FLOW CYTOMETRY
Flow cytometry is a technology that is used to analyses the physical and chemical
characteristics of particles in a fluid as it passes through at least one laser. Cell
components are fluorescently labeled and then excited by the laser to emit light at
varying wavelengths.
The fluorescence can be measured to determine various properties of single particles,
which are usually cells. Up to thousands of particles per second can be analysed as
they pass through the liquid stream. Examples of the properties measured include the
particle’s relative granularity, size and fluorescence intensity as well as its internal
complexity. An optical-to-electronic coupling system is used to record the way in which
the particle emits fluorescence and scatters incident light from the laser.
Three main systems make up the flow cytometer instrument and these are the fluidics,
the optics and the electronics. The purpose of the fluidics system is to transport the
particles in a stream of fluid to the laser beam where they are interrogated. Any cell or
particle that is 0.2 to 150 μms in size can be analyzed. If the cells are from solid tissue,
they require disaggregation before they can be analyzed. Although cells from animals,
plants, bacteria, yeast or algae are usually measured, other particles such as
chromosomes or nuclei can also be examined. Some particles such as marine algae
are naturally fluorescent, but in general, fluorescent labels are required to tag
components of the particle. The section of the fluid stream that contains the particles is
referred to as the sample core.
The optics system is made up of lasers which illuminate the particles present in the
stream as they pass through and scatter light from the laser. Any flourescent molecules
that are on the particle emit fluorescence, which is detected by carefully positioned
lenses. Generally, the light scattered from up to six or more fluorescences is determined
for two different angles. Optical filters and beam splitters then direct the light signals to
the relevant detectors, which emit electronic signals proportional to the signals that hit
them. Data can then be collected on each particle or event and the characteristics of
those events or particles are determined based on their fluorescent and light scattering
properties.
The electronics system is used to change the light signals detected into electronic
pulses that a computer can process. The data can then be studied to ascertain
information about a large number of cells over a short period. Information on the
heterogeneity and different subsets within cell populations can be identified and
measured. Some instruments have a sorting feature in the electronics system that can
be used to charge and deflect particles so that certain cell populations can be sorted for
further analysis.
The data are usually presented in the form of single parameter histograms or as plots of
correlated parameters, which are referred to as cytograms. Cytograms may display data
in the from of a dot plot, a contour plot or a density plot.
Applications
Immunophenotyping
The most common application performed on the cytometer is immunophenotyping. This
technique identifies and quantifies populations of cells in a heterogeneous sample - usually
blood, bone marrow or lymph. These cell subsets are measured by labeling population-specific
proteins with a fluorescent tag on the cell surface. In clinical labs, immunophenotyping is useful
in diagnosing hematological malignancies such as lymphomas and leukemia.
Cell Sorting
The cell sorter is a specialized flow cytometer with the ability to physically isolate cells of
interest into separate collection tubes. The cytometer interrogates and characterizes
each cell as it passes through the laser. The sorter then uses sophisticated electronics
and fluidics to identify and "kick" the cells of interest out of the fluidic stream into a test
tube.
Cell Cycle Analysis
Flow cytometry can analyze replication states using fluorescent dyes to measure the
four distinct phases of the cell cycle. Along with determining cell cycle replication states,
the assay can measure cell aneuploidy associated with chromosomal abnormalities.
Apoptosis
Apoptosis, or programmed cell death, is a normal part of the life cycle of eukaryotic
cells. Cells die for a variety of reasons: through necrosis, brought on by external
physical and chemical changes to the cell or through apoptosis, a process in which cells
initiate a "suicide" program through internally controlled factors. These two distinct types
of cell death, apoptosis and necrosis, can be distinguished by flow cytometry on the
basis of differences in morphological, biochemical and molecular changes occurring in
the dying cells.
Cell Proliferation Assays
Cell proliferation assays are widely used in cell biology to measure cellular metabolic
activity in response to stimuli such as growth factors, cytokines and other media
components. The flow cytometer can measure proliferation by labeling resting cells with
a cell membrane fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE). When
the cells are activated, they begin to proliferate and undergo mitosis. As the cells divide,
half of the original dye is passed on to each daughter cell. By measuring the reduction
of the fluorescence signal, researchers can calculate cellular activation and proliferation.
Intracellular Calcium Flux
Cells interact with one another and their environment through signal transduction pathways.
When these pathways are activated, membrane-bound calcium ion channels pump calcium into
the cell and rapidly increase the intracellular calcium concentration. The higher calcium levels
provide energy to the cell to respond to the external stimuli. The cytometer can monitor the flux
of calcium into the cell and measure the extent to which cells respond to the stimuli .
Membrane potential- Bacterial membrane potential may be analyzed using DiOC2, which exhibits
green fluorescence in all bacterial cells, but shifts to red fluorescence as the dye becomes more
concentrated in cells with larger membrane [Link] membrane potential may be
analyzed in the same manner with JC-1.
Live/dead bacteria discrimination- You can test how fast an antibiotic is killing microbes: live cells
have intact membranes and are impermeable to dyes such as propidium iodide, which only leaks
into cells with compromised membranes. Thiazole orange enters all cells, live and dead, to varying
degrees. Thus a combination of these two dyes provides a rapid and reliable method for
discriminating live and dead bacteria.
Fluorescent intensity is used to determine the amount of cellular DNA present in each cell (i.e. two
copies of a genome have roughly twice the fluorescent intensity of one copy).
sCell proliferation- Carboxyfluorescein diacetate, succinimidyl ester (CFSE) is a dye that diffuses in
to the fluorescent intensity of their parent cells.