Flow cytometry is a molecular technique that is used to analyze the physical and chemical characteristics of particles in a fluid as it passes through laser.
- It is laser-based technology used to count, sort, and profile cells in a heterogeneous fluid mixture.
- In this technique the cell components are fluorescently labeled and then excited by the laser to emit light at varying wavelengths.
- It is highly sensitive technique has ability to analyze up to thousands of particles per second as they pass through the liquid stream. The properties measured with the flow cytometry includes the particle’s relative granularity, size and fluorescence intensity as well as its internal complexity.
- A flow cytometer allow multiparametric analysis of the physical and chemical characteristics simultaneously. This makes it a rapid and quantitative method for analysis and purification of cells in suspension.
- Using this technique, the researcher can determine the phenotype, function and even sort live cells.
Fluorescence-activated cell sorting (FACS)
It is a specialized type of flow cytometry. It is 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 flourecent 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.
- Highly specific antibodies labeled with fluorescent conjugates utilizes in the FACS analysis allows us to simultaneously collect data on, and sort a biological sample by a nearly limitless number of different parameters.
- FACS has been used extensively in biomedical research, clinical diagnostics and therapeutics.
The most common usage of FACS is found in:
– Whole human blood analysis for diagnosing diseases
– Sorting different blood cell fractions for ex-vivo manipulations or transplantations
– Analysis of Immuno-phenotypic murine blood to identify transgenic/knockout animals
– Sorting and analysis of a slew of cell lines for various biological assays
– Characterization and isolation of rare cells types like adult stem cells and cancer initiating cells.
The Flow Cytometer
The flow cytometer instrument consists of three core systems: fluidics, optics, and electronics.
Fluidics: The fluidics system used to transport cells or particles in the stream of fluid though narrow channel and into the laser beam in a single align. This hydrodynamic focusing allows the analysis of one cell at a time by laser interrogation.
The size of particle which has to be analyzed in the flow cytometer is 0.2 to 150 μms in but If the cells are from solid tissue, they require disaggregation before they can be analyzed. To tag components of the particle fluorescent labels are required.
Optics: The optics system consists of various filters, light detectors, and the light source, which is usually a laser line producing a single wavelength of light at a specific frequency. It is made up of lasers which illuminate the particles present in the stream as they pass through and scatter light from the laser. In this the particles are passed through at least one laser beam. Lasers with different power levels are available at different wavelengths ranging from ultraviolet to far red. It have a variable range of interrogation by the laser beam excites any compatible fluorescent probes that are conjugated to antibodies, causing the probes to emit light or fluoresce at specified wavelengths.
Forward scatter light signals (FSC) are measured by detector in front of the light beam and detectors to the side measures side scatter light signals (SSC).
Fluorescence signal intensity emitted from positively stained cells and particles are measured by Fluorescence detectors. Within the flow cytometer, all of these different light signals are split into defined wavelengths and channeled by a set of filters and mirrors so that each sensor will detect fluorescence only at a specified wavelength. These sensors are called photomultiplying tubes (PMTs). These light signals are converted by the electronics system to data that can be visualized and interpreted by software.
Electronics: The electronics system is used to change the light signals detected into electronic pulses which can be processed by computer. The data can then be studied to ascertain information about a large number of cells over a short period. Information on the heterogeneity and different subsets within cell populations can be identified and measured. Some instruments have a sorting feature in the electronics system that can be used to charge and deflect particles so that certain cell populations can be sorted for further analysis.
The data is collected in the form of single parameter histograms or as plots of correlated parameters, which are referred to as cytograms. Cytograms may display data in the from of a dot plot, a contour plot or a density plot.
General Experimental Procedure:
The main requirement for all types of analysis is that the cells which are going to be analyzed must be in a single-cell suspension. Single cell suspension helps to avoid clogging up the system with clumps.
Peripheral blood mononuclear cells (PBMCs) isolated from whole blood through Ficoll gradient centrifugation, or RBC lysed whole blood, or non-adherent cultured cells are readily applicable for flow cytometric analysis.
Enzymatic digestion or mechanical dissociation of the tissue is necessary for the analysis of Adherent cultured cells or cells present in solid organs. Mechanical filtration should be performed to avoid unwanted instrument clogs and obtain higher quality flow data.
A wide range of fluorophores can be used as labels in flow cytometry. 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 thecell 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 or laser used to excite the fluorochromes and on the detectors available. 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. 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 microscopy 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.
Quantum dots also has ability of narrower emission peaks which can be used in place of traditional fluorophores .
In case of isotope labeling utilize 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. Mass cytometry is different from flow cytometry: cells are introduced into 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.
- The technique of flow cytometery is used in various fields such as molecular biology, pathology, immunology, plant biology and marine biology. It also has major application in medicine especially in transplantation, hematology, tumor immunology and chemotherapy, prenatal diagnosis, genetics and sperm sorting for sex preselection.
- It is effectively used for the detection of DNA damage and apoptosis.
- In neuroscience, co-expression of cell surface and intracellular antigens can also be analyzed.
- In marine biology, the autofluorescent properties of photosynthetic plankton can be exploited by flow cytometry in order to characterise abundance and community structure.
- 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.