The principles of light excitation, light scattering, and the emission of fluorochrome molecules for specific multi-parameter data generation from cells and particles are used in the process of flow cytometry to get accurate results in various biological tests.

The cells and particles used for this purpose always fall within the size range of 0.5 um to 40 um diameter. A sheath of PBS is used during the process of flow cytometry to hydro-dynamically focus the cells before intercepting a light source that is optimally focused for the test. Once this setup is complete, the process of flow cytometry can begin.

Light Interception in Flow Cytometry

During the process of cytometry, the particles and cells intercept the light source, before scattering the light and causing the fluorochromes to reach a higher energy state, due to the excitement generated by the scattering process. This excess energy is released later in the form of a photon of light with unique spectral properties specific to various fluorochromes.

As opposed to spectrophotometry, fluorescence in flow cytometry is measured per cell or particle. On the other hand, the percent transmission and absorption of specific light wavelengths are measured in spectrophotometry for samples in bulk volume. Light scattered and emitted from particles and cells is converted by optical detectors into electrical pulses.

Collimated light, also known as parallel light waveforms, is picked up during flow cytometry by confocal lenses focused at the light source and the intersection point of the cells. Optical fibers are then used to send light to different detectors. For instance, a band pass filter of 525 nm will only allow green light to pass into the detector when placed in the light path in front of the detector. The photomultiplier tube or PMT is the most common type of detector used by a majority of scientists performing flow cytometry.

The light detected by the PMTs release certain electrical pulses, which are then processed by a series of log and linear amplifiers once they leave their point of origin. Fluorescence in cells is typically measured during this process with the help of logarithmic amplification. This type of amplification serves to compress the scale of strong signals while expanding the scale of weak or non-specific fluorescence signals.

Data Storing Techniques in Flow Cytometry

An analog to digital converter (ADC) is used to process the different pulses or signals after they have all been amplified and duly expanded or compressed. The ADC allows for the systematic plotting of events on a graphical scale, such as one parameter and two-parameter histograms, during the process of flow cytometry.

The differentiation of cell populations on the basis of size through light-scattering is the primary purpose of this process. The data outputs obtained through flow cytometry are stored in the form of computer files with the FCS 3.0 standard. A listmode or histogram file may also be used to store data corresponding to one sample of the cytometry.

Flow cytometry is also sometimes used for the identification of specific cell types. This is accomplished by using fluorescent-tagged antibodies to stain a sample and hence recognize specific cellular markers. Cell population analyses, death/cell cycle analysis, proliferation, apoptosis, cytokine production, intracellular or surface proteins, and binding studies are some of the readouts that can be achieved through this process.

Flow cytometry also facilitates the analysis and purification of human PBMC, monocytes, and lymphocytes in laboratory settings. Side scatter (SSC) is used to measure internal complexity while forward scatter (FSC) can help measure the size of granulocytes and monocytes.

Choosing the Right Flow Cytometer

During the process of flow cytometry, it is important to first check the configuration of the cytometer being used, in order to determine whether or not lasers for the fluorophores being used are available. The emission and excitation spectra of every single fluorophore must also be checked for the purpose of assessing spillover. Spillover essentially occurs when fluorescence from one fluorophore spills into another's detection channel unnoticed.

Ideally, this process should be undertaken with the help of a high-quality fluorescence spectra viewer. Moreover, fluorophores should be chosen that have as little spillover as possible. Knowing about the prospective fluorophores and their relative brightness will also help the scientists select the ideal panel for the flow cytometry to be conducted. Choosing the right fluorophores with the help of a reliable fluorophore brightness chart will minimize the risk of errors in the experiment.

Understanding the cell biology of the experimental samples being used in the flow cytometry process will also maximize the chances of success. For instance, the researchers need to know their target antigen and its relative expression well in order to determine which fluorophore to use for any given marker. For highly expressed antigens, dim fluorophores are typically used, while antigens with lower expression usually require brighter fluorophores.

In Conclusion

For the best results, a reputed laboratory with a great record of successful flow cytometry experiments in its repertoire should be chosen to conduct any experiments requiring this complex and delicate procedure. A team of scientists with a broad range of skills, profiles, and experience will be best suited to maximize the chances of success and minimize the potential for error.