Flow Cytometric Instrumentation Research Articles

Advancing in phosphoflow

FLOW cytometry enables assessment of the relative concentrations of proteins on the cell surface or within the cell with single-cell resolution. Many molecules of cell signaling, however, are expressed at low levels, perhaps at hundreds to only a few thousands of copies per cell. Being below or near the edge of resolution, detection of these targets using conventional flow cytometers and staining techniques is not reliable. Flow cytometric instrument detection limits in the fluorescein channel, for example, range from 1,000–3,000 molecules per cell. Cellular autofluorescence also plagues measurement sensitivity: the 98th percentile of autofluorescence in various primary cell populations is in the range of 2,500–4,000 fluorescein molecules. Detection sensitivity may be increased by using sandwich techniques, quantum dots or far red emitting dyes, among others.

Specific and Rapid Enumeration of Viable but Nonculturable and Viable-Culturable Gram-Negative Bacteria by Using Flow Cytometry

An issue of critical concern in microbiology is the ability to detect viable but nonculturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population, and other options (direct viable count and the double-staining method using epifluorescence microscopy and inhibitory substance-influenced molecular methods) are also biased and time-consuming. A rapid approach that reduces selectivity, decreases bias from sample storage and incubation, and reduces assay time is needed. Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria. This report outlines a method to optimize staining protocols and the flow cytometer (FCM) instrument settings for the enumeration of VBNC and VC bacterial cells within 70 min. Experiments were performed using the FCM to quantify VBNC and VC Escherichia coli O157:H7, Pseudomonas aeruginosa, Pseudomonas syringae, and Salmonella enterica serovar Typhimurium cells after staining with different fluorescent probes: SYTO 9, SYTO 13, SYTO 17, SYTO 40, and propidium iodide (PI). The FCM data were compared with those for specific standard nutrient agar to enumerate the number of cells in different states. By comparing results from cultures at late log phase, 1 to 64% of cells were nonculturable, 40 to 98% were culturable, and 0.7 to 4.5% had damaged cell membranes and were therefore theoretically dead. Data obtained using four different gram-negative bacteria exposed to heat and stained with PI also illustrate the usefulness of the approach for the rapid and unbiased detection of dead versus live organisms.

Virtual‐core flow cytometry

AbstractTraditional flow cytometers use a sheath fluid to position particles or cells for cytometric measurements, but the need for sheath fluid greatly complicates flow cytometric instrumentation. A cytometric detector that is free of the requirements of sheath fluid can simplify the design of flow cytometers and can extend their use into a number of areas. We designed a flow cytometer that uses a combination of three photodetectors to sense the position of a particle in sample stream. The position‐sensitive detectors create a virtual core in the sample stream that eliminates the need for sheath fluid. In this article, we demonstrate the efficacy of a virtual‐core flow cytometer (VCFC) using test particles, immunofluorescently labeled thymocytes, and raw seawater. The VCFC performs accurate measurements that can be used for a number of uses including environmental monitoring or simple immunology tests. © 2009 International Society for Advancement of Cytometry

Automation in high‐content flow cytometry screening

High-content flow cytometric screening (FC-HCS) is a 21st Century technology that combines robotic fluid handling, flow cytometric instrumentation, and bioinformatics software, so that relatively large numbers of flow cytometric samples can be processed and analysed in a short period of time. We revisit a recent application of FC-HCS to the problem of cellular signature definition for acute graft-versus-host-disease. Our focus is on automation of the data processing steps using recent advances in statistical methodology. We demonstrate that effective results, on par with those obtained via manual processing, can be achieved using our automatic techniques. Such automation of FC-HCS has the potential to drastically improve diagnosis and biomarker identification.

On-chip fluorescence-activated particle counting and sorting system

A fluorescence-activated particle counting and sorting system is developed for lab-on-a-chip applications. This system integrates the microfluidic chip, fluorescence excitation and detection, electronic power switch control, and optical visualization. The automatic sorting function is achieved by electrokinetic flow switching, which is triggered by a pre-set fluorescent threshold. A direct current electric pulse is generated to dispense the fluorescent particles to the collection reservoir. A user-friendly software interface is developed for automatic real-time counting, sorting and visualization. The design of the disposable microfluidic chip is simple and easy for integration. This system represents a promising prototype for development of affordable and portable flow cytometric instruments.

The fragile environments of inexpensive CD4+ T-cell enumeration in the least developed countries: Strategies for accessible support

With the advent of affordable antiretroviral treatment (ART), flow cytometry has ventured out of the exclusive realms of First World research to the resource-strapped clinical environment of developing countries (DCs). Flow cytometric instrumentation for ART has become more cost-efficient, thanks to simplified, yet accurate protocols and smart technologies. These positive developments have, however, not taken shape without problems, as health care in DCs remains weak due to chronic underfunding of their primary health systems. In addition, the multiplicity of donors has created parallel infrastructures that are difficult to manage and may undermine the responsibilities of public services. Hence, there is a prevailing lack of attention to maintenance, support, and human resource development. Not uncommonly, the procurement of high-value equipment is guided by nontechnical interests with mixed results. As conventional service contracts are unpopular, the sustainability of equipment is under serious threat after warranty periods, with environmental factors such as dust and unreliable power supplies being well-known culprits. Reagent supplies and servicing constitute further challenges, where a combination of short reagent shelf life, cold-box shipping, huge distances across poor infrastructures, rigid accounting procedures, and erratic customs requirements cause significant delays and extra costs. Although excellent, highly trained or trainable local staff is available, it is frequently diverted by brain drain from the government sector to privately funded hospitals, research facilities, and overseas postings. Despite these challenges, corporate service management has commonly remained loyal to its roots in the developed world.A number of propositions address the current situation: "Reagent-rental" agreements represent an attractive alternative to service contracts, while smart instrument design has started to make inroads into more robust device concepts. To avoid logistical bottlenecks, reagents call for lyophilization and increased heat stability. Newly designed remote diagnostic tools are expected to save costs on service visits. Furthermore, web-based customer-relationship management and enterprise resource planning software is expected to ease the existing complex communication- and logistics issues. In addition, a public-private partnership is proposed that involves government, manufacturers, and local distributors with field application specialists. The latter operate crossbrand as independent subcontractors to manufacturers under a nationally endorsed cost-capping and quality assurance agreement to service all cytometric devices common in the region. These locally run networks may serve as "templates" for improved laboratory services in general, in collaboration with CD4 counting, haematology and infectious disease diagnostics.

Design and characterization of a compact dual channel virus counter

Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the "gold standard." Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels. The instrument was fully characterized, which included analysis of the data acquisition rate, sampling time, flow rate, detection efficiency, linear dynamic range, channel cross-talk, and the limit of detection. Baculovirus samples were analyzed and the results were compared with concentrations obtained by a one-channel flow cytometer and plaque assay. The dual channel virus counter yields a representative value for the concentration of active viruses in an unpurified sample when compared with plaque assay and a one-channel flow cytometer. The technique is rapid (within minutes), requires only minimal sample preparation and minimum sample size (approximately 100 microl).

Estimation of Microbial Viability Using Flow Cytometry

For microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold-standard) usage equates viability and culturability (i.e., the ability to multiply), but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells, and we can therefore exploit the flow cytometric approach to evaluate so-called viability stains and to develop protocols for more routine assessments of microbial viability. This unit is primarily commentary, but several basic protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form.

An analytical system based on a compact flow cytometer for DNA fragment sizing and single-molecule detection.

Previous reports have demonstrated accurate DNA fragment sizing of linear DNA fragments, from 564 to approximately 4 x 10(5) bp, in a flow system. B-phycoerythrin (B-PE), commonly used in conventional cytometric applications that require high-sensitivity, was the first fluorophore detected in flow at the single-molecule level. Dilute solutions of stained DNA fragments or B-PE were analyzed in a simplified, compact flow system, with enhanced performance and lower cost, utilizing a solid-state laser and a single-photon sensing avalanche photodiode detector (SSAPD). Extensive data processing and display software, developed specifically for the photon-counting data stream, extracts correlated height, width, and area features from bursts of photons due to discrete molecules passing through the sensing region in the flow channel. DNA fragment sizing in flow has now been demonstrated for SYTOX-orange-stained fragments ranging in size over 3.4 orders of magnitude, from 125 to 5 x 10(5) bp. For Lambda bacteriophage DNA (lambda DNA; 48.5 kbp) a CV of 1.2 % has been achieved. Analysis of a femtomolar B-PE solution demonstrates that the bursts of photons from individual molecules can be baseline-resolved with 0.5 mW of laser power at a signal to noise ratio (SNR) of approximately 30, with approximately 100 photons detected from each molecule. A compact, low-power, high-sensitivity system detects DNA fragments as small as 125 bp or individual B-PE molecules in a flowing liquid stream. Demonstrated linearity, sensitivity, and resolution indicate that <1.0 mW of laser power is optimal, permitting further miniaturization of the system and additional cost reduction. Comprehensive analytical software exploits the standard cytometric paradigm of multiple 2D graphs and gating to extract features from classes of individually analyzed biomolecules. This complete system is thus poised to engage high-sensitivity applications not amenable to conventional flow cytometric instrumentation.

Multiparametric analysis of apoptosis by flow and image cytometry.

Flow cytometric assays for apoptosis are now in widespread use. The multiparametric nature of flow cytometry allows multiple assays for several apoptotic characteristics to be combined in a single sample, providing a powerful tool for elucidating the complex progression of apoptotic death in a variety of cell types. This chapter describes one such assay, allowing simultaneous analysis of caspase activation, annexin V binding to "flipped" phosphatidylserine residues and membrane permeability to DNA binding dyes. This multidimensional approach to analyzing apoptosis provides far more information than single-parameter assays that provide only an ambiguous "percent apoptotic" result, given that multiple early, intermediate, and late apoptotic stages can be visualized simultaneously. This multiparametric approach is also amenable to a variety of flow cytometric instrumentation, both old and new.

Flow cytometric discrimination of various phycobilin-containing phytoplankton groups in a hypertrophic reservoir.

Knowledge of phytoplankton structure is important information in water quality control. Lake restoration and sanitation measures in particular must be evaluated on the organismic level to valuate biological effects and assess the risk of potentially toxic Cyanobacteria blooms. We used and comparatively tested three independent methods for phytoplankton analysis in a hypertrophic reservoir under restoration. Nine unialgal cultures and outdoor samples were examined by high-performance liquid chromatography pigment analysis, microscopical cell counting, and flow cytometric (FCM) light scatter and fluorescence analysis to measure the percentage contribution of the major algal groups to chlorophyll a and biovolume. The FCM instrument settings and identification criteria were developed using a single excitation wavelength at 514 nm to differentiate nine algal species representing the major groups of algae. Fluorescence was detected at 585, 620, 650, and 680 nm. The results show that FCM is the only method for determining changes in the phytoplankton composition on both a chlorophyll a and biovolume basis. Each of the three methods has specific advantages and disadvantages, and should be chosen depending on the experimental problem. FCM sorting allows the combination of all three and offers further new perspectives.

Evaluation of Laser In Situ Scattering Instrument for Measuring Concentration of Phytoplankton, Purple Sulfur Bacteria, and Suspended Inorganic Sediments in Lakes

A laser in situ scattering and transmissometry (Lisst-100) probe has been used for estimating the particle-size distribution of phytopankton, purple photosynthetic sulphur bacteria (Chromatiaceae), and suspended inorganic sediments in different lakes. Results from Lisst-100 have been compared to laboratory measurements, such as those obtained by using a Galai laser size analyzer (GL), an optical microscope (OM), and a flow cytometer (FC). Although all of these instruments were shown to provide reliable values of the particle number concentration for the given populations, the Lisst-100 was the fastest and most reliable instrument because it did not require manipulation of the samples—which is not the case of GL, OM and FC instruments—and avoided the tedious procedure of microscopic counts. The total particle volume concentration results obtained with Lisst-100 differed from those obtained with GL for populations with large and porous aggregates, such as phytoplankton cells. The difference was attributed to the breakage of fragile algal aggregates resulting from the measuring procedure used by GL. Although for suspended sediment particles both instruments gave the same results for the total particle volume concentration, the particle-size distribution obtained with GL was found always shifted to smaller diameters than with Lisst-100, probably because inorganic sediment particles present compact aggregates. When these aggregates break, they split into a high number of small particles that contribute the same to the total volume concentration as the previous aggregates. Finally, results of the total particle volume concentration with Lisst-100 were in accordance with those obtained with GL for the Chromatiaceae population, because cells remained in a dispersed phase. A good correlation was found between the total particle volume concentration of Chromatiaceae measured with Lisst-100 and the concentration of bacteriochlorophyll a (BChl a), which is the parameter habitually used to estimate the concentration of Chromatiaceae. Therefore, Lisst-100 was found to be a reliable instrument to estimate the Chromatiaceae concentration in aquatic ecosystems.

Chapter 1 A brief history of flow cytometry and sorting

This chapter discusses some of the key events in the development of flow cytometry. The flow cytometric instruments and a growing number of monoclonal antibody probes of cell structure, cell constituents, and cell function have forever changed the classification of hemopoietic and other cell types, and recast the study of cell biology. It is difficult now to find a single clinical or biomedical laboratory that does not make use of this technology. During the 1980s and 1990s, there have been dramatic advances resulting from (i) improved instrumentation, increasing the precision of measurements and the number of simultaneously measurable parameters including, most recently, combining flow with cell scanning image cytometry; (ii) new antibodies and other probes of cellular constituents, with new techniques to measure a variety of cell attributes and cell functions; and (iii) new clinical and research applications. Advances in each of these areas depend on and influences the others.

Chapter 2 Principles of flow cytometry: An overview

This chapter presents a cohesive overview of the theory of flow cytometric instrumentation; the chapter cites a selection of representative references from the literature and describes a generalized flow cytometer in an attempt to provide an introduction into the field—with the background necessary for a critical understanding of the strengths, pitfalls, and potential of this technique and its applications. Flow cytometry is a system for detecting and then analyzing the light signals generated by particles as they flow in a liquid stream past an illuminating beam. By this definition, it is apparent that a chapter providing a description of flow cytometry will involve a confluence of the disciplines of electronics (detecting); computational data analysis (analyzing); optics (light signals, illuminating beam); and fluidics (flow in a liquid stream). The power of flow cytometry comes from rapid, objective, and quantitative computing, allowing calculations, correlations, and statistical conclusions from those few numbers derived from each of many cells.

Sexing mammalian sperm for production of offspring: the state-of-the-art

Predetermination of sex in livestock offspring is in great demand and is of critical importance to providing for the most efficient production of the world's food supply. With the changes that have taken place in animal agriculture over the past generation the application of sex preselection to production systems becomes increasingly necessary. The current technology is based on the well-known difference in X- and Y-sperm in the amount of DNA present. The method has been validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content and embryo biopsy for sex determination. The technology incorporates modified flow cytometric sorting instrumentation to sort X- and Y-bearing sperm. Resulting populations of X or Y sperm can be used in conjunction with IVF in swine and in cattle for the production of sexed embryos to be transferred to eligible recipients for the duration of gestation. It can also be used for intratubal insemination and for deep-uterine and conventional insemination in cattle. This semipractical sexing method, though currently impractical for some production systems (where large numbers of sperm are required for fertilization) could be used to provide a more flexible progeny-producing option in many livestock operations. Improvements in the production rate of sexed sperm continue as new technology is developed. High-speed sorting is one of the newer technological advances and is being used in our laboratory to increase sorted sperm throughput. With our original technology we sorted 350,000 sperm/h. We now sort 6 million of each sex, under routine conditions. Sorting only the X population results in about 18 million sperm/h. Improvements in the technology will no doubt lead to much greater usage of sexed sperm, depending on the species involved. Insemination of lower sperm numbers in cattle has proven to be an effective means of utilizing the sexing technology. Solving the problems associated with inseminating low sperm numbers in the pig would be advantageous to the utilization of sexed sperm for some type of deep artificial insemination. Such a development would also enhance the economy of using lower sperm numbers with conventional artificial insemination (AI) and aid the swine industry worldwide. The use of sexed sperm for non-ordinary applications such as endangered species, laboratory animals, hobby or pet species is also of interest and will become a part of the move to be more reproductively efficient in the next millennium. Sexed sperm on demand over the next several years will provide livestock producers with many options in seeking to improve efficiency of production and improve quality of products to enhance consumer acceptability.

26] Flow cytometric analysis of transgene expression in higher plants: Green fluorescent protein

The green fluorescent protein (GFP) of Aequorea victoria provides a unique means to study living cells, because transgenic expression of the coding sequence is sufficient to produce fluorescence within the cells of many different pro- and eukaryotic species. The availability of GFP as a transgenic marker has spurred interest in methodologies and instruments that can accurately measure cellular fluorescence emission, of which flow cytometry is one. Flow cytometric instrumentation is designed to examine the optical properties of suspensions of single cells or cellular homogenates as they are constrained to pass rapidly in a fluid stream through focused illumination. The individual cells or subcellular particles scatter light and, if they contain fluorochromes, also absorb light that is subsequently reemitted in the form of fluorescence. These signals are detected using sensitive photomultiplier tubes (PMTs) and photodiodes screened by appropriate wavelength-selective filters. After conversion to voltage-versus-time pulse waveforms, the intensity values are digitized and stored for analysis or further processing; the values are stored either on a cell-by-cell basis (“listmode”), or in the form of frequency distributions. Flow cytometric data are then presented in the form of multidimensional population distributions, which are graphically represented as histograms. This method of data accumulation and display, because it preserves differences between cells, is different from that commonly encountered in biochemical determinations, which provide only population means. Flow cytometry therefore allows identification of subpopulations of cells having different optical characteristics; these typically are observed as individual clusters within two-dimensional representations. Flow cytometry is also inherently quantitative, in that the intensities of the individual light scatter and fluorescent signals are accurately recorded. Either linear or logarithmic scales can be employed, the latter being preferred when signals of large dynamic ranges are encountered.

Estimation of Microbial Viability Using Flow Cytometry

AbstractFor microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold‐standard) usage equates viability and culturability (i.e., the ability to multiply), but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells, and we can therefore exploit the flow cytometric approach to evaluate so‐called viability stains and to develop protocols for more routine assessments of microbial viability. This unit is primarily commentary, but several basic protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form.

European survey of the flow cytometric determination of CD34-expressing cells

Introduction Transplantation of circulating hemopoietic stem and progenitor cells has become a routine therapeutic modality since Siena et al. reported in 1989 [1] that CD34-expressing cells could be measured by flow cytometry in cancer patients. Following a report by our group, describing a linear correlation between CD34-expressing cells from both healthy adults and cancer patients, as measured by flow cytometry and the in vitro colony-forming units– granulocyte/macrophage (CFU-GM) clonogenic assay [2], the flow cytometric assay for CD34-expressing cells has become a widely-used clinical test. However, flow cytometric analysis for CD34-expressing cells is associated with a number of inherent difficulties. The number of these cells is small, both in terms of absolute and relative numbers, necessitating statistical analysis for their proper identification [3,4]. The CD34 antigen is formed by at least three distinct epitopes and the MAbs used to detect each of these epitopes differ in their affinity, specificity and fluorochrome labeling characteristics [5–7]. Flow cytometry is performed using many parameters which are almost exclusively operatordefined. Thus, the settings of a flow cytometer, even with regard to identical instruments from a single manufacturer, vary widely both between and within institutions [8]. Although a number of tentative guidelines and reference protocols have been published [9–14], there is no established reference protocol for CD34 determination. Recent data suggest that the determination of subsets of CD34-expressing cells might be more informative than the determination of all CD34-expressing cells [15–17]. In an attempt to obtain information about CD34 measurement, we mailed questionnaires, including listmode data files of representative measurements of CD34expressing cells to members of EBMT and ISHAGE Europe, and assessed their analyses in this report. Materials and methods Questionnaire design The first questionnaire was sent out in October 1996 to all institutions (n1⁄4 522) in EBMT and ISHAGE Europe. This queried the flow cytometric instrument and software currently used for CD34-expressing cell determination. This first questionnaire was returned by 182 participants. A second questionnaire was mailed in June 1997 to all participants returning the first questionnaire. Although some institutions returned more than one initial questionnaire, the second questionnaire was mailed to only one representative per institution (n1⁄4 152). The second questionnaire was accompanied by a disk containing list-mode data files from flow cytometric measurements of CD34expressing cells, obtained from both whole blood and a cytapheresis product.

Basics of flow cytometry.

In summary, a beginner requires fundamental knowledge about flow cytometric instrumentation in order to effectively use this technology. It is important to remember that flow cytometers are very complex instruments that are composed of four closely related systems. The fluidic system transports particles from a suspension through the cytometer for interrogation by an illumination system. The resulting light scattering and fluorescence is collected, filtered, and converted into electrical signals by the optical and electronics system. The data storage and computer control system saves acquired data and is also the user interface for controlling most instrument functions. These four systems provide a very unique and powerful analytical tool for researchers and clinicians. This is because they analyze the properties of individual particles, and thousands of particles can be analyzed in a matter of seconds. Thus, data for a flow cytometric sample are a collection of many measurements instead of a single bulk measurement. Basic knowledge of instrumentation is a tremendous aid to designing experiments that can be successfully analyzed using flow cytometry. For example, it is important to know the emission wavelength of the laser in the instrument that will be used for analysis. This wavelength is critical knowledge for selecting probes. It is also important to understand that a different range of wavelengths is detected for each fluorescent channel. This will aid selection of probes that are compatible with the flow cytometer. Understanding the complication that emission spectra overlap contributes to detection can be used to guide fluorochrome selections for multicolor analysis. All of these experiment design considerations that rely on knowledge of how flow cytometers work are a very practical and effective means of avoiding wasted time, energy, and costly reagents. Data analysis is a paramount issue in flow cytometry. Analysis includes interpreting as well as presenting data that has been stored in list-mode files. Data analysis is very graphically oriented. There are a number of types of graphic representation that are available to visually aid data analysis. Two standard types of displays are used. These data plots are one-parameter histograms and bivariate plots. A user must be familiar with these two fundamental types of display in order to effectively analyze data. Histograms are the most simple modes of data representation. Histograms allow visualization of a single acquired parameter. Mean fluorescence and distributional statistics can be obtained based on markers that the user can graphically set on the plot. Percentages of positively expressing particles relative to a control sample can also obtained in a similar manner. In addition, multiple histograms can be overlayed on one another to depict qualitative and quantitative differences in two or more samples. Two-parameter data plots are somewhat more complicated than histograms; however, they can yield more information. Two-parameter dot plots of FSC vs SSC allow visualization of both light-scattering parameters that are important for identifying populations of interest. Bivariate fluorescent plots allow discrimination of dual-labeled populations that might remain hidden if histograms were used to display fluorescent data. Two-parameter plots that combine one light-scattering parameter and a fluorescent parameter are useful for analyzing control samples to elucidate the origin of nonspecific binding. Data analysis is very graphically oriented. Experience and pattern recognition become important when using two-parameter data plots for qualitative as well as quantitative analysis. The technique of gating or drawing regions on dual parameter light-scatter plots allows one to exclude information and examine the population of interest by disallowing particles that might confound or interfere with analysis. This is one of the fundamental uses for gating. (ABSTRACT TRUNCATED)

Flow cytometry for algal studies

Laser flow cytometric instrumentation has been developed in order to make very rapid measurements on phytoplankton samples for application in water quality research. The apparatus has been specially designed to make multi-angle light scattering and autofluorescence measurements on individual cells in a flow system. Quantitative LFC measurements are made of optical characteristics on samples of a minimum of 5000 cells, with a total measurement time of < 30 s. These measurements permit the identification and enumeration of algae by species. The instrumentation has been used to make the first laboratory ecotoxicological studies on freshwater phytoplankton by flow cytometry, in which the effects of trace metals and NTA on mixed algal populations have been investigated.