Flow cytometry is a system for rapidly analyzing and differentiating cells or particles as they move in a liquid stream through single or multiple lasers. The laser beam strikes the particles and measures their optical and fluorescent characteristics through light-scattering and fluorescence emission. Light scattering is measured through Forward Scatter (FSC) which indicates the size of the cells, and Side Scatter (SSC) which indicates the cell’s granularity. On the other hand, fluorescent characteristics are measured depending on whether the molecules carry antibodies, dyes, or fluorescent proteins.

Flow cytometry provides valuable information concerning the molecular, biochemical, and biophysical aspects of cells. Therefore, it is used in various applications in multiple disciplines, including cancer biology, molecular biology, virology, immunology, and infectious disease monitoring.

Principle 

Flow cytometry measures light scattering and fluorescence emission as the cells or particles pass through the laser beam. The light is scattered in all directions. The forward scatter measures the size of the cells since the light is scattered at low angles from the axes, which is proportional to the square of the radius of a sphere. On the other hand, the side scattering of light measures the cell’s granularity since light entering the cells can be reflected and refracted by the cell’s nucleus and other components, which is proportional to the granularity of the cell. 

Fluorescent characteristics are measured by labeling the cells with fluorochrome-linked antibodies or staining them with fluorescent nuclear, membrane, or cytoplasmic dyes. A fluorescent probe is used to measure fluorescent emission, which is proportional to the amount of fluorescent probe bound to the cell. This is used to evaluate whether certain membrane antigens or receptors are present, and differentiate cell types, enzyme activity, membrane potential, pH, and DNA content. 

See our flow cytometry article here for more information.

Components of a Flow Cytometer

Flow cytometry is conducted on Flow Cytometers. It consists of a Fluidics system, an optical system, and an Electronic system. 

• Fluidics system

The fluidics system transports particles in a fluid stream to the laser beam. It includes two components: sheath fluid and pressurized lines. The sheath fluid (usually buffered saline solution) is injected into the flow chamber by pressurized lines. The suspended cells in the sample tube are also injected into the flow chamber using a pressurized airline. The sample stream becomes a sample core that is focused in the center of the sheath fluid stream where the laser beams interact with the particles, also known as a coaxial flow. This flow is based on the pressure difference between the sample stream and sheath fluid. The cells line up in a single file through the laser beam since the sample pressure is greater than the sheath fluid pressure, allowing hydrodynamic focusing of cells. 

• Optical system 

The optical system comprises excitation optics and collection optics. The excitation optics is composed of lasers and lenses that shape and focus the laser beam to the sample flow to illuminate the particles. The collection optics consists of a collection lens that collects the light emitted from the particle after interaction with the laser beam. The system also includes optical filters. A series of dichroic filters divert the collected fluorescent light to specific optical detectors, and bandpass filters determine their wavelengths, allowing each fluorochrome to be detected and measured. 

• Electronics system

The electronics system converts the fluorescent signals from the detectors into electronic signals that can be read by a computer. 

Other Types of Flow Cytometers 

Cell Sorters 

A cell sorter is a type of traditional flow cytometer that can purify and collect samples for further examination. A cell sorter allows the operator to choose (gate) a population of cells or particles that are positive or negative for the desired parameters and then direct those cells into a collection vessel. The cell sorter separates cells by generating drops by oscillating the sample stream of liquid at a high frequency. The drops are then given a positive or negative charge and sent to a designated collection vessel based on their charge after passing through metal deflection plates. Tubes, slides, or plates can be used as collection vessels.

Fluorescent Activated Cell Sorters

Fluorescent activated cell sorters (FACS) are flow cytometers capable of sorting fluorescent-labeled cells from a mixed cell population using fluorescent markers. The cells are deposited into different containers depending on specific light scattering or fluorescence patterns. 

Mass Cytometers

Mass cytometers combine flow cytometry and time-of-flight mass spectrometry. In this method, the cells are labeled with heavy metal ion-tagged antibodies instead of fluorescently-tagged antibodies used in traditional flow cytometry. Moreover, the cells are detected using time-of-flight mass spectrometry instead of FSC and SSC light detection. 

Imaging Cytometers

Traditional flow cytometry and fluorescence microscopy are combined in imaging flow cytometers (IFC). This allows for quick morphological and multi-parameter fluorescence examination of a sample at both the single-cell and population levels. IFC can detect protein distributions within individual cells, but it can also analyze vast numbers of cells like a flow cytometer. They’re particularly effective in various applications, including cell signaling, co-localization investigations, cell-to-cell interactions, DNA damage and repair, and any other application that requires coordinating cellular location with fluorescence expression on large populations of cells.

Spectral Analyzers 

Spectral analyzers measure the whole fluorescent emission spectra of each fluorophore using a series of detectors. Algorithms are then used to segregate the spectra. This enables the employment of overlapping spectra fluorophores to expand the number of parameters examined in a single experiment.

Applications of Flow Cytometry 

Flow Cytometry has several applications in multiple fields of study:

Immunology 

Immunophenotyping 

Flow Cytometry is mostly used for immunophenotyping, which analyzes multiple parameters of mixed cell populations simultaneously. In this method, cells are stained with fluorochrome-conjugated antibodies, which are targeted against cell surface antigens. 

The surface proteins and glycoproteins on leukocytes, erythrocytes, and platelets have all been extensively researched. Flow cytometric examination of erythrocytes, leukocytes, and platelets is possible due to the availability of monoclonal antibodies directed against these surface proteins.

Antigen-Specific Responses

Antigen-specific responses can be assessed by stimulating cells with a particular antigen and then monitoring cytokine production, activation, proliferation, memory, or antigen recognition via MHC multimers, which is commonly used in vaccine research. 

Molecular Biology 

Flow Cytometry is utilized in various processes of molecular biology such as

Analysis of Cell Cycle

Flow cytometry can quantify the four distinct phases of the cell cycle using fluorescent dyes to analyze replication states. The assay can evaluate cell aneuploidy associated with chromosomal aberrations in addition to determining cell cycle replication states.

Fluorescent Protein Analysis

Fluorescent proteins, such as GFP, mCherry, YFP, and mRuby, are utilized as protein expression markers. Cells are usually transfected with a plasmid containing a promoter sequence and encoding a gene of interest as well as a fluorescent protein. The fluorescent protein’s expression is utilized as an indicator for the expression of the gene of interest.

Other Applications of Flow Cytometry include  

• Absolute Cell Counting 
• Multiplexed Bead Array Assays
• Phagocytosis Assays
• Quantitative Flow Cytometry
• Small Particle Analysis and Sorting

Advantages and Disadvantages of Flow Cytometry

Flow cytometry is valuable for analyzing subpopulations of cells within a heterogeneous sample in a high-throughput fashion. It allows rapid and quantitative measurement of up to 20 parameters of cell phenotype in a highly sensitive and reproducible manner, which is possible thanks to advances in technology and fluorophore chemistry. Cells of interest can also be isolated with very high purity with flow cytometric cell sorting. Moreover, polychromatic flow cytometry’s high specificity also allows the evaluation of discrete cell subsets and rare populations.

Although flow cytometry has revolutionized scientific research and the medical world, it also has several drawbacks. The fluidics in the system can be difficult to keep consistent, with cells or debris blocking flow or generating fluctuations in flow rates that impair analysis. Contamination in the pipework can cause the system to get clogged, interrupting the flow. Consistent results necessitate laser alignment, which requires checking regularly. Flow cytometry also usually generates huge amounts of data, making analysis difficult. Moreover, the need for analyzed cells to be in a suspension makes data on tissue architecture and cell-cell interactions unavailable. In addition, analyses with more fluorophores are prone to signal spillover, and it is difficult to distinguish between cell subpopulations with similar marker expressions.

The Future of Flow Cytometry

Over the last few decades, substantial improvements in the field have allowed for large increases in the number of parameters examined in each sample while also reducing the size of these burdensome instruments. These trends are anticipated to continue in the coming years, adding to the complexity of the data acquired and necessitating the development of tools to standardize and validate such assessments.

More advancements in this field will allow for more comprehensive studies on a larger scale, increasing the number of markers that can be tested for each sample and improving data analysis tools to allow for the detailed study of huge sample numbers. Reduced instrument sizes will make it easier to position them on lab benchtops and may be used in limited formats in the field. Furthermore, greater workflow uniformity will lower user error rates and enable high-throughput clinical and diagnostic applications. Flow cytometry analyses will improve by automatic pipetting and compensation. 

References 

Adan, A., Alizada, G., Kiraz, Y., Baran, Y., & Nalbant, A. (2017). Flow cytometry: basic principles and applications. Critical reviews in biotechnology, 37(2), 163–176. https://doi.org/10.3109/07388551.2015.1128876

McKinnon K. M. (2018). Flow Cytometry: An Overview. Current protocols in immunology, 120, 5.1.1–5.1.11. https://doi.org/10.1002/cpim.40

Macey, M. G., & Macey, M. G. (2007). Flow cytometry. Berlin, Germany:: Springer.

Jahan-Tigh, R. R., Ryan, C., Obermoser, G., & Schwarzenberger, K. (2012). Flow cytometry. The Journal of investigative dermatology, 132(10), e1. )  doi:10.1038/jid.2012.282