Current Protocols in Cytometry Wiley All Volumes Togather

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Transcript of Current Protocols in Cytometry Wiley All Volumes Togather

Overview of Flow Cytometry InstrumentationFlow cytometry is a technology in which a variety of measurements are made on cells, cell organelles, and other objects suspended in a liquid and flowing at rates of several thousand per second through a flow chamber. Flow sorting is an extension of this technology in which any single cell or object measured can be selectively removed from the suspension based on the measurements made. Flow cytometry is a very broadly applicable methodology. A brief list of applications that use flow cytometers includes: Disease diagnosis Chromosome karyotyping Cell function analysis Cancer therapy monitoring Detecting fetal cells Cell kinetics Identifying tumor cells Cytogenetics Fundamental cell biology. In a flow cytometer, cells in suspension are made to flow one at a time through a sensing region of a flow chamber (flow cell) where measurements are made. An example of an early flow cytometer is the Coulter counter (APPENDIX 3A). In this device, cells pass through a small orifice across which an electric current is flowing. As a cell enters the orifice, the flow of current is reduced because the cells are largely nonconducting. Electronic circuits detect the decrease in current and thus the presence of the cell. In this way the device can count the number of cells per second passing through the orifice, and because the volume flow rate can be measured one can determine the number of cells per milliliter of sample. The Coulter counter has been in use since 1949 and is still a mainstay of the clinical laboratory. Under the right conditions (e.g., size and length of orifice, current magnitude), the reduction in current through the orifice is proportional to the size (volume) of the cell, as demonstrated at the Los Alamos Scientific Laboratory in 1962. In modern flow cytometers, cells flow through a light beam rather than through a Coulter orifice; a Coulter orifice can, however, be included in these devices. Many different types of measurements can be made on the cells, based on the size and shape of the light beam and on the dyes used to stain components of interest. The light beam can come from arc lamps (e.g., mercury), as in early flow cytomeContributed by Phillip N. DeanCurrent Protocols in Cytometry (1997) 1.1.1-1.1.8 Copyright 1997 by John Wiley & Sons, Inc.

UNIT 1.1

ters, or from lasers. Methods of measurement include absorption and scattering of the light beam by the cell, fluorescence of attached fluorescent dyes, and shape of the detected signal. Some of the properties and components that can be measured by a flow cytometer using these various methods are listed in Table 1.1.1. In principle, any component of a cell to which a fluorescent dye can be attached can be measured in a flow cytometer. If the binding of the dye is stoichiometric (i.e., amount of dye is proportional to amount of component) then the measurement can be quantitative and highly accurate (to within a few percent or better).Table 1.1.1 Properties and Components of Cells Measured in Flow Cytometry

Properties Cell diameter Dye distribution Internal structure Membrane potential Nuclear diameter Surface area Volume

Components DNA Nuclear antigens Enzymes Protein RNA Hormones Surface antigens

A flow cytometer is made up of several parts, as shown diagrammatically in Figure 1.1.1. All components of the system are necessary; the weakest part of the system defines its limitations. Other chapter units discuss the different parts of the system in detail. This overview describes the technology in general to give the reader a feeling for the interplay between the various parts of a flow cytometer. It also contains a brief history of the development of flow cytometry instrumentation.

CELL PREPARATIONObjects to be measured must be suspended in a liquid. This is simple for blood cells, for example, but cells from tissue must be disaggregated and removed from any noncellular material. For most tissues this can be accomplished by procedures as simple as mincing the tissue with a knife and pulling cells through a 19-gauge needle into a syringe, followed by passing the cell suspension through a 200-mesh nylon screen. Details for such procedures are

Flow Cytometry Instrumentation

1.1.1

found elsewhere in this publication in units that deal with specific measurement and analysis protocols (e.g., see UNIT 5.2 for general procedures for handling, storage, and preparing human tissues and APPENDIX 3B for procedures for disaggregating cultured cell monolayers). After a single-cell suspension is obtained, the cells

are stained with dyes that bind to the specific features that are to be measured.

FLOW CHAMBERAfter staining, cells are made to flow one at a time through the interrogating light beam; a laser beam is illustrated in Figure 1.1.2. To

light source (UNIT 1.5 )

cell preparation

fluidics control (UNIT 1.2 )

flow chamber (UNIT 1.2 )

detectors and signal processing (UNITS 1.3 &1.4 & Chapter 10 introduction)

sorter module (this unit, UNIT 1.2 )

analysis (UNIT 10.1)

display (UNIT 10.4)

Internet (UNIT 10.2 )

Figure 1.1.1 Schematic diagram of a complete flow cytometer system.

sample sheath

flow chamber

focusing lens

laser beam

Overview of Flow Cytometry Instrumentation

Figure 1.1.2 Longitudinal cross-sectional view of the flow chamber of a flow cytometer. The sample stream is surrounded by the sheath fluid which confines the cells (black dots) to the center of the chamber. The laser beam is focused onto the cell stream.

1.1.2Current Protocols in Cytometry

obtain the best resolution, every cell must flow through the middle of the beam and be exposed to the same intensity of illuminating light. However, the laser beam has a Gaussian intensity distribution (i.e., the intensity is at a maximum in the center of the beam and decreases exponentially in the radial direction), and this puts a severe constraint on the stability of the flow stream. The system includes two features to alleviate this problem. (1) The beam leaving the laser has a circular cross section, and a long-focal-length cylinder lens is used to spread the beam in the horizontal direction and to produce a large depth of focus, resulting in a relatively large region of constant intensity in the center of the flow stream. (2) A sheath stream is introduced to the flow chamber. This sheath has a higher flow rate (5 ml/min) than the sample (100 l/min), which serves to compress the sample stream and confine it to the center of the overall flow stream. This technique, called hydrodynamic focusing, is explained in more detail in UNIT 1.2. The end result is that cells are constrained to flow through an expanded laser beam in the center of the flow chamber. An additional constraint on the flow chamber is that it must be constructed of a material that will pass the excitation beam without appreciable scattering or absorption; this is usually accomplished through the use of quartz glass, which must be kept scrupulously clean. This is especially true when using ultraviolet light for excitation. The flow chamber can take many configurations. If a small orifice (e.g., sapphire jewel with a 70- to 100-m hole) is placed at the

chamber exit, the flow stream will be compressed and will leave the chamber at high velocity. If the chamber is then vibrated at high frequency (e.g., 20,000 Hz), the stream will break up into uniform droplets and the flow cytometer will become a flow sorter. In this configuration measurements on cells can still be made in the chamber, although the time interval between cell detection and sorting can be relatively long. However, it is more common to pass the laser beam through the fluid stream just below the jewel before the stream breaks up (see Fig. 1.1.6). Then the interval between cell detection and sorting is shorter. In the latter configuration, the material requirements on the chamber are considerably reduced; the chamber becomes what is often called a sorter nozzle and can be constructed of ceramic materials. Because the hydrodynamic focusing does take place in the nozzle, in some sense the nozzle is a chamber. The sorting configuration is described in more detail later (see Sorting).

DETECTORSAs a cell flows through the beam, light scattered by the cell and fluorescence light from dyes added to the cell are collected by light detectors, usually photomultipliers and photodiodes (see UNIT 1.4 for further discussion of photodetectors). These devices convert the light signal to an electrical signal that can be processed by the data processing and analysis unit. Photomultipliers, being very sensitive to light, are used where the light signal is weak (fluorescence), and photodiodes are used where the signal is strong (small-angle light scatter). The simplest flow cytometer would have per-

photomultiplier

filter pinhole photodiode collection lens laser beam flow chamber

Figure 1.1.3 Arrangement for a simple flow cytometer, containing a single fluorescence detector (photomultiplier) and a photodiode for detecting laser light scattered by a cell.

Flow Cytometry Instrumentation

1.1.3Current Protocols in Cytometry

haps one photomultiplier and one photodiode, as shown in Figure 1.1.3. With the appropriate electronics system, this permits one to make two simultaneous measurements on a cell. As a cell flows through the beam it scatters some of the incident light, and the light scattering is typically detected by the photodiode, which is less sensitive than the photomultiplier. This continues as long as the cell is within the beam. Thus, the length of time a cell is in the beam (and the width of the electrical pulse produced) is proportional to the width of the cell.