This page will help you get started using Facsimile which is designed as a flow cytometry teaching aid.
Background:
Flow cytometry core facilities in institutes which have some teaching responsibilities are faced with unique problems when it comes to educating new users. These problems are characterised by a lack of available instrumentation time to become familiar with the technology, lack of "action and affect" scenerios, limited sample volumes to learn instrument set-up, and the expense involved in preparing samples for purely teaching purposes. In an attempt to address these issues, we have undertaken to develop a virtual flow cytometer.
The concepts of compensation of data has often proven to be a stumbling block for people new to flow cytometry, so one of the main goals was to enable compensation of data as events appear on the displays. This software allows compensation of data and viewing of the compensation matrix which is generated.
This program is designed to with a main data display window and a data file selection window. The data files that appear for selection can be changed by editing the "index.dat" file in the data directory, and this file should be edited to add
Creating displays & 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:
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.
"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.
This page was last updated by G.W.Osborne on the 12th Feb. 2002