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.

Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA

“…Flow cytometric measurements were performed on a Los Alamos flow cytometer (Steinkamp et al 1982(Steinkamp et al , 1991). An Innova 90-5 argon ion laser (Coherent; Palo Alto, CA) operating at 488 nm (300 mW) was used as the excitation source for TOTO and YOYO, 457 nm (200 mW) for MI, and 514 nm (300 mW) for 7-AAD and EthD II.…”

<title>Resolution of heterogeneous fluorescence emission signals and decay-lifetime measurement of fluorochrome-labeled cells by phase-sensitive FCM</title>

“…Since the modulation frequency is very high, compared to the velocity of the cells across the laser beam, i.e., fluorescence signal pulse width, the signals appear as a Gaussian-shaped white blurred region when viewed and photographed with an oscilloscope (2 tsec sweep speed) (see Fig. The measured fluorescence phase shift (lag) with respect to the reference was approximately 54°, which corresponds to a fluorescence decay lifetime of 14 nsec, as determined using Equation (1). To photograph the phase shift of the high-frequency component of the modulated fluorescence signal, a Chesterfield Products 16 MHz bandpass filter was placed between the preamplifier output and the oscilloscope input and the oscilloscope sweep speed was increased to 20 nsec/div.…”

“…The major limitation of these procedures is the availability of fluorescent dyes with a common excitation region, i e , requiring only one excitation source, and emission spectra that are sufficiently separated to permit measurement by conventional multicolor detection methods that employ employ dichroic and bandpass filters (1). The major limitation of these procedures is the availability of fluorescent dyes with a common excitation region, i e , requiring only one excitation source, and emission spectra that are sufficiently separated to permit measurement by conventional multicolor detection methods that employ employ dichroic and bandpass filters (1).…”

Predictions of tracer transport in interwell tracer tests at the C-Hole complex. Yucca Mountain site characterization project report milestone 4077

“…These microspheres have been shown in laboratory experiments at Los Alamos to transport through fractures with less attenuation than ordinary carboxylated polystyrene microspheres or silica microspheres . The CML microspheres will be tagged with fluorescent dyes and analyzed by flow cytometry (Steinkamp et al 1991).…”