Principles of Flow Cytometry

Marion G. Macey

Summary

Flow cytometry is a powerful tool for interrogating the phenotype and characteristics of cells. It is based upon the light-scattering properties of the cells being analyzed and these include fluorescence emissions. This fluorescence may be associated with dyes or conjugated to mAbs specific for molecules either on the surface or in the intracellular components of the cell. Flow cytometry facilitates the identification of different cell types within a heterogeneous population. It was initially developed by immunologists wishing to separate out different cell populations for subsequent coculture experiments to determine the function of cells within the immune system. This was achieved by using fluorescence-activated cell sorting, or FACS, on the flow cytometer. The initial instruments were able to analyze one or two colors of fluorescence; today, instruments capable of analyzing 11 colors of fluorescence are available.

Key Words: Acquisition; amplification; fluorescence; histograms; light scatter.

1. History and Development of Flow Cytometry

Flow cytometry has developed over the last 60 yr from single-parameter instruments that detected only the size of cells to highly sophisticated machines capable of detecting 13 parameters simultaneously. A brief overview of the development of flow cytometry is given in Table 1.

2. Principles of Flow Cytometry

All forms of cytometry depend on the basic laws of physics, including those of fluidics, optics, and electronics (8). Flow cytometry is a system for sensing cells or particles as they move in a liquid stream through a laser (light amplification by stimulated emission of radiation)/light beam past a sensing area. The relative light-scattering and color-discriminated fluorescence of the microscopic particles is measured. Analysis and differentiation of the cells is based on size, granularity,

From: Flow Cytometry: Principles and Applications Edited by: M. G. Macey © Humana Press Inc., Totowa, NJ

Table 1

A Brief History of Flow Cytometry

Year Development

1954 An instrument in which an electronic measurement for cell counting and sizing was made on cells flowing in a conductive liquid with one cell at a time passing a measuring point was first described by Wallace Coulter (1). This became the basis of the first viable flow analyzer.

1965 Kamentsky et al. (2) described a two-parameter flow cytometer that measured absorption and back-scattered illumination of unstained cells, and this was used to determine cell nucleic acid content and size. This instrument represented the first multiparameter flow cytometer, and the first cell sorter was described that same year by Fulwyler (3). Use of an electrostatic deflection ink-jet recording technique (Sweet) (4) enabled the instrument to sort cells in volume at a rate of 1000 cells/s.

1967 Thompson (5) developed a system for the electrostatic charging of droplets which enhanced the development of cell sorters. Van Dilla et al. (6) exploited the real volume differences of cells to prepare suspensions of highly purified (>95%) human granulocytes and lymphocytes.

1983 First clinical flow cytometers were introduced.

1990 Advances in technology, including availability of mAbs and powerful but cheap computers, brought flow cytometry into routine use. Benchtop instruments developed with enclosed flow cells were developed.

1995 The ability to measure a minimum of five parameters on 25,000 cells in 1 s used routinely to enhance the diagnosis and management of various disease states and understanding of the pathogenesis of disease.

1999 Instruments equipped with lasers and capable of analyzing 11 fluorochromes developed by Bigos et al. (7).

2003 High-speed sorters using digital technology introduced.

and whether the cell is carrying fluorescent molecules in the form of either antibodies or dyes. As the cell passes through the laser beam, light is scattered in all directions, and the light scattered in the forward direction at low angles (0.5-10°) from the axis is proportional to the square of the radius of a sphere (9) and so to the size of the cell or particle. Light may enter the cell and be reflected and refracted by the nucleus and other contents of the cell; thus, the 90° light (right-angled, side) scatter may be considered proportional to the granularity of the cell. The cells may be labeled with fluorochrome-linked antibodies or stained with fluorescent membrane, cytoplasmic, or nuclear dyes. Thus, differentiation of cell types, the presence of membrane receptors and antigens, membrane potential, pH, enzyme activity, and DNA content may be facilitated.

Flow cytometers are multiparameter, recording several measurements on each cell; therefore, it is possible to identify a homogeneous subpopulation within a heterogeneous population. This is one of the most useful features of flow cytometers and makes them preferable to other instruments such as spectrofluorimeters, in which measurements are based on analysis of the entire population.

Most commercial flow cytometers have the capacity to make five or more simultaneous measurements on every cell, but some specialized research instruments have considerably greater capacity, and with three lasers it is possible to analyze up to 11 parameters (7). A typical flow cytometer consists of three functional units: (1) one or more laser light sources and a sensing system that comprises the sample/flow chamber and optical assembly, (2) a hydraulic system that controls the passage of cells through the sensing system, and (3) a computer system that collects data and performs analytical routines on the electrical signals relayed from the sensing system (Fig. 1).

The flow chamber is instrumental in delivering the cells in suspension to the specific point that is intersected by the illuminating beam and the plane of focus of the optical assembly. Flow chambers may comprise flat-sided cuvets to minimize unwanted light reflections, and, where cell sorting is required, so-called stream or "jet in air" flow cells are used.

Cells suspended in isotonic fluid are transported through the sensing system. Most instruments use a lamina/sheath flow technique (10) to confine cells to the center of the flow stream; this also reduces blockage due to clumping. Cells enter the chamber under pressure through a small aperture that is surrounded by sheath fluid. The sheath fluid in the sample chamber creates a hydrodynamic focusing effect and draws the sample fluid into a stream. Accurate and precise positioning of the sample fluid within the sheath fluid is critical to efficient operation of the flow cytometer, and adjustment of the relative sheath and sample pressures ensures that cells pass one by one through the detection point. This alignment may be performed manually on some machines, but in most it is fixed.

Water-cooled laser sources with an output power in the range of 50 mW-5 W may be used for fluorescence and light-scatter measurements. Air-cooled lasers have a maximum output of 100 mW and are now more commonly used together with laser diodes in commercial instruments. Lasers have the advantage of producing an intense beam of monochromatic light which in some systems may be tuned to several different wavelengths. The most common lasers used in flow cytometry are argon lasers, which produce light between wavelengths of 351 and 528 nm. Other lasers used include UV lasers, which produce light between 325 and 363 nm; krypton lasers, which produce light between 350 and 799 nm; helium-neon lasers, which produce light at 543, 594, 611, and 633 nm; and helium-cadmium lasers, which produce light at 325 and 441 nm.

Fig. 1. Schematic representation of a flow cytometer, including the flow cell, sheath stream, laser beam, sensing system computer, deflection plates, and droplet collection.

3. Fluorescence Analysis

Fluorescence is excited as cells traverse the laser excitation beam, and this fluorescence is collected by optics at right angles to the incident beam. A barrier filter blocks laser excitation illumination, while dichroic mirrors and appropriate filters (see Section 4.1.) are used to select the required wavelengths of fluorescence for measurement. The photons of light falling upon the detectors are converted by photomultiplier tubes (PMTs) to an electrical impulse, and this signal is processed by an analog-to-digital (A-to-D) converter that changes the electrical pulse to a numerical signal. The quantity and intensity of the fluorescence are recorded by the computer system and displayed on a visual display unit as a frequency distribution that may be single-parameter (Fig. 2), dual-parameter (Fig. 3), or multiparameter. Single-parameter histograms usually convey information regarding the intensity of fluorescence and number of cells of a given fluorescence, so that weakly fluorescent cells are distinguished from those that are strongly fluorescent.

Fig. 2. Single-parameter histogram of fluorescence and cell count illustrating a typical distribution for weakly fluorescent and strongly fluorescent cells.

Dual-parameter histograms of forward angle scatter and 90° light scatter (90° LS) allow identification of the different cell types within the preparation, based on size and granularity. Right angle and side scatter are alternative names for 90° LS (Fig. 3).

4. Light-Scatter and Fluorescence Detection 4.1. Filters

Light scattered by particles as they pass through a laser or light source must be efficiently detected, and fluorescent light of a given wavelength requires specific identification. The amount of light scattered is generally high in comparison with the amount of fluorescent light. Photodiodes are therefore used as forward angle light (FAL) sensors; they may be used with neutral density filters that proportionally reduce the amount of light received by the detector. A beam absorber (or diffuser or obscuration bar) is placed across the front of the detector to stop the laser beam itself and any diffracted light from entering the detector. The scattered light is focused by a collecting lens onto the photodiode(s) which converts the photons into voltage pulses proportional to the amount of light collected (integrated pulse). These pulses may be amplified by the operator. In some systems with multiple diodes, upper and lower light may be collected which may help separate populations of cells or particles.

Fig. 3. Dual-parameter histogram of the forward-scatter and side-scatter analysis of leukocytes from peripheral blood. The characteristic distribution of lymphocytes, monocytes, and granulocytes is shown.

Fluorescence detectors are usually placed at right angles to the laser beam and sample stream. Stray light may be excluded by an obscuration bar in front of an aspheric (objective) lens which collects the light and refracts it into a parallel beam. To detect the components of the beam, filters and dichroic mirrors are used to remove unwanted wavelengths of light and direct light to the correct detector(s). Table 2 describes some of the different types of lenses and filters, and Table 3 lists suppliers.

Figure 4 illustrates a possible lens configuration for detecting 90° LS, green (fluorescein), orange (phycoerythrin [PE]), and red (PE Texas Red [ECD]) fluorescence. Typically, the first filter used eliminates 488-nm laser light that still may have passed through. This light may then be diverted to a beam splitter or a dichroic mirror. This mirror reflects light in one band of wavelengths (usually long) while allowing another band (usually short) to pass through. It should be noted that there is no direct cutoff here between reflection and transmission. There is a middle band of wavelengths that will do both. For this reason, the color components are passed through other filters before entering the detector. These filters remove the unwanted wavelengths and allow the desired wavelengths to pass through to the detector. These filters are called band-pass filters and are designated by whether they transmit long wavelengths (long-pass) or shorter wavelengths (short-pass).

Table 2

Types of Filters

Filter type Comments

Absorbance The transition from absorbance to transmission occurs over a set range of nanometers; the filters are therefore named at the 50% transmission point. Dye in glass band-pass filters have excellent blocking properties and very high (>50%) pass of light. They are inexpensive but fluoresce and so should not be used as primary blocking filters. They are always long-pass filters (i.e., they block short wavelengths and transmit long wavelengths).

Interference These long-pass filters are manufactured by an etching process to give a raised and cut surface with ridges at set distances which cause interference in the wavelength of light transmitted. They are reflectance filters, so the shiny side is toward the laser. They do not fluoresce but have 90% efficiency at best, and they have poor trans-mittance. Also, the etching process allows light of incorrect wavelength to pass. They may be termed by the center wavelength, and band widths are usually given (e.g., 500/50).

Dichroic mirrors These are a combination of a mirror and an interference filter that need to be placed at an angle of 458 to the beam. They reflect short wavelengths and let longer wavelengths pass. They are used with other filters. They are normally long-pass filters.

Beam splitters These are metallic-coated quartz substrates and are designed to work at a 458 angle of incidence. Numbers indicate reflection/transmission values.

Band-pass These filters allow light within certain wavelengths to pass. They are interference filters with two coatings and act as a long-pass and a short-pass filter. They transmit and reflect but may suffer from attenuation.

Neutral density These attenuate all wavelengths and may be used for FALS and 90° LS.

LS, light scatter.

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