What Is Spectral Unmixing And Why It’s Important In Flow Cytometry

As the labeled cell passes through the interrogation point, it is illuminated by the excitation lasers. The fluorochromes, fluoresce; emitting photons of a higher wavelength than the excitation source. 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 421TM (purple) and Alexa Fluor 488 (green). 

Figure 1: Excitation and emission profiles of BV421TM and AF488Ⓡ 

In traditional fluorescent flow cytometry (TFF), the instrument measures each fluorochrome off an individual detector. 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 that case fluorochromes  BV421TM can be measured using a BP431/28 and AF488 a BP530/30.

Figure 2:  Sample filters used to measure BV421TM and AF488.

It is clear from these spectra, that a lot of potentially useful information is being thrown away. More importantly, it is clear that the spectra can extend over 50 or more nanometers. This requires the cytometrist to perform compensation; the mathematical correction for this spectral overlap.  

What if, instead of capturing just a portion of the signal we captured the whole spectra?  

This is the principle of Full Spectrum Cytometry (FSC). Look at these two dyes again but measure the whole spectra only, modeled on a 5-Laser Cytek Aurora, we would get the following.

Figure 3:  Full spectra measurements of BV421TM and AF488 using the Cytek Aurora spectral cytometer

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.

Table:  5-laser Aurora Configuration

Excitation Laser# of DetectorsEmission range measured(nm)
UV14365-829
Violet14420-829
Blue14498-829
Yellow Green10567-829
Red8653-829

The SONY ID7000TM —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. 

Figure 4Sony ID7000TM spectral range. 

Spectral Unmixing

Let’s suppose that we label a cell with three different fluorochromes: A, B and C.  When we look at the spectral output from a cell, we see the observed spectra shown in Figure 5.  

Figure 5:  Spectral output from a cell captured using FSC.  

With the TFF, we need single color controls to determine the intensity of each fluorochrome on the target cell.  Shown in figure 6.

Figure 6.  Control spectra for theoretical 3-color experiment. 

The question that we need to answer is how much of each of these fluorochromes is present on the cell. 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):

Table 2

So, the question is what is the relationship between the individual spectra and the composite spectra? 

We have three matrices. The ‘mixing matrix’ (M) comprises individual spectra. The amount of each fluorochrome can be represented by matrix ‘a’ and the product of these two is the observed (O) specta. Mathematically you can write it as: 

Ma=O

To determine the abundance, we rearrange the equation to solve for a:

a=OM-1

In this case you can determine that the amount of A is 10 units, B is 6 units and C is 4 units.

As with compensation, there are some specific rules for this mixing matrix:

  • Must be as bright or brighter than experimental sample
  • Need to have identical autofluorescence
  • Must have good separation between positive and negative
  • Fluorescence spectrum needs to be identical to experimental sample

An interesting caveat with the unmixing controls is that the spectrum on the control has to be the same as on the cells. Thus, in some cases, beads will not make a good control, therefore cells have to be used.  

Concluding Remarks

Full spectrum cytometry moves away from the one fluorochrome/one detector model that most traditional cytometers use. FSC collects all the photons from the emitting fluorochromes, and as long as the spectra have differences, it is possible to use the two fluorochromes in combination.  By moving away from the one fluorochrome, one detector, FSC allows for higher dimensional analysis, such as the recently published 40-color OMIP. Using the process of spectral unmixing, the control spectra can be used to determine the amount of each fluorochrome on the target cell.  As with traditional compensation, controls are critical and the rules must be followed. At the end of the day, FSC is another powerful tool to understand and characterize the biological process of cells at the whole cell level. 

To learn more about important control measures for your flow cytometry lab, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

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Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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