Flow cytometry - Wikipedia. For the mechanical instrument, see Hemocytometer. 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 by an electronic detection apparatus. It 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 is to physically sort particles based on their properties, so as to purify populations of interest. History. Patent 2,6.
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Wallace H. Mack Fulwyler was the inventor of the forerunner to today's flow cytometers - particularly the cell sorter. At that time, absorption methods were still widely favored by other scientists over fluorescence methods.
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The first label- free high- frequency impedance flow cytometer based on a patented microfluidic . At the 5th American Engineering Foundation Conference on Automated Cytology in Pensacola (Florida) in 1. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers .
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To analyze solid tissues, a single- cell suspension must first be prepared. A flow cytometer has five 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 sensinga measuring system - commonly used are 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.
Ne, He. Cd (UV)); diode lasers (blue, green, red, violet) resulting in light signalsa detector and Analog- to- Digital Conversion (ADC) system - which 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 binary computeran amplification system - linear or logarithmica 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 (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 lasers. 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 analysis. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed . Individual single cells are often distinguished from cell doublets or higher aggregates by their . Because different fluorescent dyes' emission spectra overlap.
Data accumulated using the flow cytometer can be analyzed using software, e. JMP (statistical software), Win. MDI. This is especially necessary in core facilities where usage of these machines is in high demand. Computational analysis. Automated identification systems could potentially help findings of rare and hidden populations. Representative automated methods include FLOCK .
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 . 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 technique was expanded by Len Herzenberg, who was responsible for coining the term FACS. 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 each cell of interest 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 immediately prior to fluorescence intensity being measured, 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. The acronym FACS is trademarked and owned by Becton, Dickinson and Company.
Each fluorophore has a characteristic peak excitation and emission wavelength, and the emission spectra often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available. Absolute fluorescence sensitivity is generally lower in confocal microscopy because out- of- focus signals are rejected by the confocal optical system and because the image is built up serially from individual measurements at every location across the cell, reducing the amount of time available to collect signal. This method could theoretically allow the use of 4.
Although this method permits the use of a large number of labels, it currently has lower throughput capacity than flow cytometry. It also destroys the analysed cells, precluding their recovery by sorting. Similar to ELISA sandwich assays, cytometric bead array (CBA) assays use multiple bead populations typically differentiated by size and different levels of fluorescence intensity to distinguish multiple analytes in a single assay. The amount of the analyte captured is detected via a biotinylated antibody against a secondary epitope of the protein, followed by a streptavidin- R- phycoerythrin treatment. The fluorescent intensity of R- phycoerythrin on the beads is quantified on a flow cytometer equipped with a 4. Concentrations of a protein of interest in the samples can be obtained by comparing the fluorescent signals to those of a standard curve generated from a serial dilution of a known concentration of the analyte.
Commonly also referred to as cytokine bead array (CBA). Impedance flow cytometry. They represent a well- established method for counting and sizing virtually any kind of cells and particles. The label- free technology has recently been enhanced by a . The relatively small size and robustness allow battery powered on- site use in the field. Measurable parameters.
These are conventionally abbreviated as FSC and SSC respectively. Total DNA content (cell cycle analysis, cell kinetics, proliferation, ploidy, aneuploidy, endoreduplication, etc.)Total RNA content. Transgenic products in vivo, particularly the Green fluorescent protein or related Fluorescent Proteins. Various combinations (DNA/surface antigens, etc.)Applications. Also, it is extensively used in research for the detection of DNA damage. 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. See also. ISBN0- 4.
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