An analysing flow cytometer, the optical plankton analyser (OPA), is presented. The instrument is designed for phytoplankton anaysis, having a sensitivity comparable with commercially available flow cytometers, but a significantly extended particle size range. Particles of 500 pm in width and over 1,000 pm in length can be analysed. Sample flow rates of up to 55 pl/s can be used. Also, the dynamic range of the instrument is significantly increased for particles larger than about 5 pm. The optics, hydraulics, and electronics of the instrument are described, including the best form for a low fluid shear cuvette. The new pulse quantification technique we call digital integration is presented. This technique is essential for the instrument to handle both short and very long particles with a large dynamic range. Test measurements demonstrating particle size range and dynamic range are presented. Dynamic ranges of 10,000 and 100,000 were typically observed, measuring field samples with Microcystis aerug i n o s a colonies, whereas one sample showed a dynamic range of lo6. A simple method for interpretation of time of flight (TOF) data in terms of particle morphology is presented. The specifications of the instrument are given.

No abstract

Chromosomes were isolated from a variety of human cell types using a HEPES-buffered hypotonic solution (pH 8.0) containing KCl, MgS04, dithioerythritol, and RNase. The chromosomes isolated by this procedure could be stained with a variety of fluorescent stains including propidium iodide, chromomycin A3, and Hoechst 33258. Addition of sodium citrate to the stained chromosomes was found to improve the total fluorescence resolution. Highquality bivariate Hoechst vs. chromomycin fluorescence distributions were obtained for chromosomes isolated from a human fibroblast cell strain, a human colon carcinoma cell line, and human peripheral blood lymphocyte cultures. Good flow karyotypes were also obtained from primary amniotic cell cultures. The Hoechst vs. chromomycin flow karyotypes of a given cell line, made at different times and at dye concentrations varying over fourfold ranges, show little variation in the relative peak positions of the chromosomes. The size of the DNA in chromosomes isolated using this procedure ranges from 20 to over 50 kilobases. The described isolation procedure is simple, it yields high-quality flow karyotypes, and it can be used to prepare chromosomes from clinical samples.

While performing DNA analysis with propidium iodide using the FACS-I1 cell sorter, it was noted that the fluorescence intensity was not constant during the measurements. W h e n different sheath and sample fluids were used, the fluorescence intensity of a given cell population increased or decreased during the m e a s u r e m e n t , depending on the concentration of sodium chloride in the fluids. By selecting the appropriate sheath and sample fluid combination, the drift in fluorescence intensity can be avoided.Key terms: Propidium iodide, signal stability, cell cycle, DNA analysis Propidium iodide (PI) was first described as a quantitative DNA stain by Crissman and Steinkamp (2). Since then, it has appeared to be an easy to handle dye with high specificity for DNA.A rapid method for PI staining under hypotonic conditions was described by Krishan (3) and modified by Vindelov (6) to allow DNA determinations in needle aspirates of solid tumors. Such a staining method is useful for scheduling chemotherapy in tumor-bearing patients. Cell cycle phase specific drugs should be given at the time when optimal results of the treatment can be expected. Especially for combination chemotherapy or for monitoring recruitment of cells into cycle rapidly DNA analysis is indispensable. Its usefulness has been shown by many authors (1,5). For this purpose, DNA histograms before and after treatment must be compared. Differences in the fluorescence intensity must be related to DNA concentration. Therefore, complete stability is required during the measurements. Even then, internal standards may prove to be necessary. In flow cytometry (FCM), the sample is injected in a fluid stream-the sheath fluid. Although the buffer containing the sample is usually carefully chosen, little attention has been paid to the composition of the sheath fluid.In this study, it was found that the composition of the sample and sheath fluids may influence the intensity and the stability of the PI fluorescence. Materials and MethodsCell Suspension and Fixation: The experiments were performed with thymus cells of the Brown Norway inbred rat strain. DNA Staining: The cells were taken from the pellet and stained with propidium iodide at 50 mg/liter (CalBiochem, San Diego, CA) in the presence of 0.1% sodium citrate. The cell concentration during the staining procedure was adjusted to 3 X 10' cells/ml. Before measurements were performed, the cells were washed two times (10 min at 400 g at 4°C) and resuspended in either distilled water or in a solution of the desired sodium chloride osmotarity.Fluorescence Measurements: The cells were analyzed with a FACS-I1 cell sorter (Becton Dickinson FACS Systems, Sunnyvale, CA). The system operates with an Argon laser of which the 488 nm line was used. Fluorescence above 620 nm was measured. The cells were transported to the nozzle with increased pressure ("boosting"). As soon as the first cells arrived, the measurements were started. ResultsThe instability of the PI measurement was tested under two conditions. The results a...

The interactions and binding characteristics of DNA dyes used in the flow cytometric analysis of chromatin were studied using human chromosomes and mouse thymocyte nuclei. The kinetics of dye binding and the relationship between fluorescence intensity and dye concentration are presented. Under the conditions used, Hoechst 33258, propidium iodide and chromomycin A3 reach an equilibrium with thymocyte nuclei after approximately 5 min, 20 min and more than 1 h, respectively. The same binding kinetics are observed with Hoechst 33258 and chromomycin when nuclei are stained with a mixture of the two dyes. Sodium citrate, which improves the resolution of flow karyotypes, causes a rapid increase in Hoechst and propidium iodide fluorescence, but a decrease in the fluorescence of chromomycin. The relative peak positions of chromosomes in a flow karyotype are unaffected by sodium citrate addition. The spectral interaction between Hoechst and chromomycin is quantified. There is variation among the human chromosome types in the amount of energy transferred from Hoechst to chromomycin. By measuring the Hoechst and chromomycin fluorescence of each chromosome after Hoechst excitation, it is shown that the amount of energy transferred is correlated to the ratio of the amount of Hoechst to chromomycin bound. Although the energy transfer between the two dyes is considerable, this has little effect on the reproducibility of flow karyotype measurements. The relative peak positions of all human chromosomes in a 64 X 64 channel flow karyotype, except for the 13 and Y chromosomes, vary in the order of 0.5 channel over a 16-fold change in either Hoechst or chromomycin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)