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Flow cytometry

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Flow cytometry
FACS-buisje.JPG
A flow cytometer tube with suction straw
ClassificationCytometry
AnalytesCells or particles
Other techniques
RelatedCoulter counter

Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.[1][2][3][4]

In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so light is absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer.

Flow cytometry is routinely used in basic research, clinical practice, and clinical trials. Uses for flow cytometry include:

A flow cytometry analyzer is an instrument that provides quantifiable data from a sample. Other instruments using flow cytometry include cell sorters which physically separate and thereby purify cells of interest based on their optical properties.

History

The first impedance-based flow cytometry device, using the Coulter principle, was disclosed in U.S. Patent 2,656,508, issued in 1953, to Wallace H. Coulter. Mack Fulwyler was the inventor of the forerunner to today's flow cytometers - particularly the cell sorter.[5] Fulwyler developed this in 1965 with his publication in Science.[6] The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Göhde from the University of Münster, filed for patent on 18 December 1968[7] 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.[8] 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 (fluorescence-activated cell sorting) instrument from Becton Dickinson (1974), the ICP 22 (1975) from Partec/Phywe and the Epics from Coulter (1977/78). The first label-free high-frequency impedance flow cytometer based on a patented microfluidic "lab-on-chip", Ampha Z30, was introduced by Amphasys (2012).

Name of the technology

The 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 1976 - eight years after the introduction of the first fluorescence-based flow cytometer (1968) - it was agreed to commonly use the name "flow cytometry", a term that quickly became popular.[9]

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Cytometry

Cytometry

Cytometry is the measurement of number and characteristics of cells. Variables that can be measured by cytometric methods include cell size, cell count, cell morphology, cell cycle phase, DNA content, and the existence or absence of specific proteins on the cell surface or in the cytoplasm. Cytometry is used to characterize and count blood cells in common blood tests such as the complete blood count. In a similar fashion, cytometry is also used in cell biology research and in medical diagnostics to characterize cells in a wide range of applications associated with diseases such as cancer and AIDS.

Electrical impedance

Electrical impedance

In electrical engineering, impedance is the opposition to alternating current presented by the combined effect of resistance and reactance in a circuit.

Wallace H. Coulter

Wallace H. Coulter

Wallace H. Coulter was an American electrical engineer, inventor, and businessman. The best known of his 85 patents is the Coulter principle, which provides a method for counting and sizing microscopic particles suspended in fluid. His invention of the Coulter Counter made possible today's most common medical diagnostic test: the complete blood count (CBC). The Coulter principle is used in quality control of consumer products, such as chocolate and beer, paint and toners, and was even used to analyze moon dust.

Science (journal)

Science (journal)

Science, also widely referred to as Science Magazine, is the peer-reviewed academic journal of the American Association for the Advancement of Science (AAAS) and one of the world's top academic journals. It was first published in 1880, is currently circulated weekly and has a subscriber base of around 130,000. Because institutional subscriptions and online access serve a larger audience, its estimated readership is over 400,000 people.

University of Münster

University of Münster

The University of Münster is a public research university located in the city of Münster, North Rhine-Westphalia in Germany.

Göttingen

Göttingen

Göttingen is a university city in Lower Saxony, central Germany, the capital of the eponymous district. The River Leine runs through it. At the end of 2019, the population was 118,911.

Absorption (electromagnetic radiation)

Absorption (electromagnetic radiation)

In physics, absorption of electromagnetic radiation is how matter takes up a photon's energy — and so transforms electromagnetic energy into internal energy of the absorber. A notable effect is attenuation, or the gradual reduction of the intensity of light waves as they propagate through a medium. Although the absorption of waves does not usually depend on their intensity, in certain conditions (optics) the medium's transparency changes by a factor that varies as a function of wave intensity, and saturable absorption occurs.

Fluorescence

Fluorescence

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum, while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.

German language

German language

German, or more precisely High German, is a West Germanic language mainly spoken in Western Europe and Central Europe. It is the most widely spoken and official or co-official language in Germany, Austria, Switzerland, Liechtenstein, and the Italian province of South Tyrol. It is also a co-official language of Luxembourg and Belgium, as well as a recognized national language in Namibia. Outside Germany, it is also spoken by German communities in France (Bas-Rhin), Czech Republic, Poland, Slovakia, and Hungary (Sopron).

Flow cytometers

Schematic diagram of a flow cytometer, from sheath focusing to data acquisition.
Schematic diagram of a flow cytometer, from sheath focusing to data acquisition.

Modern flow cytometers are able to analyze many thousands of particles per second, in "real time" and, if configured as cell sorters, can actively separate and isolate particles with specified optical properties at similar rates. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers high-throughput, 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 uses 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 (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (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.g., voltage, compensation) for the sample being tested, and also assists in displaying initial sample information while acquiring sample data to ensure that parameters are set correctly. 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 have been developed.

Modern instruments usually have multiple lasers and fluorescence detectors. The current record for a commercial instrument is ten lasers[10] and 30 fluorescence detectors.[11] 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.

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Microscope

Microscope

A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope.

Quantification (science)

Quantification (science)

In mathematics and empirical science, quantification is the act of counting and measuring that maps human sense observations and experiences into quantities. Quantification in this sense is fundamental to the scientific method.

Mercury (element)

Mercury (element)

Mercury is a chemical element with the symbol Hg and atomic number 80. It is also known as quicksilver and was formerly named hydrargyrum from the Greek words hydorcode: ell promoted to code: el (water) and argyroscode: ell promoted to code: el (silver). A heavy, silvery d-block element, mercury is the only metallic element that is known to be liquid at standard temperature and pressure; the only other element that is liquid under these conditions is the halogen bromine, though metals such as caesium, gallium, and rubidium melt just above room temperature.

Xenon

Xenon

Xenon is a chemical element with the symbol Xe and atomic number 54. It is a dense, colorless, odorless noble gas found in Earth's atmosphere in trace amounts. Although generally unreactive, it can undergo a few chemical reactions such as the formation of xenon hexafluoroplatinate, the first noble gas compound to be synthesized.

Logarithmic scale

Logarithmic scale

A logarithmic scale is a way of displaying numerical data over a very wide range of values in a compact way. As opposed to a linear number line in which every unit of distance corresponds to adding by the same amount, on a logarithmic scale, every unit of length corresponds to multiplying the previous value by the same amount. Hence, such a scale is nonlinear: the numbers 1, 2, 3, 4, 5, and so on, are not equally spaced. Rather, the numbers 10, 100, 1000, 10000, and 100000 would be equally spaced. Likewise, the numbers 2, 4, 8, 16, 32, and so on, would be equally spaced. Often exponential growth curves are displayed on a log scale, otherwise they would increase too quickly to fit within a small graph.

Hardware

Fluidics system of a flow cytometer

Cells must pass uniformly through the center of focused laser beams to accurately measure optical properties of cells in any flow cytometer.[12][13][14] The purpose of the fluidic system is to move the cells one by one through the lasers beam and throughout the instrument. Fluidics in a flow cytometer with cell sorting capabilities also use the stream to carry sorted cells into collection tubes or wells.[12]

Hydrodynamic focusing

For precise positioning of cells in a liquid jet, hydrodynamic focusing is used in most cytometers.[12][13][14] The cells in suspension enter into the instrument enclosed by an outer sheath fluid. The sample core is maintained in the center of the sheath fluid. The sample input rate or how fast the cells flow through to the laser interrogation can be controlled by the pressure of the sheath fluid on the sample core. Under optimal conditions, the central fluid stream and sheath fluid do not mix.

Acoustic-assisted hydrodynamic focusing

Acoustic focusing technology is used in some flow cytometers to support hydrodynamic focusing.[12][14] Acoustic waves (>2 MHz) pre-focus the sample before introduction to sheath fluid. The pre-focused sample is then injected into the hydrodynamic core and flowed through the instrument. This may help with increasing data accuracy under high sample input rates.

Optics and electronics

Optical filters

Light emitted from fluorophores are in a spectrum of wavelengths, so combining multiple fluorophores may cause overlap. To add specificity, optical filters and dichroic mirrors are used to filter and move light to the detectors such as photomultiplier tubes (PMTs) or avalanche photodiodes (APD).[12] Optical filters are designed as band pass (BP), long pass (LP), or short pass (SP) filters. Most flow cytometers uses dichroic mirrors and band pass filters to select specific bands of the optical spectrum.

Prisms, gratings, and spectral flow cytometry

Spectral flow cytometry uses prisms or diffraction gratings to disperse the emitted light of a marker across a detector array.[12][15] This allows for the full spectra from each particle to be measured. The measured spectra from single cells are subsequently unmixed by using reference spectra of all used dyes and the autofluorescence spectrum. This may allow for a wider panel design and the application of new biological markers.

Imaging flow cytometry

Imaging flow cytometry (IFC) captures multichannel images of cells.[12][16] Detectors used in imaging platforms can be equipped with charge-coupled device (CCD) or complementary metal–oxide–semiconductor (CMOS) to capture images of individual cells.

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Hydrodynamic focusing

Hydrodynamic focusing

Hydrodynamic focusing is a technique used to provide more accurate results when using flow cytometers or Coulter counters for determining the size of bacteria or cells.

Fluorophore

Fluorophore

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.

Optical filter

Optical filter

An optical filter is a device that selectively transmits light of different wavelengths, usually implemented as a glass plane or plastic device in the optical path, which are either dyed in the bulk or have interference coatings. The optical properties of filters are completely described by their frequency response, which specifies how the magnitude and phase of each frequency component of an incoming signal is modified by the filter.

Dichroic filter

Dichroic filter

A dichroic filter, thin-film filter, or interference filter is a color filter used to selectively pass light of a small range of colors while reflecting other colors. By comparison, dichroic mirrors and dichroic reflectors tend to be characterized by the colors of light that they reflect, rather than the colors they pass.

Photomultiplier tube

Photomultiplier tube

Photomultiplier tubes (photomultipliers or PMTs for short) are extremely sensitive detectors of light in the ultraviolet, visible, and near-infrared ranges of the electromagnetic spectrum. They are members of the class of vacuum tubes, more specifically vacuum phototubes. These detectors multiply the current produced by incident light by as much as 100 million times or 108 (i.e., 160 dB), in multiple dynode stages, enabling (for example) individual photons to be detected when the incident flux of light is low.

Prism (optics)

Prism (optics)

An optical prism is a transparent optical element with flat, polished surfaces that are designed to refract light. At least one surface must be angled — elements with two parallel surfaces are not prisms. The most familiar type of optical prism is the triangular prism, which has a triangular base and rectangular sides. Not all optical prisms are geometric prisms, and not all geometric prisms would count as an optical prism. Prisms can be made from any material that is transparent to the wavelengths for which they are designed. Typical materials include glass, acrylic and fluorite.

Diffraction grating

Diffraction grating

In optics, a diffraction grating is an optical component with a periodic structure that diffracts light into several beams travelling in different directions. The emerging coloration is a form of structural coloration. The directions or diffraction angles of these beams depend on the wave (light) incident angle to the diffraction grating, the spacing or distance between adjacent diffracting elements on the grating, and the wavelength of the incident light. The grating acts as a dispersive element. Because of this, diffraction gratings are commonly used in monochromators and spectrometers, but other applications are also possible such as optical encoders for high precision motion control and wavefront measurement.

Charge-coupled device

Charge-coupled device

A charge-coupled device (CCD) is an integrated circuit containing an array of linked, or coupled, capacitors. Under the control of an external circuit, each capacitor can transfer its electric charge to a neighboring capacitor. CCD sensors are a major technology used in digital imaging.

Data analysis

Compensation

Each fluorochrome has a broad fluorescence spectrum. When more than one fluorochrome is used, the overlap between fluorochromes can occur. This situation is called spectrum overlap. This situation needs to be overcome. For example, the emission spectrum for FITC and PE is that the light emitted by the fluorescein overlaps the same wavelength as it passes through the filter used for PE. This spectral overlap is corrected by removing a portion of the FITC signal from the PE signals or vice versa. This process is called color compensation, which calculates a fluorochrome as a percentage to measure itself.[17]

Compensation is the mathematical process by which spectral overlap of multiparameter flow cytometric data is corrected. Since fluorochromes can have wide-ranging spectrum, they can overlap, causing the undesirable result of confusion during the analysis of data. This overlap, known as spillover and quantified in the spillover coefficient, is usually caused by detectors for a certain fluorochrome measuring a significant peak in wavelength from a different fluorochrome. Linear algebra is most often used to make this correction.[17]

In general, when graphs of one or more parameters are displayed, it is to show that the other parameters do not contribute to the distribution shown. Especially when using the parameters which are more than double, this problem is more severe. Currently, no tools have been discovered to efficiently display multidimensional parameters. Compensation is very important to see the distinction between cells.

Analysis of a marine sample of photosynthetic picoplankton by flow cytometry showing three different populations (Prochlorococcus, Synechococcus, and picoeukaryotes)
Analysis of a marine sample of photosynthetic picoplankton by flow cytometry showing three different populations (Prochlorococcus, Synechococcus, and picoeukaryotes)

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. 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 beam[18]

The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap,[19][20] signals at the detectors have to be compensated electronically as well as computationally. Data accumulated using the flow cytometer can be analyzed using software. 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 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 [21] in Immunology Database and Analysis Portal (ImmPort),[22] SamSPECTRAL[23] and flowClust[24][25][26] in Bioconductor, and FLAME [27] in GenePattern. T-Distributed Stochastic Neighbor Embedding (tSNE) is an algorithm designed to perform dimensionality reduction, to allow visualization of complex multi-dimensional data in a two-dimensional "map".[28] Collaborative efforts have resulted in an open project called FlowCAP (Flow Cytometry: Critical Assessment of Population Identification Methods,[29]) 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.

FMO controls

Fluorescence minus one (FMO) controls are important for data interpretation when building multi-color panels - in which a cell is stained with multiple fluorochromes simultaneously. FMO controls provide a measure of fluorescence spillover in a given channel and allow for compensation. To generate a FMO control, a sample is stained with all the fluorochromes except the one that is being tested - meaning if you are using 4 different fluorochromes your FMO control must contain only 3 of them (example: fluorochromes - A, B, C, D; FMOs - ABC_, AB_D, A_CD, _BCD).

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Flow cytometry bioinformatics

Flow cytometry bioinformatics

Flow cytometry bioinformatics is the application of bioinformatics to flow cytometry data, which involves storing, retrieving, organizing and analyzing flow cytometry data using extensive computational resources and tools. Flow cytometry bioinformatics requires extensive use of and contributes to the development of techniques from computational statistics and machine learning. Flow cytometry and related methods allow the quantification of multiple independent biomarkers on large numbers of single cells. The rapid growth in the multidimensionality and throughput of flow cytometry data, particularly in the 2000s, has led to the creation of a variety of computational analysis methods, data standards, and public databases for the sharing of results.

Compensation (cytometry)

Compensation (cytometry)

In cytometry, compensation is a mathematical correction of a signal overlap between the channels of the emission spectra of different fluorochromes.

Photosynthesis

Photosynthesis

Photosynthesis is a process used by plants and other organisms to convert light energy into chemical energy that, through cellular respiration, can later be released to fuel the organism's activities. Some of this chemical energy is stored in carbohydrate molecules, such as sugars and starches, which are synthesized from carbon dioxide and water – hence the name photosynthesis, from the Greek phōs, "light", and synthesis, "putting together". Most plants, algae, and cyanobacteria perform photosynthesis; such organisms are called photoautotrophs. Photosynthesis is largely responsible for producing and maintaining the oxygen content of the Earth's atmosphere, and supplies most of the energy necessary for life on Earth.

Picoplankton

Picoplankton

Picoplankton is the fraction of plankton composed by cells between 0.2 and 2 μm that can be either prokaryotic and eukaryotic phototrophs and heterotrophs:photosynthetic heterotrophic

Picoeukaryote

Picoeukaryote

Picoeukaryotes are picoplanktonic eukaryotic organisms 3.0 µm or less in size. They are distributed throughout the world's marine and freshwater ecosystems and constitute a significant contribution to autotrophic communities. Though the SI prefix pico- might imply an organism smaller than atomic size, the term was likely used to avoid confusion with existing size classifications of plankton.

Dimension

Dimension

In physics and mathematics, the dimension of a mathematical space is informally defined as the minimum number of coordinates needed to specify any point within it. Thus, a line has a dimension of one (1D) because only one coordinate is needed to specify a point on it – for example, the point at 5 on a number line. A surface, such as the boundary of a cylinder or sphere, has a dimension of two (2D) because two coordinates are needed to specify a point on it – for example, both a latitude and longitude are required to locate a point on the surface of a sphere. A two-dimensional Euclidean space is a two-dimensional space on the plane. The inside of a cube, a cylinder or a sphere is three-dimensional (3D) because three coordinates are needed to locate a point within these spaces.

Histogram

Histogram

A histogram is an approximate representation of the distribution of numerical data. The term was first introduced by Karl Pearson. To construct a histogram, the first step is to "bin" the range of values—that is, divide the entire range of values into a series of intervals—and then count how many values fall into each interval. The bins are usually specified as consecutive, non-overlapping intervals of a variable. The bins (intervals) must be adjacent and are often of equal size.

Intensity (physics)

Intensity (physics)

In physics, the intensity or flux of radiant energy is the power transferred per unit area, where the area is measured on the plane perpendicular to the direction of propagation of the energy. In the SI system, it has units watts per square metre (W/m2), or kg⋅s−3 in base units. Intensity is used most frequently with waves such as acoustic waves (sound) or electromagnetic waves such as light or radio waves, in which case the average power transfer over one period of the wave is used. Intensity can be applied to other circumstances where energy is transferred. For example, one could calculate the intensity of the kinetic energy carried by drops of water from a garden sprinkler.

Hematology

Hematology

Hematology is the branch of medicine concerned with the study of the cause, prognosis, treatment, and prevention of diseases related to blood. It involves treating diseases that affect the production of blood and its components, such as blood cells, hemoglobin, blood proteins, bone marrow, platelets, blood vessels, spleen, and the mechanism of coagulation. Such diseases might include hemophilia, sickle cell anemia, blood clots (thrombus), other bleeding disorders, and blood cancers such as leukemia, multiple myeloma, and lymphoma. The laboratory analysis of blood is frequently performed by a medical technologist or medical laboratory scientist.

Bioconductor

Bioconductor

Bioconductor is a free, open source and open development software project for the analysis and comprehension of genomic data generated by wet lab experiments in molecular biology.

GenePattern

GenePattern

GenePattern is a freely available computational biology open-source software package originally created and developed at the Broad Institute for the analysis of genomic data. Designed to enable researchers to develop, capture, and reproduce genomic analysis methodologies, GenePattern was first released in 2004. GenePattern is currently developed at the University of California, San Diego.

Dimensionality reduction

Dimensionality reduction

Dimensionality reduction, or dimension reduction, is the transformation of data from a high-dimensional space into a low-dimensional space so that the low-dimensional representation retains some meaningful properties of the original data, ideally close to its intrinsic dimension. Working in high-dimensional spaces can be undesirable for many reasons; raw data are often sparse as a consequence of the curse of dimensionality, and analyzing the data is usually computationally intractable. Dimensionality reduction is common in fields that deal with large numbers of observations and/or large numbers of variables, such as signal processing, speech recognition, neuroinformatics, and bioinformatics.

Cell sorting by flow cytometry

Cell sorting is a method to purify cell populations based on the presence or absence of specific physical characteristics.[12][14][30] In flow cytometers with sorting capabilities, the instrument detects cells using parameters including cell size, morphology, and protein expression, and then droplet technology to sort cells and recover the subsets for post-experimental use.[12][14]

The first prototype sorter was built at the Los Alamos National Laboratory (LANL) in 1965 by physicist Mack J. Fulwyler by joining a Coulter volume sensor with the newly invented ink jet printer.[31] Live cell cell sorter or fluorescence-activated cell sorter (FACS)[a] was generated by Len Herzenberg, who subsequently won the Kyoto Prize in 2006 for his seminal work.[33]

Cell Sorting Using Flow Cytometry and Droplet Technology
Cell Sorting Using Flow Cytometry and Droplet Technology

Flow cytometry cell sorters have a collection system unlike flow cytometry analyzers. The collection process starts when a sample is injected into a stream of sheath fluid that passes through the flow cell and laser intercepts.[34] The stream then carries the cell through a vibrating nozzle which generates droplets with most containing either one cell or no cells. An electrical charging ring is placed just at the point where the stream breaks into droplets and a charge is placed on the ring based immediately prior to fluorescence intensity being measured; the opposite charge is trapped on the droplet as it breaks from the stream and the droplets are therefore charged. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based on 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. After collecting, these cells can be further cultured, manipulated, and studied.

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Cell sorting

Cell sorting

Cell sorting is the process through which a particular cell type is separated from others contained in a sample on the basis of its physical or biological properties, such as size, morphological parameters, viability and both extracellular and intracellular protein expression. The homogeneous cell population obtained after sorting can be used for a variety of applications including research, diagnosis, and therapy.

Los Alamos National Laboratory

Los Alamos National Laboratory

Los Alamos National Laboratory is one of the sixteen research and development laboratories of the United States Department of Energy (DOE), located a short distance northwest of Santa Fe, New Mexico, in the American southwest. Best known for its central role in helping develop the first atomic bomb, LANL is one of the world's largest and most advanced scientific institutions.

Leonard Herzenberg

Leonard Herzenberg

Leonard Arthur "Len" Herzenberg was an immunologist, geneticist and professor at Stanford University. His contributions to the development of cell biology made it possible to sort viable cells by their specific properties.

Kyoto Prize

Kyoto Prize

The Kyoto Prize is Japan's highest private award for lifetime achievement in the arts and sciences. It is given not only to those that are top representatives of their own respective fields, but to "those who have contributed significantly to the scientific, cultural, and spiritual betterment of mankind". The Kyoto Prize was created in collaboration with the Nobel Foundation and is regarded by many as Japan's version of the Nobel Prize, representing one of the most prestigious awards available in fields that are not traditionally honored with a Nobel.

Electric charge

Electric charge

Electric charge is the physical property of matter that causes matter to experience a force when placed in an electromagnetic field. Electric charge can be positive or negative. Like charges repel each other and unlike charges attract each other. An object with an absence of net charge is referred to as neutral. Early knowledge of how charged substances interact is now called classical electrodynamics, and is still accurate for problems that do not require consideration of quantum effects.

Electrostatic deflection

Electrostatic deflection

Electrostatic deflection refers to a way for modifying the path of a beam of charged particles by the use of an electric field applied transverse to the path of the particles. The technique is called electrostatic because the strength and direction of the applied field changes slowly relative to the time it takes for the particles to transit the field, and thus can be considered not to change for any single particle.

Labels

Use of flow cytometry to measure copy number variation of a specific DNA sequence (Flow-FISH)
Use of flow cytometry to measure copy number variation of a specific DNA sequence (Flow-FISH)

Flow cytometry uses the light properties scattered from cells or particles for identification or quantitative measurement of physical properties. Labels, dyes, and stains can be used for multi-parametric analysis (understand more properties about a cell). Immunophenotyping is the analysis of heterogeneous populations of cells using labeled antibodies[35] and other fluorophore containing reagents such as dyes and stains.

Fluorescent labels

A wide range of fluorophores can be used as labels in flow cytometry.[19] Fluorophores, or simply "fluors", are typically attached to an antibody that recognizes a target feature on or in the cell; they may also be attached to a chemical entity with affinity for the cell membrane or another cellular structure. 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.[36] The maximum number of distinguishable fluorescent labels is thought to be 17 or 18, and this level of complexity necessitates laborious optimization to limit artifacts, as well as complex deconvolution algorithms to separate overlapping spectra.[37] Flow cytometry uses fluorescence as a quantitative tool; the utmost sensitivity of flow cytometry is unmatched by other fluorescent detection platforms such as confocal microscopy. 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.[38]

Quantum dots

Quantum dots are sometimes used in place of traditional fluorophores because of their narrower emission peaks.

Isotope labeling

Mass cytometry overcomes the fluorescent labeling limit by utilizing lanthanide isotopes attached to antibodies. This method could theoretically allow the use of 40 to 60 distinguishable labels and has been demonstrated for 30 labels.[37] Mass cytometry is fundamentally different from flow cytometry: cells are introduced into a plasma, ionized, and associated isotopes are quantified via time-of-flight mass spectrometry. 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.[37]

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Flow-FISH

Flow-FISH

Flow-FISH is a cytogenetic technique to quantify the copy number of RNA or specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols.

Immunophenotyping

Immunophenotyping

Immunophenotyping is a technique used to study the protein expressed by cells. This technique is commonly used in basic science research and laboratory diagnostic purpose. This can be done on tissue section, cell suspension, etc. An example is the detection of tumor markers, such as in the diagnosis of leukemia. It involves the labelling of white blood cells with antibodies directed against surface proteins on their membrane. By choosing appropriate antibodies, the differentiation of leukemic cells can be accurately determined. The labelled cells are processed in a flow cytometer, a laser-based instrument capable of analyzing thousands of cells per second. The whole procedure can be performed on cells from the blood, bone marrow or spinal fluid in a matter of a few hours.

Antibody

Antibody

An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen. Each tip of the "Y" of an antibody contains a paratope that is specific for one particular epitope on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly.

Fluorophore

Fluorophore

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.

Cell membrane

Cell membrane

The cell membrane is a biological membrane that separates and protects the interior of all cells from the outside environment. The cell membrane consists of a lipid bilayer, made up of two layers of phospholipids with cholesterols interspersed between them, maintaining appropriate membrane fluidity at various temperatures. The membrane also contains membrane proteins, including integral proteins that span the membrane and serve as membrane transporters, and peripheral proteins that loosely attach to the outer (peripheral) side of the cell membrane, acting as enzymes to facilitate interaction with the cell's environment. Glycolipids embedded in the outer lipid layer serve a similar purpose. The cell membrane controls the movement of substances in and out of cells and organelles, being selectively permeable to ions and organic molecules. In addition, cell membranes are involved in a variety of cellular processes such as cell adhesion, ion conductivity, and cell signalling and serve as the attachment surface for several extracellular structures, including the cell wall and the carbohydrate layer called the glycocalyx, as well as the intracellular network of protein fibers called the cytoskeleton. In the field of synthetic biology, cell membranes can be artificially reassembled.

Excited state

Excited state

In quantum mechanics, an excited state of a system is any quantum state of the system that has a higher energy than the ground state. Excitation refers to an increase in energy level above a chosen starting point, usually the ground state, but sometimes an already excited state. The temperature of a group of particles is indicative of the level of excitation.

Deconvolution

Deconvolution

In mathematics, deconvolution is the operation inverse to convolution. Both operations are used in signal processing and image processing. For example, it may be possible to recover the original signal after a filter (convolution) by using a deconvolution method with a certain degree of accuracy. Due to the measurement error of the recorded signal or image, it can be demonstrated that the worse the SNR, the worse the reversing of a filter will be; hence, inverting a filter is not always a good solution as the error amplifies. Deconvolution offers a solution to this problem.

Confocal microscopy

Confocal microscopy

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.

Quantum dot

Quantum dot

Quantum dots (QDs) - also called semiconductor nanocrystals, are semiconductor particles a few nanometres in size, having optical and electronic properties that differ from those of larger particles as a result of quantum mechanics. They are a central topic in nanotechnology and materials science. When the quantum dots are illuminated by UV light, an electron in the quantum dot can be excited to a state of higher energy. In the case of a semiconducting quantum dot, this process corresponds to the transition of an electron from the valence band to the conductance band. The excited electron can drop back into the valence band releasing its energy as light. This light emission (photoluminescence) is illustrated in the figure on the right. The color of that light depends on the energy difference between the conductance band and the valence band, or the transition between discrete energy states when the band structure is no longer well-defined in QDs.

Mass cytometry

Mass cytometry

Mass cytometry is a mass spectrometry technique based on inductively coupled plasma mass spectrometry and time of flight mass spectrometry used for the determination of the properties of cells (cytometry). In this approach, antibodies are conjugated with isotopically pure elements, and these antibodies are used to label cellular proteins. Cells are nebulized and sent through an argon plasma, which ionizes the metal-conjugated antibodies. The metal signals are then analyzed by a time-of-flight mass spectrometer. The approach overcomes limitations of spectral overlap in flow cytometry by utilizing discrete isotopes as a reporter system instead of traditional fluorophores which have broad emission spectra.

Lanthanide

Lanthanide

The lanthanide or lanthanoid series of chemical elements comprises the 15 metallic chemical elements with atomic numbers 57–71, from lanthanum through lutetium. These elements, along with the chemically similar elements scandium and yttrium, are often collectively known as the rare-earth elements or rare-earth metals.

Plasma (physics)

Plasma (physics)

Plasma is one of four fundamental states of matter, characterized by the presence of a significant portion of charged particles in any combination of ions or electrons. It is the most abundant form of ordinary matter in the universe, being mostly associated with stars, including the Sun. Extending to the rarefied intracluster medium and possibly to intergalactic regions, plasma can be artificially generated by heating a neutral gas or subjecting it to a strong electromagnetic field.

Cytometric bead array

In addition to the ability to label and identify individual cells via fluorescent antibodies, cellular products such as cytokines, proteins, and other factors may be measured as well. 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 488 nm excitation source. 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

Impedance-based single cell analysis systems are commonly known as Coulter counters. 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 "lab-on-a-chip" based approach and by applying high frequency alternating current (AC) in the radio frequency range (from 100 kHz to 30 MHz) instead of a static direct current (DC) or low frequency AC field.[39][40] This patented technology allows a highly accurate cell analysis and provides additional information like membrane capacitance and viability. The relatively small size and robustness allow battery powered on-site use in the field.

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Electrical impedance

Electrical impedance

In electrical engineering, impedance is the opposition to alternating current presented by the combined effect of resistance and reactance in a circuit.

Coulter counter

Coulter counter

A Coulter counter is an apparatus for counting and sizing particles suspended in electrolytes. The Coulter counter is the commercial term for the technique known as resistive pulse sensing or electrical zone sensing. The apparatus is based on the Coulter principle named after its inventor, Wallace H. Coulter.

Lab-on-a-chip

Lab-on-a-chip

A lab-on-a-chip (LOC) is a device that integrates one or several laboratory functions on a single integrated circuit of only millimeters to a few square centimeters to achieve automation and high-throughput screening. LOCs can handle extremely small fluid volumes down to less than pico-liters. Lab-on-a-chip devices are a subset of microelectromechanical systems (MEMS) devices and sometimes called "micro total analysis systems" (µTAS). LOCs may use microfluidics, the physics, manipulation and study of minute amounts of fluids. However, strictly regarded "lab-on-a-chip" indicates generally the scaling of single or multiple lab processes down to chip-format, whereas "µTAS" is dedicated to the integration of the total sequence of lab processes to perform chemical analysis. The term "lab-on-a-chip" was introduced when it turned out that µTAS technologies were applicable for more than only analysis purposes.

Alternating current

Alternating current

Alternating current (AC) is an electric current which periodically reverses direction and changes its magnitude continuously with time in contrast to direct current (DC), which flows only in one direction. Alternating current is the form in which electric power is delivered to businesses and residences, and it is the form of electrical energy that consumers typically use when they plug kitchen appliances, televisions, fans and electric lamps into a wall socket. A common source of DC power is a battery cell in a flashlight. The abbreviations AC and DC are often used to mean simply alternating and direct, respectively, as when they modify current or voltage.

Direct current

Direct current

Direct current (DC) is one-directional flow of electric charge. An electrochemical cell is a prime example of DC power. Direct current may flow through a conductor such as a wire, but can also flow through semiconductors, insulators, or even through a vacuum as in electron or ion beams. The electric current flows in a constant direction, distinguishing it from alternating current (AC). A term formerly used for this type of current was galvanic current.

Capacitance

Capacitance

Capacitance is the capability of a material object or device to store electric charge. It is measured by the change in charge in response to a difference in electric potential, expressed as the ratio of those quantities. Commonly recognized are two closely related notions of capacitance: self capacitance and mutual capacitance. An object that can be electrically charged exhibits self capacitance, for which the electric potential is measured between the object and ground. Mutual capacitance is measured between two components, and is particularly important in the operation of the capacitor, an elementary linear electronic component designed to add capacitance to an electric circuit.

Viability assay

Viability assay

A viability assay is an assay that is created to determine the ability of organs, cells or tissues to maintain or recover a state of survival. Viability can be distinguished from the all-or-nothing states of life and death by the use of a quantifiable index that ranges between the integers of 0 and 1 or, if more easily understood, the range of 0% and 100%. Viability can be observed through the physical properties of cells, tissues, and organs. Some of these include mechanical activity, motility, such as with spermatozoa and granulocytes, the contraction of muscle tissue or cells, mitotic activity in cellular functions, and more. Viability assays provide a more precise basis for measurement of an organism's level of vitality.

Measurable parameters

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Apoptosis

Apoptosis

Apoptosis is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA decay. The average adult human loses between 50 and 70 billion cells each day due to apoptosis. For an average human child between eight and fourteen years old, approximately twenty to thirty billion cells die per day.

Caspase

Caspase

Caspases are a family of protease enzymes playing essential roles in programmed cell death. They are named caspases due to their specific cysteine protease activity – a cysteine in its active site nucleophilically attacks and cleaves a target protein only after an aspartic acid residue. As of 2009, there are 12 confirmed caspases in humans and 10 in mice, carrying out a variety of cellular functions.

Chlorophyll

Chlorophyll

Chlorophyll is any of several related green pigments found in cyanobacteria and in the chloroplasts of algae and plants. Its name is derived from the Greek words χλωρός, khloros and φύλλον, phyllon ("leaf"). Chlorophyll allow plants to absorb energy from light.

Antigen

Antigen

In immunology, an antigen (Ag) is any molecule, molecular structure, foreign particulate matter, or pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. Antigens can be proteins, peptides, polysaccharides, lipids, or nucleic acids.

Cluster of differentiation

Cluster of differentiation

The cluster of differentiation is a protocol used for the identification and investigation of cell surface molecules providing targets for immunophenotyping of cells. In terms of physiology, CD molecules can act in numerous ways, often acting as receptors or ligands important to the cell. A signal cascade is usually initiated, altering the behavior of the cell. Some CD proteins do not play a role in cell signaling, but have other functions, such as cell adhesion. CD for humans is numbered up to 371.

DNA

DNA

Deoxyribonucleic acid is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.

Copy number variation

Copy number variation

Copy number variation (CNV) is a phenomenon in which sections of the genome are repeated and the number of repeats in the genome varies between individuals. Copy number variation is a type of structural variation: specifically, it is a type of duplication or deletion event that affects a considerable number of base pairs. Approximately two-thirds of the entire human genome may be composed of repeats and 4.8–9.5% of the human genome can be classified as copy number variations. In mammals, copy number variations play an important role in generating necessary variation in the population as well as disease phenotype.

Flow-FISH

Flow-FISH

Flow-FISH is a cytogenetic technique to quantify the copy number of RNA or specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols.

Enzyme

Enzyme

Enzymes are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. Almost all metabolic processes in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called enzymology and the field of pseudoenzyme analysis recognizes that during evolution, some enzymes have lost the ability to carry out biological catalysis, which is often reflected in their amino acid sequences and unusual 'pseudocatalytic' properties.

Glutathione

Glutathione

Glutathione is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources such as reactive oxygen species, free radicals, peroxides, lipid peroxides, and heavy metals. It is a tripeptide with a gamma peptide linkage between the carboxyl group of the glutamate side chain and cysteine. The carboxyl group of the cysteine residue is attached by normal peptide linkage to glycine.

Cytokine

Cytokine

Cytokines are a broad and loose category of small proteins important in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines have been shown to be involved in autocrine, paracrine and endocrine signaling as immunomodulating agents.

Membrane fluidity

Membrane fluidity

In biology, membrane fluidity refers to the viscosity of the lipid bilayer of a cell membrane or a synthetic lipid membrane. Lipid packing can influence the fluidity of the membrane. Viscosity of the membrane can affect the rotation and diffusion of proteins and other bio-molecules within the membrane, there-by affecting the functions of these things.

Applications

The technology has applications in a number of fields, including molecular biology, pathology, immunology, virology,[41] plant biology and marine biology.[42] It has broad application in medicine especially in transplantation, hematology, tumor immunology and chemotherapy, prenatal diagnosis, genetics and sperm sorting for sex preselection. Flow cytometry is widely applied to detect sperm cells abnormality associated with DNA fragmentation[43] in male fertility assays.[44] Also, it is extensively used in research for the detection of DNA damage,[45][46] caspase cleavage and apoptosis.[47] Photoacoustic flow cytometry is used in the study of multi-drug-resistant bacteria (most commonly MRSA) to detect, differentiate, and quantify bacteria in the blood marked with dyed bacteriophages.[48] In neuroscience, co-expression of cell surface and intracellular antigens can also be analyzed.[49] In microbiology, it can be used to screen and sort transposon mutant libraries constructed with a GFP-encoding transposon (TnMHA),[50] or to assess viability.[51] 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. The main advantages of flow cytometry over histology and IHC is the possibility to precisely measure the quantities of antigens and the possibility to stain each cell with multiple antibodies-fluorophores, in current laboratories around 10 antibodies can be bound to each cell. This is much less than mass cytometer where up to 40 can be currently measured, but at a higher price and a slower pace.

Aquatic research

In aquatic systems, flow cytometry is used for the analysis of autofluorescing cells or cells that are fluorescently-labeled with added stains. This research started in 1981 when Clarice Yentsch used flow cytometry to measure the fluorescence in a red tide producing dinoflagellate.[52] The next year researchers published flow cytometric measurements of multiple algal species which could be distinguished based on their fluorescence characteristics.[53] By 1983, marine researchers were assembling their own flow cytometers[54] or using commercially available flow cytometers on seawater samples collected off Bermuda to demonstrate that phytoplankton cells could be distinguished from non-living material and that cyanobacteria could be sorted from a mixed community and subsequently cultured in the lab.[55] Flow cytometry also allowed marine researchers to distinguish between dimly-fluorescing Prochlorococcus and heterotrophic microorganisms, a distinction that is difficult with microscopy-based assessments.[56] Advances in technology now allow aquatic scientists to use flow cytometers continuously during research cruises[57] and flow cytometers are used to provide images of individual phytoplankton cells.[58][59] Marine scientists use the sorting ability of flow cytometers to make discrete measurements of cellular activity and diversity,[60][61] to conduct investigations into the mutualistic relationships between microorganisms that live in close proximity,[62] and to measure biogeochemical rates of multiple processes in the ocean.[63]

Cell proliferation assay

Cell proliferation is the major function in the immune system. Often it is required to analyse the proliferative nature of the cells in order to make some conclusions. One such assay to determine the cell proliferation is the tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE). It helps to monitor proliferative cells. This assay gives quantitative as well as qualitative data during time-series experiments.[64] This dye binds covalently with the long-lived molecules present inside the cell. When the cells divide, the molecules divide too and, the daughter cells possess half the dye than the parent population. This decrease in the intensity can be visualized by flow cytometry.[65] In literature, this powerful technique of flow cytometry and CFSE has been used to find the efficiency of T-cells in killing the target cells in cancer such as leukemia. In order to visualize the target cell death, both rapid and slow, scientists have used CFSE labelling with antibody staining of certain kinds of cells and fluorescently labelled microbeads. This also gave information regarding the proliferation of the target cells upon the treatment of certain cytokines.[66]

Measuring genome size

Flow cytometry has been used to measure genome sizes, or more precisely: the amount of DNA in a cell or nucleus. Although genomes can be analyzed with more precision by genome sequencing, this is often difficult due to a high fraction of micro-chromosomes or repetitive sequences which may be missed by sequencing (or which get filtered out during the analysis step when they cannot be assigned to chromosomes). However, flow cytometry is not perfect either. The resulting genome sizes may differ based on the dye used. An analysis of fish genomes resulted in significantly different genome sizes when propidium iodide (PI) and DAPI were used, respectively. For instance, the genome of Anguilla japonica was found to contain 1.09 pg of DNA with PI vs. 1.25 pg with DAPI. Similarly, the genome of Myxocyprinus asiaticus was found to contain 2.75 pg of DNA (PI) vs. 3.08 pg (DAPI). That is, the differences were on the order of 12-14%.[67]

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Molecular biology

Molecular biology

Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physical structure of biological macromolecules is known as molecular biology.

Immunology

Immunology

Immunology is a branch of medicine and biology that covers the medical study of immune systems in all organisms. In such we can see there is a difference of human immunology and comparative immunology in veterinary medicine and animal biosciences.

Marine biology

Marine biology

Marine biology is the scientific study of the biology of marine life, organisms in the sea. Given that in biology many phyla, families and genera have some species that live in the sea and others that live on land, marine biology classifies species based on the environment rather than on taxonomy.

Medicine

Medicine

Medicine is the science and practice of caring for a patient, managing the diagnosis, prognosis, prevention, treatment, palliation of their injury or disease, and promoting their health. Medicine encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness. Contemporary medicine applies biomedical sciences, biomedical research, genetics, and medical technology to diagnose, treat, and prevent injury and disease, typically through pharmaceuticals or surgery, but also through therapies as diverse as psychotherapy, external splints and traction, medical devices, biologics, and ionizing radiation, amongst others.

DNA fragmentation

DNA fragmentation

DNA fragmentation is the separation or breaking of DNA strands into pieces. It can be done intentionally by laboratory personnel or by cells, or can occur spontaneously. Spontaneous or accidental DNA fragmentation is fragmentation that gradually accumulates in a cell. It can be measured by e.g. the Comet assay or by the TUNEL assay.

Apoptosis

Apoptosis

Apoptosis is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA decay. The average adult human loses between 50 and 70 billion cells each day due to apoptosis. For an average human child between eight and fourteen years old, approximately twenty to thirty billion cells die per day.

Neuroscience

Neuroscience

Neuroscience is the scientific study of the nervous system, its functions and disorders. It is a multidisciplinary science that combines physiology, anatomy, molecular biology, developmental biology, cytology, psychology, physics, computer science, chemistry, medicine, statistics, and mathematical modeling to understand the fundamental and emergent properties of neurons, glia and neural circuits. The understanding of the biological basis of learning, memory, behavior, perception, and consciousness has been described by Eric Kandel as the "epic challenge" of the biological sciences.

Bacterial display

Bacterial display

Bacterial display is a protein engineering technique used for in vitro protein evolution. Libraries of polypeptides displayed on the surface of bacteria can be screened using flow cytometry or iterative selection procedures (biopanning). This protein engineering technique allows us to link the function of a protein with the gene that encodes it. Bacterial display can be used to find target proteins with desired properties and can be used to make affinity ligands which are cell-specific. This system can be used in many applications including the creation of novel vaccines, the identification of enzyme substrates and finding the affinity of a ligand for its target protein.

Clarice Yentsch

Clarice Yentsch

Clarice Morel Yentsch is a scientist, author, education and museum professional, and community benefactor. As a scientist, she pioneered the use of flow cytometry to investigate marine phytoplankton and co-founded Bigelow Laboratory for Ocean Sciences.

Genome size

Genome size

Genome size is the total amount of DNA contained within one copy of a single complete genome. It is typically measured in terms of mass in picograms or less frequently in daltons, or as the total number of nucleotide base pairs, usually in megabases. One picogram is equal to 978 megabases. In diploid organisms, genome size is often used interchangeably with the term C-value.

DNA

DNA

Deoxyribonucleic acid is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.

Cell (biology)

Cell (biology)

The cell is the basic structural and functional unit of life forms. Every cell consists of a cytoplasm enclosed within a membrane, and contains many biomolecules such as proteins, DNA and RNA, as well as many small molecules of nutrients and metabolites. The term comes from the Latin word cellula meaning 'small room'.

Source: "Flow cytometry", Wikipedia, Wikimedia Foundation, (2023, March 16th), https://en.wikipedia.org/wiki/Flow_cytometry.

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See also
Notes
  1. ^ The acronym FACS is trademarked and owned by BD Biosciences-Immunocytometry Systems, a division of Becton-Dickinson, which licensed Stanford's patents.[30][32]
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Further reading
  • Carey JL, McCoy Jr JP, Keren DF (2007). Flow cytometry in clinical diagnosis (4th ed.). Chicago, IL: American Society for Clinical Pathology (ASCP) Press. ISBN 978-0-89189-548-0.
  • Darzynkiewicz Z, Roederer M, Tanke HJ, eds. (2004). Methods in Cell Biology, Cytometry. Vol. 75 (4th ed.). Elsevier /Academic Press. ISBN 0-12-480283-4.
  • Darzynkiewicz Z, Robinson JP, Roederer M (2009). Essential cytometry methods (1st ed.). Amsterdam: Elsevier/Academic Press. ISBN 978-0-12-375045-7.
  • Darzynkiewicz Z, et al., eds. (2011). Recent Advances in Cytometry. Part A. Methods in Cell Biology. Vol. 102. Elsevier/Academic Press. ISBN 978-0-12-374912-3.
  • Darzynkiewicz Z, et al., eds. (2011). Recent Advances in Cytometry. Part B. Methods in Cell Biology. Vol. 103. Elsevier/Academic Press. ISBN 978-0-12-385493-3.
  • Lloyd D (1993). Flow cytometry in microbiology. London: Springer-Verlag. ISBN 978-3-540-19796-6.
  • Ormerod MG (1999). Flow cytometry (2nd ed.). Oxford, UK: Bios Scientific Publishers. ISBN 978-1-85996-107-0.
  • Ormerod MG, ed. (2000). Flow cytometry : a practical approach (3rd ed.). Oxford [England]: Oxford University Press. ISBN 978-0-19-963824-6.
  • Ormerod MG (2008). Flow cytometry : a basic introduction. Redhill: M.G. Ormerod. ISBN 978-0-9559812-0-3.
  • Robinson JP, et al. (1993). Handbook of flow cytometry methods. New York: Wiley-Liss. ISBN 978-0-471-59634-9.
  • Shapiro HM (2003). Practical flow cytometry (4th ed.). New York: Wiley-Liss. ISBN 978-0-471-41125-3.
  • Sklar LA (2005). Flow cytometry for biotechnology. New York: Oxford University Press. ISBN 978-0-19-515234-0.
External links

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