Flow cytometry

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2009)

Analysis of a marine sample of photosynthetic picoplankton by flow cytometry showing three

different populations (Prochlorococcus, Synechococcus, and picoeukaryotes)

Flow cytometry is a technique for counting and examining microscopic particles, such as cells and
chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection
apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical
characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the
diagnosis of health disorders, especially blood cancers, but has many other applications in both
research and clinical practice. A common variation is to physically sort particles based on their
properties, so as to purify populations of interest.

Contents
[hide]

• 1 History
o 1.1 Name of the technology
• 2 Principle of flow cytometry
• 3 Flow cytometers
• 4 Data analysis
o 4.1 Gating
o 4.2 Computational analysis
• 5 Fluorescence-activated cell sorting
• 6 Fluorescent labels
 • 7 Measurable parameters
• 8 Applications
• 9 See also
• 10 Bibliography
• 11 References

• 12 External links

[edit] History
The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang
Göhde from the University of Münster [1] and first commercialized in 1968/69 by German developer
and manufacturer Partec through Phywe AG in Göttingen. At that time, absorption methods were still
widely favored by other scientists over fluorescence methods [2]. Soon after, flow cytometry
instruments were developed, including the Cytofluorograph (1971) from Bio/Physics Systems Inc.
(later: Ortho Diagnostics), the PAS 8000 (1973) from Partec, the first FACS instrument from Becton
Dickinson (1974), the ICP 22 (1975) from Partec/Phywe and the Epics from Coulter (1977/78).

[edit] Name of the technology

The original name of the flow cytometry technology was "pulse cytophotometry" (German:
Impulszytophotometrie). Only 20 years later in 1988, at the Conference of the American Engineering
Foundation in Pensacola, Florida, the name was changed to "flow cytometry", a term that quickly
became popular.

[edit] Principle of flow cytometry

A beam of light (usually laser light) of a single wavelength is directed onto a hydrodynamically-
focused stream of fluid. A number of detectors are aimed at the point where the stream passes through
the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to
it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle from 0.2 to 150
micrometers passing through the beam scatters the light in some way, and fluorescent chemicals found
in the particle or attached to the particle may be excited into emitting light at a longer wavelength than
the light source. This combination of scattered and fluorescent light is picked up by the detectors, and,
by analysing fluctuations in brightness at each detector (one for each fluorescent emission peak), it is
then possible to derive various types of information about the physical and chemical structure of each
individual particle. 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 cytoplasmic granules or the membrane
roughness). Some flow cytometers on the market 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.

[edit] Flow cytometers

Modern flow cytometers are able to analyze several thousand particles every second, in "real time,"
and can actively separate and isolate particles having specified properties. A flow cytometer is similar
to a microscope, 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, a single-cell suspension must first be prepared.

A flow cytometer has 5 main components:

• a flow cell - liquid stream (sheath fluid), which carries and aligns the cells so that they pass
single file through the light beam for sensing
• an optical system - commonly used are lamps (mercury, xenon); high-power water-cooled
lasers (argon, krypton, dye laser); low-power air-cooled lasers (argon (488nm), red-HeNe
(633nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light
signals
• a detector and Analogue-to-Digital Conversion (ADC) system - which generates FSC and SSC
as well as fluorescence signals from light into electrical signals that can be processed by a
computer
• an amplification system - linear or logarithmic
• a computer for analysis of the signals.

The process of collecting data from samples using the flow cytometer is termed 'Acquisition'.
Acquisition is mediated by a computer physically connected to the flow cytometer, and the software
which handles the digital interface with the cytometer. The software is capable of adjusting parameters
(i.e. voltage, compensation, etc) for the sample being tested, and also assists in displaying initial
sample information while acquiring sample data to insure that parameters are set correctly. Early flow
cytometers were, in general, experimental devices, but technological advances have enabled wide-
spread applications for use in a variety of both clinical and research purposes. Due to these
developments, a considerable market for instrumentation, analysis software, as well as the reagents
used in acquisition such as fluorescently-labeled antibodies has developed.

Modern instruments usually have multiple lasers and fluorescence detectors (the current record for a
commercial instrument is 4 lasers and 18 fluorescence detectors). Increasing the number of lasers and
detectors allows for multiple antibody labeling, and can more precisely identify a target population by
their phenotypic markers. 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.

[edit] Data analysis

[edit] Gating

The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram,
or in two-dimensional dot plots or even in three dimensions. The regions on these plots can be
sequentially separated, based on fluorescence intensity, by creating a series of subset extractions,
termed "gates." Specific gating protocols exist for diagnostic and clinical purposes especially in
relation to hematology.

The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra
overlap [3], signals at the detectors have to be compensated electronically as well as computationally.
Data accumulated using the flow cytometer can be analyzed using software, e.g., WinMDI(depricated)
[3]
, Flowjo, or CellQuest Pro. Once the data is collected, there is no need to stay connected to the flow
cytometer. For this reason, analysis is most often done on a separate computer. This is especially
necessary in core facilities where usage of these machines is in high demand.
[edit] Computational analysis

Recent progress on automated population identification using computational methods has offered an
alternative to traditional gating strategies. Automated identification systems could potentially help
findings of rare and hidden populations. Representative automated methods include FLOCK in
ImmPort [4], FLAME [5] in GenePattern and flowClust [6], [7], [8] in Bioconductor. Collaborative efforts
have resulted in an open project called FlowCAP (Flow Cytometry: Critical Assessment of Population
Identification Methods, [9]) to provide an objective way to compare and evaluate the flow cytometry
data clustering methods, and also to establish guidance about appropriate use and application of these
methods.

[edit] Fluorescence-activated cell sorting

Fluorescence-activated cell sorting is a specialized type of flow cytometry. It provides a method for
sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time,
based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful
scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals
from individual cells as well as physical separation of cells of particular interest. The acronym FACS
is trademarked and owned by Becton Dickinson[10]. While many immunologists use this term
frequently for all types of sorting and non-sorting applications, it is not a generic term for flow
cytometry. The first cell sorter was invented by Mack Fulwyler in 1965, using the principle of Coulter
volume, a relatively difficult technique and one no longer used in modern instruments. The technique
was expanded by Len Herzenberg who was responsible for coining the term FACS. Herzenberg won
the Kyoto Prize in 2006 for his work in flow cytometry.

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 per 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.

[edit] Fluorescent labels

The fluorescence labels that can be used, will depend on the lamp or laser used to excite the
fluorochromes and on the detectors available:[11]

Blue argon laser (488 nm)

This is an air cooled laser and therefore cheaper to set up and run. It is the most commonly available
laser on single laser machines.
 • Green (usually labelled FL1): FITC, Alexa Fluor 488, GFP, CFSE, CFDA-SE, DyLight 488
• Orange (usually FL2): PE, PI
• Red channel (usually FL3): PerCP, PerCP-Cy5.5, PE-Alexa Fluor 700, PE-Cy5 (TRI-
COLOR), PE-Cy5.5.
• Infra-red (usually FL4; not provided by all FACS machines as standard): PE-Alexa Fluor 750,
PE-Cy7

Red diode laser (635 nm)

• APC
• APC-Cy7, APC-eFluor 780
• Alexa Fluor 700
• Cy5
• Draq-5

Violet laser (405 nm)

• Pacific Orange
• Amine Aqua
• Pacific Blue
• DAPI
• Alexa Fluor 405
• eFluor 450
• eFluor 605 Nanocrystals
• eFluor 650 Nanocrystals

[edit] Measurable parameters

This list is very long and constantly expanding.

• volume and morphological complexity of cells

• cell pigments such as chlorophyll or phycoerythrin
• DNA (cell cycle analysis, cell kinetics, proliferation, etc.)
• RNA
• chromosome analysis and sorting (library construction, chromosome paint)
• protein expression and localization
• Protein modifications, phospho-proteins
• transgenic products in vivo, particularly the Green fluorescent protein or related fluorescent *
cell surface antigens (Cluster of differentiation (CD) markers)
• intracellular antigens (various cytokines, secondary mediators, etc.)
• nuclear antigens
• enzymatic activity
• pH, intracellular ionized calcium, magnesium, membrane potential
• membrane fluidity
• apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane
potential, permeability changes, caspase activity)
• cell viability
• monitoring electropermeabilization of cells
• oxidative burst
 • characterising multidrug resistance (MDR) in cancer cells
• glutathione
• various combinations (DNA/surface antigens, etc.)

[edit] Applications
The technology has applications in a number of fields, including molecular biology, pathology,
immunology, plant biology and marine biology. In the field of molecular biology it is especially useful
when used with fluorescence tagged antibodies. These specific antibodies (which number tens of
thousands against several thousand genes) bind to antigens on the target cells and help to give
information on specific characteristics of the cells being studied in the cytometer. It has broad
application in medicine (especially in transplantation, hematology, tumor immunology and
chemotherapy, genetics and sperm sorting for sex preselection). In marine biology, the auto-
fluorescent properties of photosynthetic plankton can be exploited by flow cytometry in order to
characterise abundance and community structure. 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.

[edit] See also