Further developments of a microscope-based flow cytometer: light scatter detection and excitation intensity compensation

Abstract

A microscope-based flow cytometer, which is based on a new flow system, has been extended to include light scatter detection, dual parameter fluorescence analysis, and a device which compensates for variations in excitation light intensity. A dichroic mirror splits the fluorescence into two spectral components that are further purified by additional filters situated in front of the photomultiplier tube detectors (PMT). Light scatter detection is obtained by a dark field configuration. An objective focuses the scattered light emitted within the dark field onto an adjustable slit in front of a PMT. Resolution of light scatter histograms of 1.5-micrometer latex spheres is about CV = 2.5%. Dual parameter analysis revealed a linear correlation between the light scatter signal and fluorescence of cells stained with fluorescein isothiocyanate, indicating that the light scatter signal is closely proportional to cell dry mass. The instrumental light scatter, which is proportional to the excitation light intensity, gives rise to a DC signal. This signal regulates the fluorescence signal amplifiers so that these signals are compensated for fluctuations in the excitation light intensity.