Before describing how flow cytometer detects and processes signals, it is important to understand what happens to the laser light as it strikes the single-celled particles.
(I) Light Scatter
Light scattering occurs when a particle deflects incident laser light. The extent to which this occurs depends on the physical properties of a particle, namely its size and internal complexity. Factors that affect light scattering are the
(i) cell's membrane,
(ii) nucleus,
(iii) any granular material inside the cell,
(iv) cell shape and surface topography (11.)
(Forward and side scattering has been mentioned in earlier pages but lets talk about them in detail.)
Forward-scattered light (FSC) is proportional to cell-surface area or size. FSC is a measurement of mostly diffracted light and is detected just off the axis of the incident laser beam in the forward direction by a photodiode.
Side-scattered light (SSC) is proportional to cell granularity or internal complexity. SSC is a measurement of mostly refracted and reflected light that occurs at any interface within the cell where there is a change in refractive index. SSC is collected at approximately 90 degrees to the laser beam by a collection lens and then redirected by a beam splitter to the appropriate detector.(11.)
Light-scattering properties of a cell (13.)
Correlated measurements of FSC and SSC can allow for differentiation of cell types in a heterogeneous cell population.
For example, leukocytes can be differentiated by using FSC and SSC.
Cell subpopulations based on FSC vs SSC(14.)
(II) Optical system
The optical system consists of excitation optics and collection optics. The excitation optics consist of the laser and lenses that are used to shape and focus the laser beam. The collections optics consist of a collection lens to collect light emitted from the particle–laser beam interaction and a system of optical mirrors and filters to route specified wavelengths of the collected light to designated optical detectors.
(A) The optical bench in a flow cytometer provides a stable surface that holds the light source and the excitation and collection optics in fixed positions. The alignment of a benchtop analyzer is very stable because the flow cell is fixed in its alignment with the laser beam. This ensures that the laser intercepts the sample stream consistently from day to day.
(B) Once a cell or particle passes through the laser light, emitted SSC and fluorescence signals are diverted to the photomultiplier tubes (PMTs) and a photodiode collects the FSC signals. All of the signals are routed to their detectors via a system of mirrors and optical filters. PMTs detect fluorescence signals, which are often weak. The specificity of a detector for a particular fluorescent dye is optimized by placing a filter in front of the PMT, which allows only a narrow range of wavelengths to reach the detector. This spectral band of light is close to the emission peak of the fluorescent dye. Such filters are called bandpass (BP) filters.Other filters used in the flow cytometer are shortpass (SP) filters, which transmit wavelengths of light equal to or shorter than a specified wavelength, and longpass (LP) filters, which transmit wavelengths of light equal to or longer than a specified wavelength.(11.)
Light transmittance through longpass, shortpass, and bandpass filters(11.)
