What is flow cytometry?
Flow cytometry uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multi-parameter data from particles and cells in the size range of 0.5um to 40um diameter. Cells are hydro-dynamically focused in a sheath of PBS before intercepting an optimally focused light source. Lasers are most often used as a light source in flow cytometry.
Figure 1.1 Flow cytometers use the principle of hydrodynamic focusing for presenting cells to a laser (or any other light excitation source). The sample is injected into the center of a sheath flow. The combined flow is reduced in diameter, forcing the cell into the center of the stream. This the laser one cell at a time. This schematic of the flow chamber in relation to the laser beam in the sensing area.
As your cells or particles of interest intercept theÂ light source they scatter light and fluorochromes are excited to a higher energy state. This energy is released as a photon of light with specific spectral properties unique to different fluorochromes. One unique feature of flow cytometry is that it measures fluorescence per cell or particle. This contrasts with spectrophotometry in which the percent absorption and transmission of specific wavelenths of light is measured for a bulk volume of sample. Scattered and emitted light from cells and particles are converted to electrical pulses by optical detectors. Collimated (parallel light waveforms) light is picked up by confocal lenses focused at the intersection point of cells and the light source. Light is send to different detectors by using optical filters. For example, a 525 nm band pass filter placed in the light path prior to the detector will only allow green light into the detector. The most common type of detector used in flow cytometry is the photomultiplier tube (PMT).
The electrical pulses originating from light detected by the PMTs are then processed by a series of linear and log amplifiers. Logarithmic amplification is most often used to measure fluorescence in cells. This type of amplification expands the scale for weak signals and compresses the scale for strong or specific fluorescence signals. After the different signals or pulses are amplified they are processed by an Analog to Digital Converter (ADC) which in turn allows for events to be plotted on a graphical scale(One Parameter, Two parameter Histograms).
How to store this information:
Flow cytometry data outputs are stored in the form of computer files using the FCS 2.0 or 3.0 standard. Data corresponding to one sample can be stored as a listmode file and/or histogram file.
Table 1.1 Fluorescence spectra of commonly used fluorochromes. Excitation spectra is represented by the gray lines while emission spectra is in black. The bottom part of the table summarizes the emission wavelengths of various light sources used in flow cytometry. The 488nm line of the argon ion laser is extended over the spectra. (From Practical Flow Cytometry, Third Edition, Howard M. Shapiro. P. 245).