Flow Cytometry-Definition, Principle, Parts, Steps, Types, Uses

Flow cytometry is a technique that allows the analysis of multiple characteristics of cells or particles in a fluid suspension. It can measure physical properties such as size, shape, and granularity, as well as chemical properties such as the presence and amount of specific molecules on the cell surface or inside the cell. Flow cytometry can also sort cells based on these properties, allowing the isolation of specific subpopulations for further study or application.

Light scattering is the phenomenon that occurs when a particle deflects incident laser light. The extent and direction of light scattering depend on the physical properties of the particle, such as its size and internal complexity.

Forward-scattered light (FSC)

FSC is the light that is scattered in the same direction as the incident laser beam, but at a small angle. FSC is measured by a detector placed in line with the laser path, on the opposite side of the stream.

FSC is proportional to the cell-surface area or size of the cell. The larger the cell, the more light it scatters in the forward direction, and the higher the FSC signal.

FSC can be used to distinguish cells based on their size, such as red blood cells, platelets, lymphocytes, monocytes, and granulocytes.

Side-scattered light (SSC)

SSC is the light that is scattered at a larger angle than FSC, usually perpendicular to the laser path. SSC is measured by a detector placed at 90 degrees to the laser beam.

SSC indicates the cell granularity or internal complexity of the cells. The more organelles or structures inside the cell, such as nuclei, mitochondria, lysosomes, or granules, the more light it scatters in the side direction, and the higher the SSC signal.

SSC can be used to differentiate cells based on their internal structure, such as activated lymphocytes, apoptotic cells, bacteria, or extracellular vesicles.

The combination of FSC and SSC can provide information about the identity and morphology of cells in a heterogeneous population. For example, lymphocytes have low FSC and low SSC signals, indicating that they are small and have few organelles. Granulocytes have high FSC and high SSC signals, indicating that they are large and have many granules. Monocytes have intermediate FSC and high SSC signals, indicating that they are medium-sized and have complex cytoplasm.

The plot of FSC versus SSC is called a scattergram or a dot plot. It can be used to visualize and gate different cell populations based on their light scattering properties.

Light scattering can also be affected by factors such as cell shape, orientation, refractive index, wavelength of the laser, and angle of detection. Therefore, it is important to use appropriate controls and calibration standards to ensure accurate and reproducible measurements.

Fluorescence is the phenomenon of emitting light of a longer wavelength after absorbing light of a shorter wavelength. In flow cytometry, fluorescence is used to detect the expression of cellular molecules such as proteins or nucleic acids by using fluorescent markers that bind specifically to the target molecules.

Fluorescent markers are molecules that can absorb light energy and emit it at a different wavelength. They can be divided into two main categories: fluorescent proteins and fluorescent dyes.

Fluorescent proteins are genetically encoded proteins that can produce fluorescence when exposed to light. They are derived from natural sources such as jellyfish, corals, or bacteria, or engineered by mutagenesis or fusion. Some examples of fluorescent proteins are GFP (green), RFP (red), YFP (yellow), and CFP (cyan). Fluorescent proteins can be used to generate reporter constructs that express the protein under the control of a specific promoter or as fusion proteins with the target molecule.

Fluorescent dyes are synthetic or natural compounds that can bind to specific molecules or structures and emit fluorescence when excited by light. They can be classified into different types based on their binding specificity, such as DNA-binding dyes, antibody-conjugated dyes, viability dyes, or ion indicator dyes. Some examples of fluorescent dyes are DAPI (blue), FITC (green), PE (orange), and PI (red). Fluorescent dyes can be used to stain cells or particles with the target molecule or property.

The use of fluorescent markers in flow cytometry allows the measurement of multiple parameters simultaneously by using different combinations of markers that emit fluorescence at different wavelengths. The fluorescence signals are collected by detectors that are equipped with filters that allow only a narrow range of wavelengths to pass through. The intensity and frequency of the fluorescence signals are proportional to the amount and type of the target molecules present in the cells or particles.

However, the use of fluorescent markers also poses some challenges, such as spectral overlap and spillover spreading. Spectral overlap occurs when two or more fluorescent markers have similar emission spectra and interfere with each other`s detection. Spillover spreading occurs when a fluorescent marker emits light that is detected by more than one detector due to insufficient filtering. These issues can affect the accuracy and specificity of the flow cytometry analysis and require proper compensation and correction methods.

A flow cytometer is an instrument that uses a laser beam to analyze the physical and chemical properties of cells or particles in a fluid stream. A flow cytometer consists of three main systems: fluidics, optics, and electronics.

Fluidics

The fluidics system transports the sample to the laser beam in a narrow and focused stream. The sample is injected into a stream of sheath fluid (usually a buffered saline solution) within the flow chamber. The design of the flow chamber allows the sample core to be centered in the sheath fluid where the laser beam interacts with the particles. This process is called hydrodynamic focusing.

Optics

The optics system consists of the excitation optics and the collection optics. The excitation optics includes the laser and lenses that shape and focus the laser beam to the sample stream. The collection optics includes a collection lens that gathers the light emitted by the particles after they interact with the laser beam, and a system of optical mirrors and filters that direct the specified wavelengths of light to designated detectors.

The light emitted by the particles can be either scattered or fluorescent. Scattered light is proportional to the size and internal complexity of the particles, and it is measured by two detectors: forward-scattered light (FSC) detector and side-scattered light (SSC) detector. Fluorescent light is emitted by fluorescent markers that bind to specific molecules on or within the particles, and it is measured by photomultiplier tubes (PMTs) that are sensitive to different colors of fluorescence.

Electronics

The electronics system converts the light signals from the detectors into electronic signals that can be processed by a computer. The electronic signals are proportional to the intensity of the light signals. The FSC and SSC signals are converted into voltage pulses by photodiodes, while the fluorescent signals are converted into voltage pulses by PMTs. The voltage pulses are then converted into digital numbers by analog-to-digital converters (ADCs). The digital numbers are stored and displayed as histograms or scatter plots that represent the characteristics of each particle.

The process of flow cytometry consists of the following steps:

Sample Preparation

Before running in the flow cytometer, the cells under analysis must be in a single-cell suspension. This can be achieved by using enzymatic digestion or mechanical dissociation of solid tissues, or by filtering clumped cultured cells. The resulting cells are then incubated in test tubes or microtiter plates with unlabeled or fluorescently conjugated antibodies and analyzed through the flow cytometer machine.

Antibody Staining

Once the sample is prepared, the cells are coated with fluorochrome-conjugated antibodies specific for the surface or intracellular markers present on different cells. This can be done either by direct, indirect, or intracellular staining.

• In direct staining, cells are incubated with an antibody directly conjugated to a fluorophore.
• In indirect staining, the fluorophore-conjugated secondary antibody detects the primary antibody.
• The intracellular staining procedure allows direct measurement of antigens present inside the cell cytoplasm or nucleus. For this, the cells are first made permeable and then are stained with antibodies in the permeabilization buffer.

Running Samples

At first, control samples are run to adjust the voltages in the detectors and to set up compensation for overlapping fluorescence signals. The flow rates in the cytometer are set and the sample is run. The data is collected and stored in a computer for further analysis.

Some additional steps that you may want to add or change are:

• You may want to mention that different types of controls are used for different purposes, such as unstained cells, single-stained cells, isotype controls, fluorescence-minus-one (FMO) controls, etc.
• You may want to explain what compensation is and why it is necessary when using multiple fluorochromes that have overlapping emission spectra.
• You may want to describe how the data is displayed and analyzed using histograms, dot plots, contour plots, etc.

There are different types of flow cytometers based on the purpose and precision of the process:

• Traditional flow cytometers: These are the common cytometers that use sheath fluid for focusing the sample stream. The most common lasers used in traditional flow cytometers are 488 nm (blue), 405 nm (violet), 532 nm (green), 552 nm (green), 561 nm (green-yellow), 640 nm (red) and 355 nm (ultraviolet). These cytometers can measure light scattering and fluorescence from multiple detectors and can analyze thousands of cells per second. However, they cannot physically separate or image the cells.
• Acoustic Focusing Cytometers: These are cytometers that use ultrasonic waves to focus the cells for analysis. This prevents sample clogging and also allows higher sample inputs. These cytometers can also measure light scattering and fluorescence, but with higher sensitivity and resolution than traditional cytometers. They can also analyze cells at higher flow rates and with less sample volume.
• Cell sorters: These are a category of traditional flow cytometers that allow the user to collect samples after processing. The cells that are positive for the desired parameter can be separated from those that are negative for the parameter by using electrostatic deflection or microfluidic switching. Cell sorters can isolate cells based on their size, shape, granularity, fluorescence or other properties. They can also sort cells into different collection tubes or plates for further analysis or culture.
• Imaging flow cytometers: These are cytometers that combine traditional flow cytometry with fluorescence microscopy. Imaging cytometers allow for rapid analysis of a sample for morphology and multi-parameter fluorescence at both a single cell and population level. They can capture images of cells as they pass through the laser beam and use image analysis software to quantify various features such as shape, size, texture, intensity, colocalization and spatial distribution of fluorescent markers. Imaging cytometers can also perform cell sorting based on image features.

Flow cytometry is a versatile technique that can be used for various purposes in different fields of biology and medicine. Some of the common applications and uses of flow cytometry are:

• Immunophenotyping: This is the process of identifying and quantifying the presence of specific cell types in a sample based on their expression of surface or intracellular markers. Immunophenotyping is useful for diagnosing hematological malignancies, such as leukemia and lymphoma, as well as for monitoring immune responses, such as T cell subsets, B cell subsets, and cytokine production.
• Cell sorting: This is the process of physically separating cells of interest from a heterogeneous mixture based on their fluorescence or light scattering properties. Cell sorting can be used for isolating pure populations of cells for further analysis, such as gene expression, functional assays, or transplantation.
• Cell cycle analysis: This is the process of measuring the DNA content of cells and determining their replication status. Cell cycle analysis can be used for studying cell proliferation, cell death, and chromosomal abnormalities.
• Apoptosis: This is the process of programmed cell death that occurs in response to various stimuli or stressors. Apoptosis can be detected by flow cytometry based on changes in cell size, membrane permeability, mitochondrial potential, caspase activation, or annexin V binding.
• Cell proliferation assays: These are assays that measure the metabolic activity or division rate of cells in response to stimuli, such as growth factors, cytokines, or drugs. Cell proliferation assays can be performed by flow cytometry using fluorescent dyes that are retained or diluted by dividing cells, such as carboxyfluorescein succinimidyl ester (CFSE) or bromodeoxyuridine (BrdU).
• Intracellular calcium flux: This is the process of rapid increase in intracellular calcium concentration that occurs when cells are activated by external signals, such as ligands, hormones, or neurotransmitters. Intracellular calcium flux can be measured by flow cytometry using fluorescent indicators that change their fluorescence intensity or wavelength in response to calcium binding.
• Other applications: Flow cytometry can also be used for various other purposes, such as measuring gene expression using fluorescent reporter proteins or probes, detecting microorganisms using fluorescent antibodies or nucleic acid stains, analyzing cell morphology using imaging flow cytometry, and quantifying biomolecules using mass cytometry.

Flow cytometry is a powerful tool that can provide valuable information about the characteristics and functions of cells at the single-cell level. However, it also has some limitations, such as high cost, complex data analysis, sample preparation requirements, and potential artifacts due to autofluorescence or nonspecific binding. Therefore, it is important to optimize the experimental design and protocol to ensure reliable and accurate results.

Flow cytometry is a powerful technique that can provide rapid and detailed analysis of cells and particles in a fluid suspension. However, it also has some limitations that should be considered before using it for a specific purpose. Some of the common limitations are:

• Cost and expertise: Flow cytometry instruments are expensive and require regular maintenance and calibration. They also need highly trained technicians to operate them and interpret the data. The sample preparation and staining procedures can also be time-consuming and require specialized reagents and protocols.
• Sample quality: Flow cytometry requires the cells or particles to be in a single-cell suspension and free of clumps, debris, or aggregates. This can be challenging for some types of samples, such as solid tissues, blood clots, or viscous fluids. The sample quality can affect the accuracy and reliability of the measurements and may cause clogging or damage to the instrument.
• Fluorescence limitations: Flow cytometry relies on the use of fluorescent markers to detect specific molecules or characteristics of the cells or particles. However, fluorescence has some inherent limitations, such as:

• Spectral overlap: Fluorescent dyes emit light over a range of wavelengths, which can overlap with each other or with the background autofluorescence of the cells or particles. This can cause false-positive or false-negative signals and reduce the resolution of the analysis. To minimize this problem, careful selection of compatible fluorochromes, appropriate filters, and compensation techniques are needed.
• Photobleaching: Fluorescent dyes can lose their ability to emit light after repeated exposure to the laser beam. This can reduce the signal intensity and affect the quantification of the fluorescence. To prevent this problem, optimal laser power, exposure time, and dye concentration should be used.
• Quenching: Fluorescent dyes can also interact with each other or with other molecules in the sample and reduce their fluorescence intensity. This can affect the sensitivity and specificity of the detection. To avoid this problem, optimal staining conditions, buffer composition, and storage conditions should be used.

Spatial information: Flow cytometry does not provide information on the spatial distribution or location of the molecules or characteristics within the cells or particles. It only measures the average properties of each cell or particle as it passes through the laser beam. Therefore, flow cytometry cannot distinguish between different subcellular compartments or structures, such as nucleus, cytoplasm, membrane, organelles, etc. To obtain spatial information, other techniques such as microscopy or imaging flow cytometry are needed.

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