BD FACSDiVa 4.1

1. BD FACSDiVa 4.1 An introduction

2. FACSDiVa Software • The new digital acquisition platform introduces a complete new software concept from BD • The software runs on a Windows2000 computer, Macintosh systems are no longer available

3. FACSDiVa Software • The software uses a database server software to manage the flow cytometry data. • All data, plots, gates, instrument settings, and statistics are saved inside the database. • The user cannot specify any path for saving, everything is done automatically in the background.

4. The Browser Management of your experimental data and settings Worksheet The area for plots, histograms, Statistics and everything else Inspector Always shows the properties of selected objects Instrument Voltage settings, Threshold, Compensation The main layout

5. Sort control Defines all settings for Frequency, amplitude, and Stream deflection Acquisition Status Counters for events per second, Aborted events, Elapsed time Acquisition Control Starts and stops Acquisition and data recording. „Floating windows“ (always on top)

6. Log-in • The softwares requires a user log-in. Users can only see and manipulate their own data. • Only the Administrator can define new users and their rights. • The Administrator can „Share“ data with other users.

7. Log-in • Each time the software is started, the log-in menu appears:

8. Log-in • Depending on the log-in, the user can only see the appropriate datasets: A user can only see his own data. The Administrator can see everything

9. How to start? All data are stored in an „Experiment“ inside the database. Each time you start a new measurement, you either have to create a new Experiment or use an old one as template for new data. A new experiment can be created via the associated browser icon or via the Menu bar. New Experiment Note that you also can create folders like on a harddisk for better data management. The folders exist only in the database!

10. Global Worksheets Global Templates offer the possibility to „re-use“ plots like in CellQuest. When pressing the „NEXT“ button, the same plots are used for data display. Also saved data can be loaded into plots. For different views on the same data you can create multiple global sheets.

11. A new experiment A new experiment contains a „standard“ instrument setting and a single tube in a so-called „specimen“. When selecting "New Experiment" from the menu bar you can select a "Template" for your Experiment (created from your older experiments). A selected and activated element is always highlighted in blue in the browser window.

12. Specimens The FACSDiVa software uses „Specimens“ to associate a series of tubes as a pattern or panel. Existing Specimens can be used as a template for the next, similar tube pattern. It is possible to “pre-define” a tube list, but you can always duplicate a specimen after measurement of all tubes. Pre-definition is not required.

13. The first plots At the top of the Worksheet Window you will find the "Tool Palette" Clicking on the “DotPlot” icon activates the creation of DotPlots. Just click once on the worksheet to create a dotplot of “standard-size”. Dragging and holding the mouse creates a dotplot with the size of cour choice. In addition do dotplots, the software offers contour/density plots and histograms.

14. DotPlots The DotPlot can be activated by clicking onto the white border area. The Inspector window shows informations about the activated plot(s):

15. Data acquisition is started and stopped with the „Acquire“ Button During acquisition, data saving is initiated by pressing the „Record“ button. The „Restart“ button is available during acquisition to restart the acquisition and/or data recording. Acquiring Data Data acquisition is controlled via the „Acquisition Control“ window

16. Acquiring data The acquisition control window shows also the tube name, an event counter and the elapsed time since the acquisition has started. The user also controls which events are counted and stored: Storage Gate: Which events are recorded and saved Stopping Gate: Which cells are counted Events To Record: How many cells should be recorded (in the Stopping Gate) Events to Display: How many events are shown “on-line” in the acquisition plots

17. The instrument control The „Instrument“ window controls all parameters of the instrument. It consists of different tabs, which all relate to specific instrument functions.

18. Instrument - Parameters The Parameter tab shows the instrument parameters and their settings. The user can change the PMT voltage, the linear/logarithmic settings, and can select whether Area, Height and Width for the parameter should be acquired. The digital electronics has no parameter limitation!

19. Instrument Parameters The „Add“ and „Delete“ buttons allow the user to delete unwanted parameters, thus saving space for the recorded data sets. Note that the parameters correspond to fluorochrome names, not channel numbers. The association between channels, lasers and fluorochromes is explained later.

20. Instrument - Threshold The digital electronics allows the user to define multiple thresholds. Thresholds can be set on any parameter from any laser. Threshold can be connected with logical OR and logical AND, so that either one of the threshold conditions is fulfilled or all conditions must be met by an event.

21. Instrument - Compensation The digital electronics allows the compensation of all parameters against each other. Thus, the compensation starts to get increasingly complex in multicolor applications. The software allows „Autocompensation“ if single color stains are available, reducing the workload for instrument setup during multicolor experiments.

22. Instrument - Ratio The instrument can automatically calculate the ratio between any two parameters, even from different lasers. Calcium flux:Ratio Fluoro3/FuraRed Ratio Indo(bound)/Indo(Free) Absorption ratio for dyes excitable by different wavelengths

23. Instrument - Lasers Users should in most cases not change these settings. All values are setup during installation of your machine. Please use this tab only if a BD representative asks you to do so.

24. Instrument - Status The Status tab shows any errors that the instrument encounters. If an error occurs, the color of the tab changes to red, signalling that something is wrong. The Clear button at the lower right corner deletes the warning messages.

25. Acquisition Status The Acquisition Status window included a counter for events over the threshold (Events/Second) and a sum counter (all events since Acquire has been pressed). The Abort Rate and Abort Count show how many cells are „lost“ from the analysis because they were too close together to be separated as single events. Processed Events shows how many cells have been analysed and is always close to the Threshold Count. Elapsed Time shows how long you have been acquiring the data.

26. Working Without Global Worksheets In order to switch to the "old" DiVa way of working, click on the small green triangle in the upper left corner of the tool palette. Now you can create different plots for each tube. In addition, each tube now has its own set of population. For a long list of tubes with different analysis requirements the old way may be the better way for data analysis. However, be careful which mode is currently active: A green tab means "Global Worksheet", a grey tab "Classic Worksheet".

27. User Preferences • Tube specific Worksheet: Each Tube gets ist own Worksheet • Start Acquisition on pointer change: Saves one mouseclick on Acquisition • Show GUID: Unique ID for each tube (useful?) • Remove Tube-Specific Instrument Settings on Duplicate: Use the same Instrument Setting in an Experiment, even when one tube is changed. • Save Analysis after Recording: Copies all Plots from the Template onto the normal Worksheet after Recording • Automatically Update Instrument Setting: Use always the latest setup even in "old" experiments

28. User Preferences • Interval Gate Default Color: Histogram Gates per Default have no color to reduce clutter • Quadrant Gate color: Quadrants are also not colored to reduce data cluttering

29. User Preferences • Default Background Color since version 4.1 is White (equal to printout) • Can be set to any color by the user • In some instances, especially when updating existing databases, it may occur that you get black dots on black background, always open the Population Hierarchy to check this!

30. User Preferences • Each user can define his own default templates for new experiments. • Default Global Worksheet: BD recommends to use the global worksheet as standard method to reduce screen clutter.

31. Templates In the "BDEXPORT\Templates\Experiment" folder users can add additional folders as necessary. They are NOT created by the software. However, the software uses the folders and displays them correctly when exporting or importing templates.

32. Exporting Templates After choosing "Export" the user can select the folder in a drop-down box.

33. New Inspector view for recorded dtubes • The new inspector view for recorded tubes shows the instrument settings in a different way. • It is no longer possible to change the voltages or thresholds on saved data. • In the previous versions the corresponding buttons were just "greyed out", still leading to confusion. • As before, changing linear and logarithmic display and changing the compensation is still possible.

34. New scaling In the digital electronics there are no errors in the determination of the logarithmic value for small intensity values. Indeed, it always looks up the mathematically correct value. However, the logarithmic scale can only display numbers from 26 to 260.000 in the 4-decade display (which is recommended). In the 5-decade display it is 2.6 to 260.000 channels. The electronics actually calculates intensities from –2600 to 260.000. Everthing below 26 in the 4-decade scaling therefore is an "on-axis" event, as there is no logarithmic value to plot. The "way out": Use a different axis scaling. The "bi-exponential" sclaing is linear at the beginning and converts to logarithmic at a certain point.

35. Example 1 – Normal log-scale

36. Example 1 – Bi-exponential

37. Example 2 – Normal log-scale

38. Example 2 – Bi-exponential

39. Example 3 – Normal log-scale

40. Example 3 – Bi-exponential

41. Bi-Exponential Bi-exponential scaling shows also nicely the effect of compensation. The upper panel is over-compensated, the effect is clearly visible in the bi-expoential display. Correct Compensation can be nicely seen in the lower panel.

42. Bi-Exponential – Where to activate? The bi-exponential scaling is activated for each plot in the Inspector. Thus, it is possible to have bi-exponential and normal log scale on the same page. Populationborders drawn in bi-exponential displays cannot be displayed in "normal" plots, but are always calculated on bi-exponential scaling. The same holds true for the borders of population from "normal" log scales. They are not drawn in bi-exponential plots, but calculated based on log scale.

43. "Real" Density Plots In previous versions the "density plots" were just colored contour plots. Now "real" densities can be calculated and plotted. Note that density plots can also be calculated on bi-exponential scaling, showing the population distribution.

44. Copy and Paste... Finally, the FACSDiVa Software now supports Copy and Paste to generate PowerPoint Presentations or Word-Documents (or any other format) with flow cytometry data. It is still possible to export the graphics as files, but still only as JPG files in the screen resolution.

45. Hinged Quadrants In many cases rectangular quadrants are not the correct solution. The FACSDiVa Software now offers "hinged" quadrants, which can be moved individually to gate the cells of interest without the neeed to create four different polygon regions.

46. Hinged Quadrants In many cases rectangular quadrants are not the correct solution. The FACSDiVa Software now offers "hinged" quadrants, which can be moved individually to gate the cells of interest without the neeed to create four different polygon regions.

47. Offset Quadrants • Sometimes it may be useful to offset one side of the quadrants to adjust clearly overlapping populations. • You can either offeset quadrants or use the hinged version. It is not possible to use both versions simultaneously!

48. Enough of the Talking Just Do It!