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1. Width Extension of the sub object mask The examples below illustrate the effects of these parameters Overlap treatment Options are Segment Segment slow and remove Main object mask Distance 0 Width 1 Distance 5 Width 5 Distance 8 Width 8 When sub objects are used for analysis a further parameter becomes available in the Pa rameter tab see Chapter 3 5 Measurement Parameters Obj 1 counts if the default name for sub objects is used otherwise it would be subobjectsname counts This parameter gives the number of sub objects detected for each main object and is a parameter of the main object 20 Chapter 3 Assays OLYM PUS 3 4 Object Finder Modules 3 4 1 Entire Image This is a very simple object definition the entire image is used as object You may use this for meas urements of integral intensities of your sample i e for each image and each parameter see Chapter 3 5 Measurement Parameters a single value is calculated independent of the objects within the im age Settings Objects found Ignore Frame 0S pisels Ignore frame This is the only parameter to adjust the size of a bordering frame to be ignored The default value is 0 no bordering frame 3 4 2 Intensity Threshold As the name says the Intensity Threshold method is based on intensity values pixels with intensities above a predefined threshold will be united to one individual object The Object Finder Intensity
2. 33 T v lt gt Object type Main v Reference gate G2 v Gated populations ene OE 0 I I I I 1 I 0 0 20 40 60 80 10 0 12 0 Group Export Table 9 To export the results either select Export Table in the Populations or Measurement Results or select the results to be exported via Export Definitions as described in Chapter 4 3 3 Export Definitions 5 3 Time Lapse Analysis In this example a time lapse series is analyzed The images of the example show cells during mitosis The cells are marked with two stains GFP and TxRed The images left and right show images from the same well and position taken at two different time points t 10 t 50 In order to analyze time lapse data it is necessary to segment the images properly and to track the moving cells from frame to frame scan Analysis Software Manual Chapter 5 Example Assay Step by Step 65 1 Execute Scan gt Open navigate to the folder where the demo data are stored select the experiment_descriptor xml file scan R_Demo_Data_DVD_20070216 Slabicki_Buch holz MPI CBG CMV mRed cyclineB1GFP GFP mRed 001 experiment descriptor xml sample data kindly provided by F Buchholz MPI CBC Dresden and open it with OK 2 Execute Assay Edit Assay to open the Assay Settings window 3 Go to the Image processor module in the Image Processing tab to set Background Correc tion for all channels TxRed and GFP Press Adjust and observe in the new wind
3. It is suitable to select the parameters Area and Circularity Factor in the first histogram and to dis play Total intensity dapi vs Mean intensity TxRed in the second histogram The small image window left to the main window shows the main object recognition in the image that is cur rently being analyzed By clicking on the Main Display button you can have it displayed in the largeimage window Selected Wells cee e Selected Wells Main Display 16 24 amp Analysis Progress Analysis Progress a OO Run End Analyzing the Data The first thing to do is to get an overview of the detected main objects in our example the cell nuclei The nuclei should all have a similar size and a more or less round shape i e a Circularity Factor CF close to 1 0 Set Area as X axis parameter and Circularity Factor as Y axis parameter and examine the size shape distribution of object population in the scatter plots The bulk of the population of the example has a CF of lt 1 1 and an area of around 300 pixels in images taken with 4x magnification Clustered cells CF 1 17 Area 1590 pixels k i 250 0 500 0 750 0 1000 01250 01500 01750 0 2126 1 D Area HE Cl bel ell Hace fee Ai 1 59E 3 1 17E 0 Object x Area v Main v Y Circularity Factor v Objects that are larger and have a larger Circularity Factor may for example consist of clus tered cells that could not
4. Parameters list Each Measurement is labeled with an ID p1 p2 and is assigned to the Main Object or a Sub object and depending on the type of measurement may be assigned to a Color channel Measurement Select a parameter from the shortlist The available parameters can be adapted through Modules Object Analyzers Chapter 6 3 1 Color channel Assign a Color channel from the shortlist to the newly added parameter Object Assign an Object type from the shortlist of available object types The image mask of this ob ject defines the parameter measurement area New This allows inserting a new parameter into the list Remove This deletes the selected parameter from the list A special parameter is available in the Measurement list when Sub objects are used Obj 1 counts if the default name for sub objects is used otherwise it would be subobjectsname counts This parameter gives the number of sub objects detected for each main object and is a parameter of the main object 25 26 Chapter 3 Assays OLYMPUS 3 6 Derived Parameters The Assay Settings Derived Parameters tab allows performing calculations on the Parameters defined in the Measurements list by the use of algebraic expressions sqrt etc For a complete list see Appendix 6 4 This is especially useful for parameters assigned to different color Channels Ge Assay Settings Main Object Sub objects Parameters Derived Paramete
5. This chapter explains the features of the image displays and briefly introduces the different menu points and buttons accessible from the main user interface 2 1 Generals ee Ee 2 2 General Settings ccccccccssssssseeeeeeessseeeees 2 3 The scan R Data Structure 2 4 Managing Histograms and Scatter Plots 2 4 1 The Histogram Context Menu 2 4 2 The Region Context Menu rrrrrrrvvvvrvnnnnn 2 5 Using the Image Viewer 2 6 Adjusting the Image Dusplavs 2 7 Selecting Wells for analysis rrrrrnnnnnnnnn 3 4 Chapter 2 Main User Interface OLYMPUS 2 1 General The scan4R Analysis main graphical user interface contains four histograms and an image viewer win dow The functions of these histograms are explained in Chapter 2 4 Managing Histograms The image viewer window shows the image with the object corresponding to a data point selected in a histogram A description of the image viewer functions is given in Chapter 2 5 Using the Image Viewer Navigation through the images of a scan is possible with the tools in the Image box Color channel selection is done with respective pull down shortlists in the Display box An assay can be started and followed online with the tools and displays in the field at the lower right of the main window The menu bar at the top contains several pull down menus and commands They are listed in the fol lowing overview
6. d pa v Mame Description HO OO ml OM ler D CH E Well Results Measurement Results Populations Export Definitions Export File name ResultO0 txt Remove File Name Wells Groups ResultOO txt Wells RO BO ROL R01 in in in in ROL AND R04 ROT AND ROZ ROL AND ROS R01 AND ROS ResultO1 Et Mean Intensity GM130 Error lt New Creates a new tab delimited txt file for export Export file name Enter the name for the txt file here The default is ResultO txt Remove Removes the selected File and its selection Export Allows setting set the folder where the txt files are stored manually Otherwise the folder given in Settings see Chapter 2 3 General Manager is used Wells Groups Use this button to select if the values are to be given for individual wells or entire groups of wells All wells that have the same Name Description entry form a group Wells that do not pertain to a group will be listed independently The Name Description is set in Scan Select Wells See Chapter 2 7 Selecting Wells 55 56 Chapter 4 Analysis Results OLYM PUS 4 3 4 Export results of individual objects Analysis Export Table exports the values determined for each detected object to a spread sheet The values exported depend on the active view When also sub objects are detected not
7. 01 Definitions Name Name Operator Result max MeanIntensity GFP lifetime lifetime mean CircularityFactor mean max MeanIntensity GFP min TotalIntensity GFP i J min Area i Operator Remove Compatibility Status as Trace parameters Well Position Trace You can navigate through the set of curves using these functions and their arrow buttons Add Range Click here if you want to analyze just apart of the trace Two vertical blue bars appear in the graph to define the upper and lower limit of the range They can be moved via mouse drag The analyzed part of the curve is displayed solid The remaining part is displayed as a dashed line Click Add Range several times to create multiple ranges 2369 669 2200 2000 18002 16002 14002 12002 1000 800 600 Mean Intensity TxRed F D rou e SE g e Nee Ber E u ep eo nef PE sett my ema E 400 x DEE 30 939 Si I I I I I I I I I I I I I I o i 20 30 40 50 60 70 op 90100 110 120 130 140 Real Time Result 520 681 Two ranges applied to a trace Add Threshold Click here to open a menu which allows you set an upper or lower bound for the curve A blue bar appears that marks the upper or lower part of the curve respectively The bar can be moved via mouse drag Remove Click here to remove the Range limits 39 40 Chapter 3 Assays OLYM PUS Parameter Select the measurement parameter of which the time curve is to be shown in the gra
8. 3 EE 16 Object Finder Detecting Main Objects AAA 16 Sub object Finder Detecting Sub objects REENEN 18 Obed Finder Modules eee 20 ENUE Made EE cauwostsdntinsseieaneeateiedesend eursatet 20 Intensitv I hreshold 4 een 20 Edge Detechon 21 Measurement Parameters AEN 25 Derived Harameiers oaned a 26 MAE FO SN KEE 27 Background COn c ION een ee een 28 E ES NE NE T EE 29 Jee 0 en ea a ee er 29 GI NAG nee ee te Bett Det he 30 Vinual Channels eu een 31 Simple MAN us ee une 31 Special NMIN irinse era e NE e a E NE 33 Tracking Analyzing Time Lapse Data 35 Tracking E aalt E reirse ae a 37 Track Analysis Parameters EEN 38 beier 43 15 16 Chapter 3 Assays OLYMPUS 3 1 General An assay defines all steps necessary to extract quantitative data from the acquired images It usually starts with some sort of image processing like background correction Secondly objects have to be detected in the images The analysis for example different kinds of measurements e g area intensity shape is finally performed on these objects and results for the samples e g wells are generated The Assay Edit Assay command opens the Assay Settings window and allows applying or adapting the assay to the loaded scan The Assay Settings window contains six tabs Main Object Sub objects Parameters Derived Parameters Image Processing and Virtual Channels The tabs are arranged from left to right but you can always jump back an
9. 42 Chapter 3 Assays OLYMPUS 3 9 2 3 Derived Parameters ti Trace Parameters Original Parameters Derived Parameters Original Parameters Id Name al lifetime pl maxiMeanIntensityiS FFY p t maxiMeanintensiby GFR p t mintDer MeanIntensiky GFP p tE maxi DerimMeanintensity GFP Derived Parameters Name Id Name Formula l Peak Duration Peak Duration a Formula p4 p3 fane EN The Trace Parameters Derived Parameters tab allows to perform calculations with the parameters listed in the Definitions list described above by the use of basic algebraic expressions X sqrt etc For a complete list see Appendix 6 4 Original Parameters This is the list as created on the Original Parameters tab and includes also the fitted parameters when the operator curve fitting is applied New Click here to create a new list entry Name Set the name of the new Derived parameter here Formula Set the formula here using the ID of the original parameter s and the algebraic expression Example Imagine you want to measure the duration of a peak It starts with a pronounced increase in intensity and ends with a pronounced decrease Mathematically this would be the time between a maximum and a minimum in the first derivative of the Mean Intensity curve The Original Parameters needed are thus time of the maximum t max and time of the minimum t min of the Parameter Mean Intensity with the opt
10. 9E 2 55 9 LIED 1 4E 1 12 6 Hz Nucs 47001 59 5 1 2E 6 6 4E 2 3 3E 2 51 0 H3 34104 649 g 5 6E 2 6E 2 er eae 5 6E 2 ERE 4E 2 25 4 2 i Gallery H4 GZ 11294 215 7 2E 2 2 2E 2 30 6 hd Remove Region Box This command removes the selected entry from the Regions list and deletes the defined Region Gates It lists all gates with their Names and Definitions Name Set the name of a Gate Color Click into the box to open the dialog window to select the color of the object boundaries when displayed in the image viewer This color is also used for color gating Compare Chapter 2 4 Managing Histograms and Scatter P ots Definition A Region or a Boolean combination via AND OR or AND NOT of Regions can be set to define the Gate New Click here to add a new Gate The default Name and default Definition will be that of a region in the Regions list The Name can be modified at will the definition must be a region or a logical combina tion of regions Remove This command removes the selected entry from the Gates list Gate Application This table lists all histograms with the applied Gates and statistical information about the gated data Number of Objects Count Percentage of total number of detected objects Tot Mean Value STDV and CV for the parameters plotted along the X and Y axis Histogram Number of the histogram window in the scan4R Analysis front panel Hi to H4 respec tiv
11. Analysis Run starts the analysis with the current settings Analysis Batch Run starts multiple analysis with the current settings T k scan R Analysis CellCycleMain Analysis Scan View Modules Repositioning Settings About k 500 0 1000 0 1500 0 2000 0 2401 7 D 00 250 0 500 0 750 0 1000 01250 0 1500 0 1750 0 2000 0 D 0 0 250 0 500 0 750 0 1000 01250 0 1500 0 1750 0 2000 0 D Area _ Area Area Area 6 29E 2 Object x Area v 6 29E 2 Object x Area M 6 29E 2 Circularity Factor wv 1 14E 0 Main ai Y Mean Intensity DAPI v 2 31E 3 Main sl Y Mean Intensity DAPI v 2 31E 3 500 0 R01 475 0 450 0 425 0 400 0 375 0 350 0 325 0 300 0 275 0 250 0 225 0 200 0 1750 150 0 125 0 100 0 Image 75 0 Row Column 50 0 25 0 0 0 k 0 1 1000000 0 2000000 0 3000000 0 40000000 pg T otallD apiCorr Object x TotalIDapiCorr wv 1 43E 6 Main wi TotallDapiCorr v 1 43E 6 Selected Wells Interactive objects Main v D any End view mode 8 Population SES Analysis Progress Trace T B 342 G 322 R X 663 369 Z 1 Well Name Description B7 scan Analysis Software Manual Chapter 2 Main User Interface Analysis Open opens a previous analysis sca file Analysis Save Save as saves the current analysis sca file Analysis Export Table exports the values determined for each detected object to a spread sheet The val
12. Chapter 5 T 8 10 Example Assay Step by Step OLYM PUS f r Assay Settings Main Object Sub objects Parameters Derived Parameters Image Processing Parameters ID Measurement Channel Object al Area Center Y Center 3 Position Color Channel Circularity Factor Mai GFP Mean Intensity Total Intensity Measurement Time f aoa rer Compatibility Status Ce Confirm the Assay Settings with OK and Run the analysis After the analysis you can plot the Histograms and Scatterplots as described in Chapter 5 2 Analyzing the Data The next step after the image segmentation and the extraction of the parameters is the track ing of the objects Go to Tracking Configure Tracer Check the Enable Tracking box and select Main as tracked object Set a range of 20 pixels Confirm with OK When leaving this dialog the tracking will start The progress of the tracking is shown in the status bar Tracer Tracking Particles After the tracking is finished the Trace Parameters menu will open Here you can browse through the traces and determine the kinetic parameters that will be extracted from the curves Select the operators which will be applied to the kinetic curves of the selected Pa rameters Let s say we want to find all the cells that undergo mitosis during the measure ment When undergoing Mitosis the cells show an increase in the mean intensity of GFP First set
13. Define Parameters At startup it shows the kinetics graph of one of the measured parameters of the first track in first stage position of the first well acquired and selected for analysis See Chapter 2 7 Se ecting Wells for Analy SIS To navigate through the curves use the Well Position and Trace selectors on the right Each Trace represents the object in subsequent time frames that the tracer according to the settings in Track ing Configure Tracer detected as belonging together In the Original Parameter menu you can set the Operators that are applied to the time curves to quantify the time curves By applying an Operator min max std first etc the time curves again are reduced to a single values per curve that in turn can be displayed in a 1 D or 2 D histogram For example when TotallntensityGFP was selected in the Pa rameter tab of Edit Assay the time curve of TotallntensityGFP can now be plotted By applying the Operator max on this time curve for all traces the maximum of TotallntensityGFP will be calculated scan Analysis Software Manual Chapter 3 Assays ti Trace Parameters meet Original Parameters Derived Parameters 1271 136 Well 1250 15 v 1225 ii postion 1175 Hais 1125 1100 1050 1025 Add Threshold 996 489 7 1 Mean Intensity GFP 1 1 1 1 I I I Remove 60 70 80 90 100 110 120 130 140 Absolute time Derivative Parameter Mean Intensity GFP Smoothing sigma 0
14. Image Channel Definition Scan Settings View Conversion Results Root directory for image list generation File extension Image list SCH oc Save Rule Load Rule Cancel File extension This is set automatically when the Source image type is chosen The file extension serves to filter the content of the selected directory scan Analysis Software Manual Chapter 6 Appendix 73 Image list Retrieve This function reads the matching files into the Full file paths list The arrow buttons allow easy browsing and verification of retrieved image data 6 1 2 Image Channel Definition If the file names of the third party images contain codes for Well Position Z layer and Time scan4R will sort them automatically upon conversion into scan4R format The Image Channel Definition tab is used to define the conversion rules for this Unique Channel Identification String If the original file names contain a certain expression or Identifi cation String that classifies them as belonging to a certain color channel enter it here Channel Name Enter the Channel Name to be used in scan R here New Click here to add the newly defined channel to the Image Channel list Well Position Slide Z Time RegExp If the file names contain information about Well Position Z layer and Time in form of regular expressions RegExp scan R can group them when the corresponding code is entered here An overview of the syntax of
15. Region tool and keep in mind the name R02 Execute Analysis Assay Gating to open the Gate Manager window Regions Gates Edit Gate DO A Definition ROZ Name Color RO AND ROZ E BE Definition R01 AND R02 New Remove v well Results Click new to create a new gate You can give the new gate a meaningful name e g G1 and also select a color In the Definition line enter RO1 AND R02 to select only the objects which are contained in both regions Rename the Gate RO1 to Nuclei these are the objects defined in step 2 Then Close the Gate Manager Next draw a region around the right cloud i e the nuclei in G2 and repeat the steps de scribed in step 3 and name this gate G2 In the two remaining histograms choose Mean Intensity TxRed for the X and Yaxis This will give the distribution of objects with a certain amount of TexasRed staining in all nuclei To find out if the distribution of TexasRed is different in the G1 and G2 phase right click and se lect Set Gate G1 in the context menu in one histogram and Set Gate gt G2 in the other his togram scan Analysis Software Manual 1500 0 61 Counts 100 0 1000 0 2000 0 Mean Intensity TxRed ol Object x Mean Intensity TxRed vi 2 77E 2 Main v vi Mean Intensity TxRed 2 77E 2 250 0 G2 Counts 100 0 1000 0 2000 0 Mean Intensity TxRed D Object Mean Intensity TxRed vi 2 77E 2 Main v Y Mean I
16. be dis played with maximum and minimum brightness The intensity of the remaining range of pixels will then be scaled linearly in between The higher the numbers the stronger the resulting contrasts Absolute Define here the range of pixel counts to be displayed with maximum and minimum brightness by dragging the red horizontal lines that represent the maximum and minimum thresholds with the mouse The intensity of the remaining range of pixels will then be scaled linearly in between Gray scale palette The channel selected in the gray pull down menu in the main GUI can be displayed in different false color palettes that can be selected here Use scan settings 2 7 Selecting Wells for analysis The command Scan gt Select Wells opens the Select Wells display It features a graphical representa tion of all wells of the well plate or slide When the window is opened for the first time green circles represent wells that were selected for scan ning These wells will be considered in the analysis To change this selection double click on the wells These wells are also removed from the list on the right A second double click reactivates the well Dragging a rectangle around a group of wells likewise carries out deactivation and activation In the list on the right you can also change the name of the wells and group wells by assigning the same name Restore This function restores the initial well pattern Name Description In ce
17. curve the corre sponding image with the marked object will be shown in the image display of the main scan R Analysis window Also the data points marked with a red circle in the histograms are linked to the time curves Likewise upon clicking on an object in the image display or in a scatter plot the corresponding time curve will be shown in the Trace Viewer A dashed vertical line marks the current point in time in the Trace Viewer Upon moving the cursor left and right while keeping the mouse clicked one can thus scroll back and forth through the movie of the time lapse series L3UU U 1280 0 1260 0 1240 0 1220 0 1200 0 1180 0 Mean Intensity 1160 0 1140 0 1120 0 1100 0 1080 0 Save ae l l l I I D 10 20 30 40 50 60 70 DU 90 100 110 La Real Time Derivative Parameter Mean Intensity GFP EI Smoothing J Sigma 0 01 Show Gates F Show Trace Gallery Movie 0 1 d 15 scan Analysis Software Manual Chapter 4 Analysis Results 4 Analysis Results This chapter describes how an analysis is executed how the results can be grouped into object popu lations and how these can then be analyzed and statistically evaluated 4 1 4 2 4 2 1 4 2 2 4 2 3 4 3 4 3 1 4 3 2 4 3 3 4 3 4 RUNNING ANANAS arrene 46 VE E else ge 47 Gatesand REGIONS ran Be een 47 The Gate Manager 47 GOlOn GAMING ren ee ana 49 ER EI 52 Medas rement Resuls
18. logscale and you rescale the X Y data with different scaling factors as might happen with the automated scaling during export because of range differences there might be a shape change An alternative to FlowJo is FCS Express http www denovosoftware com site Download shtmli which is supposed to stick more strictly to ISAC FCS 3 0 standard scan Analysis Software Manual 6 3 Libraries 6 3 1 Object Analyzers Library OAL YE Object Analysers Library OA Parameter Name Center of Mass X 2 D Geoms vi Center of Mass X Mean Intensity 2 D Mean vi Mean Intensity Minimum Intensity 2 D Mean vi Minimum Intensity Max Feret Diameter Orientation 2 D Geoms vi Heywood Circularity Factor cs MI Environment vi CS Mean Intensity M EE E E Object Analysers Library OA Module OA Parameter Name Total Intensity Mean Intensity Maximum Intensity Parameter Name Total Intensity Mean Intensity Max Intensity Min Intensity Minimum Intensity Area 2 D Geoms vi Area Circularity Factor Heywood Circularity Factor 2 D Geoms vi Perimeter Max Feret Diameter elongation Facto Convex Hull Perimeter Int Standard Variation Perimeter Max Feret Diameter Elongation Factor Convex Hull Perimeter Int Standard Variation lt gt Chapter 6 Appendix Module Location i Object Analyser Module FEE Parameter Center of Mass X v Module Location C Pr
19. non separable nuclei The latter are usually much larger and less circular If sub objects are to be detected it is useful to add the parameter Obj 7 Counts to the list to determine how many sub objects are connected to each main object f r Assay Settings Main Object Sub objects Parameters Derived Parameters Image Processing Lal Farameters ID Measurement Channel Object zl pi Area Main Measurement p Mean Intensity dapi Main Drea Kg p Mean Intensity TxRed Main ENTER EE ee EEE Color channel p Circularity Factor Main dar p Max Intensity TxRed Main se p Max Intensity dapi Main Object p7 Total Intensity den Wan Man p Total Intensity TxRed Main ps x Main pid Main os Ze ff BR EEE EEE _ Compatibility Status i x 11 The Derived Parameters tab allows you to perform calculations on the previously defined pa rameters e g here you can calculate rations between different color channels For example you could evaluate mean Intensity Dapi mean Intensity TxRed scan Analysis Software Manual 9 2 12 13 14 Chapter 5 Example Assay Step by Step Now you have set up the analysis Press OK to exit the Assay Settings menu You can save your assay with Analysis Save assay Start the analysis by clicking on the Run button Prepare the histograms in the main window if you want to follow the analysis online
20. only one file is exported but for every sub object a separate list is exported For the population view the list contains for example ParameterData_Main txt ObjectID MeanlntensityDAPI Obj 1Counts ParentObjectID ParentTracelD RO1 0 174 52045 NaN 1 0 0 1 295 50986 2 1 1 1 2 220 76321 1 1 2 1 3 161 89612 NaN 1 3 0 ParameterData Obij 1 txt ObjectID Total Intensity GM130 Area Parent Object ID ParentTracelD RO1 0 201294 608 1 1 0 1 2504 12 1 1 0 2 2232 12 2 2 0 3 41191 108 4 4 0 4 4860 18 4 4 0 e Object ID ID of the detected object e Parameters e g MeanIntensityDAPI Area e Derived Parameters e Parent Object ID 1 if it is the main object if not the ID of the main object is given e ParentTracelD if tracking was performed the ID of the Trace the detected object belongs to is given If no tracking was performed the Trace ID is set to 1 e Gates If the object belongs to the listed gate the value is set to 1 if not it is set to 0 For the Trace view the list contains TracelD lifetime max Area TrackedObjType TraceLength FirstParticlelD LastParticlelD HO 0 1 31 0 1 0 0 0 1 15 78 0 15 1 1940 0 2 8 626 0 8 2 1041 0 3 19 849 0 19 3 2640 0 e Trace ID The ID of the Trace e Parameters of the Trace e g lifetime max Area e Derived Parameters e Traced Obj Type 0 main object 1 2 3 Sub object type e Trace Length Number of objects in subsequent time frames belonging to the Tr
21. operation that is to link the result of Channel 1 and 3 with Channel 3 e g DAPI FITC XTxRed from the second pull down selector Select Skip to deactivate Channel 2 or Channel 3 respectively 3 8 1 1 Example Cytoplasm not detectable on a single color channel OD scan Analysis Software Manual Chapter 3 Assays 33 In this example it is not possible to detect the cytoplasm on a single color channel because the area of the nucleus has very little cytoplasmatic staining Problem The detection is incomplete because staining is missing on the nucleus area Solution The nucleus staining and the cytoplasmatic staining are added as VC ja Channels SEC 1 hagh r 16 BG Result Full Cell Segmentation of the complete cell can be performed on the calculated new channel 3 8 2 Spectral Unmixing A major problem in live cell imaging arises from the use of different fluorochromes with overlapping spectra in one multi labeled sample impairing a number of applications Even with the use of high quality optical filters it is not satisfactorily possible to separate the spectral information The conse quence is the excitation and imaging of structures that are labeled with one of the present fluorophores when using a filter set that is actually chosen to excite and image another fluorophore With the Spectral Unmixing module it is possible to separate and resort the contribution of different fluorochromes to the total signal in each
22. points within its boundary are being considered in further steps Gates and Regions can be created using the drawing tool beside each scatter plot A Region can be defined as Gate by selecting Set Gate in the histogram context menu or by defining a gate in the Gate Manager See Chapter 2 4 Managing Histograms and Scatter Plots By clicking on the borderline the Region can be displaced By clicking one corner of the region this corner can be moved in order to include or exclude data points Right clicking on the borderline of a Region opens the Region context menu See Chapter 2 4 2 The Region Context Menu 4 2 2 The Gate Manager The Gate Manager allows converting Regions into Gates and to combine Gates by Boolean operations The Gate Manager also gives an overview of the results in the histograms H1 to H4 and the applied Gates The command Analysis Assay Gating opens the Gate Manager window that allows administrating the Gates and Regions Regions This lists all regions drawn into the histograms Show Click here to display the selected Region in the histogram selected in the Gate application list 48 Chapter 4 Analysis Results OLYMPUS tE Gate Manager Regions Edit Gate A Definition ROZ 3 ER Name ROS uch ROL AND ROZ Nuclei ROL AND ROS Definition RO Remove Well Results Gate application Applied gate Histogram Gate Name Count De Tot xMean e None Ke Hi None 52516 100 0 SB 2E 2
23. regular expressions can be found under http www regular expressions info tutorial html er Scan File Conversion Images List Image Channel Definition Scan Settings View Conversion Results Unique channel identification substring Well RegExp Red vW 0 9 Channel name Position RegExp Pe P D 9 Slidefz RegExp Color channels i SL 0 9 A Blue Green Time RegExp T 0 9 Comment RegExp Initiator Comment Regexp Terminator Comment RegExp Initiator Terminator If the file names contain information about wells scan R can use this as Well Description see for example Chapter 2 7 Se ecting Wells for Analysis Enter the first Initiator and last Terminator alphanumerical digit s in the respective boxes 74 Chapter 6 Appendix OLYMPUS 6 1 3 Scan Settings Scan settings are used to reconstruct a minimal configuration file for the data conversion scan R needs information about the used multi well plate and the arrangement of the images within the single wells in order to represent the data properly in the graphical user interface Result Dir Click on the folder button on the right of the box to open the navigation window Navigate to the storage directory and click Select Curr Dir Positions Columns Well and Rows Well Set the number of image columns and rows respectively per well Wells Columns and Rows Set the number of columns and rows respectively of the multi well
24. to control the result in the main user interface by pressing the Processed button It is useful to zoom into the image so that individual pixels can be detected visually 3 7 3 Inversion This function serves to invert the intensities of an image channel i e to convert a channel into its negative image This is necessary for example if objects are to be detected in transmission images The object detection tools of scan R are designed to detect bright objects on dark background The situa tion is reverse in transmission images Thus they have to be inverted prior to the object detection Adjust Intensity This defines the maximum intensity in the converted image l e it is the intensity that the originally darkest pixel will get in the converted image 4096 is the default and corresponds to the maximum intensity in a raw image taken by a 12 bit CCD camera 29 30 Chapter 3 Assays OLYM PUS 3 7 4 Cut Image This module allows defining regions of interest inside an image and setting all image parts outside the region to the intensity 0 Different drawing tools are available L Rectangle Define a rectangle by mouse drag Mouse dragging the center changes its position The size can be adjusted by dragging the corners S Rotated Rectangle This is similar to the rectangle tool Upon mouse over its central axes are displayed Dragging the ends of the axes turns the rectangle L7 Polygon Standard tool to draw polygons a Fr
25. unmixing is shown in the right display of the 3x3 Spectral Unmixing dialog window Unmixing dual labeled samples In the case of two channel images the third input channel will be occupied by one of the two available channels Click Ignore stain to ignore the third channel in order to properly unmix two channel images 3 9 Tracking Analyzing Time Lapse Data The tracking function of scan4R Analysis allows the analysis of objects over the course of time i e in experiments that consist of time lapse acquisitions It relates any object detected in an image to the same object in the previous and subsequent images in the time lapse series acquired at the same stage position Thus the change of the parameters that are measured according to the assay settings can be followed over the course of time To enter the Trace View select Trace in the lower left part of the front panel or in the menu bar select Tracking Trace view The front panel display changes such that the displayed objects change from Main to Trace and also the available X Y parameters in the histograms are changed according to the parameters that are defined in Tracking Define Parameters See Chapter 3 9 2 1 Original Parameters 36 Chapter 3 Assays OLYMPUS Analysis Scan Tracking View Modules Repositioning Settings About 200000 0 hr k ef ON k he 00 250 500 75 0 100 0 125 0 150 0 17510 2000 0O 0 0 5
26. your settings work with them as well When you are satisfied with your settings click Ok on the bottom right side The options at the Selection mode dropdown list are Min level Selects all level 0 particles Max level Selects all particles which do not have other particles nested inside Selected level Selects all particles with the specified level Best closures Selects for each nesting branch the particles which are best according to their closure quality scan Analysis Software Manual Chapter 3 Assays 3 5 Measurement Parameters The scan4R Object Analyzer modules in the standard configuration offer a large number of individual parameters that may be measured for each recognized object To limit memory utilization and save CPU time only the values for the parameters listed in the Assay Settings Parameters list will be ex tracted during the analysis TE Assay Settings wem Main Object Sub objects Parameters Derived Parameters Image Processing Parameters ID Measurement Channel Object Measurement p Position Main Area u 1 Main p Center V Main p5 Heywood Circularity Factor Main p Mean Intensity DAPI Mein Object pr Mean Intensity FITC Main ME p Max Intensity DAPI Main p10 Total Intensity IC Dots pll Drea Dots plz Mean Intensity FITE Dots pls Dots counts Ma Color Channel GFP Main Compatibility Status e si Cl
27. 0 0 100 0 150 0 200 0 250 0 300 0 350 0 400 0 D 1000 0 2000 0 3000 0 4000 0 5000 0 D l lifetime L as max MeanlntensitylGFP 8 mar Area EE Object u lifetime v 7 10E 1 Object u x max Meanintensity GFP D 8 09 1 Object 8 p max Area _ Mi I 1 69E 3 Trace Mi r lifetime v 7 10E 1 Trace v Y max Totallntensity TxRed vi 1 13E 6 Trace Y max Meanlntensity GFP v 8 09E 1 Display None vi Image Row Column e el k Position 0 0 1000 0 20000 30000 40000 50000 G 1 4 maxl rea L Object x max Area v 1 69E 3 is Trace w max Totallntensity TxRed v 1 13E 6 2 B Selected Wells Main Display r 83 2 Interactive objects Trace v jo view mode 9 a Population N B Analysis Progress Trace 2 0 0 0 539 95 Well Name Description B3 g When clicking on one tracked object in the image now not only the object is highlighted with a green border but also the trace it covered during acquisition will be displayed With time the object moves from the blue end of the line to the red end Like in the Population View the detected object and the corresponding data points in the histograms are directly linked Right clicking on an object in the image yields the following context menu Save aS Show Gates Show Trace Gallery Movie Save as This allows you to store the displayed image separately Show Gates It has the same function as in t
28. 034 Hamburg Germany Printed in Germany 2008 12 M 1 1 0 6
29. Analysis Software Manual Chapter 2 Main User Interface 9 2 4 1 The Histogram Context Menu Set Gate Show Region Clone From Gallery Zoom out Color Gating Settings The histogram can be managed through the context menu accessible via right click into the histogram Note that you will get the region context menu if you right click on a region border as described be low The histogram context menu contains the following commands e Set Gate Apply a gate on the selected histogram by choosing a region from the list that appears e Show region Select a region from the list that appears to generate a zoomed in view of it with the X Y parameters that were used to define this region e Clone from Select a histogram from the list that appears to duplicate the histogram e Gallery This command generates an image gallery of objects in the current histogram The number of images and the selection criteria is set according to the gallery preferences given in the Settings menu e Zoom out This command zooms out of the current view by a factor of 2 in case of linear axis scal ing e Color Gating This command causes the population of each gate to be displayed in a different color in the histogram e Settings This command opens the Histogram Properties dialog with the following settings Z Histogram Properties Axis attributes Grid cosmetics Aukoscale x Minimum SIE y Minimum Line color E Autoscale XY oF Zlz
30. Expor Functionally onenean 75 SC Ee 77 6 3 1 Object Analyzers Library OAL asia aan 77 6 3 2 Object Finders Library OFL nnnnnnnnnnnnnnnnnnnnsnnnnnnnnnnnnnsrnnrrnsnnnrresnnnneenne 78 6 3 3 Image Processing Library rervnnnnevnnnnnvnnnnnnvnnnnnnennnnnnennnnnnennnnnrennnnnsennnnusene 78 6 3 4 Virtual Channel Ubram nennen 79 6 4 Valid functions for derived Darameiers 80 Chapter 6 Appendix OLYMPUS 6 1 File Conversion scan4R Analysis is able to analyze image data acquired with a third party imaging system as long as the data are stored as monochromatic 16 bit single TIFF images The file names must allow the as signment of the images to individual positions time stamps and color channels The command Scan Custom conversion opens the Scan File Conversion dialog that allows setting up conversion rules The creation of conversion rules is a four step process which will be described in the following chapters Save rule This function saves the current conversion settings as a Custom Pattern file cr Load rule This function allows loading a stored set of conversion settings 6 1 1 Images List Root directory for image list generation Click on the folder button on the right of the box to open the navigation window Navigate into the storage directory and click Select Curr Dir Source image type Select the desired image type TIFF JPEG PNG or BMP from the shortlist TE Scan File Conversion Images List
31. FP Whereas data points gt 1 are the ones that undergo mitosis and have a higher maximum mean intensity Draw a rectangle around the data points with ratio gt 1 Right click on the border of the rectangle and select Gate The new gate longtraces AND R03 is created Mark one of the data points and the corresponding cell is shown in the image display with a green outline Right click on the image of the cell and select show trace In order to show all the traces of the gated cells deactivate single mode and as gate select longtraces AND R03 You will see that this parameters lead to a better discrimination of mitotic cells than the more simple approach used before 69 70 Chapter 5 Example Assay Step by Step t Trace Viewer Gate longtraces AND w Max displ Traces 1000 gt un D kd Cc m a Single mode EEE 1 I I I I 1 I I 1 I I I 1 1 1 I m 92 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 94 Trace Time Time scale t max MeanIntensity GFP Derivative Parameter l Mean Intensity GFP Smoothing g sigma l 0 01 0 1 OLYMPUS scan Analysis Software Manual Chapter 6 Appendix 71 6 Appendix Bt Eile Gornversion aaa 72 Oiled Images Est ine 72 6 1 2 Image Channel Detnmon nennen 73 8 1 3 Scan SENGS aa cde cde teases en e decease 74 6 1 4 View Conversion Results ccccccccccssssececceeseeeeeceeeseeeeeeeaeeeesesaseeeesseaaes 74 562 FCS
32. OLYMPUS INSTRUCTIONS scan AUTOMATED IMAGE AND DATA ANALYSIS SOFTWARE This instruction manual describes the Olympus scan Automated Image and Data Analysis Software for Life Science To ensure safety obtain optimum performance and familiarize yourself fully with the use of these products we recommend that you study this manual thoroughly before operation Together with this manual please also read the scan Automated Image Acquisition Software and Hardware manuals as well as the manuals of all other devices controlled by that software in order to under stand general operation methods Retain this manual in an easily accessible place near a system for future reference OLYMPUS SOFT IMAGING SOLUTIONS GMBH Rupert Mayer Strasse 44 D 81379 M nchen Tel 49 89 89 55 805 660 Fax 49 89 89 55 805 6606 Email info olympus sis com www olympus sis com OLYMPUS Imaging Excellence We at Olympus Soft Imaging Solutions GmbH have tried to make the information in this manual as accurate and reliable as possible Nevertheless Olympus Soft Imaging Solutions GmbH disclaims any warranty of any kind whether expressed or implied as to any matter whatsoever relating to this man ual including without limitation the merchantability or fitness for any particular purpose Olympus Soft Imaging Solutions GmbH will from time to time revise the product described in this manual and re serves the right to make such changes without obli
33. Selected 55 37 Ma Mc 24 98 15 95 Chapter 4 Analysis Results 51 Wa 10000 Counts D 2335 500 0 10000 15000 2000 0 Area Selected Gl RO1 AND ROZ AND ROS RO1 AND RO2 AND R04 2 In this example the situation is identical to the first example but the order has been changed All Pixels having a majority of G1 will now be shown in a green All G1 objects also belong to Selected but when the same number of objects belongs to G1 and to Selected the color of the data point is determined by the list order and now G1 has a higher priority than Selected Wo 24 98 D Selected 55 37 er 15 95 3000 0 ei Ma 10000 Counts E 600 0 400 0 mG Ri 160 1 S 200 0 1400 0 16582 DP 233 5 500 0 k 2600 2 D 1000 0 1500 0 2000 0 Area Gate Manager Regions Name Definition Gl RO1 AND R02 AND ROS R01 AND ROZ 3 Inthe last example the order is changed again but now also the gate Se ected is applied to the histogram Now the exception Rule 2 applies such that the Se ectedis not dominant 52 Chapter 4 Analysis Results 4 3 3789 3 Selected E cz ci E Selected 100 00 2335 500 0 10000 15000 2000 0 2600 2 Area D Definition Gil Selected G ROL AND ROZ RO1 AND ROZ AND R04 l ROL AND R02 AND ROS Well Results LYMPUS The Well Results window via Analysis Assay Gating shows the resul
34. Threshold dialog has two image viewer displays The left one shows the gray value image including all detected objects marked with a red bounding box The right one shows the binary mask of each object Settings The settings used when opening the window depend on the Settings list selected in the Main Object or Sub object tab see Chapters 3 2 Object Finder Detecting Main Objects and 3 3 Sub object Finder Detecting Sub objects and are loaded from the Object Finders Library see Chapter 6 3 2 Object Finders Library OFL Threshold This is the intensity cut off for objects Type in a value or use the arrows to adjust it Threshold Auto Click this button to automatically evaluate the image background and set a meaning ful cut off value Watershed If neighboring objects are so close to together that thresholding does not lead to a clear separation they will be detected as one object See left image pair below The Watershed algorithm separates these objects along the contractions of the detected masks See right image pair below Set the toggle button to On to use this option Ignore border object Check this box to ignore all objects that are cut off by the image border scan Analysis Software Manual Chapter 3 Assays TE Object Finder Intensity Threshold l 1344x1024 1 1 16 bit image 1 718 773 1344x1024 1 1 16 bit image 797 0 826 Settings Objects found Images 948 42 W00002 P00001 200000 T00000 dapi t
35. ace e First Particle ID ID of the first object belonging to the Trace e Last Particle ID ID of the last object belonging to the Trace e Gates If the object belongs to the listed gate ate the value is set to 1 if not it is set to 0 scan Analysis Software Manual Chapter 5 Example Assay Step by Step 57 5 Example Assay Step by Step This chapter guides you step by step through the setup and execution of an assay and the analysis of the results 5 1 Setting up and Executing an ASSAY nennen 58 59 2 ANTE Dala eee 61 53 TIm L ps e Analysis een ende 64 58 Chapter 5 Example Assay Step by Step OLYMPUS 9 1 Setting up and Executing an Assay The example scan which is used here to guide you through the setup and execution of an assay con sists of images taken from cells labeled with DAPI to mark the nuclei and with FITC to mark the micro tubules A repair protein which is active in late G2 phase stained with TexasRed 1 Execute Scan gt Open navigate to the folder where the demo data are stored select the experiment_descriptor xml file Scan R Demo Data DVD 20070216 Slabicki Buchholz MPI CBG H2AX PFA 002 experiment descriptor xml sample data kindly provided by F Buchholz MPI CBC Dresden and open it with OK scan4R Analysis loads the first image of the scan i e the image acquired at the first position of the first well in the main window of the font panel Only the first channel of the image wi
36. age Processing Library Image Processing Modules Module Name Module BackgroundCorrection RolingBall i obt ImagexYShi t vi Cubimage Cubimage vi Absorbanceln Absorbancetn vi 6 3 4 Virtual Channel Library 44 Virtual Channel Library KEE Module Simple Math Simplearithnmetics01 i Spectrallinmixing LinearUnmixing3in3out vi Chapter 6 Appendix BackgroundCorrection o d C Program Files Scan R_Package_061015 4nalysis Image IF The password protected Virtual Channel Library can be accessed through Modules gt VC Processors 19 80 Chapter 6 Appendix OLYMPUS It allows managing the set of available Virtual Channel algorithms in the module selector on the Edit Assay Virtual Channels tab Add Module Click here to add a new entry to the list Remove Module Click here remove an entry from the list 6 4 Valid functions for derived parameters abs x acos X acosh x asin x asinh x atan x atan2 y x atanh x ceil x ci x cos x cosh x cot x CSC X exp x expm1 x floor x getexp x gamma x getman x int x Inp1 x log x log2 x max x y min x y mod x y pow x y Absolute Value Inverse Cosine Inverse Hyperbolic Cosine Inverse Sine Inverse Hyperbolic Sine Inverse Tangent Inverse Tangent 2 Input Inverse Hyperbolic Tangent Round Toward Infinity Cosine Integral Cosine Hyperbolic Cosine Cotangent Cosecant Exponential E
37. al channels have to be created instead see Chapter 3 8 Virtual Channels te Assay Settings Main Object Sub objects Parameters Derived Parameters Image Processing Image processors Image processor Module Module BackgroundCorrection Bark BackgroundCorrection BackgroundCorrection Color channel FITC Ca Compatibility Status am ai Image processors list It lists the Image Processor Modules i e the processing steps to be applied on the different Color channels of the images The order of appearance in this list also sets the order of 28 Chapter 3 Assays OLYMPUS execution Moving the active entry up or down the list by using the arrow buttons changes the order of execution Module Select an Image Processor Module from this shortlist Color channel Assign a Color channel from the shortlist to the active Image Processor Module Adjust Click here to open the Image Processing window for the active Image Processor Module New This allows inserting a new Image Processor Module into the Module list Remove This deletes the active module from the Image processors list 3 7 1 Background Correction For applications involving quantification of intensities and in case of inhomogeneities it is always rec ommended to use background correction The algorithm implemented is an intensity conserving algo rithm which ensures that the signals remain unchanged and can therefore be quantified left image b
38. ally Remove Click here to delete the active entry from the VC Process List Module Background Correction XY Shift Inversion and Cut Image are the same functions as de scribed in Chapter 3 7 Image Processing Adjust Click here to open the dialog window of the selected module 3 8 1 Simple Math The Simple Math module serves to perform calculations on the image through basic arithmetic opera tions addition subtraction multiplication and division Chapter 3 Assays OLYMPUS Module Select Simple Math from the Module pull down list to set this module as active entry in the VC Process List Input Channels list All color channels of the images are possible Input Channels for the Simple Math module The channel selected as first Input Channel is always the first source of the arithmetic opera tion The other channels are possible second sources Adjust Click here to open the Simple Math dialog window It contains displays of the overlay of the Input Channels and of the Virtual Channel that is created by the selected arithmetic operation He Simple Math Settings Channel 1 2 3 Each of the Input Channels can be weighed prior to the arithmetic operations by using the sliders or entering a multiplication value between 0 and 10 into the respective boxes Arithmetic operations selectors Select the operation X that is to link Channel 1 and Channel 2 from the first pull down selector e g DAPI FITC Select the
39. an ssinae a 52 Well Results erte eebe bebe 53 Export Reie 54 Export results of individual obiects AA 56 45 46 Chapter 4 Analysis Results OLYMPUS 4 1 Running an Analysis Execute Scan gt Open and select the Experiment Descriptor xml file in the storage folder of the scan By doing so the experimental settings are loaded and the first image of the scan taken at the first position of the first well will be displayed If you want to modify or revisit an analysis execute Analysis Open instead Execute Analysis Load Assay to read in a say assay file stored in the scan4R Analy sis Assays folder If you want to modify the assay file of the current analysis execute Analy sis Edit Assay instead Set or modify the parameters in the assay as described in Chapter 3 Assays If you want to perform the analysis on a subset of the wells open the Select Wells window via Scan Select Wells and select the wells of interest In order to observe the analysis results online prepare the four histogram windows by select ing the objects and measurement parameters to be displayed Click the Run button or execute Analysis Run to start the analysis You can follow the progress of the analysis in the window at the bottom right of the main scan R Analysis inter face BI e 5 Selected Wells Main Display 16124 Analysis Progress Bss ME The window shows the Analysis Progress and displays the
40. be separated by the watershed algorithm 61 62 Chapter 5 Example Assay Step by Step OLYMPUS For further analysis only consider those objects that do not differ significantly in size and shape from the bulk To do so use the Region tool and draw a polygon around the most densely populated part of the scatter plot Close the region by double clicking Right click in the scatter plot to open the context menu and select Gate By doing so the re gion RO1 is converted in a gate of the same name R01 and you can apply this gate to all other histograms to exclude all objects outside this gate Right click again and select Set Gate none to get back all objects Right click on the border of the polygon and select Gallery to display a selection of objects inside the gate this allows you to crudely evaluate if your gating was set in a useful way In the next histogram display the Total Intensity Dapi vs the Mean Intensity TxRed To in clude only the objects in RO1 right click in the histogram and select Set Gate R01 You will observe a cluster around the total Intensity Dapi of 9x10 and another cluster around 1 8x10 The left cluster consists of the nuclei in G1 whereas the cluster with twice the dapi intensity contains the nuclei in G2 With G2 cells having twice the amount of DNA they will have twice the amount of DAPI labeling and thus roughly twice the fluorescence intensity Draw a polygon around the cluster on the left with the
41. ch are defined in Assay Settings Sub objects See Chapter 3 3 Sub object Finder Detecting Sub objects 37 38 Chapter 3 Assays OLYMPUS Tracker Settings Enable Tracking Tracked Object Type Range Pixel J 1000 Range Pixel Set here the maximum difference that is allowed to change between two frames in order to maintain a track If the difference exceeds the Range an object will NOT be related to similar objects in the previous and subsequent images of the time series OK Click here to start the tracking The advance can be followed in the status bar at the bottom right corner of the scan4R Analysis main interface Once it is completed the Trace Parameters window opens Trace Parameter Calculator Tracking Particles gm 3 9 2 Track Analysis Parameters During the tracking is run not only the X Y positions of an object over time are calculated but also the time course of the parameters previously determined in Analysis Edit Assay Parameters see Chap ter 3 5 Measurement Parameters is extracted of the data The curves for each individual object are displayed in Trace Parameters Original Parameters This menu allows defining which kinetic parame ters of these curves are extracted e g the maximum intensity the time of maximum intensity or the duration of an increase in signal 3 9 2 1 Original Parameters Open the Trace Parameters Original Parameters window directly via Tracking
42. color channel and redistribute the different color intensities It thus improves the spectral resolution of the channels considerably and facilitates for example co localization studies Get from ROI Takes the mean value within the marked region as Stain Background Ignore Stain Click here to ignore the third channel in order to properly unmix two channel images Background Spectral Unmixing yields quantitatively meaningful data only if a background subtraction is performed prior to it 34 Chapter 3 Assays YE 3x3 Spectral Unmixing 1376x1038 1 2 RGB image 138 6 42 1358 1016 Intensity clipping 1 Stain Selection Stain 1 E Stain 2 _ be Stain 3 EA _ Ignore Stain Background Output Stains OLYMPUS 1376x1038 1 2 RGB image 50 9 77 770 802 Show Details Position List WO00001 P00001 W00002 PO0001 WO00003 P00001 W00004 PO0001 W00005 P00001 W00006 P00001 W00007 PO0001 W00008 P00001 WO0009 P00001 W00010 PO0001 WOOO11 POOOD1 W00013 P00001 Z00000 T00000 Dapi tif Z00000 T00000 Dapi tif Z00000 T00000 Dapi tif 200000 T00000 Dapi tif 200000 T00000 Dapi tif Z00000 T00000 Dapi tif Z00000 T00000 Dapi tif Z00000 T00000 Dapi tif Z00000 T00000 Dapi tif Z00000 T00000 Dapi tif Z00000 T00000 Dapi tif 200000 T00000 Dapi tif Intensity clipping 1 Show details Displays the matrix created by the se
43. d adjust the settings of former steps However back ground correction in the Image Processing tab should be performed prior to object detection as it changes the intensity values scan R distinguishes between two kinds of object types an assay always defines one Main Object type and up to four Sub Object types connected to it To give an example main objects may be indi vidual cells while their sub objects are individual structures within them To represent this hierarchical structure the Assay Settings window s tabs Main Object and Sub Objects are used to adapt different set the search algorithms for main and sub objects in order to ex tract the structures of interest from the images This is done by different Object Finder Modules that implement different rules for object detection The Parameters and Derived Parameters tabs contain the information about the kind of information to be extracted from the objects e g area shape The Image Processing tab allows defining image processing steps that are to be executed before the object detection and parameter extraction In the Virtual Channels tab new channels can be created as a result of post acquisition image process ing e g spectral unmixing To access the Virtual Channels tab you have to navigate through the tabs to the right using the arrow buttons on the top right 3 2 Object Finder Detecting Main Objects The Main Object tab of the Assay Settings window provides th
44. ding histograms and scatter plots etc sca files can be loaded via Analysis Open to revisit the analysis results To clarify this if you open a Scan via Scan Open the experimental settings of a scan will be loaded by reading in the Experiment descriptor xml file Thus you get access to the raw image data In con trast to this if you open an Analysis sca you will get the results in addition to the images 2 4 Managing Histograms and Scatter Plots Histograms are used in scan R for data representation classification and navigation scan R uses 2 D and 1 D histograms following the common representation in cytometry A 1 D histogram shows the frequency distribution of only one parameter A 1 D histogram is created when the same parameter for X and Y is selected On the X axis of the histogram the selected parame ter and on the Y axis the number of counts is plotted A 2 D histogram scatter plot is a plot of one parameter of a number of objects against a second pa rameter Color coding is used as third dimension to represent the frequency of occurrences The data displayed in a histogram are assigned to an object type main sub object The detection of the objects is defined by the assay The object type can be chosen from the Object pull down menu Chapter 2 Main User Interface OLYMPUS underneath the histogram The X and Y pull down menus are then automatically updated to correspond to the measured parameters o
45. e commands to define the Main Object detection Color Channel Select the color channel on which the main object detection is to be performed Module Select the method to detect individual Main Objects from the shortlist Module Leen Edge Intensity scan Analysis Software Manual Chapter 3 Assays Tak Assay Settings EEE Main Object Sub objects Parameters Derived Parameters Image Processing Object Finder Main Color Channel DAPI Module settings Add to list Settings Settings list Reject Border Boolean TRUE Nucs20 Min Object Size Boolean TRUE Min Size 132 100 Max Object Size Boolean TRUE Max 5ize 132 100000 Image segmentation Show Search Area Boolean TRUE Show Center Boolean TRUE Li ven Thari Dei mdima Dese Danla rm TMI IC Module Intensity Pl Settings list Each Object Finder Module has a list of preset parameters The lists can be modified and stored at will Individual modifications of these settings are marked as Modified Select the settings of choice from the shortlist Adjust This command opens the configuration dialog of the selected Object Finder Module For changing the list of Object Finder Modules see Chapter 3 4 Object Finder Modules Module settings This field lists the current settings of the selected Object Finder Module Add to list This adds the modified settings to the Settings list Click the button to open the Add Set tings
46. e different displays In order to activate Color Gating right click in the histogram and from the context menu right click either activate the option Color Gating directly or open Settings and there activate the checkbox Color Gating In the Histogram Properties window that opens via Settings select Show Legend This way you can additionally show the legend of the colors in the histogram The population percentages of the dis played gates are also displayed 500 0 Selected H ci 45 12 E cz 28 81 selected 100 00 Set Gate Show Region Clone From Gallery Zoom out Color Gating Settings 00 1000000 0 20000000 SO00000 0 4000000 0 E Total Intensity DAPI The following rules apply to decide which color is shown in Color Gating If there is only one object located on a pixel Bin the gating color of that object is shown If there are multiple objects on a histogram pixel the gating color of the relative majority of the objects will be shown Exception If there is a Gate applied to that Histogram that Gate will be automatically in the background as 100 of the objects will belong to that Gate 50 Chapter 4 Analysis Results Objects not belonging to a gate are shown in black OLYMPUS If there is no majority Gate the Color order of the Gating list will be used Analysis Assay Gat ing see Chapter 4 2 2 The Gate Manager The ranking can be changed with the arrow buttons i
47. e processor Module on the Image Processing tab for each of the image channels Press Adjust and observe in the new window the background in the image before left and after right background correc tion When moving the mouse over the pixels the grey value should be reduced in the cor rected images Color channel TxRed af fk Assay Settings Sub objects Parameters Derived Parameters Image Processing virtual Channels lt gt Image processors Image processor Module Channel al Module BackgroundCorrection dapi BackgroundCorrection Se BackgroundCorrection i 7 The next step is to determine the main objects Therefore open the Main Object tab in the Assay Settings Here you have to select the channel in which you want to use for image segmentation In many typical cases this will be the channel with the DAPI labeled nuclei the shape of which makes them rather ideal targets for object detection Object Finder Main Color Channel dapi Module Intensity Settings list Adjust Image segmentation LI View 8 For image segmentation use the Intensity module Both the Edge and the Intensity modules usually give good results when detecting nuclei In other cases this is mostly a question of experience and testing Click Adjust to open the Object Finder window Here you can check and adjust the settings of the intensity detection algorithm You will already get a good result by simply pressing the Aut
48. e the yel scan Analysis Software Manual Chapter 3 Assays 23 low circle appearing in the images 3 Play around with the Selectivity slider to get just the strong edges up or also the weak edges down Try to increase the selectivity as much as possible thereby removing edges due to noise and artifacts without letting gaps in the contour of wanted particles get too large As you can see in the image above contours with closed edges are marked green whi le contours with open edges are red Edge detection Selectivity Maximum object size 1 1024 0 9 0 8 0 7 ged 100 4 0 5 0 4 0 3 0 2 0 1 0 2 4 Edge Closing Click on the image or the settings cluster on the right to get to the third step In this final step the open edges extracted in step two are now closed by combining them with other open edges You can then filter particles by size and closure quality split them with the watershed algorithm or select a hierarchy Particle detection P Close edges Filtering Ignore border objects Minimum object size 1 10 100 1000 10000 90000 Minimum closure quality 0 0 2 0 4 0 6 0 8 1 Particle hierarchy Splitting Selection mode Best Closures ze C Watershed Particle Level 24 Chapter 3 Assays OLYMPUS First decide if there may have already been sufficient particles detected in step two the green ones If you think so yo
49. eehand Standard tool to draw freehand regions The region is closed automatically once the mouse button is released Circle Standard tool to draw circles Close the circle by double click ib Ring Segment Standard tool to draw rings Once a ring is drawn the inner and outer borders can be dragged to adjust the thickness The cutting line can be dragged to convert the ring into a ring segment scan Analysis Software Manual Chapter 3 Assays 31 3 8 Virtual Channels Virtual channels are image channels that are not created via image acquisition during the execution of a scan Instead they are a result of post acquisition image processing and added as new channels to the original image data These can then be used for further analysis steps e g object detection To access the Virtual Channels tab in the Assay Settings window you have to navigate through the tabs to the right using the arrow buttons on the top right te Assay Settings Sub objects Parameters Derived Parameters Image Processing Virtual Channels VIC Process List Module al get GENEE odule Simple Math Adjust pO Remove Input Channels virtual Channels DAPI CAPI DAPT DAFI Compatibility Status a ox New Click here to create a new entry in the VC Process List Virtual Channels list Default names for the virtual channels resulting from the processing are automati cally created It can be changed manu
50. efore background correction right image after background correction Size filter This is the only parameter to be set For background correction a Size filter of 200 is the default value For most cases when a general background has to be removed it works fine However to better extract e g small particles in the nucleus a background correction filter size of 15 can be suitable cf exam ple below scan Analysis Software Manual Chapter 3 Assays j fe p 1344x1024 0 5 16 bit image 257 916 170 Intensity clipping 1 v 1344x1024 0 5 16 bit image 17 806 2 Intensity clipping 1 Images Size filter er W00001 P00001 200000 T00000 dapi tif A1 W00001 P00002 200000 T00000 dapi tif A1 W00001 P00003 200000 T00000 dapi tif 15 A1 W00001 P00004 Z00000 T00000 dapi tif 41 W00001 P00005 200000 T00000 dapi tif A1 W00001 P00006 200000 TO0000 dapi tif Example for small Size filter for background correction 3 7 2 XY Shift In certain cases it may occur that image channels are shifted relative to each other in the channel over lay This is rather often the case if an observation emission filter wheel is used and images are acquired with different emission filters This function allows correcting the shift along the X and Y axes XY Shift Set here the number of pixels the chosen channel is to be shifted along the X and Y axes relative to the other channels You have
51. ely Gate Name Gate applied to the histogram Count Number of objects in the histogram Tot Amount of objects in percent of the total objects xMean yMean Mean value of the abscissa ordinate parameter of all objects in the histogram xCV yCV Standard deviation of the abscissa ordinate parameter of all objects in the histogram scan Analysis Software Manual Chapter 4 Analysis Results xCV yCV Coefficient of variation of the abscissa ordinate parameter of all objects in the histo gram Applied Gate This command allows changing the applied gate in the selected histogram The pull down menu lists all gates from the Gates table Gallery This command generates an image gallery of all objects in the selected histogram The number of images is by default limited to 100 If the histogram contains more objects the 100 objects closest to the center of gravity of the region will be shown by default See Chapter 2 2 General Settings Export Data This command exports the data of the selected histogram as tabulator delimited table in txt format Well Results This opens the Well Results window see Chapter 4 3 Well Results 4 2 3 Color Gating Color Gating shows different populations as defined by the Gates in different colors in the one and two dimensional histograms Color Gating is a property of each individual histogram displayed thus histograms with and without Color Gating can be displayed simultaneously in th
52. es to abide by the conditions in this contract 4 OLYMPUS SOFT IMAGING SOLUTIONS is the legal owner of all copyrights and trademarks of the OSIS scan4R SOFTWARE PRODUCT and documentation National and international law protect copyrights and trademarks OLYMPUS SOFT IMAGING SOLUTIONS reserves all rights which are not explicitly expressed in written form 5 Warranty 1 OLYMPUS SOFT IMAGING SOLUTIONS guarantees for the period of 12 months after the date of purchase that the software works in all major aspects according to the descriptions in the manuals OLYMPUS SOFT IMAGING SOLUTIONS as the producer of the software provides this warranty It does not replace or restrict other warranties or liabilities provided to the User by local or other sales people or organizations OLYMPUS SOFT IMAGING SOLUTIONS does not guarantee that the software is defect free that the software fulfills the specific requirements of the User or that the OBS scan SOFTWARE PRODUCT works with other software provided by the User 2 OLYMPUS SOFT IMAGING SOLUTIONS further guarantees that the software storage devices floppy disks CD ROMs etc and the manuals are free of material defects Defective storage devices or manuals will be replace free of charge if they are returned to OLYMPUS SOFT IMAGING SOLUTIONS within 90 days of purchase and accompanied by a proof of purchase 6 Liability 1 OLYMPUS SOFT IMAGING SOLUTIONS or their sales organizations ca
53. ested especially in the data at the beginning of each track Alternatively when applying the operator t_max e g on the parameter Meanlntensity GFP the time point when the mean intensity of GFP is maximal is set to be 0 This way the time curves can be displayed synchronized ti Trace Viewer 1350 0 1325 0 1200 0 1275 0 Ti Trace Viewer 1250 0 1280 0 1260 0 1240 0 1220 0 1200 0 1 150 0 FE Gate E 1 150 0 Mikoticz hi z c 1140 0 Max displ Traces 1 1000 1120 0 1100 0 100 1080 0 1060 0 1040 0 Single mode Smoothing Sigma 1020 0 I I I I I I I I P 0 01 I I I D 10 20 20 40 EO 60 70 OU ap 100 110 120 130 140 Real Time Derivative Parameter Mean Intensity GFP a EI Smoothing J Sigma 0 01 O 1 1 15 44 Chapter 3 Assays OLYMPUS Parameter Select parameter of which the time curve is to be shown in the graph display Derivative Check this option to display the first derivative of the curve of the selected Parameter Smoothing Sigma This function applies a smoothing filter on the time curve Set the strength of the smoothing with the slider The graph display is being updated immediately Selecting objects and navigating along tracks Tracks in the Trace Viewer and the objects in the images they derive from are directly linked Upon clicking on a time
54. f the chosen object type see assay definition The X and Y pull down menus are used to change the parameters displayed in a histogram The axes of abscissa and ordinate are labeled with the chosen parameter goodMucs 500 0 goodHucs Counts k k UU 2500 500 0 750 0 1000 0 1250 0 1500 0 1750 0 2000 0 E 0 0 2000000 0 DUU CO Area Total Intensity DAPI Two buttons are located in the lower right corner of each histogram They allow toggling between the Navigation and the Region Selection modes Navigation D The Navigation button with the pointer symbol allows navigating within the data Each data point within a histogram is directly linked to the object from which it is derived A selected data point is highlighted by a red circle in all histograms in navigation mode as long as the data point is within the displayed area The corresponding object is displayed in the Image Viewer The X and Y values of the data point are displayed next to the X and Y pull down menus Holding and dragging the mouse using this tool allows to virtually following the objects changes within the parameter set The navigation tool also al lows dragging and modifying existing regions Region H The Region tool is used to draw polygons into a 2 D histogram and to set a range in 1 D histograms Regions define bi dimensional intervals within the parameter range and thus subpopulations of data points Double click in order to close a region in a 2 D histogram scan
55. gation to notify the purchaser In no event shall Olympus Soft Imaging Solutions GmbH be liable for any indirect special incidental or consequential damages arising out of purchase or use of this manual or the information contained therein No part of this document may be reproduced or transmitted in any form or by any means electronic or mechanical for any purpose without the prior permission of Olympus Soft Imaging Solutions GmbH 2006 2008 by Olympus Soft Imaging Solutions GmbH All rights reserved Manual version 2 1 December 2008 LICENSE AGREEMENT Preamble OLYMPUS SOFT IMAGING SOLUTIONS GMBH is manufacturer of the scan R software and drivers interfacing software to Olympus Soft Imaging Solutions manufactured hardware These components are referred to as OSIS scanAR SOFTWARE PRODUCT LICENSE AGREEMENT between END USER and OLYMPUS SOFT IMAGING SOLUTIONS regarding the OSIS scan4R SOFTWARE PRODUCT IMPORTANT READ CAREFULLY Below you will find the contractual agreements governing the use of the OSIS scan4R SOFTWARE PRODUCT These conditions apply to you the user and to OLYMPUS SOFT IMAGING SOLUTIONS With any of the following actions you explicitly agree to be bound by the conditions of this contract purchas ing the software opening the package breaking of one of the seals or using the software In case you do not agree with any of the conditions of this contract please return all parts of the product including manual
56. he population view When you select a gate from the list the objects that fall into the gates are marked with a box in the front panel image Show Trace This opens the Trace Viewer which displays the time curve for the selected object See Chapter 3 9 3 7race Viewer scan Analysis Software Manual Chapter 3 Assays Gallery it displays a time gallery of the selected object P Main Circularily Factor Circularily Factor EL F Po og HIEN J d 4 E a 4 A A 4 DG ee A SS eng d ke HIN P FEE Eu NO se PIP RN PIP ds KIK IL gogg d d EE d d FEI d d gg 4 d po Gallery display in time lapse mode Movie This opens the menu Trace Movie which allows you to export a movie of a single trace or a complete position Ti Trace Movie Compression Filter DY Video Encoder we Frames s SAVE 45 ZPULSCSIscan R Data Test amp DocumentationiTimeLapselGFP ar Progress 3 9 1 Tracking Configuration The Tracking Configure Tracer command opens the Tracker Settings window It also opens automati cally when the View mode Trace is activated without any tracking parameters being set already Tracked Object Type Select here the kind of objects to be tracked i e main objects or any of the sub objects if su
57. if Threshold 1393 4 A2 W00002 P00002 200000 TO0000 dapi tif amp 2 W00002 P00003 Z00000 TO0000 dapi tif amp 2 W00002 P00004 Z00000 T00000 dapi tif MERER A2 W00002 P00005 200000 T00000 dapi tif amp 2 W00002 PO0006 200000 T00000 dapi tif U Ignore border objects A3 W00003 PO0001 Z00000 TO0000 dapi tif Fill holes within objects A3 W00003 P00002 200000 TO0000 dapi tif S Ges amp 3 W00003 P00003 200000 T00000 dapi tif Minimum object size 100 pixels ti 83 W00003 P00004 Z00000 T00000 dapi tif Maximum object size 1000000 pixels A3 W00003 P00005 200000 T00000 dapi tif A3 W00003 PO0006 200000 TO0000 dapi tif B2 W00026 P00001 200000 T00000 dapi tif B2 W00026 P00002 200000 T00000 dapi tif Fill holes within objects Check this box to fill the object mask in case it contains holes Minimum Maximum object size Check these boxes and adjust the values to apply minimum and maximum size filters to the objects in order to ignore objects that are outside these size limits 3 4 3 Edge Detection The EdgeSegmentation module is a general purpose edge based particle detector The idea of the algorithm is to find a closed contour around each particle First the edges of the image are extracted For those edges which already form a closed contour the algorithm stops Since the remaining open edges may be part of a closed contour around a particle the algorithm then trie
58. image that is currently being analyzed in the thumbnail display Click the Main Display button to have this image displayed in the main mage win dow You may Pause or End the analysis by clicking the respective buttons Once the analysis is completed store the results via Analysis gt Save as scan Analysis Software Manual Chapter 4 Analysis Results 47 4 2 Managing Gates scan4R Analysis detects objects in all images acquired during a scan and performs measurements and analyses on each object found The data can be displayed in form of histograms where each color coded data point represents the results of one object or of several objects that happen to have iden tical results The results and thus the objects can be grouped by Gates and Regions The Gates allow classifying the objects according to their properties i e the parameters that were extracted for these objects Once these gates are defined they can be applied to all further measurements for auto matic quantification The gates are administered in the Gate Manager here you find also access to the Well Results menu which contains the detailed results for all wells 4 2 1 Gates and Regions scan4R distinguishes between Gates and Regions Regions define classification rules in form of poly gons or ranges in histograms Gates are composed of one such region or of several regions that are linked with Boolean operators AND OR AND NOT Once a Gate is set only data
59. in Analysis Edit Assay Assay Settings Image Processing Image processing is used to im prove the quality of the displayed image but slows down the systems image display Therefore it is especially recommended to switch it off for performance when creating galleries The image process ing is described in Chapter 4 9 mage Processing 12 Chapter 2 Main User Interface OLYM PUS Row Column Position Time Use these entries to select a specific image to be displayed The up down arrows allow fast navigation though your image data set by incrementing or decrementing the Time Position and or Well number Interactive objects Select the object type to be outlined in the display when clicking on an object or data point The image viewer is equipped with a tool bar to select different mouse tools Zoom DI The Zoom mode is used to zoom into the displayed image A click into the image causes a zoomed in view with the cursor position as the center To zoom out the Shift key must be pressed simultaneously Selection D The Selection mode allows selecting individual objects within the image via mouse click The object type to be displayed can be selected in the pull down menu in the bottom right corner of the image viewer Main objects are highlighted by a green outline The data point corresponding to the selected object is highlighted with a red circle 2 D histograms and a vertical red line in 1 D histograms Move Ki Depending on the z
60. ion Derivative The formula for the Derived Parameter would thus be p4 p3 when using the entries as shown in the screenshot scan Analysis Software Manual Chapter 3 Assays 43 3 9 3 Trace Viewer Open the Trace Viewer by selecting Tracking Show Traces in the main menu Alternatively you can right click on one detected object in the image in the Trace View and select Show Trace to see the trace of this individual object Set the number of displayed traces with the slider on the right To display only objects of a certain gate that was previously defined in the Trace View this gate can be selected in the Gate drop down menu The results of the tracking are displayed in the Trace Viewer window once the tracking has been carried out Max displ traces Use the slider to adjust the number of displayed The traces will be taken from a random selection of the specified gate Single mode Use this option to have just one trace displayed If a trace is selected via mouse click it will be this one that is being displayed Gate Select a gate to have only the corresponding traces displayed Time Scale fea Time is the default and causes that each point in time is set relative to the beginning of the time lapse series When race Time is selected each point in time is set relative to the beginning of each track when t_first is selected which may be important if an object was not tracked right from the start of the time lapse series and one is inter
61. ited table in txt format Wells Groups Use this button to select if the values are to be given for individual wells or entire groups of wells All wells that have the same Name Description entry form a group Wells that do not pertain to a group will be listed independently The Name Description is set in Scan gt Select Wells see Chapter 2 7 Selecting Wells for Analysis 4 3 2 Well Results Populations In the Populations tab the number of objects absolute and relative in all defined gates are listed A reference gate can be set to the number of objects in all other gates to the reference gate The histo gram on the right gives a graphical representation of the results Size comparison The table gives the amount of objects in absolute numbers or as percentages that pertain to each of the Gates defined in the Gates Manager See Chapter 4 2 2 7he Gate manager 53 Chapter 4 Analysis Results OLYMPUS Display Counts This toggle button switches between the listing of total numbers and the percent age of objects Object type Select the object type from the pull down list Main Sub objects Reference gate The population of the selected Reference gate is set relative to the populations of the other gates in the Size comparison table Export Table This function exports the data as tabulator delimited table in txt format Wells Groups Use this button to select if the values are to be given for individual wells or e
62. lection of the stains This matrix is used for the processing of the images The entries of the matrix can be changed manually graphical representa tion is displayed Output channels Select the stains to see the result of the spectral unmixing 1 In the Module shortlist select Spectral Unmixing and press New 2 Press Adjust to start the 3x3 Spectral Unmixing menu 3 Click on the Background button 4 Mark a background area using the drawing tool button on the bottom right corner of the im age displays 5 Click the Get from ROI button 6 Identify a structure that contains just the first fluorophore 7 Mark the structure using the drawing tool button on the bottom right corner of the image displays 8 Click on the Stain 1 button scan Analysis Software Manual Chapter 3 Assays 35 9 Click the Get from ROI button 10 Repeat steps 4 7 for the other fluorophores stains In order to perform the spectral unmixing the software has to determine the contribution of the fluorescence of different fluorophores to the different color channels To do so ideally series of mono labeled reference samples would be used In case such samples are not available for each of the fluorophores molecular structures have to be identified by the user that are certain to contain just only one of the fluorophores and that do not spatially overlap with structures containing other fluorophores The result of the spectral
63. ll be shown at first and in gray scale Move the cursor over the image as a test and observe the status bar at the lower left The channel intensity of each channel at the pixel positioned at the tip of the cursor arrow Is shown in the status bar The intensity values of the objects are usually much higher than the camera offset the dark intensity of the camera T 1244 Bi Gi Ri 4302 V383 2 1 Memorize the intensity values of the brighter structures in the image for the subsequent dis play adjustment Set the grayscale selector to None and load the dapi channel in the blue selector and the TxRed channel in the red selector Display Mone Ke Execute View gt Layout and adjust the display as explained in Chapter 2 5 Using the Image Viewer The first step in setting up the assay is to find the best settings for the object detection If you would have opened an old analysis file sca rather than the scan data only the histo grams would already show the data points of the analysis that was run before In order to get these results from scratch you first have to set up a new assay to perform a background cor rection determine main and sub objects and the parameters you want to extract from the data Execute Assay Edit Assay to open the Assay Settings window scan Analysis Software Manual Chapter 5 Example Assay Step by Step 59 6 The first step in Analysis is to select the Background Correction Imag
64. logarithm of x to the base of 10 Computes the logarithm of x to the base of 2 Compares x and y and returns the larger value Compares x and y and returns the smaller value Computes the remainder of x y when the quotient is rounded toward Infinity Computes x raised to the y power scan Analysis Software Manual rand rem x y si sec x sign x sin x sinc X sinh x spike x sqrt x step x tan x tanh x Random Number 0 1 Quotient amp Remainder Sine Integral Secant Sign Sine Sinc Hyperbolic Sine Spike Square Root Step Tangent Hyperbolic Tangent Chapter 6 Appendix 81 Produces a floating point number between 0 and 1 exclusively Computes the remainder of x y when the quotient is rounded to the nearest integer Evaluates the sine integral for any real number x Computes the secant of x where x is in radians 1 cos X Returns 1 if xis greater than 0 returns 0 if xis equal to 0 and returns 1 if x is less than 0 Computes the sine of x where xis in radians Computes the sine of x divided by x sin x where xis in radians Computes the hyperbolic sine of x Generates the spike function for any real number x Computes the square root of x Generates the step function for any real number x Computes the tangent of x where xis in radians Computes the hyperbolic tangent of x OLYMPUS OLYMPUS LIFE SCIENCE EUROPA GMBH Postfach 10 49 08 20
65. mez yMaximum Sl Text color Black on white Dots 45 x log y log 4 LinIncFact S55 1 5 x Precision at y Precision 1 EE ab 7 Aut lez x Format Decimal Y Format Pecimal visible grid uboscale x Maximum Exceeding channels eo Color Gating Bk Color Show Legend Color table bin width Binned color table KR Color scheme Selector Color d WERNE Eu SER io BR Axis attributes This field offers different choices to set the axis scaling Grid cosmetics This field offers different choices to set the grid display Autoscale This field offers different choices to set the auto scale of the axes BKColor A click on the colored field opens a window that allows selecting the background color 10 Chapter 2 Main User Interface OLYMPUS LU HOOOOOOOCOOOOCOOOCOO History System EEEE 100000 OG 09589 19 ES Selector Color A click on the colored field opens a window that allows selecting the cross hair color Region Color A click on the colored field opens a window that allows selecting the color of the region outline Color scheme This color palette defines the coloring of data points pixels in the histogram in depend ence of the number of counts events they represent For example if the Color table bin width is set to three and a data point represents 7 8 or 9 counts it will be displayed in the third color from the left Color table bin width It defines the range of co
66. n that window If a histogram is gated context menu Set Gate the objects will be shown in the color of the sub gate they belong to 1000 0 Cells types 3 20 W Typeb 29 22 E TypeC 22 39 E Typed E TypeE E TypeF fl TypeG ES TypeH Counts 01 10 10 0 100 0 Color 9 62 1 60 3 56 370 3 11 Cells 100 00 1000 0 The order mechanism of Color Display 1 D In 1 D histograms the applied Gate is always in the background The order of display is organized by areas If one of the Gates has less area above the other one it will be shown in the foreground The selector line is now position sensitive in 1 D Histograms as well Thus if you click on a green area you will get an object from the green Gate if you click on gray you will get one from the gray Gate Examples 1 The example below shows a cell cycle with G1 and G2 defined as gates with different colors The histogram is not gated thus all detected objects are shown Some of the pixels repre sent more than one object In this case all pixels that have at least one object that belongs to the gate Se ected are shown in red Only the red color is shown because there is never a ma jority of G1 or G2 because G1 and G2 are subgroups of Selected and the order in the Gate Manager is Selected above G1 and G2 The other objects are shown in Black because they do not belong to any Gate A A Selected 55 371 scan Analysis Software Manual WE
67. ngs is shown in the image above 1 Clipping On the left side select an image of your choice from the list Adjust the clipping ei ther by pressing Auto or manually by moving the green bars If one or both bars are missing just press Auto once Try to clip away any unwanted noise or artifacts while maintaining good contrast between the particles you want to detect and the background Image selection Image List B3 W00015 P00001 Z00000 T00006 TxRed tif B3 WO0015 PO0001 200000 T00007 TxRed tif B3 W00015 P00001 200000 700008 TxRed tif B3 W00015 P00001 Z00000 T00009 TxRed tif B3 W00015 P00001 200000 700010 TxRed tif B3 W00015 P00001 200000 T00011 TxRed tif B3 W00015 P00001 200000 700012 TxRed tif B3 W00015 P00001 Z00000 T00013 TxRed tif B3 WO0015 PO0001 200000 T00014 TxRed tif B3 W00015 P00001 200000 T00015 TxRed tif B3 WO0015 PO0001 200000 T00016 TxRed tif B3 W00015 PO0001 200000 T00017 TxRed tif B3 WO0015 PO0001 200000 T00018 TxRed tif B3 W00015 P00001 200000 700019 TxRed tif B3 W00015 P00001 Z00000 T00020 TxRed tif DS KJANNIC MANANI FANNNN Topp TMa kif ln mh ree 2 Edge extraction Click on the image or the settings cluster in the middle to get to the second step In this step the edges of the image are extracted First grab the Maximum object size slider and adjust it so that the largest particles you want to detect just match insid
68. ngs to a scan Tracking Trace View toggles between the Population and Trace View modes Tracking Configure Tracer opens the Trace Configuration window to select how the object tracking is to be performed Tracking Define Parameters opens the Trace Parameters window to select trace analysis operations Tracking Show traces opens the Trace Viewer that visualizes the trace graphs View gt Layout display properties for the RGB display affects only the displays View gt Parameter View lists all parameters for a selected object that were determined during analysis Modules Object Finders list and configure available Object Finder Modules Chapter 6 3 Libraries Modules Object Analysers list and configure available Object Analyzer Modules Chapter 6 3 Librar ies Modules gt Image Processors list and configure available Image Processing Modules Chapter 6 3 Libraries Modules VC Processors list and configure available Virtual Channel Modules Chapter 6 3 Libraries Repositioning gt Interactive the scan R screening system moves to the position of the selected object Settings Gives access to some preferences including galleries directories data export and settings for repositioning an reclassification Chapter 2 Main User Interface OLYMPUS 2 2 General Settings The general preferences can be defined in the Settings menu Max Gallery Objects Sets the number of objects that are displayed in a galle
69. nnnnnrennnnsrnnnnnnrennnnnrennnn 46 4 2 Managing Gates rrrrnnnnnrvvnnnnnnnvrnnnnnnnrnnnnnnnnrennnnnnnsennnnnnnsssnnnnnnsennnnnn 47 4 2 1 Gates and Regions EEN 47 4 2 2 TheGateManager 47 scan Analysis Software Manual OLYM PUS 423 COLO GaU BEE 49 A3 E EE 52 4 3 1 Measurement Hesults A 52 4 3 2 Well Results Populations AANEREN 53 433 POT DENON Leed 54 4 3 4 Export results of individual objects rrrrrnnrrvrnnnrrernnnnrevnnnnrennnnnrennnn 56 5 Example Assay Step by Step rrrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnr 57 5 1 Setting up and Executing an Aesay 58 5 2 KATE Dala ak need 61 53 llmeskapse AASS gassene Seen 64 ENEE REI ee 71 er ENN 72 OLT IMAGES VEL Lesere unde 72 6 1 2 Image Channel Definition en nnnnnnnnnnnnen nn 73 6 19 SSE ENN 74 6 1 4 View Conversion R SuIts nennen 74 62 FCS Export Functionalltysarueeeeunnunaiean ana 75 6 9 OTNES LE 77 6 3 1 Object Analyzers Library OAI 77 6 3 2 Object Finders Library OF 78 6 3 3 Image Processing Library rvvrnnnrvnnnnnrvnnnnnrvnnnnnrennnnnrennnnnrennnnnrennnn 78 6 3 4 Virtual Channel Library evrrnnnrronnnnnnnnrvnnnnnnnrrnnnnnnnrennnnnnnnrnnnnnnnnnenn 79 6 4 Valid functions for derived parameters nennen 80 scan Analysis Software Manual Chapter 1 Introduction 1 1 Introduction Chapter 1 Introduction OLYM PUS Thank you very much for purchasing Olympus Screening Station for Life Science and for your confi dence in
70. nnot be held liable for damages or injuries resulting from the use of the software or the lack of capabilities of the software unless the User can show gross negligence on the part of OLYMPUS SOFT IMAGING SOLUTIONS This applies without exceptions also to losses of produc tivity or profit interruptions in the flow of business or manufacture loss of information and other financial losses Without exceptions the possi ble liability of OLYMPUS SOFT IMAGING SOLUTIONS is limited to the amount that the User paid for the product These limitations on the liability do not influence claims for reasons of product liability 7 Contract duration legal consequences of violating the license 1 The contract is deemed to be in force for an unspecified period The User rights are automatically terminated if one of the conditions of the contracts has been violated 2 In case of a contract violation the User has to return the original storage devices and all copies thereof including all modified copies and all printed and written documentation to OLYMPUS SOFT IMAGING SOLUTIONS or the User has to destroy these items 3 In addition OLYMPUS SOFT IMAGING SOLUTIONS reserves the right to file a lawsuit to claim reparations for damages non compliance or removal of the software in case of license violations The following laws and or conditions are in effect the conditions of this contract copyright laws and the laws of the civil code scan Analysi
71. ntensity TxRed v EK E You will observe that the distributions in G1 and G2 look different In G1 there are only nuclei with a low mean intensity in TexasRed whereas in G2 there are two populations one with a lower mean intensity in TexasRed and one with a higher intensity in TexasRed Again select the polygon tool to define a region around the two populations in G2 and create two gates according to step 3 We name the nuclei with a high concentration of Texas Red G2_active and the nuclei with a low concentration G2_passive The result in the Gate Manager may look as follows Regions Gates Edit Gate ROL A Definition ROZ E ROI Name Color RO G2 i RO4 GI RO1 AND ROZ de PG ROS G2 R01 AND ROS Definition G2_active RO1 AND RO3 AND R04 G2_passive RO1 AND R03 AND ROS mm AND RO3 AND ROS 7 Leave again the Gate Manager window By right clicking on the main window you can high light the nuclei in the different gates with boxes when you activate them e g Show Gates G2 active Another option to compare the results is to right click on the border of the gate and to choose Region Gallery Plot a region gallery of G2 Active and G2 passive to compare the results of the gating In the lower part of the Gate Manager you will also find statistics of the displayed histograms For more detailed results click Well Results in the gate manager and go to the tab Popula tions Here you will find the
72. ntire groups of wells All wells that have the same Name Description entry form a group Wells that do not pertain to a group will be listed independently The Name Description is set in Scan gt Select Wells See Chapter 2 7 Selecting Wells for Analysis ir Well Results Measurement Results Populations Export Definitions Display Counts TE Name Description Main Size Comparison Reference gate Nucs AZ A3 B2 83 Gated populations I I 40 600 80 4100 120 Group Export Table cane Lol 4 3 3 Export Definitions In the Export Definitions tab the parameters that are to be exported can be defined This is especially useful for batch analysis and if not the complete results tables given in the Populations or the Meas urements tab is to be exported Select New to create a new export file The parameters to be exported have to be selected in the other tabs i e in the Populations tab or in the Measurement Results tab When you right click in these two tabs on the column names a context menu will open which allows you to export the selected column to a tab delimited txt file scan Analysis Software Manual Chapter 4 Analysis Results Size Comparison Well RO in in in in in All ROLAND Add to Rest Est 1 AND RO ROL 1371 Add to best d 2 1371 ER Add to new File Bk 1121
73. o button for determining the right threshold value and turn the Wa tershed algorithm ON You can observe the effects of the parameters in the right panel where the result of the segmentation algorithm is shown 9 If Sub objects are associated with the Main Objects check and adjust their detection in the same way on the corresponding tab in the Assay Settings window In this example skip the Sub objects tab 60 Chapter 5 Example Assay Step by Step OLYMPUS Ki Dhbect Finder inieenity Threshold Her 219 ee ae DET 8 68 Leming Tiaan karat Truet 108 FER EE t A These A n 135 amp AE ee T F TOO api E z 2 e AA Ps Pe Tap o A TO Pe 0000 TOO pn M er Dk ke el Deet N boii abad A Fine ap uf hd EN EN E ANERER EE DOEN TOR Lo P Ina rte Ger AWER JK ZER Taga ar laden 1 Fakt A ARE TH Aen ti sl Mee bech ptn Iesel AMET POT E DECKT AER As Ei cancel A A Pais HECKEN Tong D I z parat a ee TO deeg hi Ei PORN BONN ECK dee tal DAPI labeled nuclei Detected objects 10 In the Parameter tab you can choose which parameters will be calculated during the analy sis Le you need to select the measurements that are to be carried out Typical measure ments are area mean and total intensity and the circularity factor Area size and circularity factor are extremely helpful as they allow to discriminate between more or less round single nuclei and impurities or objects that consist of clustered
74. objects belonging to each individual Main Object that belong to the same Sub object type scan Analysis Software Manual Chapter 3 Assays e MEAN ID This operator averages all values of the given parameter ID of the Sub objects belonging to each individual Main Object that belong to the same Sub object type e STDV ID This operator calculates the standard deviation of all values of the given parameter ID of the Sub objects of each individual Main Object that belong to the same Sub object type The parameter resulting from a special operator expression is assigned to the Main Object New This allows inserting a new Derived Measurement into the list Remove This deletes the selected parameter from the list 3 7 Image Processing Images can be processed before being analyzed in an Object Finder Module Thus image quality can be enhanced and the object detection improved and facilitated A set of predefined image processing modules is available A variable set of image processing steps can be assigned to each color channel The configuration of the individual steps takes place via the Image Processing window accessible via Assay Edit Assay Image processing changes the image data temporarily The original image data will not be lost but remain stored However in the further course of the assay execution the analysis will always work on the processed data In order to keep the original data accessible during the assay virtu
75. ogram Files Scan R Package 081015 AnalysisjObject Analysers 2 e Object Analyser Module Perimeter Parameter Perimeter v Max Feret Diameter End Y Max Horiz Segment Length Left Max Horiz Segment Length Right Max Horiz Segment Length Row gt Bounding Rect Width Bounding Rect Height Bounding Rect Diagonal J Perimeter Convex Hull Perimeter Holes Perimeter Max Feret Diameter Equivalent Ellipse Major Axis Equivalent Ellipse Minor Axis Equivalent Ellipse Minor Axis Feret Equivalent Rect Long Side Equivalent Rect Short Side Equivalent Rect Diagonal Equivalent Rect Short Side Feret Average Horiz Segment Length Average Vert Segment Length Hydraulic Radius Waddel Disk Diameter Area Holes Area Particle amp Holes Area Convex Hull Area Imaqe Area Ki OLYMPUS Chapter 6 Appendix The password protected Object Analyzers Library can be accessed through Modules Object Analyz ers Library It allows managing the set of parameters selectable in the Edit Assay tab Parameters see Chapter 4 6 Measurement Parameters Add Click here to add a new entry to the list Parameter Select the desired parameter from the drop down menu to add it to the list 6 3 2 Object Finders Library OFL The password protected Object Finders Library can be accessed through Modules Object Finders Library It lists and allows administrating the available Object Finder Modules Each module may have different
76. on Trace Q 20 15 862 306 Well Name Description BrdU Each analysis data point is directly linked to the image object it is generated from These objects can be displayed in a close up view in the image viewer Browsing in the data set by clicking on data points with the histograms Navigation tool automatically leads to a corresponding update of the image in the viewer Additionally the Well Position Time and Well Name Description fields are updated and give information about the image origin The small arrow buttons on the left of these fields can likewise be used to navigate Values can be typed in as well Display Multi color images consist of individual color channels that were recorded with different optical acquisition settings e g different excitation filters The red green and blue pull down menus serve to select the input for the three color channels that are displayed in the respective colors In order to dis play a channel e g a transmission channel in grayscale select it from the gray pull down menu When having both grayscale and any color selection active a transmission overlay display will be used for the grayscale selection The clipping of the RGB display i e the scaling of the image display brightness can be changed via the menu point View gt Layout that opens the Image Clipping window Image Processed Click this button to toggle between the original image and the processed image as defined
77. oom factor only a part of the image will fit into the display The Move mode allows moving the visible area via mouse drag The status bar in the lower left corner shows information about the magnification of the displayed im age the current x y position of the cursor and the pixel value s at this position 2 6 Adjusting the Image Displays View Layout opens the Image Clipping window that allows adjusting the display brightness Note that these image settings affect the front panel display as well as well overviews and galleries A raw image will always have a certain background intensity Also one will avoid to over saturate images and es pecially in fluorescence applications rather use only a fraction of the camera chip capacity The con sequence is that a raw image is usually low in contrast and may even appear entirely black Clipping is applied to change the image brightness by defining a range of low pixel counts to be displayed black as well as a range of high pixel counts to be displayed with maximum brightness Clipping Type This button toggles between Dynamic and Absolute clipping scan Analysis Software Manual Chapter 2 Main User Interface e Image Clipping Selected Channel Channel view Settings Absolute SS Clipping Type Absolute Dynamic 0 1 Gray scale palette Grayscale br A Use Scan Settings Dynamic Define here how many pixels as a percentage of the total number of pixels will
78. ot be implemented because most flow cytometers produce inte ger 16 bit data in single data sets It is therefore recommended to test the FCS 3 0 compatibility before deciding which FCS program is to be used for the analysis Most manufactures provide demo pro grams for testing In order to export FCS data open the Settings menu from the menu bar Data Export Exporke Txt Scale Export Export Min Export Max Clipping O mer FCS Analysis Export Welle Export Data Type Merge Well Data Integer 32 w Chapter 6 Appendix OLYM PUS Export Data Type Here you can choose the data types exported Integer 16 Integer 32 double 64 bit floating point according to FCS 3 0 standard It is recommended to use the highest precision that is supported by the program intended to import the fcs files Scale Export Scaled On Off This button activates the rescaling procedures during FCS export Switch off the scaling if you want to compare datasets from two separate export procedures quantitatively If activated the scaling settings Min Max Clipping will be used Export Min Export Max Clipping Settings used for rescaling the original data according to x fos x PnG PnOffset with x fcs exported data points X the original data points and n parameter number PNG automatically determined linear scaling factor Gain PnOffset automatically determined scaling offset The resulting scaling offset and the scaling fac
79. our products and services The scan Analysis Software is designed for the automated analysis of images that were acquired by the Olympus scan Screening Station and with the scan Acquisition Software The software is in tended for the use in biomedical research A The scan Analysis Software the scan Acquisition Software as well as the hardware com ponents of the Olympus scan Screening Station for Life Sciences are for research use only 1 1 Abstract This user manual will guide you through the usage of the analysis software of the Olympus Screening Station scan R It will assist you in setting up efficient and reliable assays from scratch This scan4R module is intended to be used for the analysis quantification and navigation through your results The analysis module of scan R allows you to run the analysis during acquisition or in offline mode after wards Special care has been taken to guarantee correct and accurate information within this documentation although this is subject to changes due to further development of the Screening System Thus the manufacturer cannot assume liability for any possible errors We would appreciate reports of any mis takes as well as suggestions or criticism 1 2 Technical Support If you find any information missing in this manual or you need additional support please contact Olympus directly scan Analysis Software Manual 2 Main User Interface Chapter 2 Main User Interface
80. ow the background in the image before left and after right background correction When moving the mouse over the pixels the grey value should be reduced in the corrected images 4 Open the Main Object tab in the Assay Settings Here you have to select the channel which you want to use for image segmentation Select the TxRed channel which has the stronger signal for image segmentation and use the intensity module Click on Adjust The Object Finder Intensity Threshold menu opens Press Auto to set the threshold and use the water shed algorithm TE Object Finder Intemsity Ihreshold reel Themba Age ul D LAND d EI Pd oe Wanted on FSW SPO SS TTT _ Lossen kurie n gre EI SPORE D H Cp i CG JON POO eftel nif Fl hole whin objects 4 15 POD004 00000 TODNDT Tele H ot bl ren ech soe Tim parti ERREGER TEE Ted k T Maimam obiect sine 1000000 el gefellt TECK Nibe CO SPU OPC Tee ET SPORE FON DEL Tie iced ti sp PCa Bodd TEL 2 Teed E 5 Skip the sub object tab 6 Go to the Parameters tab Select here the parameters you want to detect for the main object Select Area Center X Center Y Position Well and Circularity Factor which are determined on the channel of the main object i e TxRed For the GFP Channel select Mean Intensity and Total Intensity Note that the intensities of the GFP channel are also detected on the main object which was originally segmented using the TxRed channel 66
81. ph display Derivative Check this option to calculate and display the first derivative of the kinetics time curve of the selected Parameter Smoothing Sigma This function applies a smoothing filter on the track data Set the strength of the smoothing with the slider The graph display is being updated immediately Definitions This lists the names of the values to be determined from the curves as well as the operators used In the Result column the values for the selected trace for all parameters are listed Operator Select an operator from the shortlist This will be applied on the currently selected Parameter For example if mean is applied on the Mean Intensity of the tracked objects the time average of the mean intensity of each object will be calculated The available Operators are Lifetime Gives the number of contiguous time points the particle is detected Sum Calculates the sum of the parameter value over the time Mean Calculates the mean value of the parameter Min Takes the minimal value the parameter reaches Max Takes the maximal value the parameter reaches Std Calculates the standard deviation of the values First Takes the value of the parameter at the first point of the curve Last Takes the value of the parameter at the last point of the curve T_max Takes the time point when the parameter is maximal TT min Takes the time point when the parameter is minimal T first Gives the time poin
82. plate Time lapse cycles Set the number of time points for this scan er Scan File Conversion Images List Image Channel Definition Scan Settings View Conversion Results Result directory ClConverkedscan Positions Wells Scan Columns Wel Columns E A Rows Well I A 3 6 1 4 View Conversion Results Test Upon clicking this button scan R converts the file names and lists them in the New File Name list This allows checking whether the conversion rules yield the desired result Start Click here to start the file conversion process The Start button becomes active once a conver sion rule is defined or loaded It initiates the file conversion This may be time consuming because an entire new set of images is generated and stored scan Analysis Software Manual Chapter 6 Appendix 75 er Scan File Conversion Images List Image Channel Definition Scan Settings View Conversion Results New file names Ca Start 6 2 FCS Export Functionality scan R is able to export single cell data to ISAC FCS 3 0 Standard files Please note that this standard is not fully implemented by most of the programs that specify FCS 3 0 import support Therefore some settings modifications for export might be necessary depending on the specific limitations of these programs In practice especially the FCS 3 0 requirements for double and integer 32 bit data support as well as multi dataset FCS files might n
83. r Detecting Sub objects Sub objects are structures that are directly linked to individual Main Objects The search for Sub objects takes place on an image mask derived from the corresponding Main Object This Main Object mask can be adapted for each Sub object type separately TE Assay Settings ES Main Object Sub objects Parameters Derived Parameters Image Processing Sub object finder Sub object A Mame New Color Channel Dots FITC Module settings Add to list Module Edge Max 5ize 132 100000 Settings list Show Search Area Boolean TRUE Modified 1 Show Center Boolean TRUE Show Bounding Box Boolean TRUE EI Filter Parameters Main object mask mp EdgeFinishing Keep Edges On Modify Compatibility Status Sub object finder Color Channel Module Setting list Adjust Add to list These functions are analo gous to the ones described in the previous Chapter 3 2 Object Finder Detecting Main Objects Name Give a name to each new Sub object The default name is Obj 1 Sub object list It gives an overview of the defined Sub object types The New and Remove buttons allow the insertion and deletion of Sub object types Main Object Mask Each individual Main Object found in an image creates a mask The individual Sub objects are associated with this mask rather then with the Main Object itself Imagine a Main Object is the cell nucleus and the Sub objects are structures outside of i
84. re developed by the User A distribution of the software can only be made in compiled form as part of the software developed by the User under strict observation of the conditions set forth in the written permission to the User The User must include the OSIS scan4R SOFTWARE PRODUCT copyright notification with the User s software The User has to make sure that OLYMPUS SOFT IMAGING SOLUTIONS cannot be held liable for any damages or injuries resulting from the use of the User s software that include parts of the OSIS scan R SOFTWARE PRODUCT 4 Copyright 1 OLYMPUS SOFT IMAGING SOLUTIONS or its subsidiaries remain owners of the software and it s documentation With the purchase the User obtains ownership of the diskettes or other physical storage de OLYMPUS vices excluding the software and other data contained thereon and the manuals 2 OLYMPUS SOFT IMAGING SOLUTIONS reserves the right to all publications duplication editing and marketing of the software and the software documentation Without prior written permission the User may not change translate de compile or de assemble the software copy any of the written or printed documentation of the software rent lease or license the software to a third party 3 The license property and user rights to the OLYMPUS SOFT IMAGING SOLUTIONS software disks and manuals may only be sold or transferred to a third party on a permanent basis if the third party agre
85. results for all wells and all gates You can choose if you want to display the total counts or the results as percentages You can export these results for fur ther analysis A graphical display allows you to directly compare the results for different wells Select G2 as reference gate and G2 G2_active and G2_passive as gated populations Chapter 5 Example Assay Step by Step 63 64 Chapter 5 Example Assay Step by Step OLYMPUS You will notice that in the first 8 wells the numbers of active and passive nuclei are almost identical However in the last 4 wells the number of active cells in G2 is strongly decreased Er Well Results Measurement Results Export Definitions Size Comparison ar g Group Name Description G2 G2 G2 G2 G2 A In In In In In All Nucs Gi G2 G2 active G2 1 0 0 0 0 0 0 0 2 AZ 100 0 100 0 1 7 100 0 45 5 54 3 a3 100 0 100 0 BE 100 0 51 6 48 4 B2 100 0 100 0 2 9 100 0 49 4 50 5 8 100 0 mn 15 100 0 47 9 31 6 Ke 100 0 100 0 3 6 100 0 53 0 47 7 c3 100 0 100 0 3 0 100 0 58 7 41 8 D2 100 0 100 0 2 6 100 0 54 0 49 D3 100 0 100 0 3 3 100 0 57 5 42 10 e 100 0 100 0 KS 100 0 4 5 95 1 E3 100 0 100 0 L 100 0 6 2 93 12 F2 100 0 100 0 2 8 100 0 7 9 92 is IS 100 0 11000 1 9 100 0 7 1
86. rs Image Processing Derived Parameters EN Di Dot rea SUMpl 1 Name De DotIntensity SUMIpLO DotArea Formula SUMO STOW MEAN SUM p1 1 Variable Selector No Selection Remove LOL Compatibility Status Ji j Derived Parameters list It lists the Derived Measurements to be carried out Each Derived Measure ment is labeled with an ID D1 D2 and requires a formula that entangles parameter IDs from the Measurements List Name This is the input field for setting the new Derived Measurement s name Formula This is the input field for the algebraic expression that connects the desired parameter IDs from the Derived Measurements List Variable Selector The parameters of the measurements list can be selected from this drop down menu and their corresponding ID is entered in the Formula field Vice versa if you select a parameter in the formula field you get the corresponding parameter name displayed in the Variable Selector field Special Operators It is possible that a number of individual Sub objects of the same type can be identified within the mask of a single individual Main Object A set of statistical operators allows evaluating individual parameters of the entire Sub object population of this type for the in dividual Main Objects These Operators are SUM STDV and MEANQ Statistical Operators e SUM ID This operator sums up all values of the given parameter ID of the Sub
87. rtain cases it may be advantageous to group wells see also Chapter 4 3 Weil Results Wells of one group need to have an identical entry in the Name Description column of the well list The default entry is the alphanumerical code of the well position The name can be changed at will 14 Chapter 2 Main User Interface OLYMPUS Ti Select Wells Name Description positive control negative control SO e o ee zz 6 OO e e ee zz 6 o e ee ez o OO e oO o o o e o OO e e o e o e 8 00000000 OO e o e eo o e o OO oe e oO e oO 8 e OO e e o e oO e e OO e e o o oO 8 oe v skipped well ere PP Scanned wells Cancel OK scanned well Right click on one of the scanned wells gives you the option Well Overview For a time series you will have to set the time point first The Well Overview will display all the positions you recorded for one well Selecting one of the images will load the image in the main display on the front panel Se Well No 2 A2 EIER 630480 1 scan Analysis Software Manual Chapter 3 Assays 3 Assays Assays are the recipes to extract the data of interest from the images of a scan They define which objects are to be recognized and how and which measurements are to be performed on the found objects This chapter explains in detail how assays are to be set up or modified 3 1 3 2 3 3 3 4 3 4 1 3 4 2 3 4 3 3 5 3 6 3 7 3 7 1 3 7 2 3 7 3 3 7 4 3 8 3 8 1 3 8 2 3 9 3 9 1 3 9 2 3 9
88. ry Sort mode The options are Typical Random yx and yx When Typical is set the galleries display the objects which are closest to the center of gravity of the selected region or histogram in the order of distance to that center Random displays randomly selected objects within the selected region The options yx and yx allow to create a gallery that is ordered by one parameter Default Data Directory Enter the directory where the data are read by default used for open scan Result Export Directory Enter the directory where the results are to be stored When this field is empty the results will be stored in the scan directory Population Results The results of a tracking analysis will bestored in the scan directory Trace Results Note that in earlier versions the results are stored in the scan directory Results folder e Settings Galleries Max Gallery Objects SortMode 7 Typical Directories Default Data Directory HE Result Export Directory Data Export Exporte Txt Scale Export Export Min Export Max Clipping s TET f scaed 10 64000 0 d Fes Analysis Export Wells Export Data Type Merge Well Data Integer 32 Repositioning amp Reclassification Acquisition Server Reclassification Hotkey Assignment Hotkey User Parameter Assigned value NUMPAD D UserDef 1 w localhost scan Analysis Software Manual Chapter 2 Main User Interface 7 Export2Txt The results can be exported as txt files or a
89. s fcs files For export to fcs format see Chapter 6 2 Repositioning amp Reclassification Set the Port and the Address for communication with the scan R screening system for experiments with repositioning 2 3 The scan R Data Structure scan R analysis data can be separated into the acquired images and the assay being applied on them The acquired images as a whole are called a scan it includes the individual images and their acquisi tion settings like color channels integration time plate information etc An assay describes the proc essing and analysis steps applied to extract data out of the images This separation between analysis settings and acquired images allows the reuse of once adapted analysis settings for different scans The images acquired during a scan R scan are stored as 16 bit tif files in a Data subfolder in the ex periment scan storage folder Additionally the scan settings are stored in an Experiment descriptor xml and the stage positions in the Acquisitionlog dat file The scan4R analysis software serves for the analysis of the scans The instructions assays for these analyses are stored in the scan4R Analysis Assays folder as say files These files can be loaded via Analysis Load Assay to then apply the assay on a scan data set Once an assay has been performed on a data set a sca file is generated and can be stored in the experiment storage folder These files contain all analysis data inclu
90. s Software Manual Contents 1 1HtrOQUGTION a rene desea 1 1 ADEL ae henne 2 12 Technical SUPBOr Aude 2 2 Main User INENICE vasse 3 2111 2 2 WEE 4 22 General Seting Saed 6 2 3 Thescan R Data Struct re issimo aai 7 2 4 Managing Histograms and Scatter Hlots AAA 7 2 4 1 The Histogram Context Menu REENEN 9 2 4 2 The Region Context Menu 10 2 5 Using the Image Viewer 11 2 6 Adjusting the Image Dsplavs nennen 12 2 7 Selecting Wells for analysis cccccseeseeeeeeeeeeeeseeeeeeeeeeeeeeeeeeeeeeeeeas 13 BASSaVS ee ed rein ass ga odes ors tec cae dea de este teen 15 Bet ENN 16 3 2 Object Finder Detecting Main Objects nennen 16 3 3 Sub object Finder Detecting Sub objects ccccseeseeeeeeseeeeeeeees 18 3 4 Object Finder Modules area 20 341 BUE IMAGE E 20 34 2 Intensity Threshold WEE 20 34 3 Edge Eeer a uken EN 3 5 Measurement Parameters ENEE 25 3 6 Derived Parametere 26 Sul Image EE UI 27 3 7 4 Background G rfeetion zn Na 28 Oe EI eee eege 29 Ee UNNE TON use E E E E EE 29 TA GUMMAN see 30 3 8 Virtual Channels rrnnnnennnnnonnnnrnnnnnennnnrnnnnnennnnrnnnnnennnnrnnnnnennnnsennnee 31 38 1 SME Ma e EN 38 2 Speal UNMIXINO eegenen 33 3 9 Tracking Analyzing Time Lapse Data 35 3 9 1 Tracking Configuration EE EE 37 3 9 2 TrackAnalysis Parameters cineraria a EEN 38 393 Mace VEVEN unsern 43 AAnalysis Resul E 45 4 1 Running an Analysis errnnrennnnnrvnnnnnrennnnnnvnnnnnnre
91. s to combine these open edges so that they form a closed contour as well The edge detection algorithm yields better results when objects of strongly varying intensity have to be detected In these cases the threshold detection will either lead to clusters when the threshold is set to a low value in order to detect also dim objects If a higher value for the threshold is set then the dim particles will be missed Furthermore as edge detection is intensity independent it is especially suitable for cell cycle analysis 21 22 Chapter 3 Assays te Object Finder Eden Based Sepmentation Ea EE bt mage i43 577 706 Image sector Long deler nos Selactivey 12 W000 14 P0000I ZU000N 1 R2 W0001 4 P00004 700000 CC J B2 WO00 1 4 P00005 2 PIR 0 9 2 W000 Besen T0000 f S i ii Je RGA imaga O00 755 735 OLYMPUS fu aadei za 13 lbk mane p 410 706 Pamiela nes v Close edges Piers 7 Ignore border objects Mina object ae 1 10 100 1000 10000 0000 Mirana dosare quality 0 0 2 0 4 D 0 8 t Paticle hierarchy Soli Selection mode Best Care To reduce complexity the process of finding the right settings is split up into three independent steps The three settings clusters in the Object Finder menu reflect these three steps They are traversed from left to right but you can always jump back and adjust the settings of former steps In each step the result of adjusting the current steps setti
92. s without delay Remove all software installations of the product from any computer you might have installed it on Return all electronic media of the product or completely destroy all electronic media of the product and send proof that this has been accomplished For a refund please return everything to where you purchased the product 1 Scope 1 This License agreement explicitly covers only the software diskettes or other media you received with the purchase and the software stored on these media the manuals as far as they were developed and pro duced by OLYMPUS SOFT IMAGING SOLUTIONS 2 User rights 1 OLYMPUS SOFT IMAGING SOLUTIONS permits the User for the duration of this contract to use the software on a single computer and a single terminal on that computer This license is explicitly non exclusive i e the User does not have an exclusive right to use the software As a licensed user you can copy the software from one computer to another by using a computer network or other storage devices as long as it is assured that the software can only be used on a single computer or terminal at any time and that the conditions set forth under 4 are observed 2 The User has the right to produce a copy of the software only for backup purposes 3 Additional user rights Only if OLYMPUS SOFT IMAGING SOLUTIONS provides the User with permission in written form the User can incorporate parts of the software into other softwa
93. settings lists and may thus appear several times YE Object Finders Library Settings Name Object Finder Module Module Name ParticlesSmall2 AutoThresh ParticlesMorph vi Standard01 AT vi ParticlesMorph Standard01 AT ModuleDefault Standard OnMap AT vi Standard OnMap AT ModuleDefault Standard01 OnMap vi Standard01 OnMap ModuleDefault SegmentO nCores T vi SegmentOnCoresAT TestMorph ParticlesMorph vi ParticlesMorph FullFrame FullImage vi FullImage _ ModuleDefault Edgent vi Edgeni Test Edge01 vi Edge01 Nucs20x EdgeO1 vi Edge01 ModuleDefault Standard vi Standard01 2007det StandardO1 vi Standard01 SubSmall StandardO1 vi Standard01 2D Particles amp StandardO1 vi Standardo1 2D Particles Standardo1 vi Standard01 Module Location Settings Object Finder Module Remove Add Remove Click here to add or delete a new entry to the list 6 3 3 Image Processing Library The password protected Image Processing Library can be accessed through Modules gt Image Proces SOS It allows managing the set of available Image Processing algorithms in the Image Processing modules Add Module Click here to add a new entry to the list Remove Module Click here remove an entry from the list scan Analysis Software Manual 4 Im
94. splay and analyze the curves regarding to a certain timepoint e g the time of mitosis Therefore select t_max MeanIntensity GFP as Time scale er Trace Viewer 1347 7 1320 0 1300 0 1280 0 1260 0 1240 0 Ai Gate 1200 0 AY longtraces AND w Y m N N 2 o 1 ean Intensit Max displ Traces 1000 PLES N Ca SO AY Die cor le Au Prey We 10 1 Single mode Trace Time Time scale t max MeanIntensity GFP Parameter Mean Intensity GFP Smoothing D sigma 0 01 scan Analysis Software Manual Chapter 5 Example Assay Step by Step 17 As you can see the green curve shows no mitosis but has a high mean intensity in GFP all 18 19 the time When you select the green curve and observe the corresponding object in the im ages you see that this is probably an apoptotic cell In order to avoid this you can set up a more advanced analysis e g you can set the parameters max Meanilntensity GFP and min MeanIntensity GFP in the Trace Parameters menu therefore go back again to Tracking Define Parameters Go to the Derived Parameters tab In the Trace Parameters menu and set as new derived parameter the ratio of max Meanintensity GFP and min Meanintensity GFP Name this new parameter ratio After running the trace analysis plot a histogram of ratio Set again longtraces as gate The data points around 1 show about the same minimum and maximum mean intensity of G
95. t In order to be detected the original Main Object Mask which only covers the area on the nucleus needs to be modified in order to en able the detection of the Sub objects Click the checkbox to enable the image mask modification of the Main Object Modify button Click here to open the Modify Object Boundaries dialog to adapt the main object mask to the needs of the Sub objects detection Distance The distance is measured from the outer rim of the main object mask positive and negative values are valid scan Analysis Software Manual Chapter 3 Assays 19 Er Modify Object Boundaries 1344x1024 2 1 16 bit image O 607 456 1344x1024 2 1 16 bit image 388 633 464 Settings Images BrdU W00001 P00001 200000 TO0000 DAPIL tif Distance Overlap treatment BrdU W00001 P00002 200000 TO0000 DAPIL tif 5 zl E BrdU WO0001 P00003 200000 T00000 DAPItif pixels A gt egment BrdU W00001 P00004 200000 T00000 DAPLtif BrdU W00001 P00005 200000 T00000 DAPIL tif Width BrdU W00001 P00006 Z00000 TO0000 DAPIL tif us BrdU W00001 P00007 200000 T00000 DAPLif 5 pixels BrdU W00001 P00008 Z00000 T00000 DAPL tif BrdU W00001 P00009 200000 TO0000 DAPIL tif BrdU W00001 P00010 200000 TO0000 DAPIL tif BrdU W00001 P00011 200000 T00000 DAPIL tif BrdU W00001 P00012 200000 T00000 DAPL tif BrdU W00001 P00013 200000 T00000 DAPIL tif BrdU W00001 P00014 Z00000 T00000 DAPIL tif
96. t when the trace starts T_last Gives the time point when the trace ends Num_zero_crossings Gives the number of zero crossings of the curve Num local max Gives the number of local maxima of the curve Num local min Gives the number of local minima of the curve Name The name of the analysis function is set automatically as lt Operator gt Parameter It can be changed manually New Click here to create a new list entry Remove Click here to delete the selected list entry Advanced Gives advanced options The Derivative checkbox is replaced by a menu that allows you to apply Anti Derivatives of higher order In the Operator shortlist a new option becomes available the curve fit Curve fit This Operator allows you to fit certain models to the parameter curves Upon selecting curve fit from the Operator drop down menu the Define button becomes accessible scan Analysis Software Manual Chapter 3 Assays Define This opens a menu to set the options for curve fitting As objects might move in or out of the focal plane or the value entered for Range might be set too small there might be a large number of short traces Therefore Lifetime is a useful parameter as it allows setting a gate on long traces 3 9 2 2 Curve Fitting Model type Select the type of model that is to be fitted to the curves You can select Polynomial Ex ponential General Is linear and non linear Polynomial The only parame
97. ter to be set is the Polynomial order E g the model function is t a a t a t for Polynomial order of 2 In this example the parameters a and a are fitted to the curves Exponential No parameters have to be set The model function is j a exp ct The parameters a and c are fitted to the curves General Is linear General linear least squares fit Here you can enter arbitrary Basis functions linear exponential trigonometric which will be combined linearly E g set f t 17 t 4 and f t exp t 2 2 will be combined linearly to ma f t a f J a 17 t 4 a exp t 2 2 This function represents a linear combination of a line and a Gaussian The parameters a and a are fitted Non linear Here you can enter an arbitrary model function to be fitted with up to 6 parameters You have also to enter the start values as First guess E g if you want to fit a Gaussian you set f t a exp t b 2c and enter start values for a band c You can also set the number of iterations to be run by entering a value for Max iterations e Curve fitting Model type Polynomial b Polynomial order Model Status men o The parameters that are fitted e g a a and the mean squared error of the fit mse are listed in the derived Parameters tab in the following representation curve_fit lt mode function gt Parameter a0 curve_fit lt mode function gt Parameter a1 curve fit lt mode function gt Parameter mse 41
98. the smoothing to 1 to obtain smoothed curves In order to find out the mitotic cells in a simple approach set the following definitions We need the max Meanlntensity GFP to find out the maximum intensity of the curve Then we need the lifetime to gate on the cells that are tracked over all images We are also interested in the time point when the maximum mean intensity is reached Therefore select also max MeanIntensity GFP scan Analysis Software Manual Chapter 5 Example Assay Step by Step 67 he Trace Parameters Dee rn i i 100 i i 1 1 41 ve Sieg oO Hra E r E wi 11 Close the menu with OK The analysis of the curves is performed In the lower right of the front panel the status bar displays the progress of the analysis Trace Parameter Calculator max MeanIntensity GFP 12 When the analysis is finished you can plot a lifetime histogram in the first histogram panel In order to select the cells that were detected in all timeframes draw a rectangle around the data points at 140 Right click on the rectangle and select Gate to convert the region into a gate Counts 00 250 500 75 0 100 0 126 0 150 0 175 0 200 0 lifetime Object x lifetime v RE Trace w d lifetime ON 6 10E 1 13 Go to Analysis Assay Gating and rename the Gate R01 to longtraces Leave the menu with OK Tie Gate Manager Edit Gate Name Definition Al EY ROL WI e HE Name Color long
99. to OFL window Object Finders Library see Chapter 4 5 Object Finders Library where you have to give a New Settings Name for the modified settings list E View Segmentation 1344x1024 1 2 16 bit image 17 972 804 1344x1024 1 2 16 bit image 524 12 840 Image List 5 Temp MitosisidataiBrdU w00001 P00001 z00000 T00000 DAPI tif A S Temp Mitosis data BrdU WvO0001 P00002 200000 TO0000 DAPI tiF K S Tempi Mitosis datalBrdU W00001 P00003 200000 T00000 DAPL tif S Temp Mitosis data1BrdU W00001 P00004 Z00000 T00000 DAPI tif S TempiMitosis data BrdU W00001 P00005 200000 T00000 DAPL tif S Tempi Mitosis data BrdU W00001 P00006 200000 T00000 DAPL tif S TempiMitosis datajBrdU W00001 P00007 200000 T00000 DAPIL tif S iTempiMitosistdatalBrdU W00001 P00008 200000 T00000 DAPI tif Image segmentation This function divides if activated via the check box the entire image into seg ments as many segments as there are Main Objects where each segment is assigned to the Main Ob ject in its center In other words each image pixel is assigned to the Main Object it is closest to All pixels that are assigned to the same Main Object form one segment of irregular shape and size The 17 18 Chapter 3 Assays OLYMPUS View button opens the View Segmentation window that contains on the left a display of the object circumscribing rectangles and on the right a display of the segments 3 3 Sub object Finde
100. tor are stored in the FCS file header in the keywords PNG and PnOffset If you switch on the scaling then the range of the original data will be scaled to fit into the given export range PNG and PnOffest are calculated from the original dataset and the scale settings according to Export Range Export Max Export Min Original Data Range Original Data Set Max Value Original Data Set Min Value Clipped Range Qriginal Data Set Max Value ignoring the largest clip Original Data Set Min Value ignoring the smallest clip PNG Export Range Clipped Range PnOffset Export Min PnG Original Data Set Min Value ignoring the smallest clip Analysis Export Wells FCS Settings Merge Well Data All Wells are taken together to a single dataset Single File Exports Wells into separate datasets in a single FCS file according to FCS 3 0 standard this Multi dataset format is not supported by all FCS programs Separate Files The export table function creates a single FCS file for every well i16 export FlowJo When using i16 export and importing with FlowJo FlowJo uses the gain to rescale the data but is then not able to go back to the logscale Therefore it is rec ommended to use raw mode with i16 and FlowJo To switch to logscale use then the FlowJo function Add derived and convert the required parameters to log Doubles and Multi dataset FCS Files can not be read by FlowJo Please note that if you have data within a X Y
101. traces SE Regions Gates D I Chapter 5 Example Assay Step by Step OLYMPUS 14 In the second histogram display plot max MeanIntensity GFP Right click in the histogram and select Set Gate Longtraces in order to include only the cells that were detected in all images into the analysis Draw a rectangle around the datapoints on the right These are the cells which show a high mean intensity of GFP Right click on the border of the rectangle and select Gate The new gate longtraces AND RO2 is created 15 Mark one of the datapoints and the corresponding cell is shown in the image display with a green outline Right click on the image of the cell and select show trace from the context menu The Trace Viewer opens and displays the curve of the selected cell In order to show all the traces of the gated cells deactivate single mode and for Gate select longtraces AND R02 The curves show all the same characteristics CH Trace Viewer 1347 7 1320 0 1300 0 1280 0 1260 0 Gate longtraces AND v Max displ Traces 1000 dei wv e D ES m a 100 lt fm ea aa ag ER a f I S ma o SE e I o 5 9 Ss Single mode i O 1040 0 2 100 110 120 130 Real Time Time scale Absolute time Derivative Parameter Mean Intensity GFP v CT Smoothing g sigma 0 01 16 As default the Time scale is set to Absolute time but it is also possible to di
102. ts of a complete scan analysis Statistical results of different parameters are listed for gated populations of the individual wells or of groups of wells and are displayed graphically in a histogram Additionally the results can be exported as txt file for further analysis with other software 4 3 1 Measurement Results The Measurement Results tab lists all the wells recorded in one measurement and displays the results Number of Objects of tot Mean Error Error and CV of the measurement parameter which is selected in the drop down menu Measurement The result for a combination of several wells is given in the bottom line when several wells are selected press shift and click the lines you want to com bine The results can be exported by Export Table The graph on the right gives a graphical representa tion of the results Well Group results This table gives a statistical analysis of the measurement results for each well or group of wells It lists the mean value of a parameter for the gated population in each well or group of wells as well as its error and coefficient of variation Objects Number of objects belonging to the population of the selected Gate of tot Relative size of the Gated population Mean Mean value of the selected Parameter It is shown in red in the histogram Error Absolute error of Mean It is shown in green in the bar plot Error Relative error scan Analysis Software Manual Chapter 4 Anal
103. u can skip the closing process by unchecking Close edges thereby reducing processing time dramatically All unclosed edges red are discarded then Generally the loss of particles is too high with Close edges being unchecked If Close edges is checked you can see in the middle image that the open edges red are connected to a closed contour by yellow lines In the Filtering cluster you can filter particles by location size and closure quality The clo sure quality is a rating attributed to each detected particle which describes the qual ity reliability of the respective closure Particles whose contour has already been closed in step 2 the green ones have closure quality 1 Especially when particle detection is difficult you can at least filter out most of the wrongly detected particles by moving the Minimum clo sure quality slider towards 1 By checking the Watershed checkbox you can split particles which have merged The algo rithm inspects the shape of each particle splitting it at constrictions This can be extremely useful when detecting nuclei Sometimes detected particles are nested into each other E g spots inside nuclei or nuclei inside the cytoplasm Since overlapping particles are not allowed the Particle hierarchy clus ter provides options to select the nesting or hierarchy level you are interested in See below for the function of the Selection mode options Don t forget to check other images of the scan to verify that
104. ues exported depend on the active view When also sub objects are detected not only one file is exported but for every sub object a separate list is exported The values that are exported depend on the active view population view trace view see Chapter4 3 4 Analysis Edit Assay opens the Assay settings menu Analysis Load Assay loads an existing assay say file In contrast to the sca file the say file con tains only the analysis i e the operations to perform on a data set but not the results of a specific analysis Analysis Save Assay saves the current assay say file Analysis Assay Gating opens the gate manager see Chapter 4 2 2 Analysis Exit exits the analysis Scan Open opens a scan and assigns it to the current assay The file types that can be opened are the scan R experiment descriptor files xml format and dotslide images wtp and ets format Scan gt Relink images re links acquired images to an analysis To do so navigate to the folder where the images are stored and select Current Folder Scan Custom conversion converts a third party data set into the scan R format see Chapter 6 1 Scan Select wells selects a set of wells for analysis and data navigation allows also to display a well overview i e an overview of the images that were acquired in a well see Chapter 2 7 Scan Scan Info displays the path to the image data Scan gt Settings displays the setti
105. unts to be given the same color from the Color scheme Binned color table Click this button to activate the color binning as set in Color table bin width Color Gating This command causes the population of each gate to be displayed in a different color in the histogram Show Legend This command causes a legend of the colors in the Color Gating mode to be displayed in the histogram 2 4 2 The Region Context Menu The region context menu will open if you right click on a region border Me Es Mer E E ER ron r Remove nak Tera zoom bo Gate Ha 4 Region Gallery me e Remove Deletes the selected region or gate e Zoom to Gives a zoomed view of the selected region e Gate Converts the region into an AND Gate See also Chapter 4 4 2 The Gate Manager When a gate is applied to a histogram only the data points within this gate are displayed To display all data points open the histogram context menu and go to Set gate none e Region Gallery This command generates an image gallery of objects in the selected region The number of images and the selection criteria is set according to the gallery preferences given in the Settings menu For more information see Chapter 2 2 General Settings scan Analysis Software Manual Chapter 2 Main User Interface 11 2 5 Using the Image Viewer Interactive objects Main Ke View mode Populati
106. xponential Arg 1 Round To Infinity Mantissa amp Exponent Gamma Mantissa amp Exponent Round To Nearest Natural Logarithm Natural Logarithm Arg 1 Logarithm Base 10 Logarithm Base 2 Max amp Min Max amp Min Quotient amp Remainder Power of X Returns the absolute value of x Computes the inverse cosine of x in radians Computes the inverse hyperbolic cosine of x Computes the inverse sine of xin radians Computes the inverse hyperbolic sine of x Computes the inverse tangent of x in radians Computes the arctangent of y x in radians Computes the inverse hyperbolic tangent of x Rounds x to the next higher integer smallest integer gt x Evaluates the cosine integral for any real nonnegative number x Computes the cosine of x where xis in radians Computes the hyperbolic cosine of x Computes the cotangent of x 1 tan x where xis in radians Computes the cosecant of x 1 sin x where x is in radians Computes the value of e raised to the x power Computes one less than the value of e raised to the x power e 1 Truncates x to the next lower integer largest integer lt x Returns the exponent of x Evaluates the gamma function or incomplete gamma function for x Returns the mantissa of x Rounds x to the nearest integer Rounds x to the nearest integer between x and zero Computes the natural logarithm of x to the base of ei Computes the natural logarithm of x 1 Computes the
107. ysis Results CV Coefficient of variation of Mean StdV Standard deviation of Mean It is shown in blue in the bar plot ti Well Results Measurement Results Populations Export Definitions Well Group results Object type Group ee I Description Objects Tot Error C Io Main 2594 81 3 2 84 144 56 3520 87 2 2 44 144 65 3093 78 0 2 68 148 90 Nucs 86 4 2 045E 2 4 75E 0 2 32 90 6 2 183E 2 4 75E 0 2 18 89 9 2 021E 2 4 53E 0 5 Mean Intensity TxRed 1 984E 2 4 41E 0 139 18 1 996E 2 3 99E 0 129 18 1 178E 2 3 40E 0 188 34 Well Group results 1 774E 2 5 70E 0 222 47 1 379E 2 39240 156 49 Stav 1 350E 2 3 51E 0 179 62 4E 2 Measurement S EF 2 9E 27 1 5E 2 Mean Intensity TxRed EA m FA l 1E 2 5E 1 on DR 1 020 4 0 Selected combined 11828 88 9 2 085E 2 271640 1 30 141 28 Export Table Object Type Select the object type from the shortlist Main Sub objects Gate Select a gate from the shortlist in order to get the values for the corresponding gated population Measurement Select the parameter of interest from the shortlist Selected combined Select a number of Well Group entries press shift and click in the lines you want to combine their combined results will be given here Export Table This function exports the data as tabulator delim

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