Flow cytometry WikipediaCytometer redirects here. For the mechanical instrument, see Hemocytometer. BD Biosciences provides flow cytometers, reagents, tools, and a wide range of services to support the work of researchers and clinicians who understand disease and. Looking to join one of the big pharmaceutical companies with a biotechnology career Learn about courses, programs and degrees in biotechnology. In biotechnology, flow cytometry is a laser or impedance based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them through an electronic detection apparatus. A flow cytometer allows simultaneous multiparametric analysis of the physical and 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 basic research, clinical practice and clinical trials. A common variation involves linking the analytical capability of the flow cytometer to a sorting device, to physically separate and thereby purify particles of interest based on their optical properties. Such a process is called cell sorting, and the instrument is commonly termed a cell sorter. HistoryeditThe first impedance based flow cytometry device, using the Coulter principle, was disclosed in U. S. Patent 2,6. 56,5. Wallace H. Coulter. Mack Fulwyler was the inventor of the forerunner to todays flow cytometers particularly the cell sorter. Fulwyler developed this in 1. Science. 2 The first fluorescence based flow cytometry device ICP 1. Wolfgang Ghde from the University of Mnster, filed for patent on 1. December 1. 96. 83 and first commercialized in 1. German developer and manufacturer Partec through Phywe AG in Gttingen. At that time, absorption methods were still widely favored by other scientists over fluorescence methods. Soon after, flow cytometry instruments were developed, including the Cytofluorograph 1. BioPhysics Systems Inc. Ortho Diagnostics, the PAS 8. Partec, the first FACS Fluorescence activated cell sorting instrument from Becton Dickinson 1. Bd Cellquest Pro Software' title='Bd Cellquest Pro Software' />ICP 2. PartecPhywe and the Epics from Coulter 1. Download How To Hack To Get Free Load For Smart. The first label free high frequency impedance flow cytometer based on a patented microfluidic lab on chip, Ampha Z3. Amphasys 2. 01. 2. Name of the technologyeditThe original name of the fluorescence based flow cytometry technology was pulse cytophotometry German Impulszytophotometrie, based on the first patent application on fluorescence based flow cytometry. At the 5th American Engineering Foundation Conference on Automated Cytology in Pensacola Florida in 1. Flow cytometersedit. Front view of a desktop flow cytometer the Becton Dickinson fluorescence activated cell sorter FACSCaliburModern flow cytometers are able to analyze many thousand particles per second, in real time, and, if configured as cell sorters, can actively separate and isolate particles at similar rates having specified optical properties. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers high throughput, large scale, automated quantification of specified optical parameters on a cell by cell basis. To analyze solid tissues, a single cell suspension must first be prepared. A flow cytometer has five main components a flow cell, a measuring system, a detector, an amplification system, and a computer for analysis of the signals. The flow cell has a liquid stream sheath fluid, which carries and aligns the cells so that they pass single file through the light beam for sensing. The measuring system commonly use measurement of impedance or conductivity and optical systems lamps mercury, xenon high power water cooled lasers argon, krypton, dye laser low power air cooled lasers argon 4. He. Ne 6. 33 nm, green He. Nine West Employee Handbook there. Ne, He. Cd UV diode lasers blue, green, red, violet resulting in light signals. The detector and analog to digital conversion ADC system converts analog measurements of forward scattered light FSC and side scattered light SSC as well as dye specific fluorescence signals into digital signals that can be processed by a computer. The amplification system can be linear or logarithmic. 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 e. Early flow cytometers were, in general, experimental devices, but technological advances have enabled widespread 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 ten lasers6 and 3. 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. Data analysiseditThe 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. Individual single cells are often distinguished from cell doublets or higher aggregates by their time of flight denoted also as a pulse width through the narrowly focused laser beam8The plots are often made on logarithmic scales. Because different fluorescent dyes emission spectra overlap,91. Data accumulated using the flow cytometer can be analyzed using software, e. JMP statistical software, Win. MDI,1. 1 Flowing Software,1. Cytobank1. 3 all freeware, Cellcion, FCS Express, Flow. Jo, FACSDiva, Cyto. Paint aka Paint A Gate,1. Venturi. One, Cell. Quest Pro, Infinicyt or Cytospec. Once the data is collected, there is no need to stay connected to the flow cytometer and analysis is most often performed on a separate computer. This is especially necessary in core facilities where usage of these machines is in high demand. Computational analysiseditRecent 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 1. Immunology Database and Analysis Portal Imm. Port,1. 7 Sam. SPECTRAL1. Clust1. 92. 02. Bioconductor, and FLAME 2. Gene. Pattern. T Distributed Stochastic Neighbor Embedding t. SNE is an algorithm designed to perform dimensionality reduction, to allow visualization of complex multi dimensional data in a two dimensional map. Collaborative efforts have resulted in an open project called Flow. CAP Flow Cytometry Critical Assessment of Population Identification Methods,2. Fluorescence activated cell sorting FACSedit.