Figure 3

'Next generation' spectral flow cytometry. (a) With a conventional flow cytometer, lasers excite cell-associated fluorochromes, and emitted light is filtered by a combination of dichroic mirrors (DMs) and bandpass filters (BFs) that reflect and filter light of specific wavelengths, respectively. Light within narrow selected wavelength ranges arrives at a photomultiplier tube (PMT), which converts light as photons to an electronic signal. (b) In spectral flow cytometry, laser diodes similarly provide initial excitation of reporter fluorochromes; however, emitted fluorescence passes through a prism array into a spectral PMT. Component fluorescence, including autofluorescence, is linearly unmixed by using spectral lookup tables. In spectral flow cytometry, unlike conventional cytometry, almost all light signals are analyzed, and signal-to-noise resolution is dramatically improved.