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After hydrodynamic focusing, each particle passes through one or more beams of light. Light scattering or fluorescence (FL) emission (if the particle is labeled with a fluorophore) provides information about the particle’s properties. Lasers are the most commonly used light sources in modern flow cytometry.
Lasers produce a single wavelength of light (a laser line) at one or more discrete frequencies (coherent light). They are available at different wavelengths ranging from ultraviolet to far red and have a variable range of power levels (photon output/time).
Light that is scattered in the forward direction, typically up to 20° offset from the laser beam’s axis, is collected by a photomultiplier tube (PMT) or photodiode known as the forward scatter (FSC) channel. The FSC equates roughly to the particle’s size. Typically, larger cells refract more light than smaller cells.
Light measured at an approximately 90° angle to the excitation line is called side scatter (SSC). The SSC channel provides information about the relative complexity (for example, granularity and internal structures) of a cell or particle. Both FSC and SSC are unique for every particle, and a combination of the two may be used to roughly differentiate cell types in a heterogeneous sample such as blood. However, this depends on the sample type and the quality of sample preparation, so fluorescent labeling is always preferred.
Fluorescence measurements taken at different wavelengths can provide quantitative and qualitative data about fluorophore-labeled cell surface receptors or intracellular molecules such as DNA and cytokines. Flow cytometers use separate channels and detectors to detect light emitted. The number of detectors will vary according to the instrument and its manufacturer. Detectors are either silicon photodiodes or photomultiplier tubes. Historically, silicon photodiodes were used to measure forward scatter for strong signals. More commonly now, PMTs are used even in the FSC channel. PMTs are more sensitive detectors and are ideal for scatter and fluorescence readings.
The specificity of detection is controlled by optical filters, which block certain wavelengths while transmitting (passing) others. There are three major filter types. Long pass filters allow light through above a cutoff wavelength, short pass filters permit light below a cutoff wavelength, and band pass filters transmit light within a specified narrow range of wavelengths (termed a band width). All these filters block light by absorption (Figure 2).
Figure 2: Different types of optical filters
A dichroic filter/mirror has a filter placed at an angle to the oncoming light. This type of filter performs two functions, first, to pass specified wavelengths in the forward direction and, second, to deflect blocked light at a 90° angle. To detect multiple signals simultaneously, the precise choice and order of optical filters is an important consideration (Figure 3).
Figure. 3. Schematic overview of a typical flow cytometer setup. FL, fluorescence; PMT, photomultiplier tube; SSC, side scatter; FSC, forward scatter; blue arrow, light path.
Signal and Pulse Processing
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