![]() Nearly simultaneously, light from the laser will excite all fluorophores associated with the cell, which produces a fluorescence emission. These measurements are termed forward angle scatter (FSC) and side angle scatter (SSC), depending on where the light is collected with respect to the path of the laser. This light scatter can be measured and correlated with relative cell size and structures inside the cell. When the laser light beam illuminates a single cell, some of the light will strike physical structures within the cell, causing the light to scatter. You might also hear it referred to as the laser intercept. The place where the cells interact with laser light is called the interrogation point (Figure 3). If collected under sterile conditions, these cells can be further cultured, manipulated, and studied. Cell sorters use fluidics and fluorescence components similar to those in flow cytometers, but are able to divert a specific population from within a heterogeneous sample into a separate tube, typically based on specified fluorescence characteristics. A flow cytometer is an analytical machine that does not perform cell sorting. This specialized flow cytometer is called a fluorescence activated cell sorter (FACS), a term that is sometimes erroneously used interchangeably with ‘flow cytometer’. Identification and characterization of distinct subsets of cells within a heterogeneous sample-including distinguishing central effector memory cells from exhausted T cells or even regulatory T cellsĪn additional capability of specialized flow cytometers is the ability to sort cells and recover the subsets for post experimental use.Cell cycle status-providing a powerful tool to assess cells in G0/G1 phase versus S phase, G2, or polyploidy, including analysis of cell proliferation and activation.Cell health status-from viability to late-stage apoptosis or programmed cell death.RNA-including IncRNA, miRNA, and mRNA transcripts.Protein post translational modifications-includes cleaved and phosphorylated proteins.Protein expression-throughout the entire cell, even the nucleus.The potential applications of analysis by flow cytometry are numerous, including the detection and measurement of: Potential applications for flow cytometry * Assumes reagents have been validated to work for fixed cells. Microscopy: major strength of this technique Microscopy: easily visualized, new assays allowed to develop multiparametric analysisįlow cytometry: imaging cytometers are now available that combine flow cytometry with imaging Microscopy: time consuming to identify without software or instrumentationįlow cytometry: possible with specialized assays that combine protein and RNA detection together in the same assay Microscopy: may require tissue sectioning but in situ characteristics can be detectedįlow cytometry: physical separation and collection of user-defined cell populations with specialized sorter Microscopy: requires the use of specific imaging instrumentation for high throughputįlow cytometry: requires tissue dissociation Microscopy: dependent on fluorochrome choice and exposure timeįlow cytometry: millions of cells in short time Microscopy: can be achieved with instrumentation and software, or can sometimes be done manually but is time consumingįlow cytometry: dependent on the fluorochromes, experimental design, and instrumentation Microscopy: up to 6 parameters with special instrumentsįlow cytometry: easily obtain statistics using embedded software Number of parameters detected on single cell Microscopy: relatively inexpensive but again, the more complex the experiment the more likely the instrument and analysis software will be more expensive Instrumentation/software complexity and expenseįlow cytometry: the more parameters needed the more complex and expensive the instrumentation/analysis software will be Science, like the rest of life, is all about trade-offs! See Table 1 for a comparison of flow cytometry and microscopy techniques. Because of its speed and ability to scrutinize at the single-cell level, flow cytometry offers the cell biologist the statistical power to rapidly analyze and characterize millions of cells, albeit at the expense of the morphological characteristics and subcellular localization that microscopy can provide. Today’s instruments offer an increased number of detectable fluorescent parameters (from 1 or 2 up to ~30 or more), all measured at the same time on the same cell. Since that time, innovations from many engineers and researchers have culminated in the modern flow cytometer, which is able to make measurements of cells in solution as they pass by the instrument’s laser at rates of 10,000 cells per second (or more). Flow cytometry is a cell analysis technique that was first used in the 1950s to measure the volume of cells in a rapidly flowing fluid stream as they passed in front of a viewing aperture. ![]()
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