis 20090630 analysis a 6 30 20 20090701 whole plate analy a 7 1 200 20090701 whole plate morla 17200 20090701 Mon 6 29amto a 7 1 200 a a a 6 30 20 Completed Label 20090612 Co cutture 6 30 20 a UEA
176. ins errors Mame Facltasel Hidoxyurea a Errors Example View Edit Analysis Object Counting Summary Name Errors Example Created 3 2 2009 11 01 4M by admir Finished 3 2 2009 11 07 AM with errors Analysis Parameters Segmentation Adaptive Beta Refin Background Intensity 200 AO Edgy Object Counting Analysis admin Foreground Intensity 60 0 AL Completed with errors Manual Adjustment 0 0 AL Processing Errors Image not found 10 30 2008 5 19 PM Processing failed 10 30 2008 5 19 PM Figure 9 9 Analysis Job with Errors 221 Page N 9 3 6 Open and Manage Analysis Jobs Completed Analysis Jobs will appear in the window under the Analysis Jobs Open and Manage Tasks Bars To open a job press the Open button at the bottom of the window Note that only completed Analysis Jobs can be opened Ongoing jobs can only be joined Open an Analysis Job Pressing the Open button will load the job Opening a completed Analysis Job will resemble launching an Analysis Preview In both cases the Object Segmentation Mask will become available under the A and B Channel Settings and Object Properties can be displayed Additionally the Analysis Filter Settings Tasks Pane will become active and can be opened However the completed Analysis Job is different in several respects e A green banner appears at the top of the Vessel View window and it displays the job name e Every image of every scan listed in the T
177. is Jobs Additional image types will be available for images that have been processed See Section 9 3 IncuCyte FLR Specific Software Applications page 204 These image types can only be exported from open Analysis Jobs Object Counting Object Counting Using Current Display Settings 8 bit TIFF Object Counting Segmentation Mask 8 bit TIFF 151 Page Object Counting Labeled Mask 16 bit lossless TIFF Note that lossless TIFF is a less commonly used format Angiogenesis Angiogenesis Using Current Display Settings 8 bit TIFF Angiogenesis Tube Segmentation Mask 8 bit TIFF Angiogenesis Tube Skeleton Mask 8 bit TIFF Angiogenesis Labeled Mask 16 bit lossless TIFF Export Movies The Sequence Types available for exporting fluorescent movies are the same as those for non fluorescent movies One thing to keep in mind when generating fluorescent movies is that the fluorescence intensity of the objects and or background can change over time If the User does not want these changes to be reflected in the movie s then the Auto Scale Always check box should be selected prior to exporting fluorescent movies See Always Autoscale page 203 After selecting the appropriate Sequence Type select the Image Type Fewer image types are available for movie export than for image export and the descriptions are fairly self explanatory Some additional image types are available for processed Analysis Jobs 8 7 8 Search Comple
178. isting archive files To add additional archive data to an existing file 116 Page FLR simply browse to the desired existing iaf destination file when selecting the archive destination and select Save A window will appear indicating that the selected file will be Appended To Appending a file will NOT result in any previously existing data being overwritten lt is NOT possible to append to a vessel archive Manipulation of Archived Data Archived scans can be opened and manipulated to the same extent as scans saved on the controller However manipulations of archived data will be slower than that of data remaining on the controller hard drive especially if the archive is large Thus while regular archiving is essential for long term IncuCyte use it is recommended that scans remain on the controller as long they are being actively manipulated To open archived data see Open an Archive under Section 8 6 7 page 117 Manipulation of Archived Data FLR Fluorescent scans can be archived and manipulated but it is NOT possible to run Analysis Jobs from archives In order to run an Analysis Job from scans that have been archived the scans must first be restored using the Restore Archive function See Restore from Archive page 114 See Section 9 3 page 204 for information regarding FLR specific applications and analysis Although analysis jobs cannot be run from archived scans any analysis jobs that were completed prior to a
179. itate a direct comparison For example the same cells were plated in two separate flasks at the same starting confluence at the same time How does their growth compare Unfortunately the mean growth curve of each flask must be generated separately creating two separate graphs However it would be optimal if both traces could be combined onto a single graph How can this be accomplished The two graphs in question are displayed in Figure 8 134 and Figure 8 135 181 Page r d J g ESS N Senn wi A gt _ gt E Ta a rm CIENCI lt Graph Col 1 Mean vs Time DER File View Edit Flask 1 Flask 1 gt o E D oO D a han gt a o D oO 6 Thu 9 Sun Apr 2006 Calendar Mode Drag and Drop Manual Alignment Auto Alignment Right click and hold on this graph and drag onto another to create a graph overlay Figure 8 134 Graph for Flask 1 Graph Col 2 Mean ys Time DER File View Edit Flask 2 Flask 2 Confluence v1 1 Percent 6 Thu 9 Sun Apr 2006 Calendar Mode Drag and Drop Manual Alignment Auto Alignment Right click and hold on this graph and drag onto another to create a graph overlay Figure 8 135 Graph for Flask 2 As indicated in the Drag And Drop box at the bottom of the graph window simply right click and hold on the graph to be dragged and then use the mouse to drag it onto the target graph In this case the
180. l etc displayed in Figure 8 86 were filled in via the remote interface See Section 8 5 6 page 75 129 Page Selected Vessel July 22 1234567 8910111213141516 3 8 Confluence v1 3 rnm um pP Properties Metrics vessel Scan Type Standard Label My Label Cell Type CHO Passage vessel Type BO Falcon 175 User Mame Wlacdate Figure 8 86 Properties EX Metrics Tab The Metric that will be primarily used is Confluence The remaining Metrics Image Mean Focus Position and Exposure time are diagnostic tools used to confirm that the software is functioning within specified parameters These tools will not be relevant to the User under normal circumstances Whichever Metric is selected here will be displayed on the View Completed Scans screen NOTE Metrics other than Confluence are ONLY available at the Administrator permission level Definitions of the Metrics Spreadsheet e Sector or Well Denotes the sector or well to which the data in that column belong e Mean Displays the Mean values for all images in each well or sector e Median Displays the Median values for all images in each well or sector NOTE If there is only one image per Well or Sector then the Mean and Median values will be the same 130 Page N e Std Dev Denotes the global Standard Deviation of all sectors wells for that vessel e 1of _ Indicates the Metric value for each image of each sector or we
181. l be overlapped Figure 8 143 and Figure 8 144 Graph All Mean vs Time Pie Wew Ect Aversge Smooth Growth Over Time Flask1 Flask2 Figure 8 143 Graph All Mean vs Time File View Eck Average Smooth Growth Over Time Flask Flask2 z 8 Confluence v1 0 Percent 8 8 Figure 8 144 The same two graphs with Calendar Mode Unchecked 188 Page N Auto Alignment NOTE Also see Preferences Outlier Removal and Drag and Drop Auto Alignment page 184 At the bottom of the graph window find the Auto Alignment tab This tab can always be opened but the Auto Alignment function is only active when the Calendar Mode button is NOT checked In Figure 8 145 the auto alignment value is set to 14 Drag and Drop Manual Alignment Suto Alignment Use the Apply button to align the plots Use the Clear button to align the plots by their First point Figure 8 145 The Auto Alignment Tab when the graph contains a single trace Drag and Drop Manual Alignment Suto Alignment T TE Apply dear Use the Apply button to align the plots Use the Clear button to align the plots by their Apply First point Figure 8 146 The Auto Alignment Tab when the graph contains multiple traces When the IncuCyte software is first opened this value will be set to the default number 22 However if the value is changed from this default the new value will be displayed until it is changed again by the
182. l jumps between frames When very precise repeated imaging is required for instance when generating a movie use the Essen ImageLock plate By selecting an ImageLock plate and running it in ImageLock mode the highest fidelity movie will be generated lf an ImageLock plate is selected as the vessel type under the Physical Layout tab the Type box will be active Figure 8 34 While Phase Contrast ImageLock will be the default setting an ImageLock plate can be run in either Phase Contrast ImageLock or Phase Contrast mode Middle Tray Tray Type Microplates amp Vessel Type 96 well Essen imagelock ss Scan Pate Senge Paten New Select Al Select None Figure 8 34 Running a Plate in ImageLock Mode lf an ImageLock plate is run in standard Phase Contrast mode the result will be comparable to that of a normal non ImageLock plate NOTE It is recommended that ImageLock plates ONLY be run in ImageLock mode ImageLock Scan Length Because the scanning process is so precise in ImageLock mode the scan length will be longer than that for a plate run in Phase Contrast mode 85 Page ImageLock Scan Patterns The scan patterns available for the ImageLock 24 and 96 well plates differ from those available for standard 24 and 96 well plates A maximum of three images is possible per well and the scan pattern is arranged in a straight line through th
183. lable for fluorescent scans that are not available for non fluorescent scans Most FLR specific Metrics are only available for Job Previews or Completed Analysis Jobs further reading on this topic is recommended See Section 9 3 9 page 231 8 10 4 Export Metrics It is possible to export the Metrics associated with a particular vessel directly to another document or to a selected file There are a variety of options regarding the format in which the data are exported To export vessel Metrics select the Export button rather than the Graph button at the bottom of the graphing window Choosing the Export button will open a new window in which details of the selected export format can be chosen See Figure 8 124 The scans for which data will be available for export are selected using the Time Tree in the graphing box The default settings are the first and last scans available for the selected vessel If a vessel was scanned 20 times but only the data from the first 10 scans need to be exported then use the mouse or arrow buttons to select the desired Time Range If only the data from a single scan need to be exported choose Single Time instead of Time Range from the drop down menu above the Start Time radio button lf Time Range is selected then the data set that is exported will be a continuous set within the given time span It is NOT possible to simultaneously export data from individual temporally discontinuous scan times If discrete
184. le for multiple wells or sectors They are NOT available for multiple images within a single well or sector Two types of Error Bars are available Standard Error Standard Deviation Calendar Mode 12PM IPM SP hel 5 Wed JAM BAe 4 Tue Mar 2006 Time Calendar Mode 11 15 06 PM on Tuesday March 04 2008 6 46 Figure 8 141 With Calendar Mode Selected At the bottom right corner of the graph is the Calendar Mode check box If the Calendar Mode box is selected the x axis of the graph will be displayed as year month day and time Figure 8 141 lf Calendar Mode is NOT selected the x axis will be displayed in units of hours Figure 8 142 10 12 20 Ze Time Hours Calendar Mode Figure 8 142 Calendar Mode NOT Selected 187 Page When should Calendar Mode be used If multiple graphs were created over the same time period they can be dragged to create overlays in the Calendar Mode setting This overlay facilitates a comparison of cell growth in absolute time However what if the same experiment was performed on different days In this case if both graphs are dragged together and then displayed in Calendar Mode they will not overlap at all because they have no dates times in common To display these graphs properly overlapped view the graph overlay with the Calendar Mode button NOT checked Now both graphs will be displayed as confluence relative to hours irrespective of the specific days and times and they wil
185. leted Scans screen e Double Left click on a point from an IncuCyte graph 131 Page ESSEN BIOSCIENCE Tite Ve file tied Vaee Ae Ta Ed z 3 Trag and Orop Manual Algrmment Auto Algrmert Graph on Graph sy Right click and hold on this graph and drag onto another to Dee etree Lh zaj Figure 8 87 Double left click on one of the points on the IncuCyte graph to open the Vessel View window for that point 132 Page ESSEN BIOSCIENCE UN Grab outside border of the Vessel view window to resize the entire window Vessel View T00 PH on 2772009 Lbs ered lange Quire inna ya 2 Export Curent image me poper Expert marie or keyp pet 7 zii pi Sadinnuiiu Ardha punet weesel E 6 Fri Se ie OF Sat poe 770 AM K E i i f 7200 PA a HH on HE Click on the far left border of a rime LETTI ka the window to reveal the ie Ee Bz ingelih Utilitiesin the Tasks Pane eo Pripet Graph E pirt These same Utilities can be accessed by selecting the 39 Utilities pull down menu a I E stowkegend T ii i Grab the Separator Bar with the mouse and drag to change the ratio of the Upper to Lower panes Figure 8 88 The Vessel View Window A variety of tasks can be selected using the Utilities pull down menu or by clicking on the far left border of the Vessel View window to open the Tasks Pane See Figur
186. liated with those vessels are archived along with the vessel These jobs can be opened and manipulated as would be the case for a non archived vessel It is not exclusively available after a job has been opened The data for the archived Analysis Job will no longer take up valuable controller disk space All the same the data will still have to be stored in an alternate location 223 Page N 9 3 7 The Object Counting Analysis Filter Pull Down Menu The Analysis Filter pull down menu is similar to the Parameters pull down menu under the Object Counting New Analysis Task Bar The Parameters Pull Down Menu page 208 The Object Counting Analysis Filter pull down menu is displayed in Figure 9 11 lt is currently set to Manual Selection Analysis Filter Settings x EF Object Counting Analysis Filter Manual Selection wa la Save Filter i Area pr Eccentricity Mean intensity Summed intensity nic De One POS One GO Onn P hm Fs mae SO mae Ome S Figure 9 11 The Object Counting Analysis Pull Down Window Initially two options will be available under the Analysis Filter pull down menu Manual Selection and Unfiltered These two options will ALWAYS be available After one or more Filter Settings has been saved these alternate Filter Settings will also be available in the pull down menu Selecting saved Filter Settings will populate the corresponding Object Property value s and the settings will autom
187. ll e Min and Max Displays the minimum and maximum values for that vessel Condensed Metrics The Metrics can be viewed both in a full or a condensed format In the condensed format the individual image data are not displayed Toggle between the condensed and non condensed view format by selecting it from the View pull down menu at the top of the screen See Figure 8 81 page 126 Tasks Available by Right Clicking on a Selected Vessel Right click on a vessel in the View Completed Scans screen and a selection of Tasks related to that vessel will become available These Tasks include View Vessel Select this Task to open the Vessel View window Graph Export Graph Export data from the vessel See Section 8 10 page 165 and Section 8 10 4 page 172 Archive Vessel Archive the vessel See Archive Vessels page 114 Delete Vessel Delete the vessel See Delete Vessels page 116 8 7 3 Vessel View Overview The Vessel View offers expanded tools related to viewing and manipulation of images data There are 6 ways to open the Vessel View window e Press the View Vessel button under the Image Metrics Properties box Figure 8 82 e Double click on a vessel in the Find Scanned Vessels Screen e Double click on a vessel in the View Completed Scans Screen e Select a vessel in the View Completed Scans Screen and then choose Vessel View from the Scans pull down menu at the top of the screen e Right click on a vessel in the View Comp
188. ll require the entire IncuCyte drawer any vessels that have already been selected in regular sized trays will be eliminated if a triple wide tray is subsequently selected Under these circumstances a warning window will appear Click on Yes to continue with the selection of the triple wide tray or click No to cancel 63 Page ESSEN BIOSCIENCE UN Change Layout A Using this tray will remove vessels From other trays because of its size Would you like to continue Figure 8 8 The Triple Wide Tray Warning Window Adding Vessels to the Tray A tray type must be selected and then one or more vessel spaces within that tray type must be further selected in order for the Vessel Type menu box to become active See Figure 8 9 Rear Tray Tray Type Tray 1 Vessel Type fh Coming 25 A Sean Type Sala Scan Pattem EE Empty Figure 8 9 Vessel Type pull down menu The top left soace has been selected Selected vessel spaces are highlighted yellow The Vessel Type box is now active and the option for that space can be displayed Use the mouse to click on a vessel space to highlight select that space If the mouse is clicked outside of the highlighted space the space will become un highlighted The space can be selected again with the mouse Vessels can only be added to highlighted spaces Two or more spaces within a single tray can be simultaneously highlighted by clicking on one or more spaces holding down
189. lly adhering to the surface This problem is exacerbated by swirling the plates and or placing freshly plated cells directly into the incubator It is recommended that cells sit at room temperature until they are well enough seated to resist clumping Then put them into the incubator Bubble on the bottom of the well on the far left side of the image Cells appear rounded because they were just seeded and have not yet fully attached Bubbles on the bottom of the well are distinguishable from floating bubbles because the cells will be clustered around the outside of a bubble on the bottom Test compound has come out of solution and formed crystals in the media o d0 9 8 oW gt GO S569 o 9 r 9 9 236 Page A D IncuCyte photographed condensation present on the outside ESSEN BIOSCIENCE bottom of the plate instead of the cells Be sure to avoid condensation on the insides and outsides of all vessels prior to placing them into the IncuCyte See top right hand corner Bubbles on the surface of the media throw shadows onto the cells The cells in the left portion of the image are in focus while the cells in the right portion are not This is an indication that the flask is not seated flat in the tray 23 Page A A Striations are visible on the bottom of the well This is an artifact of the way the microplate was created and it is NOT related to IncuCy
190. location and then email it to Essen BioScience as requested Optics Test EX The Optics Test for the IncuCyte EX can be run as described for the Standard Model Optics Test with one exception because the EX has a single built in tray that cannot be removed run the test with the tray empty i e no vessel in the tray Camera Test The Camera Test is another trouble shooting function that would be requested by an Essen BioScience employee To run a camera test press the Run button in the Camera Test box A window will appear to confirm that the camera test has begun Hit OK The status of the camera test will be indicated at the top of the IncuCyte screen In Figure 8 58 the camera test is 4 complete File view Preferences Help 1210104 admin a Status Testing 4 Figure 8 58 Camera Test Status The results of the camera test can be viewed under the Logs tab in Log Types Diagnostic See Section 8 6 8 Diagnostic page 121 8 6 6 Update NOTES The Update tab is ONLY available at the Administrator level New Licenses are issued on a per instrument controller basis Therefore ifa new license is purchased it can only be used on the instrument onto which it was installed 106 Page From time to time various updates will become available to improve or expand upon IncuCyte function In order to perform any of these updates Essen BioScience will dispatch an email containing the attached update
191. ls see Section 5 3 2 page 21 Figure 8 46 shows the Accounts screen as seen at the Administrator level At the User level the Accounts screen is the same except that only the Change Password Section is active There are two major functions available on this screen Change Password To change a password type the current password and then the new password twice Click the Change Password button A window will appear confirming that the password has been changed NOTE The only password that can be changed is the password for the User who is currently logged on Even an admin level User CANNOT change the password of other Users Create User New Users can be created using the Create User function Figure 8 47 95 Page Device Accounts Tests Update Scans Logs Change Password Current Password New Password Repeat New Password Change Password Current Users Create New User User ID Password Repeat Password Permission Level v Delete User Figure 8 46 Accounts Tab at the Administrator Level Current Users Create Mew User Liser ID Fred Password serene o Repeat Password tween O Permission Level Delete ser Administrator Figure 8 47 Create New User Create a new User by following these steps 1 Type the new User s name in the User ID box in Figure 8 47 the new User s name is Fred Typ
192. luorescence Uncalibrated 16 bit lossless TIFF Fluorescence Calibrated 32 bit raw floating point values Phase Contrast using Current Display Settings JPEG Phase Contrast Original JPEG Phase Contrast 8 bit TIFF The two image types identified with an asterisk might not be as common as the other types but they should be interpreted by most image processing software packages Users who wish to export images for presentation applications such as MS Office should avoid the less common formats Two additional image formats are available that are exclusively compatible with MetaMorph software e MetaMorph Single Multi plane Meta Series TIFF e MetaMorph ND File Sequence One thing to keep in mind when generating a sequence of fluorescent images is that the fluorescence intensity of the objects and or background can change over time If the User does not want these changes to be reflected in the images then the Auto Scale Always check box should be selected prior to exporting a fluorescent image set See Always Autoscale page 203 To apply the Auto Scale Always settings to exported images the User MUST select either Blended Composite using CURRENT DISPLAY SETTINGS or Fluorescence using CURRENT DISPLAY SETTINGS NOTE When using Fast Fluorescence fluorescent images are smaller than normal half size in both dimensions and any exported images or movies will display the resulting resolution Export Images from Processed Analys
193. luorescent IncuCyte requires a special fluorescence calibration procedure See the Essen BioScience IncuCyte FLR Application Note for more information FLR 102 Page A calibration kit will be provided with the purchase of the IncuCyte FLR and the instrument will be calibrated at the time of installation It is currently recommended that the instrument be recalibrated approximately twice a year however if the User suspects a drift in the calibration signal then the system can re calibrated at any time The fluorescence calibration kit includes liquid sufficient for multiple calibrations as well as three calibration slides These calibration slides are disposable and should only be used once New calibration kits can be purchased from Essen BioScience See Section 13 IncuCyte Catalog page 244 Follow these steps to perform the fluorescence calibration Place a microslide tray in the front position of the IncuCyte Fluorescence calibration requires use of the front tray position and the remaining positions can be occupied with trays at the time of calibration Load a calibration slide with supplied dye Use 40 ul dye per slide Place the pipet tip at one end of the slide window and expel the liquid dye The liquid will wick into the slide window After all the dye has been expelled use the pipet to transfer a small amount of dye to the other end of the window to be sure it is completely filled Avoid getting any dr
194. ly plots can be dragged into a different software program The data can be dragged either in the form of the completed plot or in the form of raw data Sometimes for example it might be convenient to copy the raw data into EXCEL To do this simply right click on the plot to be dragged be sure that Drag and Drop Raw Data to Document The raw data will be copied into the new spreadsheet and can be manipulated within the EXCEL software See Figure 7 14 52 Page N Date Time 4 5 2006 15 45 4 5 2006 18 45 4 5 2006 21 45 4 6 2006 0 45 4 6 2006 3 45 4 6 2006 6 45 4 6 2006 9 45 4 6 2006 12 45 4 6 2006 15 45 4 6 2006 18 45 4 6 2006 21 45 4 7 2006 0 45 4 7 2006 3 45 4 7 2006 6 45 4 7 2006 9 45 A 7 2006 12 45 4 7 2006 18 45 The raw data can also be exported into EXCEL using the Export button See Section 8 10 3 page 172 for more information on exporting raw data Elapsed Column 1 Column 2 D m ww O l 21 24 EF 30 33 36 a9 42 o1 2 116157 2 073041 6 748407 7 93768 9 286036 10 79348 12 91238 15 41242 18 40523 22 04792 25 11257 30 45502 33 27099 40 49081 46 00566 51 601444 63 54694 3 62277 6 483157 7 917222 6 310006 9 822143 11 6282 13 6361 15 60134 19 5973 23 03909 28 80244 34 0718 39 10634 44 93753 30 74551 56 49158 69 3348 Confiuence v1 1 Percent Growth Over Time Column 1 Column Column3 F Column x ColumnS Columnb 100 7 Fri amp Sat 3 Su
195. ly be graphed exported from the Vessel View window Select the Graph Export tab in the Vessel View window and then press the Graph Export button For unprocessed vessels this Graph Export window will be the same as described for non fluorescent scans Section 8 10 2 page 170 except that one additional Metric will be available Fluorescence Image Mean The mean of all the Pixel Intensity values within a given area of a fluorescently scanned image 230 Page UN Time Plot ESSEN BIOSCIENCE Export of fluorescence scan graph data is the same as export of non fluorescent graph data Graph Export Time Plots of Processed Jobs After processing an Analysis Job new Metrics will be available for graphing and export See the Essen BioScience Object Counting Application Note for more information regarding these Metrics Open the Vessel View window and press the Graph Export button to open the corresponding window See Figure 9 16 Note that Custom Region is also available for graph export of processed Analysis Jobs Image Mean Confluence v1 5 in i j E z H i Focus Position o 7 20 PM l E 03 Sat a Exposure Time Fluorescence Mean E 04 Sun Autofocus Sharpness 4 05 Mon Avg Object Summed Intensity Avg Object Mean Intensity Avg Object Area Statistic Histogram Object Area Object Mean Intensity Region Custom Region Object Summed Intensity a ject Count pe
196. m gt fi o o a O Jan 2008 Calendar Mode Drag Drop and Alignment Settings Figure 8 131 Flask All Sectors The Group None option was selected so there is a separate trace for each sector Each trace represents the image mean for that sector Graph Customization Initial default labels along with other graph attributes can be personalized Left click twice on the graph background area and the Customization Window will appear Figure 8 132 This window can be used to set a variety of graph attributes including title subtitle axis font etc Select the various tabs to explore all available graph related features 179 Page All Mean vs Time Customization General Plot Subsets Axis Fort Color Style Main Tithe 4ll Mean ve Time Sub Title Border Stile Numeric Precision No Border Line n O1 O2 3 Shadow O 30 Inset Viewing Style Grid Lines Color Both OY Oxs O None O Monochrome C Grid in front of data Monochrome Symbols Font Size Lange Medium O Small Figure 8 132 Graph Customization Individual well sector names however CANNOT be changed using the Customization window To assign new labels to wells or sectors select the Edit option at the top left of the graph window This will open the Titles and Labels window Figure 8 133 This window provides an opportunity to change the Title and Subtitle as well as the means to assign ne
197. m in Section 8 5 7 81 Page Related Notices If adding removing a vessel or changing its scan pattern type changes the set vessel schedule a warning will appear either on the scan timeline overlapping scans or next to the Vessel Scheduling button If this warning appears go back to the Vessel Scheduling window to view the revised schedule and edit it if necessary The Vessel Scheduling warnings refer to changes from the last applied schedule not to any changes made after the Apply button was clicked Vessel scheduling may result in overlapping scans if the original scan interval is too short In this case change the length of the scan interval on the timeline in the Schedule Upcoming Scans screen SCHEDULING TIPS Adding cooling times During a cooling time IncuCyte is simply idle As such you may open the instrument add or remove vessels trays from the instrument feed cells etc while IncuCyte is cooling Viewing the vessel schedule For the best view of the entire schedule expand the Vessel Scheduling window horizontally This will expand the timeline boxes so more information about each vessel is visible NOTE Using the Auto Cool option is recommended and allows for the greatest amount of scan time possible 82 Page LN 8 5 9 24 Hour Repeating a a 3 o a L i Lie ESSEN BIOSCIENCE Scan on Demand In addition to the 24 Hour Repeating scan schedule users also have the
198. m with the mouse Selected Vessel EX In the case of the IncuCyte EX the barcode readout is displayed under the Selected Vessel instead of its position in the drawer In Figure 8 90 the barcode label is ACF7234523HZ The barcode will also be displayed in the EX Vessel View window 134 Page Selected Vessel OF Pe stoe SHS 12345 67 6 9 101112 1314 15 16 EE T E 45 6 Confluence v1 2 A i C E E F G H l d kE Figure 8 90 The Selected Vessel Barcode in EX Image Tab The image for the currently selected well or sector is displayed under the Image tab Use the brightness and contrast sliders to adjust image quality Double click on the Image in the Vessel View window use the Large Image Shortcut icon or select the Utility View Large Image to increase the currently displayed image to full screen size Figure 8 88 Zoom Use the Zoom Slider bar to zoom into and out of the image Dragging the slider bar will zoom to the center of the image however the area of the zoomed image being displayed can be changed by grabbing the image and dragging it with the mouse Alternatively it is possible to zoom directly to a specific area within the image by scrolling the mouse wheel over the area of interest Show Hide Legend Select the Show Legend box to display the size ruler at the bottom left hand corner of the image Uncheck the box to remove the ruler The legend can be viewed in the Vessel View window bu
199. macends ra E aaa Nai 17 4 2 1 Verifying the Direct Network Connection using Windows Vista or Windows 7 IS 4 2 2 Verifying the Direct Network Connection using Windows XP ccccccsseccccccceeeeneeeeseeeseeens 19 INSTALLATION OF THE INCUCYTE CONTROL SOFTWARE 20 5 1 INCUCYTE CONTROL SOFTWARE INSTALLATION USING WINDOWS XP 20 32 INCUCYTE CONTROL SOFTWARE INSTALLATION USING WINDOWS VISTA AND WINDOWS7 20 5 3 ONE ING sso perc E E oes eee eyo E A ceeteeene eects 21 5 3 1 Setting the Date and Time ccccccccccccssseeeccccceeeeeaeeesseeceeeeeaeeeeeeeeeeeeaaaeeeeeeeeeeeaaaaeeeeeeeeeaaaeees 21 5 3 2 Establishing User ACCOUNUS occcccccccsssscccccccccccseessccceeeenesseseeeceeesaaeseeeeeeeesaaaaeseeeeeeesaaaaaseeeess 21 5 4 REMOTE ADMINISTRATION OF THE CONTROLLER eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 23 PREPARING FOR CELL CULTURE MONITORING eeeeessseseccccssssscccccsssssceeesssse 25 6 1 SELECTING AND PLACING TRAYS AND VESSELS INTO THE INCUCYTE iiine 25 6 1 1 6 1 2 6 1 3 6 1 4 6 1 5 6 2 6 3 6 4 6 5 7 1 Tedd flee 7 1 3 7 1 4 Liked 7 1 6 fe Tae 7 3 7 3 1 7 4 7 4 1 7 4 2 7 4 3 7 4 4 8 1 8 2 8 3 8 4 8 5 6 5 1 6 5 2 6 5 3 8 5 4 8 5 5 8 5 6 8 5 7 8 5 8 6 5 9 6 5 10 6 5 11 6 5 12 6 5 13 8 6 8 6 1 8 6 2 8 6 3 8 6 4 8 6 5 8 6 6 SCLC LIONS ener ern RET Cree Perc ia i 2J Selecting Trays for the IncuCyte EX cc scscsasssatnssins
200. mage composite overlay of the two images from Channels A and B The User selects a Channel A image and then a Channel B image and then uses the A B slider to select the blending ratio When the slider is positioned at the far left the image displayed is 100 of Channel A and 0 of Channel B Analogously when the slider is at the far right 100 of Channel B is displayed A 50 50 blend is achieved when the slider is centered In addition to blending Fluorescence and Phase images together the Fluorescence Threshold Blending feature defines an intensity threshold for combining the Fluorescence image with the Phase Contrast image pixels above the threshold are taken from the Fluorescence image while pixels below the threshold are taken from the Phase Contrast image In this way fluorescent areas of an image show up brighter than when simply blended with the phase image while the Phase Contrast portion of the image retains its original brightness and contrast settings Initiate Fluorescence Threshold Blending either by left clicking on the A B slider icon or by selecting it in the View menu at the top of the Vessel View window Notice the A B slider now reads Threshold instead of Blend under the A B icon With the slider all the way toward the Fluorescence channel the Fluorescence image appears with the current Max Min settings Dragging the slider toward the Phase Contrast channel will allow only those brighter areas of the Fluoresc
201. menu item has a check mark next to it Type the following in the Address box at the top of the window ICXXXXX Click on the Go button to the right of the address bar If the controller is found on the network you will be prompted for a User name and password Press the Cancel button to exit If the controller is not found then a Cannot Find message will be displayed after a brief search period 4 2 Static IP Based Networks In order to reconfigure the IncuCyte controller for a static IP address a direct connection must first be made to the controller The connection is made to the auxiliary port network interface which ships configured with the following static IP address 192 168 128 130 To use this port to connect to the controller a single PC should be connected to the controller box This can be accomplished by either a direct connection between the auxiliary port and the PC using a crossover Ethernet cable or by connecting the PC and the controller to a network hub or switch using standard cables See Figure 4 1 There are two possible configurations of this 2 device network NOTE There must be only 2 devices on this simple network 17 Page i PC 192 168 128 1 fel PC 192 168 128 1 Crossover Cable lt Controller 192 168 128 130 Hub Switch Ca Standard Cables F Controller 192 168 128 130 a Direct Connection b Hub Switch Connection Figure 4 1 Connection to aux
202. meter set in the Job Range window In Figure 9 6 the parameters were entered under the Manual Selection option However if saved Parameters were being applied the name of the saved Parameters would be displayed here 21 Page lt is important to remember that the Segmentation and Refinement Analysis Parameters CANNOT be adjusted once an Analysis Job has been launched completed Launch an Analysis Job Press the OK button at the bottom of the Job Range window and the job will be launched Several things will happen when a new Analysis Job is launched The Job Name and Job Type will appear under the Analysis Jobs Open and Manage Task Bar An icon will appear to the left of the job name indicating that the job is still incomplete See the Example job in Figure 9 8 A new Job Processor window will open indicating the status of the analysis This window will indicate the number of images processed and the number of images remaining The IncuCyte controller will join the analysis when it is available for instance if it is not scanning When an Analysis Job is launched the computer used to launch the job will participate in processing the data However the corresponding IncuCyte controller can also participate in processing The Job Processor window reflects processing taking place on the local PC but there is no window that reflects the status of controller based processing Join an Analysis It is frequently possible to
203. n e Deferred A vessel is in this position but IncuCyte did not scan it at this time point Flask 1 CHO Passage 4 Flask 2 es CHO Passage T Figure 8 83 Empty flask spaces in an IncuCyte tray Note the scan pattern indicated by dots within the flask 8 7 2 Selected Vessel Next to the vessels is the Selected Vessel box Highlighting a particular vessel on the tray will result in a larger expanded version in this window If no vessel is selected the Selected Vessel box will be empty See Figure 8 82 There are three tabs at the bottom of the Selected Vessel Box e Image e Metrics e Properties Image Tab Large views of some selected vessels are included in Figure 8 84 and in Figure 8 85 In the large view the flask is divided up by squares into sectors Sectors with dots in them represent sectors that were scanned By highlighting sectors containing dots you can view the image s of the cells in that sector when the Image tab is selected as in Figure 8 84 In some cases multiple images have been obtained for each sector or well In that case each image within the sector or well can be viewed by using the Image 1 of __ buttons below the image In Figure 8 84 only one image was obtained per sector Image 1 of 1 so the scroll box is inactive The image can be viewed in a full screen version by double clicking on it with the mouse 127 Page N ESSEN BIOSCIENCE Image Prop
204. n Figure 7 14 Graph dragged into EXCEL as Raw Data left and as Image right Summary To drag one plot onto another within the IncuCyte graphing software select Drag and Drop as Graph on Graph 2 To drag the graph image into MS Word or MS Outlook select Drag and Drop as Image to Word or Outlook 3 To drag and drop the image into other software programs select Drag and Drop as Image to Other Document 4 To drag and drop other raw data select Drag and Drop Raw Data to Document 5 After a Plot Overlay has been created all dragged traces can be subsequently deleted using the Delete button under the Manual Alignment However the original trace s for the plot that the other traces were dragged onto CANNOT be deleted tab 7 4 2 Creating Average Graphs When multiple traces are included on a single plot either as a result of dragging or by graphing all wells sectors these traces can be combined to create a single Average Graph To do this select the Edit pull down menu from the top of the Graph window and then select Average Lines Together Select OK in the dialog box that appears and a new Average graph will be created in a new window Note that the Average Graph function does not generate a graph with 53 Page error bars See Section 8 10 5 Error Bars page 187 for information on error bars within a single vessel The Average graph function can be also used to combine graphs from different
205. n of fluorescent foreground and non fluorescent background objects This process is called Segmentation The User can adjust and optimize the parameters utilized to assign Segmentation to an image lt is important to choose Segmentation Parameters that most accurately represent those components of the image that will be subject to analysis Segmentation Mask Segmentation can be visualized in the form of a Segmentation Mask Areas of an image that have been identified as belonging to the foreground will be indicated with a white mask Areas that have been identified as belonging to the background will be black In Figure 9 3 a Segmentation Mask right has been generated from a raw fluorescent image left The Segmentation parameters have been adjusted such that the mask accurately represents the fluorescent components of the image Figure 9 3 Application of Segmentation Mask The raw image is on the left and the corresponding Segmentation Mask is on the right 9 3 1 The IncuCyte FLR Tasks Pane Open the Vessel View window and then open the Tasks Pane by clicking on the left edge of the window See Figure 9 4 Use the Tasks Pane to preview and run Analysis Jobs 9 3 2 Task Bars Available in the IncuCyte FLR Tasks Pane Four Task Bars are available in the FLR Tasks Pane 205 Page Utilities The Utilities Task Bar in the IncuCyte FLR Vessel View Tasks Pane is the same as in the non fluorescent IncuCyte See Ta
206. n settings prior to export The preview allows you to view a still movie frame at the exported size as well as in a full screen mode that fills your monitor This is often important when considering the size at which the final movie will be played Careful consideration should be given to the final size of each movie frame which will have a huge impact along with the number of frames on the ultimate size of the output movie file Once you are satisfied with the customization click OK to exit the Export Designer When all the parameters have been chosen press the Export button The Export button will indicate that number of movies files that will be exported under the selected conditions In Figure 8 101 only a single movie has been selected for export A new window will appear indicating the status of movie generation Multiple movies will be exported one immediately after the next until all movies have been exported and compressed FLR 8 7 7 Export Images and Movies FLR Export Images The steps required to select and export fluorescent images are the same as those required for non fluorescent images However some additional Image 150 Page Types are available in the case of fluorescently scanned vessels A Summary of all available image types is listed below Blended Composite Using Current Display Settings JPEG Fluorescence Using Current Display Settings JPEG Fluorescence Uncalibrated 16 bit grayscale source PNG F
207. nce wl 3 OO 23 5 Confluence w13 Figure 8 2 View Completed Scans Screen with scans completed 8 1 Connections Box The Connections box is located at the top left corner of Figure 8 2 Also see Figure 8 3 It shows the instrument name ICXXXXX and the User name or the name of the archive If the User is connected to only one instrument or archive file then selecting the Connections Box pull down menu will not reveal any additional options However it is possible to be logged onto multiple instruments archives simultaneously In Figure 8 3 the User MeMe is logged into one instrument 1010094 and two archive files X It is possible to toggle between these multiple connections by using the Connections Box pull down menu 5 7 Page IncuCyte File View Scan Preferences He 1210094 MeMe I 10034 Meme eo Larchive iaf 7 view Completed Scans Figure 8 3 The Connections Box Three connections are open one instrument l1C 10094 and two archives 8 2 Menu Bar File Use the File Menu to open and close a connection to an instrument or archive Section 8 1 open a previously saved graph and exit the software View Use the View menu to select what information is displayed for each vessel on the screen See Figure 8 81 Select the characteristics displayed under the View Completed Scans Screen Scan Use the Scan menu to search through scans and perform specific actions on a selected vessel includi
208. ncuCyte will warm slowly as a result of the scanning process and prolonged scan times can result in a temperature increase of the instrument and subsequently of the incubator Longer scan times can be set if the increase in temperature that might occur is not considered to be a critical factor Generally speaking larger incubators are more resistant to temperature changes than smaller ones but the cells within the instrument will become warmer either way To minimize temperature change use the Vessel Scheduling feature Section 8 5 8 96 well plates have an increased initial scan length 96 well plates have a particularly small imaging area and in order to generate a quality image the camera must be very precisely located above each well To ensure proper placement the very first scan of a 96 well plate will require extra time 50 total scan time to assure optimum well camera alignment Subsequent scans will NOT require extra time Whenever a scan is applied that contains 96 well plates a pop up window will appear reminding the User that the first scan will be longer by __ minutes the exact number of minutes depending on the specific scan pattern In order for the scan to proceed the OK button must be selected confirming that the User is aware of the increased scan time This longer initial scan length ONLY occurs with 96 well plates Note that the increased initial Scan Time for 96 well plates does NOT apply to the IncuCyte HD
209. ng 00006 51 199 200 MICTOSCOPEC ceecceeeeeeeeeeees 9 10 13 14 29 235 Minimum Intensity cccccceeeeceeeeeeeeeeeees 203 Mouse Over Pixel Location Readout 136 ING TW OE ey eneen 16 17 18 19 New DOX cisicvaccaseestaitcanncvedeneneagicoeioonievemeseeaee 36 73 WOO EA T 36 43 73 74 Object ATEA rrrainis 231 232 233 Object Centers s 22cddoniesndcsancadidiancoosersecntte 214 215 Object Count per Image ssseseeseeeeeeeeees 232 Object Count per MM oo eeeeeeeeeeeeeeees 252 Object Count per Well eeeeeeesesseeeeeeeees 252 Object Counting 151 152 206 207 208 218 224 229 Object Counting Application 51 204 231 Object Mean Intensity ee eeeeeeeees 231 232 Object PLODCILY xccccirnsacedecannevativonnedessstenais 224 225 Object Summed Intensity 0008 231 252 Object Summed Intensity per mm 292 ODICE 208 Older Style Tray Siscsssssercizoscnscecssccecsasaesadionionceaaes 62 Open an ALchive ccccccccssssssssssssseeeeeeeeees 117 Open Connection cccccccccscseeseeeeeeees 117 118 Open ended JOD ccceccccccsessssssssssssseeeeeeeees 216 Optics Si sie spterienaseasecisderdatanbentasanerieedabuct 104 105 Outlier Removal 54 55 156 184 185 189 196 overlap sirisser 39 42 79 80 188 190 Parameters 160 165 205 208 210 211 213 215 217 218 222 223 224 227 Passage ca casoianc
210. ng be perfectly uniform Scheduling daytime scans for just before work begins at lunchtime break times and again just after working hours may produce the best results This also ensures that samples in the IncuCyte will be available during normal working hours in the lab Another useful rule to follow is don t over scan your samples We recommend keeping scan times under 45 minutes and duty cycles under 50 As an example 40 minute long scans scheduled every 3 hours results in a duty cycle of 22 2 Under these conditions any local temperature rises will typically be less than 1 degree C and relatively short lived The IncuCyte is capable of collecting nearly 1500 images in 40 minutes so another good reason to avoid such over scanning is to limit the amount of data collected This will keep hard drives from filling up unnecessarily and keep the system more responsive We have found that between 0 5 and 1 images per square centimeter is usually sufficient to characterize the growth of cells in most tissue culture vessels For example 48 images will usually characterize a 175 flask very well With good technique that results in uniform seeding even fewer images can be used Also with some slower growing cell types scanning every 4 or even 6 hours may be sufficient 235 Page Me ESSEN BIOSCIENCE 11 Troubleshooting 11 1 Images and What They Mean Cells tend to clump together immediately after plating and before fu
211. ng browsing between scans Vessel View and Graph and Archive or Delete Vessel View Analysis Jobs brings up a window displaying all analysis jobs performed on an IncuCyte FLR Double click to open a desired job See Section 9 3 FLR Specific Software Applications Preferences The Preferences menu contains options for viewing exporting data and setting the speed of the Slideshow option in Vessel View Section 8 7 6 Slideshow Mode Help The Helo menu contains links to the User Manual IncuCyte hardware and software specifications and a list of unavailable vessel trays Section 8 5 1 Note To easily send instrument specifications to Essen BioScience for technical support select About and then Copy To Clipboard to create a text file of IncuCyte specifications that can be emailed directly to Essen For additional support information refer to Section 11 2 page 241 58 Page A ESSEN BIOSCIENCE 8 3 Next Scan Time To the right of the Connections box the Device Status is indicated This typically lists the next time a scan will be initiated If scanning is active it will list the progress of the scan If a device test is in progress the status is indicated here The Next Scan Time will not typically be displayed on the IncuCyte EX because the next scan time is determined by the automated cell handler However it is important to remember that even though no Next Scan Time is displayed it does NOT mean
212. ning 430165 Corning 35mm TC Treated Culture Dish BID a a ooo rpiCOatead Ste al h nN O O AQ STE 627160 CELLSTAR Dish PS 35x10mm 8 7cm2 ST TC Vented pis 150318 5 x 10mm Dishes Nunclon A Polystyrene 153066 i a tO Q 7 DFS mm aD N Ne Aatan NIA NAO nlac NIC nace Amm niaee N im 3 35 x 10mm Dishes Nunclon A Polystyrene with Airvent S JR irin 25 y a Die 83 1800 003 UREGr ch r 93040 35dia x 11mm TC Treated Dish Growth Area 9 2cm 60mm Dishes Brand Catalog s Dish Name Tray Type 353002 BD Falcon 353004 430166 628160 CellStar Dish PS 60x15mm 21cm2 ST TC Vented 174888 60 x 15mm Dishes Nunclon A Polystyrene po TPP CT 98060 60d x 16mm TC Treated Dish Growth Area 22 1cm 100mm Dishes Brand Catalog s Dish Name Tray Type 353003 Stand Dish Style Standard TO S 9 O 353803 Standard Dish Style Primaria TC ____ _ _ o S OS 9 O 354450 __ Collagen 100 mm Culture Dishes 354451 Fibronectin 100 mm Culture Dishes S 9 O BD Falcon 354469 Poly D Lysine 100 mm Culture Dishes 354600 BD Biocoat Matrigel Matrix Thin Layer 100 mm Culture Dishes 354455 Poly D Lysine Laminin 100 mm Culture Dishes er es 255 Page 100mm Dishes Brand BD Falcon Greiner Nunc 150mm Dishes Brand Catalog s 354634 354653 356450 356469 356653 3262 3296 430167 430293 664160 664940 664950 150350 150679 172958 Catalog s 430599 639160 168381 Dish Name Tray Typ
213. ntil the option is changed backgrot To Microsoft Word Outlook or Publisher To Microsoft PowerPoint Excel or WordPad Figure 8 107 Image Drag and Drop Destination 8 8 Find Scanned Vessels Select the Find Scanned Vessels task bar to open the corresponding software screen Use this screen to search for previously scanned vessels See Figure 8 108 IncuCyte 1 10094 admin gt Next Scan 1 hour 23 minutes 6 32 PM Search Scans By Label a Search By ne ee mas When was the vessel scanned _ 7 6 Confluence v1 3 ached ia Upcoming acan D Columns 92 Vessels Shown Sea Administer IncuCyte e Start A a Label Cell Start Date Time Label Cell Type 0 T 25 ta Corning black w SVEC4 10fA 3 Paclitaxel HT 1080 2 20 2008 10 3 Cytochalasin D HT 1080 2 19 2008 11 1 BD NEW 353289 CHO Image 1 of 4 rere 3 32 PM HUVEC Ta Completed Scans 2 29 2008 3 32 PM HUVEC Thu 500 2008 t f February Ea Metrics Properties 2 29 2008 3 32 PM et Thu 400 S 29 Fri 2 28 2008 3 05 PM Cytochalasin D HT 1080 3 32 PV 2 28 2008 3 05 PM Pacliataxel HT 1080 gigs pHi 2 26 2008 11 5 HUVEC amp 3811 amp March 2 25 2008 12 0 T 75 2 25 2008 12 0 T 75 2 25 2008 12 0 100mm Dish 0 100mm Dish 0 T 25 0 T 25 0 T 25 Figu
214. nts Tests Update Scans Logs Motion Calibration Tray Position Front Y Test Calibrate and Confirm Run Confirm Only i l Vi Quick Test Optics Tests View historic tests and start new optics tests Go 384 well Plate Calibration Tray Position Front id D Run Fluorescence Calibration Run a fluorescence calibration i Run Figure 8 54 384 well Plate Calibration The results for the calibration can be viewed under the Logs tab Log Type Diagnostic Unless there is an error message in the log file the calibration was successful Calibrate EX The IncuCyte EX does not require a special calibration tray The built in vessel tray also serves as the calibration tray To calibrate the EX select the Tests tab under the Administer IncuCyte task bar Figure 8 55 Calibration is identical to that of the Standard Model IncuCyte except that the Tray Position cannot be selected because the EX contains only a single tray View the calibration results in the Logs tab under Log Type Diagnostic Device Accounts Scan Patterns Tests Update Scans Logs Motion Calibration Tray Position Test Calibrate and Confirm Run Confirm Only VY Quick Test Figure 8 55 EX Calibration Screen Fluorescence Calibration FLR only Because all IncuCyte FLR s are HD enabled instruments they must undergo standard tray calibration as well as HD calibration Additionally the f
215. o a txt file to email to Essen BioScience Support AL5 0 132 0 18 Al6 0 148 0 18 A17 0 0993 0 18 A18 0 145 0 18 Al9 0 168 0 18 A20 0 16 0 18 A2I 0 177 0 18 A22 0 205 0 18 A23 0 162 0 18 A24 0 139 0 13 B1 0 0666 0 185 C1 0 0666 0 219 T Entries To Dsplay 00 Refresh PM Add Maintenance Log 241 Page LN D ESSEN BIOSCIENCE 5 Click Yes to export the entire logs file Export All Q You are not currently viewing the entire log e 4 Do you want to export the entire log Name the text file after you company or entity 7 Email the text file along with a brief description of the issue to local support Local support can be found at www essenbioscience com contactUs html gt 242 Page a ESSEN BIOSCIENCE 12 Specifications IncuCyte ia Microsoft Windows XP IncuCyte HD Microsoft Windows XP IncuCyte FLR Microsoft Windows XP IncuCyte EX Microsoft Windows XP Operating Systems Microsoft Windows Vista Microsoft Windows Vista Microsoft Windows Vista Microsoft Windows Vista Windows 7 Windows 7 Windows 7 Windows 7 0 93um 0 93um w RERA rh 0 93um MAES WESOMIION 1280 x 1024 Pixels 1280 x 1024 Pixels o ene rn 1280 x 1024 Pixels 1280 x 1024 Pixels Objective 20X 20X 20X or 10X 20X P Phase JPEG Native Image Format JPEG JPEG Fluorescence 16 bit PNG JPEG Exported
216. o work Wells may be deselected in the same manner To easily select or deselect entire rows or columns click the row column label along the border of the plate Select and deselect all wells in the plate by clicking on the All button on the top left of the plate Immediately to the left of the All button is a blank plate icon that will also deselect all wells Figure 8 92 137 Page LN ESSEN Bik CIEN Y gt i gt OF J oo Adding Well Items Before any information can be applied to a well on the plate map it must first be loaded into one of three lists to the left of the plate map Compounds are any treatments to a well where concentrations or dilutions must be specified Cells specifies type passage and seeding density for cells present on the plate Growth Conditions are additions such as growth medium where concentrations of a reagent will not vary or need not be specified on the plate map Compounds To add a compound to the plate map click the New button below the blank compound list In a new window enter a name to appear on the plate map and optionally a longer description to appear in the list of compounds Figure 8 93 Additionally select a color in which the compound will appear on the plate map when creating a dilution of a compound higher concentrations will appear as darker shades of the indicated color D HEIS C A gla l B Click and drag to select area
217. od 24 6 M geen nr Ren Rone E re 8 2 3 UNPACKING AND CHECKING THE INCUCYTE ooo eee cceeeeeeeeeeeeeeeeeeeseeeeeeeeeeeeeeeeeeeeeeeeees 9 Hdd IncuCyte EX Type C Drawer Brace Removal ssscss seven secnstateunotioesncudlatcensieeramendlatiusonie ll INSTALLATION OF THE INCUCYTE HARDWARE sssssssssssssescccccsesees 13 3 1 POSITIONING THE CONTROLLER sasescvnassandevoncedelvsntietadonntvenevenertadusveneasvupentelanateansxesestalensinnss 13 3 2 PLACING THE MICROSCOPE INTO THE NCUBATOR eeeeeeeeeseeererererererererererererereeerereees 13 3 3 SWITCHING THE INGUC YTEM ON AND IEP vcsccssscccasinnoredss ensnnctisnoreickepntncdannerevehesenendiewenest 14 3 4 WARMING UP THE MICROSCOPE were seyasosralenecesetanwaceltreertehasieaelaroureseeuseeieltrereeterasinactareerets 14 3 4 1 Atto Warm Up sissrirsireiinpireeiiireieieiineiinere ipiis iiaa eiii idearen ierik 14 3 4 2 IncuCyte Power Supply Filter cccccccccccccccccccccssseesssseseeecccceeeensaaaaaaeasssseeeeeeeeeeseeeaqaaags 15 ESTABLISHING NETWORK CONNECTIONS ssssssssececcccccccosssssssssccecececsssssssssoe 16 4 1 DHCP BASED NETWORK S cc ncee aes cansveceunspucenat sear n NoE 16 4 1 Verifying the IncuCyte Controller Network Connection using Windows Vista or AS E a E E AE AAN E E EAN TE T 16 4 1 2 Verifying the IncuCyte Controller Network Connection using Windows XP 66 17 4 2 STATIC IP BASED NETWORKS scsejdeeasiesavedoscncceaesoveecdsanpaseas nestneds se
218. oea 154 Show Hide Legend csssseeeeeeeeeeeeeeees 135 Skele EO arira O E 152 SMOOth u eee ce eecceeceeceecceccecceccescccescescens 185 196 Standard Deviation 02 008 131 187 232 Standard Error ce eeceeccecceececceeceecees 187 232 STOP DUO eee E 39 SLOP DUON ceccetevexsnnndcnonceteeapestadoncaas dates 14 39 121 Summed Intensity os0hecvocesewextecadiazccedeunmceeatermecs 225 synchronization 21 90 91 92 93 94 Tasks Pane 0000n0n0n0nn 133 141 205 206 208 technical SUPPOTt cccceeeeeeeeeeeees 20 58 241 Tests 30 32 33 87 97 102 103 106 121 200 The Archive Folder and the Archive iaf File 119 The IncuCyte FLR ee 7 8 201 205 TIR sce sciessoe sa ieriaseeceeict E E 142 Time ET Ce iaronn 43 111 113 134 Time Z ONC ericeira 21 91 92 93 94 Timeline enisinia 38 76 77 79 80 81 258 Page traces 52 53 178 181 182 183 189 190 191 192 194 Tray Type socascnssexccanstarieaeinadessesueteedeaannts 26 61 62 trays 9 25 27 28 29 35 43 61 97 99 107 Unapplied Analysis Preview ccccccceee 215 Wr GUS ID vsisi ssi 41 42 84 Oa T a PTE EE EEE E E A 229 Update Database i ceesecesesseeeeeeeeeeeeeenaes 107 Update Device iewecneercvernsasnncoreatsanipnestcnscnadecenanies 107 UUO ossea 115 133 141 206 vessel 25 36 43 44 64 65 67 68 70 73 74 86 107 125 127 136 154 178
219. of the background and of fluorescent areas to determine the settings that best accommodate your image Whether Min Max settings are set manually or via the Autoscale function they will remain unchanged until manually readjusted or re autoscaled on a different image Therefore the Min Max settings applied to the original image will be applied to all subsequent images that are viewed within the same vessel at all scan times or even to images from different vessels If images are combined to generate movies each frame of the movie will contain an image with the same Min Max settings Always Autoscale There are times when the autoscaled Min Max settings for one image will no longer represent the best settings for other images If the Always box next to the Auto Scale button is checked then every time a new image is selected it will be automatically Auto Scaled rather than using the values from the previous image lf a movie is generated each image within the movie can be individually autoscaled In Figure 9 1 the Auto Scale Always box has been selected Note the Always button ONLY applies to Auto Scaled values For more information regarding generation of autoscaled movies see Section 8 7 7 Export Images and Movies FLR page 150 9 2 2 Phase Contrast Settings Phase contrast image Brightness and Intensity can be adjusted using the appropriate slider bars 203 Page N 9 2 3 The A B Slider Dragging the A B Slider generates a single i
220. og Type Diagnostic UEN EIS Device Accounts Tesj Update Scans Logs Camera Tests Log 7 9 2009 3 51 07PM gt gt gt Rear Tray Passed Cal Confirm lt lt lt gt gt Start 07 09 09 15 32 13 Figure 6 6 Calibration NOTE Calibration can ONLY be performed by a User at the Administrator Permission level Place the Calibration Tray into one of the tray positions the order in which tray positions are calibrated is not important but all 3 must be calibrated Navigate to the Tests tab under the Administer IncuCyte task bar The Front Middle and Rear tray positions can be selected using the pull down menu Each Tray Position must be individually calibrated and confirmed The first time the instrument is calibrated run the Calibrate and Confirm Quick Test See Section 8 6 5 page 97 The Results of the Calibration and Confirm test can be viewed in the Logs tab Select Logs and then use the pull down menu under Log Type to select Cor 30 Page N Diagnostic The calibration results will be displayed in the large window below If no results are visible press the Refresh button at the bottom of the screen to update the log The results will be displayed as coordinate values The Log will indicate whether the test passed or failed Check to be sure the test passed for all three tray positions before proceeding to Section 7 page 35 If the calibration was not successful cell culture monitoring will not produce qualit
221. ompt will appear after login but before the software is launched The exact verbage of the prompt will depend on which settings are in a state of disagreement Figure 5 1 displays the window that will appear if an individual at the User or Guest permission level logs onto the software and Time Zone synchronization is required A slightly different message will appear if the individual is at the Administrator permission level Time fone Synchronization Required A The Time Zone on controller TC10094 needs to be synchronized Please inform your IncuCybe administrator as soon as possible Figure 5 1 Time Zone Synchronization Window at the User or Guest Permission Level NOTE 1 We strongly recommend that ONLY individuals at the Administrator permission level implement changes to the controller time settings 2 Time or Time Zone synchronization is ONLY required if the IncuCyte software issues a prompt such the example displayed in Figure 5 1 Detailed instructions regarding synchronization of the controller and local settings can be found in Section 8 6 2 page 90 5 3 2 Establishing User Accounts Essen BioScience will establish the initial Administrator account Additional Users can be created using the Create User function All IncuCyte Users must have an account on each instrument they wish to use However we recommend that the number of Users with Administrator level permission be kept to a minimum 21 Page To
222. on so in that case select the approved plate from the same manufacturer lt is helpful to pre warm plates prior to using them for calibration If room temperature plates are placed directly into the warm incubator condensation can form on the bottoms of the plates and compromise the calibration results 31 Page ONLY clear bottom COMPLETELY EMPTY opaque walled 384 well plates with square wells from the approved list should be used for the 384 well Plate Calibration procedure see Figure 6 8 Follow these steps to run the 384 well plate calibration procedure Put two 2 empty 384 well plates into a microplate tray both spaces in the microplate tray must be occupied Select the appropriate tray position in the software all three tray positions need to be calibrated Press Run The calibration for each tray position takes only a few minutes Repeat for all three tray positions 384 well Plate Calibration Tray Position Front z i Run Figure 6 8 384 well Plate Calibration The results for the calibration can be viewed under the Logs tab Log Type Diagnostic Unless there is an error message in the log file the calibration was successful 6 4 Calibration EX The IncuCyte EX does not require a special calibration tray The built in vessel tray also serves as the calibration tray To calibrate the EX select the Tests tab under the Administer IncuCyte task bar Calibration is identical to that of the Stand
223. onent of vessel scanning necessitates strict control of administrative functions on the EX We HIGHLY recommend that the number of EX Users with access to the Administrator permission level be kept to an absolute minimum Ideally only those individuals who interface with the automated cell handler would also have Administrator level IncuCyte access IMPORTANT We highly recommend that the number of EX Users with access to the Administrator permission level be kept to an absolute minimum 89 Page f Accounts Scan Patterns Tests Update Scans Logs Set Device Time Local Time 12 30 2009 12 20 14PM Eastern Standard Time Daylight Savings ex20005 time 12 30 2009 12 21 06 PM Eastern Standard Time Daylight Savings Synchronize Device System Control Power off the device Shutdown Restart the device Restart Export Export scan diagnostics Diagnostics Export application events Events Export setup files Setup Files Export database Database Export device test logs Tests Figure 8 39 Administer IncuCyte EX 8 6 2 Device Tab Set Device Time The IncuCyte controller settings will be synchronized with the local computer at the time of installation It is unlikely that any further synchronization will be required after this point Should a disparity arise however the Set Device Tab can be used to adjust for these differences
224. only the View Completed Scans task list bar is available No scans can be set and no administrative functions can be performed from an archived data screen All IncuCyte features that are available under the View Completed Scans screen outside of an archived screen are also available from archived data 118 Page N ESSEN BIOSCIENCE IncuCyte Sele Xr Flasks 01 14 08 iaf gt Next Scan 1 hour 23 minutes 5 08 PM Search Scans By Task List 1 15 2008 5 30 00 PM Coming 25 Coming 25 75 9 Confluence w13 TN 4 iit Completed Scans E 2008 S January 14 Mon 15 Tue 2330 4M 5 30 AM 8 30 AM 11 30 4M 2 30 PM Image Properties Metrics 11 30 PM 16 Wed 230 AM 5 30 AM B2 Image 1 of 4 i e i v z Coming 150 m17 f Th _ 68 9 Confluence w13 Figure 8 72 The Archived Scans Screen Closing an Archive To close the archive screen but not exit the IncuCyte program select the Close Connection option from the file pull down window at the top left corner of the screen The Archive Folder and the Archive iaf File The data archive consists of two components the iaf file and the folder The folder contains the actual data and the iaf file contains the pathway to reach those data Each is useless without the other Whenever archived data are moved from one location to another BOTH the folder and the iaf file MUST be moved together
225. oplates black clear Microplate Collagen 384 well Microplates white clear Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Corning 384 Well Flat Clear Bottom Black Polystyrene TC Treated Microplates Microplate Corning 384 Well Optical Imaging Flat Clear Bottom Black Polystyrene TC Treated Microplate O 504 VOLEC UUIeC d STRHINENMNEe ANICTODIALeC HD 78116 781091 781092 781093 781098 781182 789071 G MGB101 1 2 LG MGB101 1 2 LG L MGB101 1 2 LG CC L MGB101 1 2 LG PDL L MGB101 1 2 LG FN L MGB101 2 2 LG MGB101 2 2 LG L MGB101 2 2 LG CC L MGB101 2 2 LG PDL L MGB101 2 2 LG FN L MGB101 1 1 LG MGB101 1 1 LG L MGB101 1 1 LG CC L MGB101 1 1 LG PDL L MGB101 1 1 LG FN L MGB101 2 1 LG MGB101 2 1 LG L MGB101 2 1 LG CC L MGB101 2 1 LG PDL L MGB101 2 1 LG FN L Cellstar 384W Plate PS F Bottom CLR ST 10 PCS Bag 254 Page 384 Well Plates Brand Catalog s Plate Name Tray Type HD J WVU T VY OIl VV q il OI NJIN OO _ PLL L AL d FLL 5 iss PUAN NJI NS VOULU MV LWI ile HUI UNVIALG VO VV OIL DIAGUA VUICAI DULL a L9 LLN 1S S VUILUIGC 1S P erile j IVITO I UW LLG Carrier 384 384 well microplate black clear bottom TC treated sterile 40 box Microplate N 6007430 ell 6007431 CellCarrier 384 384 well microplate black clear bottom TC treated sterile 5 box 6007434 CellCarrier 384 384 well microplate black clear bottom TC treated
226. or a Total of Hours Figure 8 27 Set Timeline Scan Intervals NOTE Two scans CANNOT be set to occur at the same time Scan Duration The duration during which scanning will occur can be set using the For a Total Of option If a total of 24 hours is set as in Figure 8 27 Set Timeline Scan Intervals scanning will occur around the clock without interruption from one day to the next If a shorter duration is selected scanning will occur only during that duration from start time to stop time However if the scan setting is not changed the same scans will occur for the same duration on the next day etc until the scans are deleted from the instrument X I 10am 12pm 2pm 4pm Figure 8 28 Scans set to start at 12pm every 30 minutes for a total duration of 2 hours In Figure 8 28 scans have been scheduled to start at 12pm every 30 minutes for a duration of 2 hours The instrument will scan each day between 12pm and 2pm until the scans are deleted There is a maximum amount of scan time recommended during any given time interval If the number of set scans exceeds this amount then a warning window will appear when the scans are applied In this case the number of set scans should be reduced 78 Page Scan Length The scan length is determined by the number of images that will be obtained during the scan As a general rule it is recommended that total continuous scan time not exceed 45 minutes This is because the I
227. or your local power 13 Page Figure 3 1 Controller Rear Panel 3 3 Switching the IncuCyte On and Off The power switch on the IncuCyte can be found under the front panel cover The system can be started or stopped by quickly depressing and then releasing the power switch When the system is running it may take 30 seconds for the system to save any settings and data before it powers down If the system fails to power down press and hold the power switch for 10 seconds IMPORTANT The IncuCyte should be powered on immediately after installation into the incubator It is recommended that the unit be powered on at all times when it is in the incubator to minimize the formation of condensation on the system Proceed to the next Section Warming up the Microscope to learn how to warm up the microscope 3 4 Warming up the Microscope 3 4 1 After the system has been on for 1 minute a warm up cycle should be run to get all of the components in the system up to temperature quickly and avoid condensation This can be done by pressing and holding the Stop button on the front of the microscope for more than 5 seconds The warm up cycle will begin after a 5 second pause after the button is released and will continue for approximately 30 minutes The green activity light on the front of the IncuCyte will be lit during the warm up If this light does not come on repeat the press and hold sequence Auto Warm up In the event
228. ord 10 Page 2 3 1 IMPORTANT Use only the AC power cord supplied or an equivalent cord with an IEC 320 C13 standard connector approved for operation at 5 amps and the appropriate voltage for your local power Also ensure the local line voltage falls within the range printed on the IncuCyte Controller Z Figure 2 2 IncuCyte Controller Packaging If the device is damaged in shipping please call Essen BioScience immediately You may then be asked to return the unit When returning an instrument please replace the shipping bolt Hand Tighten Only and pack it in the original containers as shown in Figure 2 1 and Figure 2 2 IncuCyte EX Type C Drawer Brace Removal The IncuCyte EX Type C instrument is shipped with a drawer brace that must be removed after removal of the shipping bolt See Figure 2 3 Follow the steps below to remove the drawer brace With the instrument powered on press and release the red Stop button on the front panel The drawer will automatically extend Using a number 1 or number 2 Phillips screwdriver remove the two 6 32 Phillips pan head screws indicated in Figure 2 3 The drawer brace is now free to remove as well Press and release the red Stop button to retract the drawer The two screws and drawer brace as well as the shipping bolt should be kept together and not discarded These items will be required if the instrument should require shipment again at a later date 11 Page
229. ordinates for the selected Pixel will always be displayed Which additional values will be displayed depends on the current selections in the A and B Channels In Figure 9 12 Channel A has been set to Fluorescence and Channel B has been set to Object Segmentation Mask When a Channel is set to Fluorescence the only value associated with the image aside from the coordinates is the Pixel Intensity So for the Channel A component of the display only the value for Pixel Intensity is indicated When a Channel is set to Segmentation Mask then all values for all Object Properties are available Therefore the value for each Object Property for the selected Object is indicated in the Channel B component of the display Review values for multiple Objects within the image to determine which values would be most appropriate with regard to adjusting Filter Settings Any Objects that are excluded from the image as a result of adjusting the Filter Settings can be identified with a color In Figure 9 13 the minimum Object Area has been set to 200um and the Filter Color has been set to blue Therefore all Objects in the image with an area of less than 200um are colored blue All blue Objects have been excluded from analysis 225 Page Image Properties Graph Export Fluorescence v Minimum Intensity 5 2 3 Maximum Intensity 66 3 3 D Aways As y Object Segmentation Mask v Use Filter Color E C Show Object Centers Show Legend um
230. organized under several headings 61 Page Double Click on a Row To Select the Vessel Flasks Plates Dishes Slides Type Grea cr Catalog s Flask Tray Type Corning g3 3059 3067 3068 3069 3070 RoboFlask Microplates Corning 29 3055 3056 Corning 2o 4 Tra 1 Corning 162 S150 3151 Corning 162 Tray 4 BD 150 354486 354495 354538 354645 354 BD Falcon 150 Tray 5 BL 175 BO Falcon 175 Tray 5 3292 431079 431080 431085 43130 Corning 175 Munc 175 Easy Flask 175 Mune 175 156502 176883 178695 Munclon 175 TPP 150 90150 90151 TPP 150 lt i For older style trays that require disks to hold vessels see the Help Unavailable Trays menu in the main window Figure 8 6 Find Vessel for Upcoming Scans e Type Flask Microplate or Dish brand name e Area Flasks and Dishes Area refers to the growth area for a flask or dish e Wells Plates Number of wells in the plate e Catalog s Different catalog numbers can be available for a single flask dish or microplate type depending on cap type packaging etc e Flask Plate Dish Common name for the flask plate or dish This is the name that will appear in the Vessel Type pull down menu e Tray Type Indicates the Tray Type that should be selected for the desired flask or dish There is no Tray Type column under the Plates tab because all plates use the Microplate Tray type Unavailable Trays New versions of several IncuCyte trays have been
231. ouse and vessel image to select all desired wells or sectors All images within a particular well or sector will be included in the analysis Wells sectors selected for analysis will be highlighted yellow In Figure 9 7 the wells in columns 1 2 and 3 have been selected Wells in columns 4 5 6 will NOT be included in the analysis e Regions of the vessel can be selected by clicking and dragging with the mouse e Individual wells or sectors can be selected by individually clicking on them with mouse e Right click over in a scanned vessel sector to activate the selection Deselection menu e Choose an option from the menu to select deselect individual rows columns or all e Previously selected wells sectors can be individually deselected by clicking on them with the mouse Notes Enter any notes here These notes are NOT read only this is in contrast to the notes entered in the properties box associated with a scanned vessel Therefore it is possible for the original author or any other person to modify or delete these notes at any time even after the analysis has been completed Name the Analysis Give your Analysis Job a name After the Analysis has been launched it will appear under the Analysis Jobs Open and Manage Task Bar in the Vessel View Tasks Pane Analysis Parameters A summary of the Segmentation and Refinement Analysis Parameters that will be applied to the analysis are displayed along with the name of the Para
232. pattern will repeat until scanning is terminated by the User Whenever a scan is applied to 96 well plates a pop up window will appear letting the User know that the first scan will be longer by __ minutes the exact number of minutes depending on the specific scan length In order for the scan to proceed the OK button must be selected confirming that the User is aware of the increased scan time This longer initial scan length ONLY occurs with 96 well plates See Section 8 5 7 Scan Length for more information on this topic Note that the longer initial scan time for 96 well plates does NOT apply to the IncuCyte HD or the IncuCtye FLR Vessel Scheduling When scanning multiple vessels the Vessel Scheduling button at the bottom of the Schedule Upcoming Scans screen allows the User to insert cooling idle times between vessel scans and customize the order in which IncuCyte scans vessels Using the Auto Cool function is recommended and allows for the most efficient vessel scheduling For a more detailed view see Section 8 5 8 Vessel scheduling When the Physical Layout Properties and Scan Time s have been set select the Apply button at the bottom of the screen A window will appear confirming that scanning parameters have been applied To terminate scanning delete all the Scan Bars and then hit Apply A scan can also be manually terminated by pushing the Stop button on the front of the IncuCyte Pushing the Stop button w
233. patterns in the EX navigate to the Administer IncuCyte Task Bar See Figure 8 17 New Scan Patterns can be created and edited by EX 70 Page N ESSEN BIOSCIENCE Users at the Administrator permission level but they CANNOT be deleted Scan Patterns can ONLY be deleted by the automated cell handling system NOTES In the IncuCyte EX the Administer IncuCyte Task Bar is ONLY available at the Administrator permission level Scan Patterns in the EX can be created and edited but they CANNOT be deleted through the IncuCyte EX local interface This is true even for Users at the Administrator permission level To create a new Scan Pattern first select the desired Vessel Type See Figure 8 18 2 IncuCyte File Preferences Help Ex20004 admin 7 Status Scanning 70 Task List Device Accounts Scan Patterns Tests Update Scans A Find Scanned Vessels Vessel f well BD Falcon v A view Completed Scans 5 Sa Administer IncuCyte Available Patterns ae o l Device Status Press Save when finished editing Hold down the Alt Key to remove wells Click and drag to select and deselect groups of wells Images per Well Estimated Time To Scan 00 01 40 2 42 PM New Reload Next Scan No Upcoming Scans i Refresh Patterns Status Scanning 70 Free Space 972 59 GB 97 Figure 8 17 The Scan Patterns Tab under the Administer IncuCyte EX Task Bar 71 P
234. perature 108 Page N ESSEN BIOSCIENCE 11 39 AM xt Scan 3 00 PM admin Ahe SDICIA 19 41 DV 12 5 2009 12 10 PM Figure 8 60 The Device Status box in IncuCyte FLR fir30001 Temperature History Data not available when scanning in rear DP ide EEE Active 12PM SPM 11 Fri 3AM 10 Thu Dec 2009 2009 12 10 11 03 0 0 C Figure 8 61 The Temperature History window The Three Basic Scans Tab Functions Use the Scans tab screen Figure 8 62 to archive data There are 3 related functions available on this screen e Archive Scans e Delete Scans e Restore from Archive The desired Task can be selected using the Select a Task pull down menu 109 Page ey ESSEN BIOSCIENCE 2 Choose a Time Day Month or Year Selecting a scan time will archive that scan Selecting a day will archive all scans in that day Selecting a month will archive all scans in that month etc 2009 3 Browse to a Destination Destination Figure 8 62 The Scans Tab Archive Scans for Data Management The IncuCyte controller hard drive has limited space and may require a data management plan to archive and delete information from the system This plan may vary depending on usage and number of users The recommended best practice is to archive individual experiments once completed see Archive Vessels After the scans have been archived a user with administrative permissions can then delete the experimen
235. ple to 25 See Figure 8 148 Graph Sector C3 Mean vs Time DEOR Fie View Edt Averaga Smooth Growth Over Time Flask amp Flask2 40 Time Hours 72 34 Hours 0 61 Drag and Drop Manua Alignment Ato Alignment Apol Use the Apply button to align the plots Use the Clear button to align the plots by ther y First port Figure 8 148 Resetting the Auto Alignment to 25 Now the traces overlap appropriately and growth can be more readily compared Note however that the first point for both graphs is no longer set to 0 time as before Because the graph for Flask 2 was dragged onto the graph for Flask 1 Flask 1 is the reference flask for this graph Therefore the hours setting for Flask 1 remains at 0 but the hours setting for Flask 2 is adjusted to compensate for the difference in starting confluence So Flask 2 growth now starts at 12 hours 190 Page lf growth comparison is only relevant from the point of intersection on then the early low confluence points for Flask 1 don t need to be displayed Since it is clear at exactly which time point intersection occurs the graph for Flask 1 can be re graphed using the first intersection point as the first start time point Repeat overlaying the graphs and now the result displays both traces starting at O time at the point of intersection see Figure 8 149 Graph Sector C3 Mean vs Time i i i Smooth Growth over Time Flask1 amp Flask2 Figure 8 14
236. plies a single User selected threshold value for all images included in the analysis Pixels with intensities less than this value will be classified as background Pixels with intensities greater than or equal to this value will be classified as foreground Segmentation Adaptive Adaptive Segmentation uses an algorithm to determine a good threshold for each image and it also uses different thresholds within an image This option allows the software to compensate for variations in the background fluorescence both within and between images lf Adaptive Segmentation is selected the User must choose values for Background Intensity Value of the fluorescent image outside of an object All areas within a fluorescently scanned image are assigned a fluorescence value represented as pixel intensity The User must decide which fluorescence values should be classified as background Foreground Intensity Value of the fluorescent image inside an object Manual Adjustment Value added to the threshold determined by the adaptive algorithm before the threshold is applied to the image Positive Manual Adjustment will help to remove background both areas adjacent to Objects and areas distant from Objects Negative Manual Adjustment will assist with the identification of faintly fluorescent Objects The Background and Foreground Intensities determine the range over which the software searches for a threshold and they do not need to be very precise
237. r Use the slider to realign all three traces on the graph See Figure 8 153 NOTE The advantage of the Manual Alignment slider over the Auto Alignment function is that Manual Alignment facilitates alignment of individual traces one at a time whereas Auto Alignment resets all traces simultaneously Graph Well B4 Mean vs Time File View Edit Average Smooth Growth Over Time Well amp Well2 WellS gt a E o o pms D a gt w o o 3 e i fa O Time Hours Drag and Drop Manual Alignment Auto Alignment a J Figure 8 152 Manual Alignment set to 23 Note that Well 1 is selected in the Trace Selection Box to the right of the Manual Alignment slider 193 Page Graph Well B4 Mean vs Time File View Edit Average Smooth Growth Over Time Weli amp Well2 Well3 gt a D o a a gt fi o E o t fa Oo p Figure 8 153 All three traces are aligned at the same starting concentration The Manual Alignment selection also facilitates deletion of selected traces First select the trace to be deleted by using the Trace Selection Box Now simply click on the Delete button The Delete function can be used to delete a trace from any graph containing multiple traces not just graphs created by dragging For instance all 12 wells of a 12 well plate have been graphed using the Graph All Wells selection However the trace from Well 6 needs to
238. raphed Single Column or Row In this case the only region of interest is a specific Column or Row within the microplate Selecting one of these regions will open a new drop down menu to facilitate further selection of the specific column or row of interest Single Columns and Rows offer only two grouping options All or None Either all the wells within the selected Column or Row can be combined to generate a single trace or none of the wells within the selected Column or Row will be combined resulting in multiple traces one for each well Single Well No grouping options are available for graphing a single well Scroll through the possible wells to find the well that is desired for graphing This selection will generate a single trace for the selected well Error bars are not available when a single well is graphed Single Well 96 and 384 Well Plates High density microplates microplates containing 96 or 384 well wells have a modified selection format for graphing a single well Because scrolling through 169 Page all 384 wells to select just the single well desired for graphing would be inconvenient in the case of high density plates it possible to independently select both the Row letter and the Column number Graphing Flasks and Dishes Flasks and Dishes are simpler to graph than microplates They contain only two possible regions e All Sectors e Single Sector Region All Sectors If All Sectors are selected the gr
239. rchiving will still be available after the vessel scan has been archived Thus it is possible to archive analysis jobs as well as vessels Default Name Archived data will always be given the default name Archive Open an Archive Archives can be opened the same way as opening an IncuCyte with a User account Simply click on the IncuCyte icon or select Open a Connection under the File pull down menu When the Open Connection window appears select Browse to an Archive and locate the desired archive file Figure 8 70 Unlike opening a connection with an IncuCyte a password is NOT required to open an archive 117 Page ESSEN BIOSCIENCE lt Open Connection J X MncuCyte INCUCYTE ARCHI Figure 8 70 The Open Connection Box As mentioned previously when an archive is saved both a folder and a file will appear in the target location Both will be assigned the default name archive although the file will bear the name archive iaf IncuCyte Archive File To select the archive from the Browse location double left click on the iaf file Figure 8 71 Figure 8 71 Open Archive To open the archive click on the archive iaf file The path to the archived data will then be displayed in the Browse window Select the Browse button and IncuCyte will open the screen for the desired archive Figure 8 70 In the Archive Screen the path name to the archive file is listed at the top left corner of the screen and
240. re 8 108 The Find Scanned Vessels Screen The Find Scanned Vessels screen contains 4 components e The Search box e The Search Results displayed below the Search box e The Selected Vessel box 158 Page N ESSEN BIOSCIENCE e The Time Tree for the selected vessel 8 8 1 Searching Sf 3 2008 Sf 3 2008 Figure 8 109 The Search Box Press the Options button to refine the time frame of your search In Figure 8 108 the Options button has not been selected Figure 8 109 shows a close up of the expanded search opportunities that are enabled following selection of the Options button Choose either a general time frame e g Past Month or use the Between radio button to define more specific temporal boundaries 3121 2008 w i March 7008 Sun Mon Tue Wed Thu Fri Sak ob 27 oa 29 1 4 5 6 7 amp 11 12 13 14 15 if 19 20 Ra 2 25 26 27 25 29 Figure 8 110 Selecting the Precise Time Frame of the Search Search Terms Select the Search criteria by Parameter Label Cell Type or Passage and enter one or more search terms into the For box 159 Page Search By Refine Search By YY Figure 8 111 Search Parameters ONLY Label Cell Type and Passage can be searched using this function To search other scan related terms such as those related to Properties Passage etc use the Search window at the top of the View Completed Scans screen see Section 8 7 9 page 153 Search terms can include numbers lette
241. re 9 17 Object Area Histogram Export of fluorescence scan graph data is the same as export of non fluorescent graph data The FLR Histogram View Pull Down Menu Two options are available in the FLR Histogram window View pull down menu Y Axis Display Select Count or Percent for the Y Axis Display In Figure 9 17 the Y Axis displays Count Show Analysis Filter 233 Page ESSEN BIOSCIENCE LN Choose whether to display any Analysis Filter Settings in the Graph window 234 Page N 10 Getting Optimum Performance from your IncuCyte The IncuCyte is a phase contrast microscope and as such requires an unobstructed path to the specimen from both above and below Keep in mind the following warnings to avoid image degrading problems e Keep vessels clean e Avoid condensation e Avoid bubbles e Avoid media hanging on top of flasks e Dont scratch vessels e Avoid writing labels on the tops of vessels e Avoid touching the tops and bottoms of vessels Handle them from the side as much as possible Vibration is another factor that can negatively affect image quality The incubator that contains your IncuCyte should be on a sturdy stand If possible try to schedule scans at times when the incubator will not be accessed as opening and closing the incubator door can cause image blurring even on a sturdy stand Although it is desirable to not have large gaps in data collection it is not necessary that the sampli
242. references Help Ex20003 admin z Ready To Scan Search Scans By Bar Code ae Selected Vessel Seachby a CI nd Scanned Vessels O8y03m14d17hOSn gtF as TERET ES When was the vessel scanned a Refine Search By ft c 68 8 Contilience v1 2 i Columns 500 Vessels Shown Start Date Time Bar Code Cell Type eC allele om OSy03m14d17h0 zamo chilly 3 14 2008 2 08 PM O8y03m14d14h0 gt cHo2 Completed Scans 3 14 2008 2 02 PM 08y03m14d14h0 gtf cho 2008 T T March 3 14 2008 12 2 LabelTest2 My Label CHO 14 Fri varam wera 3 14 2008 12 1 LabelTest My Label CHO NES er p 3 14 2008 12 0 D3HD47383 My Label cho 3 14 2008 11 2 CHGE638232 My Label CHO 3 14 2008 10 4 24583457 My Label CHO 3 11 2008 1 53 PM DCF7234523HZ Test Flask CHO 3 11 2008 1 48 PM CCF7234523HZ Test Flask CHO 3 11 2008 1 44 PM BCF7234523H2 Test Flask CHO 3 11 2008 1 39 PM ACF7234523HZ2 Test Flask CHO 3 4 2008 12 14 PM amar 74 My Label CHO 3 4 2008 12 13 PM amar 73 My Label CHO 3 4 2008 12 12 PM 4Mar_72 My Label CHO D6 Image lofi T j T Sl 3 4 2008 12 11 PM 4Mar_71 My Label CHO 3 4 2008 12 10 PM 4Mar_70 My Label CHO 3 4 2008 12 09 PM 4Mar_69 My Label CHO 3 4 2008 12 08 PM 4Mar_68 My Label CHO Graph Export s i 4Mar_67 My Label CHO Figure 7 9 The Find Completed Vessels Screen for the IncuC
243. ress the Refresh button before assuming the existing pattern is still accurate Scan patterns can be changed by other Users and this will not be apparent unless the Refresh button has been selected Press the Reload button to return to the last scan pattern that was previously saved 72 Page A ESSEN BIOSCIENCE 8 5 5 Setting Vessel Properties After completing the Physical Layout select the Properties tab This Section facilitates entry of vessel properties such as Cell Type Label etc Vessels selected in the Physical Layout screen will appear in the properties screen Physical Layout l Properties No Cell Type ms Passage 1 iS PlateMap EXE pees NO Cell Type Passage 1 Scratch Wound Properties V Wide Scratch Wound Images i Figure 8 20 Upcoming scans with a New vessel In Figure 8 20 there were no previous vessels of this type same brand of 6 well plate in this location for the previous scan so the Properties Screen indicates that the selected vessel is New and the Label Cell Type and Passage boxes are active The Type box will be inactive unless the selected vessel is an Essen ImageLock plate see Section 8 5 10 page 85 for more information on ImageLock plates The name of the current User and the time date automatically appear in the Notes box If the same vessel type in the same position was already present for a previous scan then the Properties Screen indicates
244. ries of scans is selected using the Shift Key 111 Page H January 09 ed 10 Thu 11 Fri 12 Saty 0 19 OM 11 19 4M 2 19PM 13 Sun i4 Mon Figure 8 64 A collection of scans is selected using the Ctrl key After scan times have been selected use the Browse to a Destination box to select the new location for the archived data Select the Browse button Choose the desired location and then click Save The new destination will now be displayed in the Browse to a Destination box Figure 8 65 Note Select the Estimate Archive Size button to display the approximate file size of the archive file on the disk 3 Browse to a Destination Destination 7 UsersiEX4MPLE SRCHIVE iaf Figure 8 65 Browse to a Destination Now select the Archive Scans button A window will appear to confirm that archiving is desired Once the archiving process has been initiated it cannot be interrupted Press OK Another window will appear that displays archive status Archiving can require a significant amount of time if large numbers of scans are being archived After archiving is complete a new Archiving Complete window will appear Any problems encountered during the archiving process will be displayed in this window In Figure 8 66 no errors were encountered during archiving 112 Page Archiving complete here Were no errors in archiving Would you like to delete the scans that were just archive
245. rs and special characters Press the GO button to initiate the search The Search function operates as follows e One or more individual terms entered into the For box will find any scan that contains any one of those terms in the specified Search By field e Enclosing two or more terms in quotes will display any scan that contains those exact terms in that exact order e The symbol can used as a wild card in the middle of a Search term For instance the Search S N will return the results for Scan or Super scan The terms entered into the Search Box act as a filter In other words whatever terms are entered for searching are used to exclude entries that do NOT contain the relevant terms Therefore if no terms have been entered the search results will display all scans that have occurred during the selected time frame see Figure 8 108 NOTE Searches are NOT case sensitive Example Searches e Search Label test 96 The results include all scans with labels that contain EITHER test or 96 7 vessels See Figure 8 112 160 Page LN ESSEN BIOSCIENCE Columns Start Date Time Label apan Type Cell Type 284 Vessels Shown Bosna sewers ira 3 iyaa 1 ertest Frenne OO 129 008 12996 wal W Satin Wound ar00 1279 0080 129 96 wal SW Scan Wound HT 000 nies amies ae ea e Std irmos fr OOo omoa asee ormsa asee ionamosos Std onormsos stand ionamsa 10 15 2009 11
246. s Click to toggle individual well selection Use the tabs below to populate the selected wells Right click a selection to edit well contents Compounds Growth Conditions 55FP 1000 SSP 300 SSP 100 nM nM Compounds used in this plate Stauros porine SSP 10 nM Short Name ODOMSO for display in plate map Description Dimethyl sulfoxide 99 8 E Ae Figure 8 93 Adding Well Items to the Definitions lists After creating a compound description select the desired area of the plate and click Add __ A new window will appear allowing the User to select a desired concentration and optionally a dilution series First select the units in which concentration will appear by clicking the drop down menu at the top of the window the default setting is mg mL and enter a numerical concentration to the left If only a single concentration is desired click OK and the compound will appear on the plate map To create a dilution series check the box next to Create a dilution to activate dilution options In the drop down menu immediately to the right select divide by or subtract and enter value by which to dilute Alternatively select manually enter to enter a range of concentrations separated by spaces or 138 Page semicolons 1000 300 100 30 To arrange the dilution series on the plate select the direction in which the dilution series runs by selecting a direction under
247. s Preview using Current Image Launch New Analysis Save Analysis Parameters The Parameters Pull Down Menu lf no Analysis Parameters have been saved then the only option under the Parameters pull down menu will be Manual However it is possible to select and then save Analysis Parameters Saving Analysis Parameters will increase the options available under the Parameters pull down window Any saved Analysis Parameters will now be available They can be selected and opened A new option will appear in the pull down window called Manage Analysis Parameters Select this option to delete or rename saved Analysis Parameters When Manual is selected the User is free to adjust the Segmentation and Refinement settings If saved Parameters have been selected then the Segmentation and Refinement values will be populated accordingly However adjusting any of the Segmentation and or Refinement Parameters will cause the saved Parameter selection to jump back to Manual The Segmentation Pull Down Menu Segmentation refers to the separation of foreground and background components of an image and it leads to the identification of Objects within the image area Objects belong to the foreground 208 Page N Object An isolated region within the image that stands out from the background Two options are available with regard to Segmentation Fixed Threshold Adaptive Threshold Segmentation Fixed Threshold Choosing Fixed Threshold ap
248. s outer perimeter one top left one top right one bottom right In Figure 6 3 the Alignment Windows are indicated by arrows Figure 6 3 Microplate Tray Indicating Location of Alignment Windows 2 Page Figure 6 4 Location of Alignment Posts on the Drawer These posts must be placed through the Alignment Windows as shown The 3 tray Alignment Windows fit over 3 posts in the corresponding locations within the IncuCyte drawer When placing trays into the drawer make sure that the drawer Alignment Posts pass through the tray Alignment Windows The tray should sit very flat and the posts should prevent any lateral movement See Figure 6 4 6 1 4 Placing Trays into the IncuCyte EX The EX is equipped with a single tray that is built into the instrument Therefore it is not necessary to place trays into the IncuCyte EX 6 1 5 Placing Vessels into the IncuCyte After the selected trays have been placed into the IncuCyte drawer it is time to add the desired vessels Be sure the selected vessels sit completely flat within the tray If the flask is not seated flatly image quality will be compromised Some trays are designed to hold multiple versions of the same vessel e g the same flask size made by different manufacturers Therefore the fit of certain vessels within certain trays might not be snug around the edges Nonetheless the fit should be adequate for IncuCyte function The quality of images and sub
249. s only available in the JPEG format Phase Contrast Original JPEG Export the image as it was originally acquired by the IncuCyte microscope in the format of a JPEG file Phase Contrast Original 8 bit TIFF Export the image as it was originally acquired by the IncuCyte microscope in the format of a TIFF file Note that TIFF images can only be exported as originally acquired NOTE TIFF images can only be exported as originally acquired Images in addition to the image currently displayed in the Vessel View window can also be exported Export Current Image Single Image To export only the SINGLE image that is currently displayed in the Vessel View window select Export Current Image A new window will open See Figure 8 96 Select the appropriate image type in the Export Single Image window using the Image Type drop down menu 142 Page C_ Export Current Image Image type i Phase Contrast using current display settings JPEt Filename Keep full size image dimensions Customize with image designer Figure 8 96 Export Current Image Window Export utilizing the Phase Contrast using Current Display Settings will export an image that maintains the brightness and contrast settings currently displayed in the Vessel View By selecting the Keep full size image dimensions option the entire image acquired at its native size is exported using the current brightness and contrast settings Furth
250. save all parameter values that are currently selected A new window will appear to give this set of parameters a name Saved Parameters will be available under the Parameters pull down menu Additionally Saved Parameters can be renamed or deleted by selecting Manage Analysis Parameters from the Parameters pull down menu Vessel View FLR Banner The colored banner at the top of the Vessel View window is color coded to reflect changes in the current viewing mode See Figure 9 5 210 Page File Utilities View 5 2008 October aan L a Gai D a D GRR HR 8 19 PM Or a aH 11 19 PM 31 Fri November B2 Image 1 of 9 1 4 Image Properties Graph Export Fluorescence Minimum Intensity 5 2 F Maximum Intensity 66 3 7 Ama Figure 9 5 Open Analysis Job has Green Banner Changes in the banner color are intended to alert the User to which mode is currently selected These modes will be explained in more detail in the following sections however a summary of the banner colors is as follows Standard Mode Blue Job Preview Mode Beige Unapplied Preview Mode Yellow Complete Analysis Job Green In Figure 9 5 the banner is green indicating that a complete Analysis Job has been opened The name of the Analysis Job is Paclitaxel 9 3 4 Preview Using Current Image Analysis Preview Mode Use this button to launch an analysis of a single currently selected image Depending on the size of the data set runnin
251. saved Filter Setting will potentially affect other users who are under the assumption that the setting is still saved and will be applied when they launch a new job Delete Filter Settings that Have been Pinned All saved Filter Settings can be selected and deleted Deleted Filter Settings will no longer be available under the Analysis Filter pull down menu UNLESS they were previously pinned to a job Filter Settings that have been pinned to a job will remain intact and they will remain pinned to the job until they are specifically unpinned from that job See Figure 9 15 In this figure the setting Example Analysis Filter Settings A e Object Counting Analysis Filter Manual Selection yt Al Save Filter Li tp Area un E ccertri AAEE selection i Unfiltered min 200 min Example Filter deleted but pinned T mas Figure 9 15 Deleted Filter Still Pinned to Job Filter has been deleted However subsequent to deleting the Example Filter setting a job was opened to which this setting had previously been pinned Now when the job is opened the saved Example Filter is still available however the fact it that was deleted is indicated in parenthesis If Example Filter is no longer desired it can be unpinned However after it has been unpinned from this job it will no longer be available in the pull down menu 9 3 9 Graph Export Data from Fluorescent Scans Unprocessed Vessels Data from fluorescent scans can on
252. scope to home and then open the drawer when the light turns off The light turning off indicates that the drawer is now unlocked and can be opened The Stop Button EX The front drawer of the EX CANNOT be opened manually like on the Standard Model IncuCyte To open the front drawer on the EX press the Stop button briefly and then release it Again there will be a short delay before the door opens Be patient and do not press the Stop button and hold it down continuously 35 Page N 7 1 2 Physical Layout Select Tray Type Vessel Type Scan Pattern and Scan Type It is NOT possible to schedule scans on the IncuCyte EX using the IncuCyte software The only aspect of scan selection available to EX Users is the selection and creation of scan patterns For more information on this function see Section 8 5 4 page 70 Scan Pattern The scan pattern describes the number and locations of images that are generated for each well microplates or sector flask dish The Scan Pattern is displayed within the software as a pattern of green dots where each dot represents one image Sector In microplates the scan pattern is defined as the number of images per well However because flasks and dishes don t contain wells they are artificially divided into units called Sectors In flasks and dishes the scan pattern is defined as the number and locations of images for each Sector Use the Tray Type pull down menu to select a Tra
253. scovered in the right position of t Device 7 26 2008 11 0 Scan started Device 7 26 2008 8 16 Scan complete Device LILI Entries To Display 1000 w Figure 8 75 Log in Grid View Use the blue scroll bar at the bottom of the screen to reveal the additional hidden Is Error column 123 Page N ESSEN BIOSCIENCE Log Types Entry Types VIEWS Filter Log DateTime Entry SOUrce anina ennn ams _ Benam pE 13 Scan aborted because stop button was pressed Device 20 2008 3 20 PM Stop button pressed at Front panel Device 220 2008 1 04 PM 18 Scan aborted because stop button was pressed Device Figure 8 76 The Log entries were filtered using the word STOP 8 7 View Completed Scans 8 7 1 Overview When this screen is open there is a Completed Scans box located beneath the Task List bar The Completed Scans box lists the dates and times of all completed scans Dates are preceded by a or icon Selecting the icon will open the next set of subdates times available for that date Selecting the icon will close them again To view the scan for a specific time highlight the time itself The corresponding scan will be displayed and the date and time will also be indicated in bold at the top of the page Scans can only be displayed one at a time At the bottom of the completed scans window Figure 8 77 Figure 8 78 Figure 8 79 the arrow buttons can be used to browse thro
254. screen to visualize them all 8 8 2 The Selected Vessel Box The Selected Vessel Box in the Find Scanned Vessels screen is no different from the same box in the View Completed Scans screen Highlight vessels in the search results to display the corresponding Selected Vessel Box 163 Page LN ESSEN BIOSCIENCE 8 9 Find Scanned Vessels EX IncuCyte File View Scan Preferences Help EX20003 admin gt Ready To Scan Search Scans By Bar Code aa Selected Vessel Search By TE D3HD47383 i When was the vessel scanned 1234567 8910111213141516 A view Completed Scans i es Y an 56 1 Confluence v1 2 Ag Administer Incucyte Columns 500 Vessels Shown gt Start Date Time Bar Code Label 3 14 2008 12 0 DIHD47383 My Label CHO 3 14 2008 11 2 CHGE638232 My Label CHO Completed Scans o 3 14 2008 10 4 24583457 My Label CHO 2008 h 3 11 2008 1 53PM DCF7234523HZ Test Flask S14 Fri a Raat 3 11 2008 1 48 PM CCF7234523H2 Test Flask DQ S 5 ae 3 11 2008 1 44 PM BCF7234523HZ Test Flask 3 11 2008 1 39PM ACF7234523HZ _ Test Flask 3 4 2008 12 14 PM 4Mar_74 My Label 3 4 2008 12 13 PM lamar 73 My Label 3 4 2008 12 12 PM 4Mar_72 My Label 3 4 2008 12 11 PM 4Mar_71 My Label 3 4 2008 12 10 PM 4Mar_70 My Label 3 4 2008 12 09 PM 4Mar_69 My Label 3 4 2008 12 08 PM amar _68 My Label 3 4 2008 12 07 PM 4Mar_67 My Label Cell Type AE SArwhe Oe
255. se both for images that are being previewed and also images from completed Analysis Jobs However there are some important differences to keep in mind Adjust Filter Settings in Analysis Preview Mode Filter Settings in Preview Mode apply only to the currently selected image However if a different image is selected the User will be exited from Preview 226 Page Mode and the Segmentation and Refinement Parameters as well as Filter Settings must be reset for the new image Adjust Filter Settings on a Completed Analysis Job When Filter Settings are adjusted within a completed Analysis Job the settings will be applied only to the currently selected image However if a new image is selected the Filter Settings will automatically be applied to the new image as well Additionally the software will remember the results from the previous image Thus the reprocessed data from each consecutively selected image will be added and temporarily stored All the same any images that have NOT yet been selected will not reflect the adjusted Filter Settings The software warns the User that Filter Settings have been applied to only a subset of the data by changing the vessel image color to red and displaying a corresponding message at the top of the Vessel View window See Figure 9 14 In this example Image 1 from Well A1 is the only image that has been selected If you navigate to the 22 Page N ESSEN BIOSCIENCE x E 2008 A sub
256. se the IncuCyte control software before making any changes to the time time zone settings on your local computer Time Zone and Daylight Savings Time lf the Daylight Savings and or Time Zone settings of the controller and the local computer are not in agreement a message window will appear after login but before the software is launched e Time Zone In this case the Time Zone on the local computer and the controller server for the IncuCyte are not properly synchronized One of two messages will be displayed depending on whether you are at the Administrator Figure 8 40 or User permission level Figure 8 41 Time fone Synchromzation Required The Time Zone on controller 1710094 needs to be synchronized Please use Administer IncuCyte gt Device gt Synchronize as soon as possible Figure 8 40 Prompt to Synchronize Time Zone at the Administer Permission Level 91 Page Time Zone Synchromzation Required The Time Zone on controller 1010094 needs to be synchronized Please inform your IncuCybe administrator as soon as possible Figure 8 41 Prompt to Synchronize Time Zone at the User or Guest Permission Level e Navigate to the Administer IncuCyte Task Bar and open the Device tab Figure 8 42 In this example the local Time Zone is Central Standard Time but the device 1C10104 Time Zone is Eastern Standard Time Note that the actual times 3 28 pm appear to be in agreement despite the fact that t
257. seal cap Collagen 75 cm Flask with vented cap O Oml Canted Neck Standard TC Filter Cap 13 247 Page i 75 cm Flasks Brand Catalog s Flask Name Tray Type 356488 Gelatin 75 cm Flask with vented cap BD Falcon 356524 Poly D Lysine 75 cm Flask with plug seal cap 356537 Poly D Lysine 75 cm Flask with vented cap 356654 Gelatin 75 cm Flask with plug seal cap 3275 Modified Triangular Straight Neck Cell Culture Flask with Phenolic Style Cap 3276 Modified Triangular Straight Neck Cell Culture Flask with Vent Cap 430198 75cm Flask 430199 75cm Flask eee 3290 CellBIND Rectangular Canted Neck Cell Culture Flask with Vent Cap g 3375 Rectangular Canted Neck Cell Culture Flask with Phenolic Style Cap 3376 Rectangular Canted Neck Cell Culture Flask with Vent Cap yp 15 15 15 15 14 14 14 14 15 15 15 Rectangular Canted Neck Cell Culture Flask with Vent Cap 15 Rectangular Canted Neck Cell Culture Flask with Plug Seal Cap 15 15 14 14 14 14 14 id 14 14 17 17 17 17 17 17 658170 250ml Canted Neck TC Flask 658175 250ml Canted Neck TC Flask Filter Cap Lo Profile Greiner CellStar T75 Flask 250mL ST CN Filter Cap 75 cm Poly D Lysine Coated TC Flask Collagen Type 1 Coated TC Flask 3110 075 75cm Tissue Culture Flask Nunclon A Straight Neck Standard TC Plug seal Cap Nunclon A Straight Neck Standard TC Vented Filter Cap Nunc EasyFlask Poly D Lysine Nunc EasyFlask Collagen
258. sector 2 If each point on a graph represents multiple wells or sectors then selecting Mean or Median refers to the Mean or Median of all the wells sectors that comprise that data point Only the image MEANS of multiple wells sectors will be combined Therefore selecting Mean or Median for multiple wells sectors will generate a graph consisting of the trace Mean or Median of the image MEANS Select Start and End Time The Time Range over which the data are to be graphed can be selected using the Start and End Time buttons to the right of the Time Tree In order to graph the Time Range option must be selected in the box above the Start Time radio button The alternate selection Single Time is only applicable to data export The default settings are the times and dates of the first and last scans in the series The chosen Start and End times will be highlighted in blue and pink respectively After the Start or End Time radio button is selected the time can be changed by e Directly selecting a time with the mouse e Using the arrow buttons to sequentially scroll through the times until the chosen set point has been reached Graphing Microplates The Microplate Graph Users can use the Microplate Graph function to graph all wells or selected wells in a microplate format That is all wells are plotted individually as they would appear in a microplate 6 well through 384 well The colors of the Microplate Graph would be directly correla
259. sels are scanned in fluorescence mode both an HD and a fluorescence image will be generated Features of the IncuCyte software that are unique to the FLR will be described using text with a green background and an icon to the left as demonstrated in this paragraph Additionally there are some applications specific to the IncuCyte FLR These applications will be described later in the manual Section 9 IncuCyte FLR page 201 2 1 6 Special Model IncuCyte Instructions This manual includes general instructions for the Standard Model IncuCyte supplemented with additional sections that discuss instructions or conditions specific to the HD enabled system or to the special models IncuCyte EX and IncuCyte FLR We recommend that the User read all the instructions for the Standard Model IncuCyte and then follow up by reading any extra sections that apply to the IncuCyte HD or to the special model IncuCytes 2 2 Safety has Priority Please note the following directions for safe and problem free operation of your IncuCyte Read through these operating instructions carefully e It is essential to follow the instructions in Section 3 page 13 when putting your new IncuCyte into operation e The unit must only be connected to a receptacle outlet with a grounding connection e Use only the AC power cord supplied or an equivalent cord with an IEC 320 C13 standard connector approved for operation at 5 amps and th
260. sequent confluence measurements obtained using the IncuCyte depend on having a clear unobstructed light path through the 28 Page N particular well or flask Therefore before use carefully inspect the vessels selected for the experiment The following is a list of important issues to avoid e Vessels that have scratches on the tops lids for microplates dishes or on the bottoms e Drops of media or other liquid on the outside tops lids of vessels If this occurs carefully clean with ethanol e Media hanging on the inside of tops lids of vessels e Condensation on top outside of vessel e Touching either the tops or bottoms of vessels during manipulations Hold vessels by the sides e Writing labels on the tops of vessels The writing will interfere with the images Label vessels on the sides or on the very edges of the tops where imaging will not occur e ltis VERY IMPORTANT that all lids are tightly fastened onto the flasks If the lids are loose it can cause the flasks to become displaced within the tray e Be sure all vessels are sitting COMPLETELY flat in the trays 6 2 System Calibration Before using the IncuCyte for the first time a calibration procedure must be performed This procedure ensures that the motion of the IncuCyte microscope is accurate and repeatable such that images are obtained within their proper target locations This is especially important when acquiring images in 96 and 384 well plates
261. set of filtered metrics has been generated Use Apply Filter on the October Graph Export tab to filter the available metrics for this scan E 30 Thu 119 PM 31 Fri H Movember Al Image 1 of 9 Metric Object Count per well na Ei Graph E sport A Apply Filter to Entire Table PS Sector Mean Median Std Dev 1 of 1 Min blax Vessel 2900 2900 2900 2900 2900 2900 Ente Wife ee Figure 9 14 Filter Settings Applied to a Subset of the Data Note the green banner at the top of the Vessel View window indicating that a completed Analysis Job has been opened Graph Export tab it will be very clear that for the Metric Object Count per Well data only exist for this single image To apply the selected Filter Settings to additional images select them individually As the images are selected the data table in the Graph Export tab will be expanded to reflect the addition of new data To apply the settings to all images simultaneously push the button Apply Filter to Entire Table Note that the warning in Figure 9 14 will only appear if Filter Settings are adjusted AND the selected Metric is specific to job analysis For instance the default Metric will always be Confluence even if an Analysis Job has been opened Because adjusting the Filter Settings does not affect the Confluence Metric no warning will appear until a Metric has been selected that is affected by adjusting the Filter Settin
262. sis Filter Settings Pane has been opened Channel A is set to Fluorescence and Channel B is set to Object Segmentation Mask In this figure the Show Object Centers box is not checked Object Segmentation Mask There are two check boxes available when Object Segmentation Mask is selected in the A or B Channel pull down window Use Filter color Check this box if you want a color to be applied to all Objects that will be excluded from analysis via the Filter settings To select a specific color for the Filter Color click directly on the colored box this box is set to blue in Figure 9 6 see Section 9 3 8 Filter Settings page 224 for further information on applying Filter settings Show Object Centers Check this box to show the center of each Object The center of each Object will be indicated with a red X When you first launch Analysis Preview Mode this 214 Page box will be checked However if you uncheck the Show Object Centers box the centers will not be shown the next time Preview Mode is launched Analysis Filter Settings Segmentation and Refinement Parameters identify potentially relevant Objects within an image area In Figure 9 6 the Segmentation Mask has identified all the Objects that meet the criteria for inclusion as defined by their fluorescence intensities and proximities However the User might choose to use additional criteria to eliminate some of the Objects from graphical analysis and or exported data
263. sk Microplates Corning 25 3055 3056 Corning 25 A Tray 1 TENG 16 1315 151 Corning 162 Tray 4 60 150 354486 354495 354538 354645 354 BD Falcon 150 Tray 5 BD 175 353028 353045 353112 353118 354 BD Fakon 175 Tray S Corning 175 3292 431079 431080 431085 43130 Corning 175 Tray S Nunc 175 159910 159920 Easy Flask 175 Tray S Nun 175 156502 178883 178893 Nunclon 175 Tray S v lt gt For older style trays that require disks to hold vessels see the Help Unavailable Trays menu in the main window Figure 6 2 Select Find under the Tray Type pull down menu to obtain a complete list of compatible vessels and their corresponding Tray Types The example above indicates that the Corning 162cm flasks with catalog numbers 3150 or 3151 require Tray Type 4 In some cases all the catalog numbers for a particular vessel type will not be visible at one time This is the case for the BD 150cm2 vessel To view the remaining catalog numbers place the mouse at the Catalog s Flask title border and drag to the right 26 Page 6 1 2 Selecting Trays for the IncuCyte EX The IncuCyte EX is equipped with a single tray that is built into the instrument Therefore it is not necessary to select trays for the IncuCyte EX 6 1 3 Placing Trays into the IncuCyte All trays need to be placed into the IncuCyte drawer in the proper orientation To ensure proper orientation each tray contains three Alignment Windows at it
264. sks Pane page 133 Analysis Jobs Open and Manage All Analysis Jobs that have been run or are currently running will be displayed in this window Opening and Managing Analysis Jobs will be discussed further in Analysis Jobs Open and Manage page 206 Object Counting New Analysis Optimize preview the parameters for Object Counting and launch a new analysis See Section 9 3 5 page 215 The opened Object Counting Task Bar is displayed in Figure 9 4 206 Page Vessel View 7 00 AM on 3 2 7009 T Vessel View 7 00 AM on 3 2 2009 Tasks Tasks x Utilities _ Utilities Analysis Jobs Open and Manage Analysis Jobs Open and Manage A Object Counting New Analysis A Object Counting Mew Analysis l Params Manual Selection E Parame Manual Selection we Segmentation Adaptive v1 0 Segmentation Fired Threshold ka Background Intensity Fixed Threshold Foreground Intensity Refinement Mone Manual Adjustment Refinement Edge Split Edge Sensitivity 4 i 4 Las Preview using Curent image ESS Preview using current image Launch new analysis Launch new analysis AC Save analysis parameters d Save analysis parameters Figure 9 4 The IncuCyte FLR Tasks Pane Three Task Bars are visible Utitilies Analysis Jobs Open and Manage and Object Counting New Analysis
265. spactoe ate oats seat acne ote 149 Background Intensity ccccceeeeeeeeeeeeeees 209 bannet cece eee e ee 210 211 212 222 223 228 Calendar Mode ecceeceeeeeee 187 188 189 Calibrate DEVICE sivsciviviedcteitcnvedeesscersedvenseieteecdss 32 Calibrate EX oiicvanssonsieanteacacancabenspetatvaneessenwacctaas 102 Calibrate FLR 2 senssancencsoasanascabineivtoteantessenvaaceans 102 Calibrate IncuCyte HD eens 100 Calibration c eee eee eee 29 30 31 60 97 98 Calibration 29 30 97 98 99 121 122 Calibration slides cceeecceecescesceeceees 33 103 eG E ee ccceteesteiees 36 73 74 Change Password cccccccsssssssseseseeeeeeeees 22 95 CHANGE SPALL iien 155 156 Close Connection Sscsdecsiadveeedescedscavarsdesseswedewen 119 Closing an Archive ccccccccssssssssessseeeeeeeeees 119 Condensed Metrics cscceeceeceeceeees 127 131 controller 8 9 10 13 16 17 18 19 21 23 24 90 91 93 106 107 111 113 117 Create New U Sei vscacvicccehienncerstereses 21 22 95 Custom Scan Fields cc cece ceecccescceecceeseeees 75 Custom Vessel Fields 75 164 165 Data Management c cc ceececcccecceesseeseeeeeeees 107 Daylights Savings Time ceeeseeeeeees 91 93 Delete Scans svsaidiceseaucasasGvsdicrevaenvaionds 109 113 114 Delete USC ears Th ecard vac Mei otters esennceasiiats
266. sseececceaaaaaseeecceessaagaeeseeeeeaas 53 Epor of Graph Dalil iisisersipesss iaren wine ni aaascisisc eae a 54 Saving and Printinge Graphs 2 2ci 2065sccessessaderasscaneravaiadce csasauaeeasndeeeiadeaea Hd Ree 55 SOFTWARE REFERENCE sssccisessiiiscsseerssecsdaestinsreasitavnacsannnaidenniseietiniinainasss 56 CONNECTIONS BOX sees ne a EEEE EE EE 57 DENDA e E E E E 58 NET CAN IM E E E E 59 TATE a E E E 59 S HEDOLING G CAN ppsa meen errr E E E S E er 60 PE E EE AEIR OAE E A E T ae ce E T terete 61 Setting Vessel Properties FLR siscccenconcuuswsservieossdeudsaxisteientesdsdeentedeseessddudecedetsvsossachieentarsees 66 Sene Scant P ACIN oaas as EEEE EEEE EEEE REEE EEEE 67 Senne Scan T alera TA resar ien Eaa E EENE EENE EEE EEE 70 Setting Vessel Proper S winncuasexsatuitenconcssivsinttavseasedludsestativis atonaealeincedsileateddederedeaetsadendhdiexteestes 73 Setting Vessel Properties PX seccsnvadsacenchitpnxbeasitensesdutssxiadedeasesaeisisdeasieosdssdecetatiedsotescvieindeesiss 75 S CAN Cl VIVES A E AAE EA 76 E 1 AEAEE EOE A EEE A O 8l SEON TIE sipe E E 83 maie Lock AMON E esr EEEa 85 marel ock VOU T LEren an E E sa taciouas asda AEE SO OL a hag erat ce ecco ashe saree g E A A nosed 86 LOTOU E TE A A 86 ADMINISTER INCU VTE oriee a a aine 87 Administer IneuCyle MEX isipite niipea cai iania ii iar ani eiii 59 OPA el aid 071s eee mneere eC TT TRCEETeE ECCT ETe MT TTNT CT TTT Tr MreTrte Tarr rer rreT rere Terectrr rer tre reer rT erat errer ret et 90 TR
267. ssel Properties are displayed image tab images 1 4 can be selected If here Use the View menu at the top only one image were obtained per sector then this box would be labeled 1 of 1 and the box would be inactive of the screen to select which properties to display Figure 7 5 The View Completed Scans Screen 45 Page a ESSEN BIOSCIENCE 7 2 2 View Completed Scans EX IncuCyte File View Scan Preferences Help Ex20003 admin Ready To Scan Search Scans By Bar Code Task List 3 14 2008 2 02 00 PM A Find Scanned vessels z j Selected Vessel EOE O8y03m14d14h00n gtF a Administer IncuCyte Completed Scans February E March 01 Sat E 02 Sun H 03 Mon H 04 Tue H 11 Tue 3 14 Fri 10 45 AM 10 47 AM Image Properties Metrics Vessel Scan D6 Image 1 of 1 Graph Export Figure 7 6 View Completed Scans Screen in the IncuCyte EX When the Standard Model IncuCyte software is launched it opens by default to the View Completed Scans screen however when the IncuCyte EX software is launched it opens by default to the Find Scanned Vessels screen This difference reflects the more vessel centric nature of the IncuCyte EX Nonetheless the View Completed Scan screen can be selected in the EX by choosing the second task bar from the top See Figure 7 6 Note that in the EX only a single tray is visible and the Schedul
268. sssteatsserbieeseisdeciierss 246 APPENDIX INCUCYTE SUPPORTED VESSELS sssssssssssseeeeceeeeees 247 16 D N ESSEN BIOSCIENCE 5 Page N 1 Warranty Your Essen BioScience Inc Essen IncuCyte product is warranted against defects in material and workmanship for a period of one year following delivery to the Buyer Essen warrants to its original Buyer only The Buyer must notify Essen in writing within fifteen 15 days following discovery of the defect During the warranty period Essen will at its option either repair or replace products that prove to be defective For warranty service or repair this product must be returned to the Essen factory Buyer shall prepay shipping charges to Essen and Essen shall pay shipping charges to return the product to the Buyer However Buyer shall pay all shipping charges duties and taxes for products returned to Essen from another country 1 1 Limitation of Warranty The foregoing warranty shall not apply to defects resulting from improper or inadequate maintenance by Buyer Buyer supplied software or interfacing unauthorized modification misuse or operation outside of the environmental specifications NO OTHER WARRANTY IS EXPRESSED OR IMPLIED ESSEN BIOSCIENCE INC SPECIFICALLY DISCLAIMS THE IMPLIED WARRANTIES OR MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE 1 2 Exclusive Remedies THE REMEDIES PROVIDED HEREIN ARE BUYER S SOLE AND E
269. stions related to your local network configuration and security software 23 Page IMPORTANT Always use the Disconnect option and not the Log Off option when terminating the Remote Desktop Connection via the controller Start menu 24 Page N 6 Preparing for Cell Culture Monitoring Now once the IncuCyte is fully installed connected and User accounts have been established you re almost ready to start cell culture monitoring Just a few remaining points need to be covered before we can get to this final step These include e How to select and place trays into the IncuCyte e How to add flasks plates or dishes to the trays e And importantly how to calibrate the instrument 6 1 Selecting and Placing Trays and Vessels into the IncuCyte 6 1 1 Selecting Trays Tissue culture vessels are held within the IncuCyte drawer in trays The IncuCyte drawer can hold a maximum of three trays and each tray will hold different numbers of vessels depending on the tray type Vessel A vessel is a general term used throughout this manual that refers to a cell culture flask plate or dish A table of all available trays and compatible vessels is listed in the Appendix at the end of this manual This same list can also be accessed through the IncuCyte software by selecting Find from the Tray Type pull down menu in the Schedule Upcoming Scans screen see Section 8 5 1 page 61 The IncuCyte can be configured wi
270. t in its entirety If this recommendation is not feasible archives can be made on a weekly or monthly basis It is not recommended to archive more than three months worth of data to a file share across a network Due to network limitations and traffic the archive may timeout resulting in an incomplete file copy 110 Page Archive Scans Use the Archive Scans task to move scan data from the IncuCyte controller hard drive to a new destination All scans available for archiving are displayed in the Time Tree in the Choose a Time box To select a single time for archiving highlight that time with the mouse To select a series of scan dates times for archiving highlight the first desired scan time with the mouse hold down the Shift key and then select the final desired scan time All scans including and between the first and last selected scan times will be highlighted for archiving To select several scan times for archiving that do not occur in a series highlight the first desired scan time hold down the Ctrl key on the keyboard and then select all remaining desired times As indicated in the Scans Screen selecting a day month year etc will archive all scans within that selected timeframe In Figure 8 63 the scans for Saturday January 12th 2008 2 19am through January 12th 2008 11 19pm have been selected for archiving January W 09 iwed 10 Thu 11 Frit i Sat 13 Sun 14 Mon Figure 8 63 A se
271. t it will not be included in movies or exported images In Figure 8 88 the Legend is turned on Measurement Mode Use Measurement Mode to make linear measurements within the image To turn Measurement Mode on or off click on the ruler icon next to the Show Legend box at the bottom of the Vessel View window See Figure 8 91 and Figure 8 88 Show Legend Figure 8 91 The Measurement Icon is a blue ruler located to the left of the Show Legend box 1385 Page When Measurement Mode is on the background color of the ruler icon will change color and a dark bar will appear in the bottom right hand corner of the image Use the mouse to draw a line across the image The length of the line in um will appear in the dark bar at the bottom of the screen Measurement Mode can be used in full sized and zoomed images Mouse Over Pixel Location Readout Holding the mouse over any region of the image will display the exact coordinates for the currently selected pixel Properties Tab Selecting this tab displays the properties of the selected vessel These are the same properties as discussed in Properties page 129 with the addition of one additional Property Vessel Location Properties Tab EX Recall that vessel Properties can be viewed but not added or edited in the IncuCyte EX The built in Properties Cell Type Passage etc are available under the Properties tab See Section 8 5 6 page 75 for more information Graph Export Tab
272. t with customization Once this Sequence Type is selected click on Image Designer Crop Scale Legend etc above the Export button at the bottom right of the Movie Image Set Export window A new window appears that is virtually identical to the Export Designer shown in Figure 8 97 As discussed in that section the designer is used to interactively crop and scale the image and to add a legend or timestamp to each image if desired There is an important preview tool found in the Export Designer and we strongly recommend that the designer and previewer be used prior to launching a full image set export with customization 148 Page Export Movies Overview A single movie can be created and exported or multiple movies can be created and exported in bulk Because a movie by definition is comprised of a minimum of two images at consecutive time points movies CANNOT be generated if the Single Time option is selected in the pull down menu adjacent to the Time Tree Movies can be created in one of 2 common formats Sequence Type e Windows Media Video e Windows AVI Video Similar to image export movies can be exported using the Current Display Settings or the Phase Contrast Original settings Similar to export of single images scan patterns with an excess of 9 images per well sector require selection of a single image per well sector or all images simultaneously Slideshow Mode As a quick alternative to creating a
273. te functioning Try switching to a different plate or maybe to a different brand of plates ESSEN BIOSCIENCE Cells were scanned shortly after being seeded and some cells are still floating represented by large out of focus circles Severe scratches on the bottom of the flask 238 Page N ESSEN BIOSCIENCE This is another example of condensation on the bottom of the wells Out of focus cells are visible through the condensation microns The flask was not seated properly and the image is uniformly out of focus There is a scratch on the bottom of the well The IncuCyte focused on the scratch instead of the cells so that the scratch is in focus and the cells are out of focus 239 Page N ESSEN BIOSCIENCE A large bubble is floating on the surface of the media creating a highly distorted image This image of confluent cells has virtually no contrast A large droplet is probably suspended from the top of the vessel destroying the contrast Alternatively a meniscus is creating the problem Sometimes in shallow cell culture vessels the media can form a bridge between the top and bottom of the vessel This bridge will act as a lens and destroy the image quality 240 Page A ESSEN BIOSCIENCE 11 2 Contacting Technical Support If you are experiencing technical difficulty with the IncuCyte or there is a Device Error in the status bar follow the instruc
274. te clear 253 Page iy 384 Well Plates Brand BD Falcon Corning Greiner Matrical 0 U eCrnine T Catalog s 354662 354663 354664 354666 354667 354831 354832 354833 354835 354836 354837 356660 356662 356663 356664 356666 356667 356694 356696 356697 356704 356705 356831 356832 356833 356835 356836 356837 356931 356932 356933 356935 356936 356937 3662 3663 3664 3683 3701 3707 3712 3985 DO JO Plate Name Tray Type Poly D Lysine 384 well Microplates Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Microplate Collagen 384 well Microplates Microplate Collagen 384 well Microplates black clear Microplate Poly D Lysine 384 well Microplates Microplate Collagen 384 well Microplates clear Microplate Collagen 384 well Microplates black clear Microplate Poly D Lysine 384 well Microplates black clear Microplate Collagen 384 well Microplates white clear Microplate Poly D Lysine 384 well Microplates white clear Microplate Collagen 384 well Microplates clear Microplate Poly D Lysine 384 well Microplates black clear Microplate Poly D Lysine 384 well Microplates clear Microplate Collagen 384 well Microplates Microplate Poly D Lysine 384 well Microplates clear Microplate Poly D Lysine 384 well Micr
275. ted using a sequence of images within a selected time frame Both Image and Movie export functions need to be accessed through the Vessel Window by e Selecting the Utilities Pull Down menu or e Revealing the individual Utilities in the Vessel View Tasks Pane see Figure 8 95 Vessel View 10 15 AM on 2 25 2009 Tasks k Utilities Eii Graph Export _Ltilities View large current image Export current image Export movie or image set Export current image Export movie or image set View large current image Archive current vessel Archive current vessel Figure 8 95 A cutout from the Vessel View showing the utilities accessed by the Utilities pull down menu or in the Tasks Pane left 141 Page Export Images Overview Images can be exported as either a JPEG or TIFF file The Image can be exported as one of three image types e Phase Contrast using the current display settings JPEG e Phase Contrast Original JPEG e Phase Contrast Original 8 bit TIFF Phase Contrast using the current display settings JPEG This function will export the image using the currently displayed image settings for instance if the brightness and or contrast settings have been adjusted Zoomed images can also be exported by selecting the Maintain CurrentView Zoom check box but note the special circumstances that might apply See Figure 8 96 Export of images using Current Display Settings i
276. ted scans can be searched by using the Search function See Section 8 7 9 page 153 8 7 9 The Scans Pull Down Menu The Scans pull down menu at the very top of the page is only active in the View Completed Scans screen See Figure 8 102 Each function within the menu is listed in addition to the corresponding key presses that can used to access the function directly At the Administrator level all functions are available however at the User Guest levels the Next and End Scan options are inactivated The functions available are as follows 152 Page N ESSEN BIOSCIENCE IncuCyte ile View Scan Preferer ag Help Flr30001 fadmin Search Ctrl ShiFt 5 Show Log Cbrl ShiFE L First Scan Ctrl Shift F Previous Scan Ctrl Shirk P Next Scan Ctrl Shift h End Scan Ctrl Shift E view Vessel Ctrl Shift Graph Export Ctrl ShiFk 6 Archive Vessel Ctrl Shift 4 Delete Vessel Ctrl Shift 0 Jump To Stark Ctrl ShiFk Change Start Cbrl ShiFk C a E 5 Figure 8 102 The Scans Pull Down Menu Search Completed scans can be searched in one of two ways e Using the Search box at the top right hand corner of the screen e Using the Search option under the Scans pull down menu The Scans pull down menu offers a more comprehensive selection of search criteria than the Search box in the corner of the screen See Figure 8 103 Search by Label Cell Type Passage User Name or Notes The Search option is NOT available
277. th any combination of trays and vessels from this list While the table in the Appendix contains all vessel types and trays available at the time this software version was released it is likely that new vessels and trays will be added to the list as they appear on the market and as customer demand dictates These additions can be downloaded into the IncuCyte vessel database See Section 8 6 6 page 106 regarding the procedure for updating the vessel database In order to further facilitate tray selection each tray will have its Tray Type number stamped on the bottom portion of the tray Additionally the shape of each tray will be displayed when it is selected within the software See Figure 6 1 For more information see Section 8 5 1 Physical Layout page 61 25 Page A ESSEN BIOSCIENCE Physical Layout Properties Rear Tray Tray Type Vessel Type Scan Type Scan Pattem New Figure 6 1 Tray Type 1 The physical appearance of Tray Type 1 displayed within the IncuCyte software Schedule Upcoming Scans screen The four spaces for T 25 flasks are indicated as Empty Grab border with the mouse and drag to the right to expand list of catalog s Or hover over to view data Double Click on a Row Io Select the Vessel Flasks Plates Dishes Sides Type Area cm Catalog s Flask Tray Type A GreinerjGNF 84 779160 AutoFlask Microplates Corning 93 3059 3067 3068 3069 3070 RoboFla
278. that the changes made have been applied i e saved Now the IncuCyte is ready to start scanning The time for the next scheduled scan will appear at the top of the IncuCyte screen IMPORTANT The Apply button MUST be selected before new parameters will be saved and scanning will be initiated 80 Page Processing Metrics and Scan Overlap After scanning has been completed the IncuCyte must process the data in order to generate images and corresponding metrics confluence and associated statistics Processing of metrics will only commence after scanning is finished Therefore the results of the scan will not be immediately available after scanning is complete The duration of metrics processing will depend upon the number of images obtained If the between scan interval is very short then it is possible that the next scan will start before metrics processing is complete In this case the processing will be interrupted to enable the next scan However the interrupted processing will continue after scanning has been finished If there is a series of such scans then the processing will require extra time to catch up after completion of the final scan 8 5 8 Vessel Scheduling To avoid prolonged scans greater than 45 minutes or to customize the order in which vessels are scanned click the Vessel Scheduling button at the bottom of the Schedule Upcoming Scans screen The Vessel Scheduling window displays a list of all scheduled
279. that the manual warm up process is omitted for instance if the IncuCyte is temporarily removed from the incubator and the person replacing it forgets to run the manual warm up procedure afterwards then an auto warm up process will be initiated The auto warm up is NOT intended as a substitute for the manual warm up cycle but rather it is a backup process that occurs in those instances where the manual warm up is accidentally omitted When auto warm up starts the microscope is powered on and the front panel LED lights up until the camera achieves the requisite temperature If the microscope is placed into the incubator cold then the auto warm up process is automatically triggered Auto warmup keeps the camera powered on for up to 90 14 Page minutes until it reaches a temperature slightly above that of the standard incubator temperature Auto warm up cannot occur if the controller is not powered on The auto warm up cycle will be interrupted by any and all device activities such as a manual warm up a device test or a scan However auto warm up will resume if the camera has not achieved its target temperature after the interrupting device activities have been concluded 3 4 2 IncuCyte Power Supply Filter The filter for the IncuCyte controller power supply is located behind the front panel see Figure 3 2 Although this filter is easily accessible it is NOT a consumable part and Users should not attempt to exchange it t
280. the SHIFT or Ctrl key and then selecting the next space s with the mouse Alternatively all soaces can be simultaneously highlighted by pressing the Select All button To add a vessel select the Vessel Type pull down window This menu will list all compatible vessels by their common names Choose the desired vessel and it will populate all highlighted spaces in the tray Select All Activate highlight all spaces Select None Deactivate unhighlight all spaces Remove Vessel To remove a vessel right click on it and select Remove Vessel 64 Page N Y p if BS Ess BIOSCIE Rear Tray Tray Type Tray 1 Vessel Type Coming 25 A 5r A Scan Type Phase Contrast Scan Pattem Sample Pattem New Select Al Select None _ Figure 8 10 Upcoming Scans Tray after adding a flask Clicking the Select All button will highlight all spaces in a tray If a vessel type is then chosen that same vessel type will be added to all the spaces If multiple spaces have been simultaneously selected using either the SHIFT or Ctrl key these spaces will also be assigned the same vessel type To choose different vessel types within a single tray each must be added individually The Vessel Type pull down menu includes an option for Generic This option is available for those instances when a specific vessel desired by the User is not available through the vessel pull down menu Under these circumstances add the desir
281. the Graph option from the Scan pull down menu Section 8 7 9 page 152 e Right clicking on a selected vessel in the View Completed Scans screen See Tasks Available by Right Clicking on a Selected Vessel page 131 Note that graphing from the Vessel View window offers some additional options that are not available via the View Completed Scans screen To access graphing functions through the Vessel View window select the Vessel View Graph Export tab see Figure 8 120 and then press the Graph Export button Pressing this button will open a new window See Figure 8 122 page 170 Metric Image Mean Focus Position Exposure Time Autofocus Sharpness Statistic Mean 4 30 PM 7 30 PM 10 30 PM Start Time 20 Sun 1 30 AM 4 30 AM 7 30 AM 10 30 AM lt Time Range 1 30 PM 4 30 PM 7 30 PM 10 30 PM 21 Mon k To ain End Time 4 30 AM 1 21 2008 1 30 PM 7 30 4M 10 30 AM Region All Wells Group A v Figure 8 119 The Graphing Box as Displayed in the View Completed Scans Screen 166 Page ESSEN BIOSCIENCE Vessel View 2 40 PM on 2 25 2009 1C10094 admin E 2009 February 25 Wed 2 ada OOG AL G 6 119 ofDNA Bi 53 OB Comttence vit we ee we wit Clee lectlecslecslecsies Al Image 1 of 4 i Graph Expott Sector Mean Median
282. the Schedule Upcoming Scans task bar This will bring up the corresponding Schedule Upcoming Scans screen Figure 7 2 page 37 Make sure that the vertical 24 Hour Repeating tab has been selected The following parameters need to be set in order to set up a scan The Scan Active Light and Stop Button Scan Active Light Note the green Scan Active light on the upper right corner of the IncuCyte microscope This light will be on if a scan is in progress It will also be lit under some other circumstances including the IncuCyte Self Test Section 8 10 6 page 199 and other tests such as the Camera Test or Optics Test When the Scan Active Light is on the IncuCyte tray drawer will be locked shut Do NOT try to force the door open while the light is on To interrupt any ongoing IncuCyte activity press the Stop button See below The Stop Button The Stop button is located on the front of the IncuCyte microscope next to the Scan Active light Press the Stop Button to interrupt any ongoing IncuCyte activity The most common reason to press the Stop button will be to interrupt an Ongoing scan and open the drawer If a scan is ongoing and the Stop button is pressed the camera will terminate scanning and return to its home position This homing process will result in a short delay between pushing the button and drawer accessibility If you want to interrupt the scan and open the drawer press the Stop button wait for the micro
283. the Synchronize button and then press OK in the new window that appears The times will be is instantly synchronized without leading to any interruption in the connection Device System Control Shutdown Use the Shutdown button to power down the instrument This is the recommended method for shutting down IncuCyte and is analogous to the Shutdown command on your computer Restart Click on the Restart Device button to reboot the IncuCyte controller Under normal circumstances the only time the User will restart the device is during the initial installation procedure according to instructions However some IncuCyte functions will also cause a restart Such as synchronizing the device time zone daylight savings Export This is a trouble shooting function that an Essen BioScience representative will request in the event of a problem The export will include or more of the following e Diagnostics e Events e Setup Files e Export Database 94 Page N Appropriate export instructions will be provided e Tests 8 6 3 Accounts Tab The next tab in the Administer IncuCyte screen is for Accounts Every IncuCyte User must have an account created on each machine in order to access the instrument Accounts can only be created and deleted by an Administrator A new account can be created for a User at one of three permission levels e Guest e User e Administrator For more information on User permission leve
284. the scan times were not so long the additional scan time would simply remain The scan could proceed under these circumstances but the 2 hour time interval would contain an extra scan between 4 and 6 pm 2 scans are clumped together at the 4 pm time point Figure 8 30 zE I 4am 6am 8am 10am 12pm 2pm 4om 6pm 8pm 10pm 12am Figure 8 30 Scan overlap eliminated with shorter scan time Scan Length FLR Fluorescence amp Phase Contrast scans are approximately twice as long as standard scans because the vessel will be scanned in both fluorescence and phase modes Thus each image can be viewed as a fluorescent or HD image See Section 9 2 page 202 NOTE For a record of the temperature within an IncuCyte FLR click the graph box at the bottom left of the screen next to the current temperature reading in the Device Status box See IncuCyte FLR Temperature History Scan Overlap lf two Scan Bars are set at the same time or if the Scan Bars are dragged so that they overlap the offending Scan Bars will be highlighted in red In Figure 8 29 there are two scan times to the left that overlap If the User attempts to apply these scan times a pop up window will appear indicating that there is an overlap problem Overlapping scans must be eliminated before scanning can occur After scan times have been set click on the Apply button at the bottom of the screen to initiate the new scan schedule A new window will pop up indicating
285. the tray types In addition the user must swap out the correct trays within the IncuCyte microscope system itself paying special attention to the location and description of the vessels that are removed so that they can be correctly replaced once the Scan on Demand has been finished In addition to a Label users can also supply a Unique ID to each vessel This Unique ID can either be generated manually by typing or pasting information into the Unique ID field or a Unique ID correlating to the current year day time will be assigned to the Scan on Demand vessel This information can be used to link Subsequent scans together However it is important to note that if the user fails to type in or select using the load button the correct Unique ID scans will not be linked together and cannot be accomplished post scanning 12am Zam dam bam Sam 10am 12pm 2pm dpm 6pm apm 10pm 12am Figure 8 32 Scan on Demand time bar illustrating acceptable scan Within the Scan on Demand scheduler the 24 Hour Repeated Scan schedule including all of the time bars will be faded to the background but will be clearly visible See Figure 8 32 The current time will be indicated as in the 24 Hour Repeated scheduler but will be followed by the Scan on Demand Time bar in yellow Similar to the 24 Hour Repeating Schedule the width of the time bar will correlate to the amount of time required to scan the vessel inserted into the Scan on Demand Tray 12am 2am dam
286. tion Tray is included with the IncuCyte for this purpose Figure 8 49 The calibration tray is the same size and is inserted in the same fashion as the regular trays Calibration is required before the instrument can be used for the first time in order to establish proper internal alignment Over time however proper 97 Page N calibration may become somewhat compromised especially if the instrument is physically moved If there appear to be any problems with image alignment during scans it is recommended that calibration be confirmed CALIBRATION TRAY Figure 8 49 Calibration Tray To calibrate complete the following procedure Place the Calibration Tray into one of the tray positions the order in which tray positions are calibrated is not important but all 3 should be calibrated For the example in Figure 8 50 start with Tray Position Front The Front Middle and Rear tray positions can be selected using the pull down menu Each Tray Position must be individually calibrated and or confirmed Select Test There are 3 test options to choose from Calibrate Device Tray Position Test Calibrate and Confirm Calibrate and Confirm Run this test the first time the instrument is calibrated Press Run A window will appear to confirm that the Calibration Tray has been loaded into the appropriate position Select Yes and a new window will appear confirming that calibration has begun press OK The IncuCyte will cal
287. tions below to export and email a log file to Essen BioScience technical support 1 Log into the IncuCyte software GUI with an account that has administrator or user permissions as shown in Figure 11 1 2 Click on Administer IncuCyte button on the left hand side of the software Click on the Logs tab on upper part of the screen 4 Then click the Export button FLR30003 admin gt Next Scan 2 hours 11 minutes 5 00 PM Search Scans By Label for GO File View Scan Preferences Help Task List erortonl aa o EES 17 2010 12 54 01 PM Scan started T 35 8 17 2010 12 51 49 PM admin Applied Active Upcoming Scan 2 17 2010 12 46 52 PM Drawer closed_ a 17 2010 12 46 25 PM Drawer open 17 2010 12 46 19 PM Drawer closed 17 2010 12 45 59 PM Drawer open 17 2010 12 41 02 PM gt gt 384 wel Plate Calibration completed successfuly lt lt Device Status gt gt 384 wel Plate Calbration of the Rear Tray Start 08 17 10 12 38 56 Wel Offsets for Cutout 0 Al 0 0799 0 17 A2 0 12 0 17 A f 0 101 0 17 A4 0 114 0 17 AS 0 0948 0 17 A6 0 0725 0 17 A 0 0799 0 17 AB 0 123 0 17 A9 0 163 0 17 A10 0 156 0 17 A11 0 142 0 17 A12 0 147 0 17 A13 0 166 0 17 A14 138 0 17 AL5 0 142 0 17 AL6 0 153 0 17 AL7 0 157 0 17 Al8 0 129 0 17 AI9 0 175 0 17 A20 0 203 0 17 A21 0 214 0 17 A22 0 156 0 17 AZ3 0 172 0 17
288. tive to the colors selected in the Plate Map Editor For more information on Microplate Graphs please refer to Section 8 10 6 page 199 If individual graphs are desired users can divide the plate into regions and graph on separate axes Regions of a microplate vary from large to small encompassing the entire plate single columns single rows or individual wells With the exception of individual wells graphical results can be viewed as grouped or not grouped 168 Page N Regions and Grouping Select the Region pull down menu to display all possible microplate regions See Figure 8 121 Region All Wells Single Column Group Single Row Si ng le Well Figure 8 121 Select Region All Wells Select All Wells to simultaneously graph the Mean or Median for all wells within a microplate The Region All Wells meaning the entire plate provides the largest number of grouping possibilities See below e Group All All the wells of the microplate will be combined into a single trace e Group Columns All the wells of the microplate will be graphed as columns For example a 24 well plate will generate a graph of 6 columns e Group Rows All the wells of the microplate will be graphed as rows For example a 24 well plate will generate a graph of 4 rows e Group None In this case none of the wells will be grouped A 24 well plate will generate a graph of all 24 wells Error bars are not available when single wells are g
289. ty of all objects within an image divided by the image area in mm Export of fluorescence scan graph data is the same as export of non fluorescent graph data The FLR Time Plot View Menu Two options are available in the FLR Time Plot window View pull down menu Error Bars Toggle Error bars on off If Error Bars are selected choose between Standard Error or Standard Deviation Show Analysis Filter Choose whether to display any Analysis Filter Settings in the Graph window Graph Export Histograms of Processed Jobs lt is possible to generate histograms from Object Counting data Histograms can only be generated for a single time point so select Single Time instead of Time Range and then select the Histogram radio button If the Histogram radio button is selected without first choosing Single Time the selection will automatically be reset from Time Range to Single Time The Histogram window is now active Three Metrics are currently available for Histograms Object Area um Object Mean Intensity Object Summed Intensity These same Metrics are available for Time Plots except that in the case of Time Plots the plot represents average values over time Histograms display the distribution of the Metric at a single time point over the designated spatial grouping As for Time Plots Histogram spatial distribution can be anything from a single image to every scanned image To customize your Histogram press the Histogram Bin Settings
290. uCyte and the IncuCyte EX The software functions available at the various permission levels are summarized in the tables displayed in Figure 5 2 and Figure 5 3 N Additional information on creating User accounts can be found in Section 8 6 3 page 95 5 4 Remote Administration of the Controller The IncuCyte controller consists of a single board computer running a Windows based embedded operating system This computer is dedicated to controlling the IncuCyte hardware and providing remote access to the data collected by the instrument To insure reliable and proper operation of your IncuCyte the factory settings of the controller operating system should not be altered However we recognize that there may be instances when it is desired or even required to log onto the embedded controller desktop Some examples include 1 Renaming the controller from the factory set name 2 Configuring the primary Ethernet port with a static IP address 3 Installing anti virus and or firewall software 4 Installing Essen recommended Windows updates The IncuCyte controller has been shipped with a unique Administrator password for accessing the controller s Windows desktop Essen BioScience maintains a record of this password so we strongly discourage you from renaming the Administrator password We also recommend that this password be protected and that the controller operating system be accessed only out of necessity Altering t
291. ugh the scan times Figure 8 77 Scroll up and down one data time at a time Figure 8 78 Jump to Last Scan Figure 8 79 Jump to First Scan In the center of the screen the selected scanned vessels can be viewed The three filled trays are indicated by black arrows in Figure 8 80 124 Page ESSEN BIOSCIENCE Figure 8 80 Trays are indicated by black arrows Sector Shading is unselected and the sectors are all white The following images information will be visible on this screen e Each scanned vessel e Within each flask dish or well the scan pattern is indicated as a pattern of dots e A box is displayed on each scanned vessel listing the vessel s properties These properties can include the Label Cell Type Passage Metric and Sector Shading The number of characteristics displayed within this box can be adjusted under the View pull down menu at the top of the screen Figure 8 81 125 Page N ESSEN BIOSCIENCE IncuCyte File View Scan s Preferences Label Cell Type Passage Metric Error Bars Sector Shading Condensed Metrics Figure 8 81 Select the characteristics displayed under the View Completed Scans Screen e Sector Shading A graphical representation of the sector or well Metric this will generally be confluence value can be viewed by selecting Sector Shading from the View pull down menu see Figure 8 81 Each sector or well will be shaded grey The darker the shade o
292. unction can simultaneously filter the results within both the Label and the Cell Type columns For example Refine the search in Figure 8 114 by the term Weekend The Refine Search results are displayed in Figure 8 115 Now the Vessel with the label Test has been eliminated and only 5 vessels are displayed instead of 6 Search By For tt When was the vessel scanned Refine Search By 5 Vessels Shown Columns Start Cate Time End Date Time Label Cell Type 1 4 2008 5 38 PM 1 4 2008 5 38 PM 1 4 2008 6 23 PM 1 4 2008 6 23 PM 1 4 2008 6 23 PM 1 7 2008 2 00 PM 1 4 2008 6 00 PM 1 4 2008 6 00 PM 1 7 2008 2 00 PM 1 7 2008 2 32 PM Weekend Test Weekend Test Weekend Test Weekend Test Weekend Test ALO Figure 8 115 Refine Search by weekend 162 Page N In summary the rules for the Refine Search By function are as follows e Only a single term can be searched e tis NOT possible to use the symbol as a wild card e The Refine Search function ONLY filters the results DISPLAYED as a result of the initial coarse search e The Refine Search function ONLY filters the Label and Cell Type columns It does not apply to the Start Date Time End Date Time and Passage columns To search according to dates or passage use the Passage option in the coarse search and or define the specific dates of interest Search Results The search results will include any vessel that was part
293. ure 8 13 Right click to select or deselect a Scan Pattern by All Row or Column Alternatively multiple wells can be selected by clicking and dragging with the mouse The selected number of images per well sector will appear in each well or sector The scan pattern can also be removed by clicking and dragging while holding down the Alt key See Figure 8 14 After a new scan pattern has been selected the Save button will become active Click it to save the new pattern Pattern Editing Press Save when Finished editing Hold down the Alt Key to remove wells Click and drag to select and deselect groups of wells Images per well Figure 8 14 Close up of the Pattern Editing box within the Scan Pattern Manager Note the instructions for Pattern Editing lt Scan Pattern Manager for IC10104 Tray Vessel Available Patterns FEXAMPLE Pattern Editing Press Save when finished editing Hold down the Alt Key to remove wells Click and drag to select and deselect groups of wells Images per well i v Carca Figure 8 15 Scan Pattern Manager Window before saving 69 Page Once a new scan pattern has been created it can be renamed or deleted so the Rename and Delete buttons will become active for the first time Remember that the Sample Pattern cannot be renamed or deleted After the new EXAMPLE pattern has been saved it will appear as an active selectable option in the Available Patterns box When finished cre
294. ured Use the Windows XP instructions for the controller configuration After configuring the primary Ethernet port log off of the controller s desktop dismantle the 2 device network and reconfigure the PC Ethernet port to its original settings 19 Page N 5 Installation of the IncuCyte Control Software The IncuCyte control software is used to both configure the instrument to collect data and for post collection viewing and analysis of these data This software is designed to run on PC s running Windows XP or Windows Vista and requires an Administrator account to install Insert the IncuCyte CD and the installer should automatically run If it does not run open a window to the CD drive and run the IncuCyteSetup exe file NOTE This version of the IncuCyte Control Software is not backwards compatible with IncuCyte microscopes that are running earlier versions of the software While this version CAN be used to open data sets archived from earlier versions it CANNOT communicate with an IncuCyte controller that is running earlier software versions Contact Essen BioScience technical support for instructions for updating controller software 5 1 IncuCyte Control Software Installation using Windows XP 1 Review the license agreement To continue installation you must Agree 2 Choose a destination folder and Install 3 If prompted install NET Framework 3 5 4 Install the Windows Media Player Codecs
295. ves when excessive noise prevents useful interpretation of the results The smoothing feature should be used with discretion in order to prevent overt distortion of the data Estimate Rate of Change When rate of change may assist data analysis such as with cell migration assays see Scratch Wound Assay Application Note this option will provide an estimated rate for each point on the graph 197 Average Lines Together Create a graph showing the average of all plots on the current graph This options can also be used to average plots from different vessels by first combining plots from different graphs using the Drag Drop feature and then averaging the resulting graph NOTE Error Bars are NOT available on graphs created using the Average function Each of these functions opens a new graph window preserving the original 196 Page N ESSEN BIOSCIENCE it z i arin Qin Cred at Ore Harnad Agree Auto Agree Anon ee ee oS Crag et Orem ad Agreement Ato Aayan aaheouh ee rier Figure 8 157 Results of graph Smoothing The top left most graph is the original The middle graph was smoothed with 3 data points selected The bottom graph smoothed with 9 data points selected Congr Ab Weds Mean a Fone f Fie tdt Vew Toots AU SAREN WANA COMA AIWA BIDA Y NO WONM 1000M 108 perez Peers reser retest eeee Aye at PETEAR S We a j f Figure 8
296. view Mode or from Standard Mode by pressing the Launch New Analysis button in the Vessel View Tasks Pane A new window will open See Figure 9 7 215 Page ey ESSEN BIOSCIENCE m ry h H ER Select this Row Select All Deselect this Column Deselect this Row Deselect All Figure 9 7 Launching a New Analysis Preparing to Launch Press the Launch New Analysis button and make the appropriate selections in the drop down selection window The options include a specified time range a single time point or an open ended job An open ended job signifies that the experiment is currently ongoing and that you want each subsequent image in the sequence to be automatically analyzed per your job settings This allows automated processing of images in real time so that one big job does not need to be completed at the end of the experiment However it is critically important that users continue to qualify their segmentations masks in subsequent images to verify that the analysis parameters completely represent the fluorescence observed in the images 216 Page Select Spatial and Temporal Range of the Analysis Job The Job Range window allows the User to select the temporal and spatial ranges that the final analysis will encompass select Temporal Range of the Analysis Use the Time Tree to select the desired scan times The analysis must include a consecutive sequence of scans Select Spatial Range of the Analysis Use the m
297. w __ Select All Select None 24 Hour Repeating Middle Tray Tray Type None A m Vessel Type None x Scan Type Scan Pattem New Select All Select None Front Tray Tray Type None gt Vessel Type None x Scan on Demand unavailable AY Scan Type SMpty Scan Pattem New Select All __ Select None 8 5 1 Figure 8 5 The empty Schedule Upcoming Scans screen Physical Layout Tray Type Use the pull down menu to select a Tray Type The Tray Types are interchangeable and a maximum of 3 trays fit into the drawer at any given time Trays do not have to be present in all available spaces for the IncuCyte to function There are different Tray Types to accommodate the different available flasks and dishes however a single Tray Type accommodates all available microplates If no tray will be used select None All 3 trays must be individually selected If unsure which Tray Type to use select Find from the Tray Type pull down menu Find Selecting Find will bring up a new window listing all compatible flasks dishes and microplates along with their appropriate Tray Types Double clicking on a highlighted row will open the corresponding tray in the IncuCyte screen The contents of the Find window are organized under separate tabs for Flasks Plates Dishes and Slides The vessels within each tab are further
298. w Completed Scans screen vessel properties appear in a tab to the right of the screen under the Selected Vessel Layout and in front of each vessel 74 Page on the screen The Properties can also be displayed in the View Completed Scans screen however this is an optional feature The properties if any that are displayed for the completed scans can be adjusted using the View pull down menu at the very top of the screen The View menu will only be active on the View Completed Scans screen See Section 8 7 1 page 124 All lean wa Time Data from Expenment 1 Data from 7i Sat Experiment 2 Tara Ti A PS om brier Pelee Th 1A T Wio epee Figure 8 22 The New box was not checked at the beginning of a scan for a new vessel Experiment 1 Therefore when the flask is graphed the graph for the previous vessel Experiment 2 is connected to the graph for the new vessel 8 5 6 Setting Vessel Properties EX Vessel Properties CANNOT be set through the IncuCyte EX local interface Rather they must be set through the automated cell handler The automated cell handler assigns values to the built in IncuCyte vessel property windows such as Cell Type Label etc However the EX offers two additional tabs Vessel and Scan each of which offers a maximum of 5 Custom Vessel Fields and 5 Custom Scan Fields respectively Custom Vessel Fields are vessel wide properties that do not change from scan to scan An example would be
299. w well and secior labels All default or previously set labels will appear in the Plot Labels box while the currently highlighted label will appear below To change a label simply highlight the desired Plot Label with the mouse and then type the new label into the box underneath Do not click OK until you are ready to close the Titles and Labels window See Figure 8 133 Clicking on the Advanced button will bring up the Customization window Figure 8 132 Additional customization options can be accessed by right clicking on the graph background 180 Page Titles and Labels Graph Subtitle Plot Labels Coming 25 Coming 25 Coming 25 Coming 25 Coming 25 Coming 25 Coming 25 Coming 25 Coming 25 Coming 25 B Figure 8 133 Titles and Labels Window Drag and Drop Alignment Settings Task Pane The Drag and Drop Alignment Settings can be visualized by opening the Tasks Pane at the bottom of the Graph Window Click on the arrow on the blue bar at the bottom of the window to open or close the Tasks Pane In Figure 8 130 the Tasks Pane is open and in Figure 8 131 the Tasks Pane is closed Dragging Graphs There are two different circumstances under which graphs can be dragged using the IncuCyte software Dragging Graphs within the IncuCyte Software Create Plot Overlays Under some circumstances it will be convenient to combine traces created in separate graphs onto one single graph in order to facil
300. wer 15F 26 1 Sakuragaoka cho Shibuya ku Tokyo 150 8512 Japan Office Telephone 81 3 5456 5481 Office Fax 81 3 5456 5511 Instrument and Reagent Inquiries jpsales essenbio com Instrumentation Service josupport essenbio com 246 Page N 15 Appendix IncuCyte Supported Vessels Please note that all 384 well microplates and a subset of 96 well plates are only compatible with the IncuCyte HD system 96 and 384 well plates have an extra column to the far right HD If this column is checked then the corresponding plates are ONLY compatible with an IncuCyte HD device Brand Catalog s Flask Name Tray Type 70ml Canted Neck Standard TC Phenolic Cap 7Oml Canted Neck Standard TC Plug seal Cap 70ml Canted Neck Standard TC Vented Cap 70ml Poly D Lysine 25 cm Flask Plug seal Cap 70ml Collagen 25 cm Flask Vented Cap 70ml Collagen 25 cm2 Flask Plug seal Cap 70ml Fibronectin 25 cm2 Flask Plug seal Cap BD Falcon 354533 Z70ml Laminin 25 cm Flask Plug seal Cap 70ml Collagen IV 25 cm Flask Plug seal Cap 70ml Poly D Lysine 25 cm2 Flask Vented Cap 70ml Poly D Lysine 25 cm Flask Plug seal Cap 70ml Collagen 25 cm2 Flask Vented Cap 70ml Collagen 25 cm Flask Plug seal Cap Oml Poly D Lysine 25 cm2 Flask Vented Cap Oml Canted Neck Standard TC Plug seal Cap Oml Canted Neck Standard TC Vented Cap 3055 riangular Angled Neck Cell Culture Flask with Phenolic Style Cap 3056 Triangular Angled Neck Cell Culture Flask with
301. with a Pixel Intensity of 104 3 or greater will be displayed as equally bright To increase the dynamic range of brightness displayed for areas at 104 3 AU and above increase the value of the Maximum Intensity 202 Page Minimum Intensity Any portion of the image with a pixel intensity value less than or equal to the Minimum Intensity will be assigned a relative brightness equal to the darkest possible value in the image in this case black Maximum Intensity Any portion of the image with a pixel intensity greater than or equal to the Maximum Intensity will be assigned a relative brightness equal to the brightest possible value for the display in this case bright green All portions of the image with pixel intensities between the Minimum and Maximum Intensity values will be assigned intermediate degrees of brightness The fluorescence Min Max Intensity settings can be adjusted using the Auto Scale function or via Manual Adjustment Setting Fluorescence Intensity Values using the Auto Scale Button Autoscaling tries to assign optimum Min Max settings for a particular image Autoscaling is easy just press the Auto Scale button and in many cases the Auto Scale settings will be adequate Setting Fluorescence Intensity Values using Manual Adjustment Either type new values directly into the Min Max intensity boxes or and press the Enter key or use the arrows to scroll through values Use the mouse to view pixel intensities
302. y 21 Tray which holds up to eight 35mm dish tissue culture dishes 1221 0349 TM na i j IncuCyte Micro Plate Tray Tray B up to two tissue culture multi well 5025 0116 IncuCyte Calibration Tray 5025 0106 IncuCyte Calibration and Microslides Tray Tray which holds up to four tissue culture Iae 1221 0275 50 pack of 24 Well ImageLock plates 24 Well plates which facilitate precise repeated 4365 imaging 50 pack of 96 Well ImageLock plates 96 Well plates which facilitate precise repeated 4379 imaging 245 Page N ESSEN BIOSCIENCE 14 Ordering Contact www essenbio com U S CONTACT INFORMATION Essen BioScience Inc 300 West Morgan Road Ann Arbor Michigan 48108 USA Telephone 734 769 1600 Fax 734 769 7295 Instrument and Reagent Inquiries sales essenbio com Instrumentation Service ussupport essenbio com Discovery Services ics essenbio com EUROPE CONTACT INFORMATION Essen BioScience Ltd BioPark Broadwater Road Welwyn Garden City Hertfordshire AL7 3AX United Kingdom Office Telephone 44 0 1707 358688 Office Fax 44 0 1707 358687 Instrument Sales 44 0 7515 947101 Instrument and Reagent Inquiries eusales essenbio com Instrumentation Service eusupport essenbio com Discovery Services ics essenbio com Regional Support Germany and Austria 49 1525 3870559 France Luxembourg Fr Speaking Belgium Spain Italy 33 6 50 33 26 85 JAPAN CONTACT INFORMATION Essen BioScience K K Cerulean To
303. y Type After a Tray Type has been selected highlight one or more empty vessel spaces within the tray The Vessel Type pull down menu below will now become active Use the Vessel Type pull down menu to select a vessel e Once a vessel has been selected the Scan Type and Scan Pattern menus will become active For information on scan types see Section 7 1 5 page 42 Section 7 1 5 page 42 and Setting Scans in the IncuCyte FLR page 37 Use the Scan Type pull down menu to choose Phase Contrast or Fluorescence amp Phase Contrast see Section 8 5 1_ Adding Vessels to Use the Scan Pattern menu to select a Scan Pattern When the software is first launched the only scan pattern available will be the sample pattern To create new scan patterns see Section 8 5 3 Setting Scan Patterns page 67 Select the Properties Tab Enter the properties of each vessel These include e Vessel Label e Cell Type e Cell Passage e Any relevant notes e g concentration at which cells were seeded etc The first time a scan is scheduled all vessels entered will be new so the New box will be inactive Figure 7 2 Later however a new scan might be set up using the same vessel types in the same positions that were used in the previous scan s In this case the New box will be active Check the New box and then enter the new properties for the new vessel s To clear all vessel properties select the Clear Vessel Properties button 36 Page
304. y be routed up a corner of the incubator so as not to interfere with the installation of shelves above the microscope A split stopper supplied with the unit can be used to plug the access hole around the cables Shelves can be reinstalled above the unit as close to the incubators as the mounting brackets allow 1 WARNING The top cover of the microscope should NEVER be used as a shelf and should NEVER be used to support a shelf 2 After the microscope is placed in the incubator proceed IMMEDIATELY to the next two sections To avoid large amounts of condensation on the unit it is desirable to connect and power the unit within a few minutes of installation For connecting the cabling see the rear panel of the IncuCyte controller Figure 3 1 There are two cables that connect the IncuCyte controller to the microscope These cables which are fixed to the microscope should be connected to the controller before the unit is powered on The larger circular style plug on the microscope main cable should be connected to the mating socket on the back of the controller The second cable is a FireWire interface to the digital camera in the microscope This should be plugged into the mating FireWire connector on the rear of the controller The power cord for the unit can now be installed Use only the AC power cord supplied or an equivalent cord with an IEC 320 C13 standard connector approved for operation at 5 amps and the appropriate voltage f
305. y results 6 3 Calibration of the IncuCyte HD The standard calibration procedure previously described in Section 6 2 is adequate for scanning all vessels in the IncuCyte HD with the important exception of 384 well plates Because scanning of 384 well plates requires such a high degree of precision it also requires a special calibration procedure All three IncuCyte tray positions front middle rear must be individually calibrated before scanning of 384 well plates is initiated In order to calibrate the IncuCyte for 384 well plate scanning complete the standard calibration procedure as described in Section 6 2 The additional 384 well Plate Calibration requires a single microplate tray and two EMPTY clear bottom black walled or white walled 384 well plates with square wells Figure 6 7 displays a list of 384 well plates that have been tested and approved for 384 well Plate Calibration Manutacturer Wall Color Catalogue Number BD Biosciences 353962 Greiner 781091 Greiner GNF 789070 G Matrix 4330 312003 Porvair 311003 Figure 6 7 384 Well Plates Approved for HD Calibration We recommend that customers select the same plate type for calibration that they also plan to use most frequently for scanning If the desired plate type is not among our approved list of candidates then select the plate type that most closely resembles your plate of interest Clear walled plates CANNOT be used for 384 well Plate Calibrati
306. y spots in the window as this will interfere with proper calibration Place the calibration slide in the FAR LEFT CUTOUT POSITION of the microslide tray The orientation of the slide within the cutout position is not important However it is ESSENTIAL that the calibration slide is the ONLY slide in the microslide tray Verify that the rest of the tray is empty Select the Tests tab under the Administer IncuCyte tasks bar Then locate the Fluorescence Calibration test and press Run A new window will open to confirm that the IncuCyte is properly configured to run the test Press the Yes button to start the calibration Figure 8 56 The calibration procedure will begin Fluorescence Calibration requires 15 minutes Calibration status will be indicated at the top of the IncuCyte software window and in the status box at the bottom left 103 Page ESSEN BIOSCIENCE LN Device Accounts Tests Update Scans Logs _ Motion Calibration Instruments Fluorescence Calibration Slide in the far left Please verify that The Microslides tray is loaded in the front position A loaded Essen Instruments Fluorescence Calibration Slide is placed in the far left All other cutout positions in the tray are empty Are you ready to start the calibration amona ee Run a fluorescence calibration i Rn Figure 8 56 Fluorescence Calibration Calibration Results When calibration is complete the results can be view
307. yte EX 7 4 Graphing 7 4 1 Create a Graph After scan data have been collected they can be used to generate graphs of the selected metric over time The IncuCyte graphing feature can be accessed in one of 4 ways e By selecting the Graph Export button in the Selected Vessel box in the Completed Scans screen This will open the Graphing Box e By selecting the Graph tab in the Vessel View window Section 8 7 3 page 130 e Choosing the Graph option from the Scan pull down menu Section 8 7 9 page 152 e Right click on a vessel in the View Completed Scans screen See Tasks Available by Right Clicking on a Selected Vessel page 131 The Graphing Box will now appear See Figure 7 10 Follow the instructions to create a graph The newly created graph can be subsequently customized according to desire See Figure 7 11 49 Page gt ESSEN RIOSCIENCI Select the Metric Generally this will be Confluence Metric Confluence v1 3 Image Mean Focus Position Exposure Time Autofocus Sharpness Select Time Range for graphing or singe time point 2008 be the very first and very last scans o available forthe vessel However use the radio buttons to the right of the i Saini Time Tree to change these times as 18 Fri 1 30 AM 4 30 AM 7130 AM 10 20 AM Choose the Region of interest either 1 20 PM desired the entire vessel or selected wells 4 30 PM En

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