The ‘mixing matrix’ (M) comprises individual spectra. So, the question is what is the relationship between the individual spectra and the composite spectra? If we extract the intensity values of each detector for each individual spectra and the observed spectra as well we will have the following data (table 2): The question that we need to answer is how much of each of these fluorochromes is present on the cell. Control spectra for theoretical 3-color experiment. With the TFF, we need single color controls to determine the intensity of each fluorochrome on the target cell. Figure 5: Spectral output from a cell captured using FSC. When we look at the spectral output from a cell, we see the observed spectra shown in Figure 5. Let’s suppose that we label a cell with three different fluorochromes: A, B and C. The SONY ID7000 TM -their newest spectral cytometer- which can have up to 7 lasers, uses a combination of a 32-channel PMT array and individual PMTs to capture the spectra as shown below. Table: 5-laser Aurora Configuration Excitation Laser Rather than having one detector for one fluorochrome, this system has 64 detectors capturing the whole range of emission possible off that excitation laser, as shown in the table below. Look at these two dyes again but measure the whole spectra only, modeled on a 5-Laser Cytek Ⓡ Aurora, we would get the following.įigure 3: Full spectra measurements of BV421 TM and AF488 Ⓡ using the Cytek Ⓡ Aurora spectral cytometer This is the principle of Full Spectrum Cytometry (FSC). What if, instead of capturing just a portion of the signal we captured the whole spectra? This requires the cytometrist to perform compensation the mathematical correction for this spectral overlap. More importantly, it is clear that the spectra can extend over 50 or more nanometers. It is clear from these spectra, that a lot of potentially useful information is being thrown away. Figure 2: Sample filters used to measure BV421 TM and AF488 Ⓡ. In that case fluorochromes BV421 TM can be measured using a BP431/28 and AF488 Ⓡ a BP530/30. Since the detectors we use - photomultiplier tubes (PMT) and avalanche photodiodes (APD) - will turn any photon of light that hits the detector into a photocurrent, the signals going to the detectors are regulated using filters. In traditional fluorescent flow cytometry (TFF), the instrument measures each fluorochrome off an individual detector. Figure 1: Excitation and emission profiles of BV421 TM and AF488Ⓡ This is typically modeled using spectral viewers such as in the figure below, which shows the excitation (dashed lines) and emission (filled curves) for Brilliant Violet 421 TM (purple) and Alexa Fluor 488 Ⓡ (green). The fluorochromes, fluoresce emitting photons of a higher wavelength than the excitation source. As the labeled cell passes through the interrogation point, it is illuminated by the excitation lasers.
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