Improved multilaser/multiparameter flow cytometer for analysis and sorting of cells and particles

Abstract

An improved multilaser instrument has been developed for quantitative analysis and separation of biological cells and particles. Argon ion, krypton ion, and dye lasers are employed as excitation sources to sequentially illuminate cells labeled with multiple fluorochromes as they pass through an improved flow chamber that incorporates an electronic cell‐volume sensor and an optical measurement region. Detectors located on the axis of each excitation beam are used to measure axial light loss and forward light scatter. Multicolor fluorescence is measured using a five‐channel detector located orthogonal to the laser beam(s)‐cell stream intersection(s). Sequential measurements are made on a cell‐by‐cell basis to provide pulse height, area, and width signals that are made coincident in time by analog delay modules to increase data throughput. Analog electronics are used to compute real‐time ratios, sums, and differences of signals. Up to eight signals are acquired and displayed as single‐parameter frequency distribution histograms and bivariate diagrams using a microcomputer. Processed signals also activate cell sorting according to computer‐controlled preselected parametric criteria. The unique measurement capabilities and other new features designed into this instrument represent marked technological improvements over our previous system.