Chipping Up the Old FACS
Francis L. Battye
The Walter & Eliza Hall Institute
The performance of older cell sorters can often be improved to a level approaching or equaling that of current model instruments. Optical, mechanical and electronic enhancements to a FACS II (circa 1977) have given it significantly improved sensitivity and general functionality.
Optical Coloured glass and dielectric film optical filters of general-purpose types have been discarded in favour of purpose-designed filters matched to the fluorochromes in use. Threshold sensitivity for Fluorescein is now of the order of 600 MESF (Molecules of Equivalent Soluble Fluorescein). The (supposedly) red-enhanced S-20 photomultiplier tube has been replaced by an S-11 type for Phycoerythrin detection. Side scatter and blue (Höchst or Coumarin) fluorescence detectors have been added (bialkali types), as has a side-on, multialkali detector with very high sensitivity for Propidium Iodide or Duochrome.
Mechanical: An extended optical bench has been fabricated to allow dual laser operation and suitable holders for the extra detectors and for the added beam splitting mirrors were designed with the geometrical requirements of the dual emissions in mind. There are now six detectors mounted.
Electronic: Pre-amplifiers for the additional detectors and a compensation network for up to four overlapping colours have been added. Four-decade logarithmic amplifiers have given increased data compression and have allowed standardization of data with that from FACScan and FACStar. Sample-and-holds, comparators, timing electronics and front panel window setting controls have been added to allow sorting on six dual laser parameters. Since the sort logic downstream from the window comparison has not been altered, the sort purity has not been compromised.