How Flow Cytometers Work & How We Save You Time & Money

The fluidics system within the analyzer aligns and transports particles in a fluid stream through the laser beam’s path for identification. When a sample in a solution is injected into the instrument, the individual cells are randomly distributed in a three-dimensional space. For accurate data collection, it is important that each specific cell passes through the laser beam on its own, with each cell passing through single file.

Most flow cytometers accomplish this by injecting the sample stream into a stream of faster flowing sheath fluid within the flow chamber, along with additional flow cells. The flow of the sheath fluid accelerates the particles and restricts them to the center of the sample core so that a single stream of particles is created. This process is known as hydrodynamic focusing.

After hydrodynamic focusing, each particle passes through one or more beams of light. The excitation by laser causes the fluorescent reagents bound to cells or particles to emit light, which is detected according to their specific wavelength range. Light scattering, through a forward scatter channel (FSC) or fluorescence emission provides information about the particle’s properties, and cell granularity or internal structural complexity is provided via a side scatter channel (SSC).

When using a combination of fluorescent dyes, reagents should have distinct emission spectra. For example, MACSQuant instruments will use various dichroic mirrors and optical filters to direct light of specific wavelengths to the according fluorescence detectors. This arrangement creates “fluorescence channels.” Various filters are used to control which wavelengths enter the fluorescence channels. These filters are referred to as long-pass, short-pass, and band-pass according to their optical properties.

• Long-pass: blocks light up to a certain wavelength.
• Short-pass: transmits light up to a certain wavelength.
• Band-pass: transmits light within a defined wavelength range.

Fluorescent measurements taken at different wavelengths can provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors or intracellular molecules, such as DNA and cytokines. The argon-ion laser is most commonly used in flow cytometry. This is because the 488 nm light that it emits excites more than one fluorochrome, while the laser and the arc lamp are the most popular light sources in modern flow cytometry.

Once a cell or particle passes through the laser light, emitted side scatter and fluorescence signals are diverted to the photomultiplier tubes (PMTs) and a silicon photodiode collects the forward scatter signals.

All of the signals are routed to their photodetectors via a system of mirrors and optical filters. Optical filters control the specificity of detection by blocking certain wavelengths and transmitting others.

To visualize the data collected, values of each measured optical parameter are plotted in various ways. The plots used can be univariate or bivariate. In the case of flow cytometry, univariate plots are represented as histograms while bivariate plots are represented as dot plots.

Univariate plots (histograms) visualize one optical parameter, such as fluorescence or light scattering, against event number. For each event, each measured parameter’s signal intensity values are stored in a data file and, as the events accumulate, a curve is generated to illustrate the distribution of events according to their individual signal intensity.

Bivariate plots (dot plots), on the other hand, allow for the simultaneous visualization of two optical parameters on a single graph. As more events are plotted, ones that share similar intensity values accumulate in clusters. These clusters can often represent specific cell populations.

Most cell sorter instrumentation can utilize sample volumes ranging from several microliters to hundreds of microliters. If you have a small volume of precious cells, consider using a microfluidics-based flow cytometry chip.

The development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry.

Automated sample introduction into the analyzer allows for walk-away cell viability assays to be performed, among other analytical experiments. The addition of automation also allows for a larger numbers of samples to be analyzed, using a variety of formats, from single test tubes to multi-well plates.

Single tubes are often contained within rectangular grid arrangements or carousel formats to match laboratory equipment used for sample preparation.

Immunophenotyping is just one of the many uses of flow cytometry, although it may be the most common. It is used in the identification of markers on cells, particularly the immune system, and allows researchers and scientists to study the protein expressed by cells.

Cell identification is based on the markers or antigens present on a cell’s surface, nuclease, or cytoplasm, and helps identify the lineage of cells using antibodies that detect the markers on each cell (thus the “immuno-” prefix).

Immunophenotyping can be as simple as identifying a cell using a single marker or as complex as applying homing profile, activation states and cytokine release all in one panel. Because of this, experimental protocols often combine surface and intracellular staining. Immunphenotyping is routinely used in basic research as well as clinical applications to diagnose disease or to monitor and evaluate residual disease.

Some examples of immonphenotyping include differentiating between acute myeloid and lymphoid leukemia, as well as B and T cell lymphoid neoplasms, which includes chronic lymphocytic leukemia and lymphoma. This method is also used for reactive and neoplastic expansions of lymphocytes, predicting prognosis in lymphoma, and in the identification of lymphocyte subsets.