Flow cytometry: instrumentation and application in phytoplankton research

In flow cytometry, light scattering and fluorescence of individual particles in suspension is measured at high speed. When applied to planktonic particles, the light scattering and (auto-)fluorescence properties of algal cells can be used for cell identification and counting. Analysis of the wide size spectrum of phytoplankton species, generally present in eutrophic inland and coastal waters, requires flow cytometers specially designed for this purpose. This paper compares the performance in phytoplankton research of a commercial flow cytometer to a purpose built instrument. It reports on the identification of phytoplankton and indicates an area where flow cytometry may supersede more conventional techniques: the analysis of morphological and physiological characteristics of subpopulations in phytoplankton samples.     

Cluster cytometry for high-capacity bioanalysis

Flow cytometry specializes in high-content measurements of cells and particles in suspension. Having long excelled in analytical throughput of single cells and particles, only recently with the advent of HyperCyt sampling technology, flow cytometry's multiexperiment throughput has begun to approach the point of practicality for efficiently analyzing hundreds-of-thousands of samples, the realm of high-throughput screening (HTS). To extend performance and automation compatibility, we built a HyperCyt-linked Cluster Cytometer platform, a network of flow cytometers for analyzing samples displayed in high-density, 1,536-well plate format. To assess the performance, we used cell- and microsphere-based HTS assays that had been well characterized in the previous studies. Experiments addressed important technical issues: challenges of small wells (assay volumes 10 μL or less, reagent mixing, cell and particle suspension), detecting and correcting for differences in performance of individual flow cytometers, and the ability to reanalyze a plate in the event of problems encountered during the primary analysis. Boosting sample throughput an additional fourfold, this platform is uniquely positioned to synergize with expanding suspension array and cell barcoding technologies in which as many as 100 experiments are performed in a single well or sample. As high-performance flow cytometers shrink in cost and size, cluster cytometry promises to become a practical, productive approach for HTS, and other large-scale investigations of biological complexity.     

Epi-illumination optical design for fluorescence polarization measurements in flow systems.

An epi-illumination design for fluorescence polarization measurements is introduced in flow cytometry with the optical axis orthogonally aligned to the cell stream. Various optical components and designs are discussed with respect to their influence on polarization measurements. Using the epi-configuration, paired measurements with the direction of polarization of the exciting light changed orthogonally are proposed for the compensation of system anisotropies and electronic mismatch. Large aperture corrections are employed for the excitation as well as for the emission pathway. Additional parameters such as fluorescence at 90 degrees, multiangle light scattering, and high precision cell-sizing by internally calibrated time of the flight measurements, as described previously, remain available with the design proposed here. Fluorescent latex microspheres, stained intracellular DNA, and algae have been used to test performance.     

Single cell analysis using surface enhanced Raman scattering (SERS) tags

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis.     

High resolution dual laser flow cytometry.

Flow cytometers based on optical sensing utilize external light sources and fluorescent dyes to measure one or more specific components or properties of individual cells or subcellular particles in liquid suspension. To provide for independent excitation of two dyes used in double staining experiments we have constructed a high resolution flow cytometer that uses two laser beams to provide two wavelengths of excitation. These beams are separated spatially so that cells flow through them sequentially, with a time separation of about 20 musec. Since the dyes are excited sequentially their emission occurs at different times and their emission spectra may overlap without causing any difficulty in analysis. We have developed new light collection optics that permit up to four measurements to be made on each cell. This approach greatly increases the number of dye combinations that can be used in flow cytometry, thus removing a significant limitation of single illumination instruments.     

Light scattering measurement in an arc lamp-based flow cytometer

The epi-illumination optics employed in most arc lamp-based flow cytometers may be modified so as to produce a dark-field configuration which facilitates highly sensitive detection of both forward and large angle light scattering in an instrument with a "jet on open surface" flow chamber. Forward scattering is detected at angles upwards from about 2 degrees, while large angle scattering includes angles above 18 degrees. Theoretical considerations suggest that large angle scattering measured around 20 degrees may be as efficient as that measured at 90 degrees for the purpose of distinguishing cells on the basis of intracellular structure. This was supported by the finding that dual parameter light scattering histograms of leukocyte suspensions obtained with the arc lamp-based instrument were closely similar to such histograms recorded with a laser-based instrument with the large angle detector at 90 degrees. Different species of bacteria could be distinguished by means of the dual parameter light scattering device, as could different species of sea algae. The sensitivity of the device is sufficient to measure 0.2 microns polystyrene particles in both forward and large angle scattering.     

流式细胞术

流式细胞术是一种综合应用光学、机械学、流体力学、电子计算机、细胞生物学、分子免疫学等学科技术,对高速流动的细胞或亚细胞进行快速定量测定和分析的方法。它一秒钟能分析几千个细胞,并同时测定细胞的多个参数,广泛应用于生物医学的许多领域,如测定细胞的特征(形态、膜电位等)和细胞内pH,细胞DNA、蛋白质含量、表面受体、Ca2+等。对生物工程学来说,了解细胞的这些参数尤为重要,因为它们能比用传统技术测得的数据更好地描述细胞群体。从流式细胞仪对细胞多种参数的测定及原理,到它在生物工程学中的应用等方面进行了介绍,并讨论了流式细胞术的局限性和面临的挑战。     

Ultrasonic particle-concentration for sheathless focusing of particles for analysis in a flow cytometer.

BACKGROUND: The development of inexpensive small flow cytometers is recognized as an important goal for many applications ranging from medical uses in developing countries for disease diagnosis to use as an analytical platform in support of homeland defense. Although hydrodynamic focusing is highly effective at particle positioning, the use of sheath fluid increases assay cost and reduces instrument utility for field and autonomous remote operations. METHODS: This work presents the creation of a novel flow cell that uses ultrasonic acoustic energy to focus small particles to the center of a flowing stream for analysis by flow cytometry. Experiments using this flow cell are described wherein its efficacy is evaluated under flow cytometric conditions with fluorescent microspheres. RESULTS: Preliminary laboratory experiments demonstrate acoustic focusing of flowing 10-microm latex particles into a tight sample stream that is approximately 40 microm in diameter. Prototype flow cytometer measurements using an acoustic-focusing flow chamber demonstrated focusing of a microsphere sample to a central stream approximately 40 microm in diameter, yielding a definite fluorescence peak for the microspheres as compared with a broad distribution for unfocused microspheres. CONCLUSIONS: The flow cell developed here uses acoustic focusing, which inherently concentrates the sample particles to the center of the sample stream. This method could eliminate the need for sheath fluid, and will enable increased interrogation times for enhanced sensitivity, while maintaining high particle-analysis rates. The concentration effect will also enable the analysis of extremely dilute samples on the order of several particles per liter, at analysis rates of a few particles per second. Such features offer the possibility of a truly versatile low-cost portable flow cytometer for field applications.     

A new multiparameter flow cytometer: optical and electrical cell analysis in combination with video microscopy in flow

BACKGROUND: Flow cytometers, which are commercially available, do not necessarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluorescence analysis for the simultaneous assessment of different physiologic parameters (intracellular pH and the intracellular concentration of calcium ions, etc). METHODS AND RESULTS: We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters including fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric imaging. The instrument is equipped with three different light sources. A tunable argon-ion laser generates efficient excitation of the most standard fluorescent probes in the visible spectral range, and an arc lamp provides the means for ultraviolet excitation at low cost. Because of the spatial filtering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ratiometric fluorescence analysis. A flow video microscope integrated into the optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effects, the motion of the object to be imaged (speed approximately 8 m/s) is frozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provides an illumination time of 18 ns (full width half maximum). Test results obtained from latex spheres and cells are shown. CONCLUSIONS: Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry.     

Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria

BACKGROUND: The stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena. METHODS: Flow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host. RESULTS: Neither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae. CONCLUSIONS: Flow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.     

One-dimensional acoustic standing waves in rectangular channels for flow cytometry

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry.     

Flow cytometry applications in the food industry

Flow cytometry has become a valuable tool in food microbiology. By analysing large numbers of cells individually using light-scattering and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate microorganisms; to distinguish between viable, metabolically active and dead cells, which is of great importance in food development and food spoilage; and to detect specific pathogenic microorganisms by conjugating antibodies with fluorochromes, which is of great use in the food industry. In addition, high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of food microbiologists in this technique. This mini-review gives an overview of the principles of flow cytometry and examples of the application of this technique in the food industry.     

Current and future applications of flow cytometry in aquatic microbiology

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.     

Flow cytometric measurement of rates of particle uptake from dilute suspensions by a ciliated protozoan

Flow cytometry is used to measure rates of ingestion of particles from dilute monodisperse suspensions by the ciliate Tetrahymena pyriformis. The particles used are polystyrene microspheres containing a fluorescent dye. Measurements were made directly, that is, by determining the fluorescence intensities from microspheres ingested by cells in samples collected from the experimental feeding apparatus. The fact that fluorescence intensities from individual cells can be grouped into discrete classes based on the numbers of fluorescent particles associated with the cells makes it possible to calibrate the flow cytometer and convert fluorescence measurements into numbers of particles ingested by average cells. At low particle concentration or high ciliate concentration, ingestion data must be corrected for depletion of particles during the assay, and a method for doing this is described. Experiments at various ciliate concentrations show that ingestion rates are not affected by this concentration. The methods developed should allow measurements of rates of ingestion of particles from concentrated and polydisperse suspensions. For such measurements, nonfluorescent particles together with a fraction of fluorescent tracer particles would be used.     

Cell biologists sort things out: Analysis and purification of intracellular organelles by flow cytometry

Flow cytometry was established originally for measuring DNA content and for the analysis of cell-surface markers in combination with cell sorting. During the past two decades, it has added new dimensions to various areas of immunology and medicine. Increased sensitivity and precision of flow cytometers, accompanied by the development of new fluorescent dyes and probes, has led to new applications in molecular cell biology and genetics. This article focuses on applications of flow cytometry in analysis and sorting of intracellular organelles.     

Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations

Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.     

Flow cytometry of mammalian sperm: progress in DNA and morphology measurement.

Variability in DNA content and head shape of mammalian sperm are potentially useful markers for flow cytometric monitoring of genetic damage in spermatogenic cells. The high refractive index and extreme flatness of the sperm heads produce an optical effect which interferes with DNA measurements in flow cytometers which have dye excitation and fluorescence light collection normal to the axis of flow. Orientation of sperm in flow controls this effect and results in coefficients of variation of 2.5% and 4.2%, respectively, for DNA measurements of mouse and human sperm. Alternatively, the optical effect can be used to generate shape-related information. Measurements on randomly oriented sperm from three mammalian species using a pair of fluorescence detectors indicate that large shape differences are detectable. Acriflavine-Feulgen stained sperm nuclei are significantly bleached during flow cytometric measurements at power levels routinely used in many flow cytometers. Dual beam studies of this phenomenon indicate it may be useful in detecting abnormally shaped sperm.     

An adapter for defined sample volumes makes it possible to count absolute particle numbers in flow cytometry.

A device is described which makes it possible to count absolute particle (cell) numbers per volume by flow cytometry. It can easily by adapted to several types of flow cytometers, especially to the Coulter EPICS V and EPICS 750 series. A volume adapter has been installed in place of the normal sample handling system without any further modifications of the instrument or the data acquisition program. The adapter consists of a special pipette with two opto-electronic detectors for the beginning and end of the measuring period. These switch on/off a shutter for the illuminating laser beam so that acquisition of the data is controlled indirectly. Sample volumes of 50 microliters were measured at flow rates up to 10(3) particles/s. Calibration beads as well as blood cells were enumerated according to FALS (forward angle light scatter), to SSC (90 degrees light scatter), and to fluorescence parameters. The results were compared to the evaluation made on a Coulter counter or in a Neubauer chamber of a light microscope. Using a concentration of 1 x 10(5)-5 x 10(5) particles/ml, the absolute numbers of particles were determined with a high reproducibility and an estimated error rate of 2-5%.     

Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD₇₅₀) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD₇₅₀ measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.