A new flow-system instrument for quantitative analysis and sorting of microscopic particles, particularly biological cells, based on multiple measurements of physical and biochemical properties has been developed. Cells stained with fluorescent dyes in liquid suspension enter a unique flow chamber where electrical and optical sensors measure cell volume, single- or two-color fluorescence, and light scatter, and emerge in a liquid jet that is broken into uniform droplets. Sensor signals are electronically processed several ways for optimum cell discrimination and are displayed as pulse-amplitude distributions using a pulse-height analyzer. Processed signals trigger cell sorting according to preselected parametric criteria. Sorting is accomplished by electrically charging droplets containing the cells and electrostatically deflecting them into collection vessels. This instrument is described in detail with illustrative examples of experiments using polystyrene fluorescent microspheres, cultured human cells, and human leukocytes.

A differential light scattering photometer has been developed for rapid size analysis of single particles in flow. A fluid stream carrying individual particles in single file intersects a focused laser beam at the primary focal point of an annular strip of an ellipsoidal reflector situated in a scattering chamber. The light scattered from polar angles theta = 2.5-177.5 degrees at azimuthal angles phi = 0 and 180 degrees , spanning a circle of 355 degrees , is reflected onto a circular array of 60 photodiodes. The signal processing electronics and computer storage can accept 32 signals/particle at rates up to 1000 particles/sec. Photometer performance is tested by comparing measured responses from individual spherical particles with angular scattering patterns calculated for the particular detector geometry. These patterns exhibit the required symmetry in the two half scattering planes. Response measurements for eight samples with particle diameters of 1.1, 2.7, 5.0, 7.9, 10.0, 12.5, 15.6, and 19.5 microm are consistent with calculated size-response curves. The composition of a mixture of five components with particle diameters of 1.1, 5.0, 10.0, 15.6, and 19.5 Am is determined from an analysis of light scattering measurements at various forward-scattering angles.

A flow-system cell-analysis instrument is described in which cells from a heterogeneous population are characterized by their light-scatter patterns alone. As the cells pass at high speed through a focused helium/neon laser beam, the scatter pattern from each cell is sampled simultaneously at up to 32 angles between 0° and 30° with respect to the laser beam axis, and the scatter pattern for each cell is transferred to a computer. A mathematical clustering algorithm is used to determine the number of classes into which the cells can be divided, and a linear separation algorithm is used to find the boundaries between the classes. Preliminary results on exfoliated cells from gynecological specimens are presented. This technique may be useful for automated prescreening of gynecological specimens.

LACEL is a newly developed, fast, generalpurpose data acquisition and processing system for flow cytometric applications. The system's modular electronics allows flexibility in system configurations. The system can process as many as eight input analog parameters and can transfer 16-bit words between the user's electronics and the computer with standard input/output interfaces. The system's %fold coincidence logic capability can be set to operate with the noncoincidental timing that may occur in multiparameter flow measurements. As many as four parameters can be used to establish amplitude and timing criteria for each of two sorting directions. Two experiments can be on line with the computer at one time.

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.