Instrumentation Development: User Participation

Francis L. Battye

The Walter & Eliza Hall Institute

For flow cytometry, as in other experimental research, there is a constant interaction between the scientific endeavour and the technology that is used in the experimentation. Where the scientific requirement leads the technology, or where the application is too esoteric to interest instrument manufacturers, users must play an active role in instrument development. At WEHI, several lines of development have been pursued to tailor the instrumentation to the flow cytometry requirements.

Computer Data Acquisition: A pdp11 data acquisition system was designed for the (1977) FACS II. The version introduced early this year has the following features:

• Up to 8 parameter data acquired and displayed as histograms, dot plots, two-parameter 3-D displays or three parameter 3-D stereo dotplots.
• Full analytical function for comparing new data with old.
• Automatic display sequence executed after acquisition.
• Conversion of bit-mapped graphics screen to DEC sixels format for serial transmission via DECserver to shared laser printer.

  Fast Small Sample Handling: A device was devised for fast input of small volume samples. The current model's features are:

• Sample turnover time of 20 seconds or less without intermixing
• Sample volumes of 20ul - 200ul (or more).
• Effective biohazard containment.
• Uses cytometer's own pressure controls.

  Dual Lasers and Multiple Detectors: With the demand for Texas Red and APC analysis the FACS II, earlier modified to a dual laser, 4-detector system, has been taken one step further:

• Extended optical bench with Innova-90, Innova-70 and dye laser
• Replacement of optical filters to improve sensitivity.
• Addition of PMT mountings to create a 7-detector array.
• Electronics to handle 6 parameters, 3 of which may be delayed.

  Data Networking: The large volume of data (300-400 Mbyte per month) from three flow cytometers, is stored on a single unit where it is accessible for archiving (as is now required) or analysis. An ethernet connection joins all flow cytometry lab computers with the WEHI mVax cluster. Software components are:

• FACStar+ VAXstation II DECNET (cluster member)
• FACS II pdp11/73 TCP/IP: Process Software
• FACScan HP9000/310 TCP/IP: Fusion network software
• Analysis uVax nodes TCP/IP: DEC UCX

  Data Analysis Software: The uVax analysis program WEFCS is now friendlier, with a menu-oriented front end. Features are:

• Up to 8-parameter data from FACStar, FACScan, FACS II, or FCS 1.0, (possibly also 2.0) accepted.
• Displays of histograms, contours, probability density, 3-D, third parameter colour coded dot plots and statistics.
• List mode file "editing" or conversion to other modes.
• Laser prints without terminal graphics capability.
• Keystroke programming for immediate or overnight execution.