A FLOW CYTOMETER DESIGNED FOR FLUORESCENCE CALIBRATION

In the development of suitable standards and calibration materials for fluorescence measurement, it becomes necessary to make accurate fluor escence measurements of these materials on flow cytometers. The result s of such measurements may be affected by numerous sources of error; p rominent among which are deviations of logarithmic amplifiers (bg amps ) from ideal response. To minimize the deleterious effects of log amps and multicolor fluorescence compensation circuitry on measurements, m e built a flow cytometer with electronics incorporating high-precision peak detectors usable over a range from below 2 mV to 10 V, and we de veloped data acquisition software that transfers held peak values to a commercial 16-bit data acquisition system mounted in a personal compu ter running Windows 95, Fluorescence compensation is done in software, and transformation of the compensated data from a 16-bit linear to an 8-bit, 4-decade logarithmic scale is accomplished using a look-up tab le. Although dynamic range may be restricted by noise in the data acqu isition system, high sensitivity can be achieved by photomultiplier tu be gain adjustment, and it is Likely that the use of a lower noise dat a acquisition system and/or digital processing of pulse information wi ll enable operation over the full 4-decade dynamic range. Even at its current performance level, our instrument provides substantially bette r linearity over most of the scale than can be obtained using conventi onal electronics incorporating log amps; me believe this characteristi c is critical for use in standards development. (C) 1998 Wiley-Liss,In c.