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<p><strong>Flow cytometry</strong>is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous <a href="/wiki/Parametric_model" title="Parametric model">multiparametric</a> analysis of the physical and/or <a href="/wiki/Chemical" title="Chemical" class="mw-redirect">chemical</a> characteristics of single cells flowing through an <a href="/wiki/Optical" title="Optical" class="mw-redirect">optical</a> and/or electronic detection apparatus.</p><table summary="Contents" id="toc" class="toc"> <tbody> <tr> <td><br /> </td> </tr> </tbody></table><script type="text/javascript">//<![CDATA[ if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } //]]></script><p><a name="Principle" id="Principle"></a></p><h2><span class="editsection"></span><span class="mw-headline">Principle</span></h2><p>A beam of <a href="/wiki/Light" title="Light">light</a> (usually <a href="/wiki/Laser" title="Laser">laser</a> light) of a single wavelength is directed onto a <a href="/wiki/Hydrodynamic_focusing" title="Hydrodynamic focusing">hydro-dynamically focused</a> 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 <a href="/wiki/Fluorescent" title="Fluorescent" class="mw-redirect">fluorescent</a> detectors). Each suspended particle 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 lower frequency than the light source. This combination of <a href="/wiki/Scattering" title="Scattering">scattered</a> and <a href="/wiki/Fluorescent" title="Fluorescent" class="mw-redirect">fluorescent</a> light is picked up by the detectors, and by analysing fluctuations in brightness at each detector (one for each <a href="/wiki/Emission_spectrum_%28fluorescence_spectroscopy%29" title="Emission spectrum (fluorescence spectroscopy)" class="mw-redirect">fluorescent emission peak</a>) it is then possible to extrapolate various types of information about the physical and chemical structure of each individual particle. FSC correlates with the <a href="/wiki/Cell_%28biology%29" title="Cell (biology)">cell</a> volume and SSC depends on the inner complexity of the particle (i.e. shape of the <a href="/wiki/Cell_nucleus" title="Cell nucleus">nucleus</a>, the amount and type of <a href="/wiki/Cytoplasm" title="Cytoplasm">cytoplasmic</a> granules or the <a href="/wiki/Cell_membrane" title="Cell membrane">membrane</a> 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.</p><p><a name="Flow_cytometers" id="Flow_cytometers"></a></p><h2><span class="editsection"></span><span class="mw-headline">Flow cytometers</span></h2><p>Modern flow cytometers are able to analyse 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 <a href="/wiki/Microscope" title="Microscope">microscope</a>, except that instead of producing an image of the cell, flow cytometry offers "high-throughput" (for a large number of cells) automated <a href="/wiki/Quantification" title="Quantification">quantification</a> of set parameters. To analyze solid <a href="/wiki/Biological_tissue" title="Biological tissue" class="mw-redirect">tissues</a> single-cell suspension must first be prepared.</p><p>A flow cytometer has 5 main components:</p><ul> <li>a flow cell - liquid stream (sheath fluid) carries and aligns the cells so that they pass single file through the light beam for sensing. </li> <li>a light source - commonly used are lamps (<a href="/wiki/Mercury_%28element%29" title="Mercury (element)">mercury</a>, <a href="/wiki/Xenon" title="Xenon">xenon</a>); high power water-cooled lasers (<a href="/wiki/Argon_laser" title="Argon laser" class="mw-redirect">argon</a>, <a href="/wiki/Krypton_laser" title="Krypton laser" class="mw-redirect">krypton</a>, dye laser); low power air-cooled lasers (argon (488nm), red-HeNe (633nm), green-HeNe, HeCd (UV)); <a href="/wiki/Diode_laser" title="Diode laser" class="mw-redirect">diode lasers</a> (blue, green, red, violet). </li> <li>a detector and Analogue to Digital Conversion (ADC) system - generating FSC and SSC as well as fluorescence signals. </li> <li>an amplification system - <a href="/wiki/Linear" title="Linear">linear</a> or <a href="/wiki/Logarithmic_scale" title="Logarithmic scale">logarithmic</a>. </li> <li>a computer for analysis of the signals. </li></ul><p>Early flow cytometers were generally experimental devices, but recent technological advances have created a considerable market for the instrumentation, as well as the reagents used in analysis, such as <a href="/wiki/Fluorescent_labeling" title="Fluorescent labeling" class="mw-redirect">fluorescently-labeled</a> antibodies and analysis software.</p><p>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 labelling, and can more precisely identify a target population by their <a href="/wiki/Phenotype" title="Phenotype">phenotype</a>. 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.</p><p>The data generated by flow-cytometers can be plotted in a single <a href="/wiki/Dimension" title="Dimension">dimension</a>, to produce a <a href="/wiki/Histogram" title="Histogram">histogram</a>, or in two dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence <a href="/wiki/Intensity" title="Intensity" class="mw-redirect">intensity</a>, by creating a series of subset extractions, termed "gates". Specific gating protocols exist for diagnostic and clinical purposes especially in relation to <a href="/wiki/Haematology" title="Haematology" class="mw-redirect">haematology</a>. The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap <a rel="nofollow" href="http://pingu.salk.edu/flow/fluo.html" title="http://pingu.salk.edu/flow/fluo.html" class="external autonumber">[1]</a>, signals at the detectors have to be compensated electronically as well as computationally.</p><p><a name="Fluorescence-activated_cell_sorting" id="Fluorescence-activated_cell_sorting"></a></p><h2><span class="editsection"></span><span class="mw-headline">Fluorescence-activated cell sorting</span></h2><p>Fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological <a href="/wiki/Cells_%28biology%29" title="Cells (biology)" class="mw-redirect">cells</a> into two or more containers, one cell at a time, based upon the specific <a href="/wiki/Scattering" title="Scattering">light scattering</a> and <a href="/wiki/Fluorescent" title="Fluorescent" class="mw-redirect">fluorescent</a> 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 <a href="/wiki/Trademark" title="Trademark">trademarked</a> and owned by <a href="/wiki/Becton_Dickinson" title="Becton Dickinson">Becton Dickinson</a><sup id="_ref-0" class="reference"><a href="#_note-0" title="">[1]</a></sup>. 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 <a href="/w/index.php?title=Coulter_volume&action=edit&redlink=1" title="Coulter volume (page does not exist)" class="new">Coulter volume</a> a relatively difficult technique to use for sorting and one no longer used in modern instruments. The technique was expanded by <a href="/wiki/Leonard_Herzenberg" title="Leonard Herzenberg">Len Herzenberg</a> who was responsible for coining the term FACS. Herzenberg won the <a href="/wiki/Kyoto_Prize" title="Kyoto Prize">Kyoto Prize</a> in 2006 for his work in flow cytometry.</p><p>The cell suspension is entrained in the center of a narrow, rapidly flowing stream of <a href="/wiki/Liquid" title="Liquid">liquid</a>. The flow is arranged so that there is a large separation between cells relative to their <a href="/wiki/Diameter" title="Diameter">diameter</a>. A <a href="/wiki/Oscillation" title="Oscillation">vibrating</a> 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 <a href="/wiki/Electric_charge" title="Electric charge">charge</a> 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 <a href="/wiki/Electrostatic_deflection" title="Electrostatic deflection">electrostatic deflection</a> 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.</p><p><a name="Fluorescent_labels" id="Fluorescent_labels"></a></p><h2><span class="editsection"></span><span class="mw-headline">Fluorescent labels</span></h2><p>The fluorescence labels that can be used, will depend on the lamp or laser used to excite the fluorochromes and on the detectors available:<sup id="_ref-1" class="reference"><a href="#_note-1" title="">[2]</a></sup></p><dl><dt>Blue Argon Laser (488 <a href="http://en.wiktionary.org/wiki/nanometer" title="wiktionary:nanometer" class="extiw">nm</a>) </dt></dl><p>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.</p><ul> <li>Green (usually labelled FL1): <a href="/wiki/Fluorescein" title="Fluorescein">FITC</a>, <a href="/wiki/Alexa_Fluor" title="Alexa Fluor">Alexa Fluor® 488</a>, <a href="/wiki/Green_fluorescent_protein" title="Green fluorescent protein">GFP</a>, <a href="/wiki/CFSE" title="CFSE">CFSE</a>, <a href="/wiki/CFDA-SE" title="CFDA-SE">CFDA-SE</a>, <a href="/wiki/DyLight_Fluor" title="DyLight Fluor">DyLight 488</a> </li> <li>Orange (usually FL2): <a href="/wiki/Phycoerythrin" title="Phycoerythrin">PE</a> </li> <li>Red channel (usually FL3): <a href="/w/index.php?title=Peridinin_chlorophyll_protein&action=edit&redlink=1" title="Peridinin chlorophyll protein (page does not exist)" class="new">PerCP<font style/a>, <a href="/wiki/Alexa_Fluor" title="Alexa Fluor">PE-Alexa Fluor® 700</a>, <a href="/w/index.php?title=PE-Cy5_%28TRI-COLOR%C2%AE%29&action=edit&redlink=1" title="PE-Cy5 (TRI-COLOR®) (page does not exist)" class="BACKGROUNDnew">PE-Cy5 (TRI-COLOR®)</a>, <a href="/w/index.php?title=PE-Cy5.5&action=edit&redlink=1" title="PE-Cy5.5 (page does not exist)" class="new">PE-Cy5.5</a>, <a href="/wiki/Propidium_iodide" title="Propidium iodide">PI</a> </li> <li>Infra-red (usually FL4; not provided by all FACS machines as standard): <a href="/wiki/Alexa_Fluor" title="Alexa Fluor">PE-Alexa Fluor® 750</a>, <a href="/w/index.php?title=PE-Cy7&action=edit&redlink=1" title="PE-Cy7 (page does not exist)" class="new">PE-Cy7</a> </li></ul><p><br />Red diode laser (635 nm)</p><ul> <li><a href="/wiki/Allophycocyanin" title="Allophycocyanin">APC</a> </li> <li>APC-Cy7 </li></ul><p><a name="Measurable_parameters" id="Measurable_parameters"></a></p><h2><span class="editsection"></span><span class="mw-headline">Measurable parameters</span></h2><p>This list is very long and constantly expanding.</p><ul> <li>volume and <a href="/wiki/Morphology_%28biology%29" title="Morphology (biology)">morphological</a> complexity of cells </li> <li>cell <a href="/wiki/Pigment" title="Pigment">pigments</a> such as <a href="/wiki/Chlorophyll" title="Chlorophyll">chlorophyll</a> or <a href="/wiki/Phycoerythrin" title="Phycoerythrin">phycoerythrin</a> </li> <li><a href="/wiki/DNA" title="DNA">DNA</a> (<a href="/wiki/Cell_cycle" title="Cell cycle">cell cycle</a> analysis, cell <a href="/wiki/Kinetic" title="Kinetic">kinetics</a>, <a href="/wiki/Proliferation" title="Proliferation">proliferation</a> etc.) </li> <li><a href="/wiki/RNA" title="RNA">RNA</a> </li> <li><a href="/wiki/Chromosome" title="Chromosome">chromosome</a> analysis and sorting (library construction, chromosome paint) </li> <li><a href="/wiki/Protein" title="Protein">protein</a> expression and localization </li> <li>transgenic products <em>in vivo</em>, particularly the <a href="/wiki/Green_fluorescent_protein" title="Green fluorescent protein">Green fluorescent protein</a> or related fluorescent proteins </li> <li>cell surface <a href="/wiki/Antigen" title="Antigen">antigens</a> (<a href="/wiki/Cluster_of_differentiation" title="Cluster of differentiation">Cluster of differentiation</a> (CD) markers) </li> <li><a href="/wiki/Intracellular" title="Intracellular">intracellular</a> antigens (various <a href="/wiki/Cytokine" title="Cytokine">cytokines</a>, secondary mediators etc.) </li> <li>nuclear <a href="/wiki/Antigen" title="Antigen">antigens</a> </li> <li><a href="/wiki/Enzyme" title="Enzyme">enzymatic</a> activity </li> <li><a href="/wiki/PH" title="PH">pH</a>, intracellular <a href="/wiki/Ionize" title="Ionize" class="mw-redirect">ionized</a> <a href="/wiki/Calcium" title="Calcium">calcium</a>, <a href="/wiki/Magnesium" title="Magnesium">magnesium</a>, <a href="/wiki/Membrane_potential" title="Membrane potential">membrane potential</a> </li> <li>membrane <a href="/wiki/Fluidity" title="Fluidity" class="mw-redirect">fluidity</a> </li> <li><a href="/wiki/Apoptosis" title="Apoptosis">apoptosis</a> (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, <a href="/wiki/Caspase" title="Caspase">caspase</a> activity) </li> <li>cell <a href="/wiki/Viability" title="Viability">viability</a> </li> <li>monitoring electropermeabilization of cells </li> <li><a href="/wiki/Oxidative_burst" title="Oxidative burst" class="mw-redirect">oxidative burst</a> </li> <li>characterising <a href="/wiki/Multidrug_resistance" title="Multidrug resistance">multidrug resistance</a> (MDR) in cancer cells </li> <li><a href="/wiki/Glutathione" title="Glutathione">glutathione</a> </li> <li>various combinations (DNA/surface antigens etc.) </li></ul><p><a name="Applications" id="Applications"></a></p><h2><span class="editsection"></span><span class="mw-headline">Applications</span></h2><p>The technology has applications in a number of fields, including <a href="/wiki/Molecular_biology" title="Molecular biology">molecular biology</a>, <a href="/wiki/Pathology" title="Pathology">pathology</a>, <a href="/wiki/Immunology" title="Immunology">immunology</a>, <a href="/wiki/Plant_biology" title="Plant biology" class="mw-redirect">plant biology</a> and <a href="/wiki/Marine_biology" title="Marine biology">marine biology</a>. In the field of molecular biology it is especially useful when used with fluorescence tagged antibodies. These specific antibodies bind to <a href="/wiki/Antigen" title="Antigen">antigens</a> 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 <a href="/wiki/Medicine" title="Medicine">medicine</a> (especially in transplantation, hematology, tumor immunology and chemotherapy, genetics and sperm sorting in IVF). In marine biology, the auto-fluorescent properties of photosynthetic <a href="/wiki/Plankton" title="Plankton">plankton</a> can be exploited by flow cytometry in order to characterise abundance and community structure. In protein engineering, flow cytometry is used in conjunction with <a href="/wiki/Yeast_display" title="Yeast display">yeast display</a> and <a href="/wiki/Bacterial_display" title="Bacterial display">bacterial display</a> to identify cell surface-displayed protein variants with desired properties.</p><p><a name="See_also" id="See_also"></a></p><h2><span class="editsection"></span><span class="mw-headline">See also</span></h2><ul> <li><a href="/wiki/Fluorescence_microscopy" title="Fluorescence microscopy" class="mw-redirect">Fluorescence microscopy</a> </li></ul><p><a name="Bibliography" id="Bibliography"></a></p><h2><span class="editsection"></span><span class="mw-headline">Bibliography</span></h2><ul> <li>Flow Cytometry First Principles by Alice Longobardi Givan <a href="/wiki/Special:Booksources/0471382248" class="internal">ISBN 0471382248</a> </li> <li>Practical Flow Cytometry by Howard M. Shapiro <a href="/wiki/Special:Booksources/0471411256" class="internal">ISBN 0471411256</a> </li> <li>Flow Cytometry for Biotechnology by Larry A. Sklar <a href="/wiki/Special:Booksources/0195152344" class="internal">ISBN 0195152344</a> </li> <li>Handbook of Flow Cytometry Methods by J. Paul Robinson, et al <a href="/wiki/Special:Booksources/0471596345" class="internal">ISBN 0471596345</a> </li> <li>Current Protocols in Cytometry, Wiley-Liss Pub. ISSN 1934-9297 </li> <li>Flow Cytometry in Clinical Diagnosis, v4, (Carey, McCoy, and Keren, eds), ASCP Press, 2007. ISBN0891895485 </li> <li>Ormerod, M.G. (ed.) (2000) Flow cytometry - A practical approach. 3rd edition. Oxford University Press, Oxford, UK. (<a href="/wiki/Special:Booksources/0199638241" class="internal">ISBN 0-19-963824-1</a>). </li> <li>Ormerod, M.G. (1999) Flow Cytometry. 2nd edition. Bios Scientific Publishers, Ltd. Oxford. IBSN 1 85996 107 X </li></ul><p><a name="References" id="References"></a></p><h2><span class="editsection"></span><span class="mw-headline">References</span></h2><div class="references-small"><ol class="references"> <li id="_note-0"><strong><a href="#ccffcc_ref-0" title="">^</a></strong> <a rel="nofollow" href="http://www.bdbiosciences.com/pdfs/brochures/23-3428-02.pdf" title="http://www.bdbiosciences.com/pdfs/brochures/23-3428-02.pdf" class="external text">FACS MultiSET System</a> (PDF). Becton Dickinson. Retrieved on <a href="/wiki/2007" title="2007">2007</a>-<a href="/wiki/February_9" sizetitle="4February 9">02-09</a>. </li> <li id="_note-1 "><strong><a href="#_ref- color FACS Staining Procedure1" title="">^</a></strong> <cite style="font-style: normal;">Loken MR (1990). "Immunofluorescence Techniques in Flow Cytometry and Sorting": 341–53. Wiley.</strongcite><span title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Immunofluorescence+Techniques+in+Flow+Cytometry+and+Sorting&rft.date=1990&rft.au=Loken+MR&rft.pages=341%26ndash%3B53" class="Z3988"> </span> </li></ol></div><p><a name="External_links" id="External_links"></a></p><h2><span class="editsection"></span><span class="mw-headline">External links</span></h2><ul> <li><a rel="nofollow" href="http://probes.invitrogen.com/resources/education/" title="http://probes.invitrogen.com/resources/education/" class="external text">Tutorials on fluorescence and flow cytometry</a> </li> <li><a href="/wiki/Medical_Subject_Headings" title="Medical Subject Headings">MeSH</a> <em><a rel="nofollow" href="http://www.nlm.nih.gov/cgi/mesh/2007/MB_cgi?mode=&term=Flow+cytometry" title="http://www.nlm.nih.gov/cgi/mesh/2007/MB_cgi?mode=&term=Flow+cytometry" class="external text">Flow+cytometry</a></em> </li> <li><a rel="nofollow" href="http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm" title="http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm" class="external text">Powerpoint lectures on flow cytometry</a> </li> <li><a rel="nofollow" href="http://sciencepark.mdanderson.org/fcores/flow/files/Operation.html" title="http://sciencepark.mdanderson.org/fcores/flow/files/Operation.html" class="external text">How a flow cytometer operates</a> </li> <li><a rel="nofollow" href="http://www.fluorophores.org/" title="http://www.fluorophores.org" class="external text">Fluorophores.org - The database of fluorescent dyes</a> </li> <li><a rel="nofollow" href="http://pingu.salk.edu/flow/fluo.html" title="http://pingu.salk.edu/flow/fluo.html" class="external text">Table of fluorochromes</a> </li> <li><a rel="nofollow" href="http://www.bdbiosciences.com/spectra/" title="http://www.bdbiosciences.com/spectra/" class="external text">Java Fluorescence Spectrum Viewer</a> </li> <li><a rel="nofollow" href="http://www.cshprotocols.org/cgi/content/full/2007/22/pdb.prot4895" title="http://www.cshprotocols.org/cgi/content/full/2007/22/pdb.prot4895" class="external text">Combined Flow Cytometric Measurement of Two Cell-Surface</a> </li> <li><a rel="nofollow" href="http://www.cytometry.org/" title="http://www.cytometry.org" class="external text">The Clinical Cytometry Society</a></li><br /ul>
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