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Definition of Flow Cytometry
- Flow cytometry is a laser-based technique that detects and measures the physical and chemical properties of cells or particles in a heterogeneous fluid mixture.
- Flow cytometry has grown in popularity over the years since it allows for the quick examination of many cell properties (both qualitative and quantitative).
- This technique may detect particle size, granularity or internal complexity, and fluorescence intensity, among other things.
- These properties are measured through an optical-to-electronic coupling device, which identifies the cells based on laser scattered by the cells.
- A flow cytometer, despite its name, does not always deal with cells; it frequently deals with cells, but it may also deal with chromosomes, molecules, and a variety of other particles that can be suspended in a fluid.
Principle of Flow Cytometry
Flow cytometry's core idea is based on the detection of light scattered by particles and the fluorescence detected when these particles are passed in a stream via a laser beam.
- The flow cytometer is made up of three major systems: the fluidic, optical, and electronic systems.
- The fluidic system is in charge of cell transport. It transports cells one at a time through pressured channels carrying sheath fluid to the interrogation point, where the laser contacts the sample. The fluidic system's sample flow rate may be changed to optimise analysis. A slow flow rate, for example, reduces the size of the sample stream, boosting the accuracy and uniformity of sample detection.
- The flow cytometer's optical system is in charge of lighting and light gathering. Excitation lasers, lenses, and filters make up this system. The lasers ensure that cells in the interrogation point are stimulated with a consistent wavelength of light. For example, argon lasers produce light at 488 nm and may be used to excite fluorophores having absorption maxima at 488 nm, such as iFluorTM 488 (Cat# 1023) and FITC (Cat# 135).
- Fluorescence and scattered laser light are emitted by the cells as they travel through the laser. The collecting optics, which include lenses and filters, separate and guide certain wavelengths of fluorescence and scattered laser light to the proper detectors. These detectors collect emitted fluorescence and scattered laser photons, transform them into photocurrents, and send them to the electronics system to be digitised and processed for further investigation.
- Flow cytometers have progressed tremendously since their inception in the 1970s till now. Early prototypes were single-laser cytometers that could only measure size. While today's cytometers include several laser and filter combinations to allow for multicolor analysis, some cytometers can detect up to 14 parameters at the same time. As a result, while selecting fluorophores for multicolor analysis, apparatus configuration " particularly the lasers and filters " must be carefully considered.
Scattering of Light
- When a particle deflects incoming laser light, light scattering occurs. The amount to which this occurs is determined by a particle's physical qualities, specifically its size and internal complexity.
- The forward-scattered light (FSC) is proportional to the cell's surface area or size. It detects rays that are barely off the axis of the incident laser beam scattered in the forward direction by a photodiode and measures primarily diffracted light.
- The cell granularity or internal complexity is shown by side-scattered light (SSC). SSC is a measurement of mostly refracted and reflected light that happens at any contact within the cell where the refractive index changes.
- FSC and SSC measurements are used to differentiate cell types in a heterogeneous cell population.
Fluorescence
- Fluorescent markers are employed in a system to identify the expression of biological molecules such as proteins or nucleic acids.
- The fluorescent compound absorbs light energy throughout a spectrum of wavelengths that is unique to that chemical.
- This light absorption raises one electron in the fluorescent molecule to a higher energy state.
- The excited electron rapidly decays to its ground state, releasing surplus energy in the form of fluorescence, which detectors gather.
- Different fluorochromes can be used to differentiate discrete subpopulations in a mixed population of cells.
- When paired with FSC and SSC data, the fluorescence pattern of each subpopulation may be used to determine which cells are present in a sample and count their relative percentages.
- The detected light signals are then converted by the electronics system into electronic signals that the computer can process.
Flow Cytometry Instrumentation/Parts
A flow cytometer is composed of three major systems: fluidics, optics, and electronics.
Fluidics
- The fluidics system's objective is to move particles in a fluid stream to the laser beam. The sample is injected into a stream of sheath fluid (typically a buffered saline solution) within the flow chamber to accomplish this.
- The flow chamber's design allows the sample core to be centred in the sheath fluid's centre, where the laser beam interacts with the particles.
- The sample suspension is focused by injecting it into the centre of a sheath liquid stream. The flow of the sheath fluid pushes the particles and confines them to the sample core's centre.
Optics System
- The cytometer's optical system is made up of excitation optics and collecting optics.
- The laser and lenses used to shape and concentrate the laser beam to the flow of the sample comprise the excitation optics.
- The collection optics consist of a collection lens that collects light released after the particle interacts with the laser beam and a series of optical mirrors that diverts the collected light's specific wavelengths to designated optical detectors.
- After a cell or particle passes through the laser light, the side rays and fluorescence signals are directed to photomultiplier tubes (PMTs), and the signals are collected by a photodiode.
- To obtain detector specificity for a given fluorescent dye, a filter is put in front of the tubes, allowing only a small range of wavelengths to reach the detector.
Electronics system
- The electrical system turns the detector signals into digital signals that can be read by a computer.
- When light signals reach one side of the phototransistor or photodiode, they are translated into a relative quantity of electrons that are multiplied to produce a larger electrical current.
- The electrical current is transformed to a voltage pulse by the amplifier.
- The peak of the pulse is reached when the particle reaches the centre of the beam, resulting in the greatest amount of scatter or fluorescence.
- The pulse is then converted to a digital number by the Analog-to-Digital Converter (ADC).
Flow Cytometry Protocol/Procedure/Process/Steps
Flow cytometry is made up of the following steps:
Preparation of Samples
- The cells being studied must be in a single-cell suspension before being passed through flow cytometers.
- Before analysing clumped cultured cells or cells existing in solid organs, they should be transformed into a single cell solution by enzymatic digestion or mechanical dissociation of the tissue, respectively.
- Mechanical filtering should therefore be used to eliminate undesirable instrument blockages and acquire higher quality flow data.
- The cells are then treated in test tubes or microtiter plates with unlabeled or fluorescently conjugated antibodies before being evaluated using a flow cytometer.
Antibody Staining
- Following preparation of the sample, the cells are coated with fluorochrome-conjugated antibodies specific for the surface markers seen on various cells. This can be accomplished using direct, indirect, or intracellular staining.
- Cells are treated with an antibody that has been directly coupled to a fluorophore in indirect staining.
- The fluorophore-conjugated secondary antibody recognises the main antibody through indirect staining.
- Intracellular staining enables for direct assessment of antigens present inside the cell's cytoplasm or nucleus. The cells are permeable first, then stained with antibodies in the permeabilization solution.
Running Samples
- First, control samples are conducted to adjust the detector voltages.
- The cytometer's flow rates are set, and the sample is run.
Types of Flow Cytometry
Flow cytometers are classified into many categories based on their function and precision:
1. Traditional flow cytometers
- Traditional cytometers are the most prevalent cytometers that use sheath fluid to concentrate the sample stream.
- Traditional flow cytometers often employ lasers with wavelengths of 488 nm (blue), 405 nm (violet), 532 nm (green), 552 nm (green), 561 nm (green-yellow), 640 nm (red), and 355 nm (ultraviolet).
2. Acoustic Focusing Cytometers
- Ultrasonic waves are utilised in these cytometers to concentrate the cells for examination.
- This reduces sample clogging while also allowing for larger sample inputs.
3. Cell sorters
- Cell sorters are a type of classic flow cytometer that allows the user to collect samples after they have been processed.
- Cells that are positive for the target parameter can be distinguished from those that are negative.
4. Imaging flow cytometer
- Imaging cytometers combine standard cytometers and fluorescent microscopy.
- An imaging cytometer enables for quick morphological and multi-parameter fluorescence examination of a sample at both the single cell and population levels.
Applications/Uses
Flow cytometry is employed in a variety of domains such as molecular biology, pathology, immunology, virology, plant biology, and marine biology. Some examples of frequent applications are:
- It is used in clinical labs to identify malignancy in body fluids such as leukaemia.
- Cytometers, like cell sorters, can be used to physically separate cells of interest in different collecting tubes.
- It may be used to identify the presence of DNA using fluorescent markers.
- Flow cytometers can analyse replicating cells at four distinct phases of the cell cycle by employing fluorescent dye.
- Acoustic flow cytometers are used to investigate multidrug resistant microorganisms in blood and other materials.
- Flow cytometers can identify the various phases of cell death, apoptosis, and necrosis based on distinctions in morphological and biochemical alterations.
Limitations
- This method does not offer information on protein intracellular location or distribution.
- Debris accumulates over time, which might lead to erroneous findings.
- The pre-treatment associated with sample preparation and staining takes time.
- Flow cytometry is a costly procedure that necessitates the use of highly trained workers.
