Flow cytometry is a technology that allows scientists to rapidly analyze individual cells suspended in a fluid. This method enables the gathering of information about millions of cells within a sample, providing insights into their physical characteristics and the presence of specific markers. Its capabilities are widely applied across various fields, advancing understanding of health and disease. It is important in the study of the immune system, revealing cellular interactions and responses.
Understanding Flow Cytometry
Flow cytometry is a technique used to count, sort, and identify cells in a liquid sample, analyzing their characteristics. It involves suspending cells in a fluid, which are then passed one by one through a focused laser beam. As each cell passes through the laser, it scatters light and, if labeled, emits fluorescent signals.
The scattered light and fluorescent emissions provide information about each cell. Forward scatter (FSC) measurements relate to cell size, while side scatter (SSC) indicates internal complexity. Fluorescent signals, generated by tags attached to cellular components, reveal molecules on or within the cell. Measuring multiple parameters simultaneously for millions of cells offers a comprehensive cellular profile.
How Flow Cytometry Works
The operation of a flow cytometer relies on a system of fluidics, optics, and electronics. The process begins with the fluidics system, where cells from a sample are injected into a sheath fluid. This sheath fluid hydrodynamically focuses the cells into a single-file stream, ensuring each cell passes individually through the laser. This alignment ensures accurate measurement.
Once aligned, the cells enter the optics system, where they encounter one or more lasers. These lasers, emitting light at specific wavelengths (e.g., 488 nm blue, 633 nm red, 405 nm violet), illuminate each cell. As the laser light strikes a cell, some light is scattered, and if the cell has been labeled with fluorescent dyes, these dyes absorb the laser light and then re-emit light at longer, characteristic wavelengths.
Fluorescence allows for the identification of cell types and their features. Researchers attach fluorescent tags, known as fluorochromes, to antibodies that bind to proteins on the cell surface or inside the cell. For example, an antibody with a fluorochrome like FITC (Fluorescein Isothiocyanate) might bind to a protein, causing the cell to emit green light when hit by a blue laser. Different fluorochromes emit different colors, allowing simultaneous analysis of multiple cellular features.
Scattered light and fluorescent signals are collected by lenses and directed to detectors. Photomultiplier tubes (PMTs) convert light signals into electrical pulses. These pulses are proportional to light intensity, providing quantitative data on cell size, internal complexity, and labeled marker expression. These electrical signals are converted into digital data, then displayed and analyzed by specialized software.
Role in Immunology
Flow cytometry is significant in immunology, allowing dissection of the immune system’s cellular landscape. It allows researchers and clinicians to identify, quantify, and characterize immune cell populations, offering insights into immune responses in health and disease. Immune cells often express unique combinations of surface proteins, which can be targeted by fluorescently labeled antibodies.
A primary application is phenotyping and enumerating immune cells. For instance, flow cytometry can distinguish and count different T cell subsets, such as CD4+ helper T cells and CD8+ cytotoxic T cells, based on specific surface proteins like CD4 and CD8. It can also identify B cells, natural killer (NK) cells, monocytes, and dendritic cells, each defined by their distinct surface marker profiles. This cellular mapping helps understand immune system composition.
In the diagnosis and monitoring of immune disorders, flow cytometry provides measurable data. For example, in HIV infection, tracking the absolute count of CD4+ T cells is a standard procedure for assessing disease progression and the effectiveness of antiretroviral therapy. Similarly, in hematological malignancies like leukemia and lymphoma, flow cytometry identifies malignant cell populations based on abnormal surface marker expression, aiding diagnosis, classification, and monitoring of minimal residual disease.
Flow cytometry is also widely employed in immunological research to understand immune responses. It helps scientists understand how immune cells respond to infections, how vaccines elicit protective immunity, and the mechanisms underlying autoimmune diseases. For instance, researchers can use it to measure the proliferation of T cells after vaccine stimulation or to detect immune cells producing specific cytokines, which are signaling molecules that regulate immune responses. This functional analysis provides information on cell activity beyond just identification.
Interpreting the Insights
The output from a flow cytometer is presented as visual plots for interpreting cellular characteristics and populations. The most common is the scatter plot, also known as a dot plot. In these plots, each dot represents an individual cell, positioned by the intensity of two measured parameters. For example, forward scatter (cell size) is often plotted against side scatter (internal complexity), enabling discrimination of broad cell populations like lymphocytes, monocytes, and granulocytes.
Fluorescent signals are also displayed on scatter plots, with two different fluorescent markers plotted against each other. This identifies specific cell subsets that co-express proteins. For instance, a plot might show cells expressing both CD3 and CD4, identifying CD4+ T helper cells as a distinct cluster. Dot density in a region indicates the abundance of that cell population.
Another common display format is the histogram, illustrating the distribution of a single parameter across a cell population. A histogram shows the intensity of a specific fluorescent marker on the x-axis and the number of cells expressing that intensity on the y-axis. This assesses the expression level of a protein across a group of cells, such as a receptor on B cells.
Gating is a technique for interpreting flow cytometry data. Gating involves drawing a boundary, or “gate,” around a population of interest on a scatter plot or histogram to isolate those cells for further analysis. By sequentially gating on different parameters, researchers can narrow down and analyze specific cell subsets, quantifying their numbers and characterizing their properties. This iterative process provides insights into the cellular composition and functional state of a sample.