Flow cytometry
Principle
Flow cytometry is a technique for counting, examining, and sorting microscopic particles
suspended in a stream of fluid. It allows simultaneous multiparametric analysis
of the physical and/or chemical characteristics of single cells flowing through
an optical and/or electronic detection apparatus.
Fig. 1 Principle
of fluorescence cytoflow meter with 3 emitted wavelengths, forward and side
scatter.
A beam of light (usually laser light) of a single wavelength is directed
onto a hydro-dynamic focussing stream of fluid. With hydrodynamic
focusing a "wall" fluid called the sheath fluid is being pumped
through. The sample is injected into the middle of the sheath flow. If the two
fluids differ enough in their velocity or density, they do not mix: they form a
two-layer stable flow, with the sheath enveloping the sample in a linear, steady flow. The diameter of the sample
flow is of μm magnitude.
Each suspended particle passing through the beam scatters
the light (see Light:
scatter) in some way (Fig. 1). The scatter is in line with the
light beam (Forward Scatter or FSC) and perpendicularly to it (Side Scatter
(SSC). Fluorescent chemicals found in the particle or attached to the particle
may be excited to emit light (see Fluorescence). This combination of scattered
and fluorescent light is picked up by the detectors and the evoked signals are
analyzed.
FSC correlates with the cell volume and SSC depends on
the inner complexity of the particle (i.e. shape of the nucleus, the amount and
type of cytoplasmatic granules or membrane roughness).
Some flow cytometers have eliminated the need for
fluorescence and use only light scatter for measurement. Other flow cytometers
form images of each cell's fluorescence, scattered light and transmitted light.
Application
Applications include research in molecular biology
(fluorescence tagged antibodies in transplantation, hematology, tumor immunology
and chemotherapy, genetics and sperm sorting in IVF), pathology, immunology,
plant biology and marine biology (auto-fluorescent properties of photosynthetic
plankton, Fig. 2). In protein engineering, flow cytometry is used in
conjunction with yeast display and bacterial display to identify cell
surface-displayed protein variants with desired properties.
A flow cytometer has 6 main components:
·
a flow cell: a liquid stream carries
and aligns the cells so that they pass one by one through the light beam for
sensing;
·
a light source: various types of
high pressure lamps and at present mostly lasers are used (HeNe for 632 nm,
red; Ar for488 nm, blue-green; Kr for 341 nm, blue light);
·
dichroic filters (see Light: beam splitter)
for the various emitted wavelengths;
·
detectors (photomultiplier tubes) and
ADC (analogue to digital converter) system generating FSC and SSC as well as
fluorescence signals.
·
an amplification system.
·
a computer for analysis of the
signals.
Modern flow cytometers are able to analyze several
thousand particles/s, in "real time", and can actively separate and
isolate particles having specified properties. A flow cytometer is similar to a
microscope (see Optical
microscopy), except that instead of producing an image of the
cell, flow cytometry offers "high-throughput" (for a large number of
cells) automated quantification of set parameters. To analyze solid tissues
single-cell suspension must first be prepared.
Fig. 2
Analysis of a marine sample of photosynthetic picoplankton by flow
cytometry showing three different populations
Commercial flow can be used with a large number of reagents,
such as fluorescently-labeled antibodies and analysis software.
Modern instruments usually have multiple lasers and many fluorescence
detectors. Increasing the number of lasers and detectors allows for multiple
antibody labeling, and can more precisely identify a target population by their
phenotype. Certain instruments can even take digital images of individual
cells, allowing for the analysis of fluorescent signal location within or on
the surface of cells.
The data generated by flow-cytometers can be plotted 1D
(lower row of Fig. 2 with number versus, here, fluorescence), 2D (upper left
panel Fig. 2 with fluorescence versus side scatter) or even 3D. The plots are
often made on logarithmic scales. Because different fluorescent dyes' emission
spectra overlap, signals at the detectors have to be compensated electronically
as well as computationally.
The fluorescence labels that can be used will depend on
the lamp or laser used to excite the fluorochromes and on the detectors
available.
More
Info
Fluorescence-activated cell sorting (FACS)
Fluorescence-activated cell sorting is a specialized type
of flow cytometry with sorting a heterogeneous mixture of biological cells into
various containers, one cell at a time, based upon the specific scattering and fluorescent
characteristics of each cell.
The cell suspension is entrained in the center of a
narrow, rapidly flowing stream of liquid. The flow is arranged so that there is
a large separation between cells relative to their diameter. A vibrating mechanism
causes the stream of cells to break into individual droplets. The system is
adjusted so that there is a low probability of more than one cell being in a
droplet. Just before the stream breaks into droplets the flow passes through a
fluorescence measuring station where the fluorescent character of interest of
each cell is measured. An electrical charging ring is placed just at the point
where the stream breaks into droplets. A charge is placed on the ring based on
the immediately prior fluorescence intensity measurement and the opposite
charge is trapped on the droplet as it breaks from the stream. The charged
droplets then fall through an electrostatic deflection system that diverts
droplets into containers based upon their charge. In some systems the charge is
applied directly to the stream and the droplet breaking off retains charge of
the same sign as the stream. The stream is then returned to neutral after the
droplet breaks off.
An alternative sorting method is magnetic bead sorting. Instead of fluorescent molecules, antibodies are
conjugated to iron particles. After staining, cells
can be purified using a strong magnet. It is less expensive than FACS and large
numbers of cells can be used but it is less sensitive than FACS to rare populations
of cells
Literature
http://biomicro.sdstate.edu/younga/VET791/VET791.pdf