Cytometry in flow

The cytometry in flow is a technique making it possible to make ravel particles, molecules or cells at high speed in the beam of a Laser. The re-emitted light (by diffusion or fluorescence) makes it possible to classify the population according to several criteria and to sort them. The first cytometers in flow were invented in the years 1950.

The cytometry in flow (CMF) is defined like the precise study of isolated particles (cells, Bactérie S…) pulled by a liquid flow. It is a technique of individual, quantitative and qualitative characterization of suspended particles in a liquide.
It consists in analyzing the signals Optique S or Physique S emitted by a particle cutting the beam of light of a laser or an arc lamp. The measured signals are primarily relatifs :

with the intrinsic optical properties of the particles which correspond to the phenomena of light diffusion related on dimensions of the particle, their internal structure or the car fluorescence of certain cells like the plants, the phytoplankton, etc
with the induced optical properties of fluorescence obtained by specific markings of structures or functions cellular.

These signals separated by optical filters are collected by photomultiplier S, are amplified, digitized, treated and stored by a computer.

This process of individual analysis (cell by cell) is multiparametric and can be carried out at the speed of several thousands of events a second. The computer calculates the statistical data associated with the distributions with the measured parameters and represents them in the form of histograms (a parameter) or cytogrammes (two parameters) on one or more populations whose cellular properties are thus evaluated.

The tri function of the cytometers in the most advanced flows makes it possible to physically sort one or two cellular populations defined by their optical properties.

Signals collected

The optical signals collected have an intensity correlated with the particulate properties.

The diffused light

The diffused light informs about the morphology and the structure of the particle. If the diffusion of the light is measured in the axis of the incidental ray, the intensity of the signal can be correlated with the size and cellular viability.

Under an angle of 90°, measurement corresponds to the intracellular structure of the cell (refringency of the Cytoplasme, morphology, nucléo-cytoplasmic ratio). The simultaneous use of these two parameters makes it possible to distinguish, in a peripheral blood for example, the plate S, the Lymphocyte S, the Monocyte S and the Polynucléaire S.

The absorptive light

This measurement evolves/moves proportionally with the diameter of the cell (presumedly spherical) and with the index of absorption of the components post will bio-intra cellular.

Emitted fluorescence

This Fluorescence can be spontaneous, but generally, it is brought to the cell by a fluorochrome. The fluorochrome absorbs the energy of the Laser and re-emits the energy absorptive in the form of photons a wavelength more élevée :

fluorochromes with clean affinity for a component cellulaire : for example for measurements of DNA, ARN, Protein S, pH, Calcium contained in the cellule ;
fluorochromes coupled to a specific ligand. This specificity can be brought by a coupling of the fluorochrome to a Anticorps or a specific ligand supercopter of a cellular component.

Principal applications

The Hématologie was one of the first medical disciplines to profit from the clinical applications of the CMF. Some of these applications are now used regularly for the diagnosis or the therapeutic follow-up of various affections. These applications as well relate to the functional study of healthy cells as the description of the pathological character of the analyzed cells.

In Cancerology, the detection of the pathological cell is the most developed application. This detection rests primarily to the measure of abnormal contents of DNA in the core of the tumoral cell.

The Immunologie uses the CMF for the detection or the identification of the sub-types of the cells implied in immunity.

The cellular Cycle represents the entirety of the period of division, i.e. the whole of the biochemical and morphological events which are responsible for the cellular proliferation. The CMF offers a methodology fast and simple to implement for the analysis of the cellular cycle. It makes it possible to follow the distribution of the cells in the various phases of the cycle according to various stimuli or the addition of certain drugs. It also makes it possible to see the presence of cells with abnormal contents of DNA.

Many studies in Pharmacologie call also upon techniques of CMF : development or study of antimitotic drugs, Immunothérapie.

In Oceanography, the cytometry in flow became a method of routine to count different the populations from the photosynthetic Picoplancton on the basis from fluorescence from the pigments such as the Chlorophylle. After marking of the samples with markers of the DNA such as SYBR-Green, one can also enumerate Bactéries and Virus.

Other research calls upon CMF : analysis of the Chromosome S, the vegetable Physiology (ploïdie, contained in DNA, for the selection of the most resistant plants), etc

External bond

International The Society for Analytical Cytometry
Purdue University - Cytometry Laboratory, a site with much of detailed informations

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