This page will help you get started using Facsimile which is designed as a flow cytometry teaching aid.

Latest Stuff: 15-July-03

Click here to play an AVI format movie ( ~2.7Mb) showing multicolour "drag and drop" gating.

Files can now be opened directly from disk.

Facsimile now supports a tree view of files opened from disk or available in an administrator controlled list of teaching reference files.
Click here to see a large example of the tree view in action

Statistics can now be generated for regions and gates

Facsimile will now read BD generated 18 bit data files. The program will prompt the users for which parameters they wish to display on a log scale.

The program still runs as an Applet for the mouse genome database.

Creating displays and Changing parameters:

A recommended initial procedure is to choose any file from the Data file selection window.
A file loading progress bar will initially appear, followed by a dotplot display of the first two parameters in the choosen file.

To change the parameters in the display, simply click and hold the right mouse button (Macintosh : COMMAND CLICK) down on the axis parameter label, a popup window will appear, and select another parameter to display.

To create additional plots, select the "Plots" menu and the type of plot to display, at this time limited to dotplots or histograms. If the option "verbose plot creation" is checked (see "Edit -Options" menu item), you will be prompted with a selection window of available parameter from the currently loaded file.

The instrument settings NOTE: If you load and display multiple files, to draw additional plots from a particular file, first select the file once more in the "Data files" selection window BEFORE selecting the "Plots" menu and display type. Once you have loaded a file, it stays in memory until the software is closed.

Simple example of Data "Acquisition" and it's "Utility":

Please open the two files "BLOCK" and "NOBLOCK" from the Data file selection window and display a dotplot of FSC-H vs SSC-H.
Now, select the Acquire menu, and select the "float" option. A floating window will appear to enabling the starting, stopping and restarting of the active data display which displays data in an identical fashion to that which occurs on a flow cytometer.
Click the "Acquire" button to begin "acquiring" data. Note that a pulse height display appears in the "Float" window which is generated from events being displayed. (NOTE: High, Medium and Low default flow rate buttons are currently disabled)
Now watch the dotplots closely and you will see how the Forward and Side Scattered light patterns change when a partial clog or blockage appears in the data stored in the file "BLOCK".
This is an example of how Facsimile can simulate things which occur on a real instrument, and teach new users how to recognise when a partial blockage has occurred. The flow rate number of dots displayed and so on can all be adjusting to suit the user (see View menu: Modifying displays, "flow rates" etc: below).

Example of "Real time" compensation:

The basic procedure to follow is:

1. Select "Simulate" menu and Acquire item
2. Select a file you wish compensate containing data from fluorscent signals
3. Select the "Adjust" menu and the Compensation item
4. As soon as Compensation is selected, "slider bars" appear on the X and Y axes of the active dot plot and "crosshairs" become active when the mouse is moved across the active plot.
5. Position the crosshairs such that they suround the unstained population and click to "lock" the crosshairs. Once the quadrant crosshairs are locked, statistics are generated for the active plot and they appear in the main statistics floating window.
   Note that quadrant statistics show arithmetic means and median values for each quadrant Upper Left (UL) etc. For these types of analysis, the median fluorescence intensity is a suitably robust statistic to give an a good indication of whether the compensation is correctly applied.
6. Adjust the slider on the axis of the parameter you wish to compensate. For example, the slider which appears on the X axis of the dotplot when moved to the right will "subtract" an increasing amount of parameter X from parameter Y.
   Note that a right click on the active plot at this time will yield a popup menu where, if you select the "Compensate" item you can choose to
   1. Auto Update: the plot while compensating. If this item is not checked, and it is by default, then one needs to press the "C" icon in the lower left corner of each plot to apply the latest changes to the display.
   2. Slider Scale: changes the full scale that the slider acts upon.
   3. Compensation Matrix: displays the compensation matrix below the active plot. Also allows direct user entry of amounts to compensate various parameters by. You really have to try this one out to compensate many parameters simultaneously...
7. Keep adjusting the slider until the mean/median of the labelled cells, in the upper left or upper right quadrants, has a value close to that of the events in the lower left quadrant, with respect to the same axis
8. Remember the object of compensation (for 2 parameters), is that parameter X -positive cells (e.g.FITC, FL1-H) should look no more positive in parameter Y (e.g. PE, FL2-H) than do negative cells.
9. Information on this and other combinations of fluorochromes can be viewed at TSRI Java spectrum viewer;
   

The main reference for the logic behind N colour compensation is
"Fluorescence Spectral Overlap Compensation for Any Number of Flow Cytometry Parameters". C Bruce Bagwell and Earl G. Adams. 1993 Annals of the New York Academy of Sciences, Clinical Flow Cytometry, Volume 677

Also worth a look if you are interested in an "early" reference about fluorescence compensation is Loken, M.R et al. 1977 J.Histochem.Cytochem. 25:899-907

View menu: Modifying displays, "flow rates" etc:

A number of adjustments can be made to change the appearance of data displayed using Facsimile all of which are accessed via the "View" menu and selecting the "Options" item. A "Settings" window will appear.

Instrument tab selected:
which allows you to change the number of dots in the dot buffer, the event delta, and the "flow rate" (which is specified in milliseconds). By modifying these parameters one can usually achieve a rate of dot display which is satisfactory. If things don't seem to be happening fast enough for your liking, try setting the event buffer size to 1000 or even 1500, and event delta to 200.
"Verbose plot creation" when checked, prompts the user to select parameters when creating a new plot, as opposed to using the first parameters in the file.

Display tab selected:
From the pull down menu select the part of the plot you want to change the colour of, for example Dot Plot changes the dot colour and then select a new colour. Changes are applied immediately to the plots.