Flow cytometry data acquisition

The light scatter assay may be used to determine absolute numbers of viable cells if flow cytometry data from cell suspensions of known concentration are used to construct a standard curve. For that purpose, cell concentrations should be determined in a series of graded, standard cell suspensions with the use of a Coulter counter. A plot of those standard concentrations versus the number of events (Hght scatter signals) acquired during a specified acquisition interval in the flow cytometer may then be used to interpolate cell concentrations for test samples that have been assayed by the light scatter procedure. 

For classification in routine lymphocyte subset analysis, expert analysis is often fully adequate. For polychromic flow cytometry, particularly when both lineage and functional phenotypes are being evaluated, more sophisticated analysis may be required. Indeed, more sophisticated analysis can define subpopulations that cannot be discriminated by any combination of two-dimensional projections of multicolor data (Zamir et al., 2005). One of the first steps in enabling more sophisticated downstream analysis is ensuring acquisition of robust data. Some of the recently described analytical methods for high-dimensional flow cytometry data are listed in Table 4.2-1. 

Laser microbeams offer several advantages over other fluorescence excitation techniques. In spectrofluorometry, observations are often made on a population of cells in a cuvette, resulting in a combined signal that lacks information about individual cellular responses. In flow cytometry, many individual cells are measured, but there is no temporal resolution since each cell is observed only once, and there is no spatial resolution since the entire cell is illuminated as it passes through the laser beam (see Chapter 30). In conventional fluorescence microscopy, individual cells can be monitored over time, and information about the two-dimensional spatial distribution of fluorescence can be obtained. However, some samples may be more susceptible to photobleaching by the arc lamps used for excitation, and the temporal resolution is limited to video-rate data acquisition (30 frames/s) (see Chapter 14). 

Acquisition In flow cytometry, acquisition refers to the process of recording the intensity of the photodetector signals from a particle in the transient memory of a computer. Once acquired, the data from a group of particles can be stored permanently on a storage medium from which it can be subjected to analysis. Acquisition and then analysis (in that order) are the two central steps in the flow cytometric procedure. 

Threshold The threshold is an electronic device by which an ADC can be made to ignore signals below a certain intensity. A forward scatter threshold is most commonly used in flow cytometry to exclude very small particles, debris, and electronic or optical noise from acquisition into a data file. 

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