This Is How Full Spectrum Cytometry Works

The process of fluorescence starts with a photon of light, which causes the excitation of a compound that releases the absorbed energy by emitting a photon of light. This process is very rapid (typically occurring in nanoseconds), and the emitted photon is of higher wavelength (thus lower energy) than the exciting photon. Here is your guide to how full spectrum cytometry works.

Tools, such as spectral viewers, allow researchers to visualize the spectral profiles of different fluorochromes. Take the fluorescence emission of AlexaFluorⓇ488, which is displayed using the Biolegend spectral viewer.

Figure 1:  Emission profile of AlexaFluorⓇ488. The blue line is the 488 nm excitation line.  

As can be seen, the maximal excitation of this molecule is ~517 nm, but the possible range of emitted photons can reach to over 600 nm.  

In the design of most traditional fluorescence flow cytometry, each detector has a specific filter in front of it that controls the specific wavelength of captured photons. The blue line represents a 520/20 bandpass filter that allows light between 510-530 nm to be measured.  

What if, instead of capturing only 20 nm of light from the emission of this molecule, it was possible to capture the entire spectrum of the emitted fluorescence? If this were possible, more information about each molecule could be captured, allowing for new combinations of fluorochromes that are not traditionally used together. It also would allow us to use more fluorochromes per experiment. 

‘Spectral cytometry’ or better named, Full Spectrum Cytometry, takes advantage of this idea, and has been commercialized by three companies – Sony, Cytek, and Propel, the most recent entry into this field. A quick search on PubMed shows that the first articles on this topic appeared in 1980 and have been on a growth trajectory since then, as shown below. 

Figure 2:  Growth of articles in PubMed with the terms ‘Spectral Flow Cytometry’ or ‘Full Spectrum Cytometry.’

Take a look at the Sony system, which uses a prism-based separation of the incoming light, sending it to a 32 channel PMT.

Figure 3:  The SONY full spectrum cytometry technology. From the Sony webpage.  

The Cytek system, the Aurora, uses multiple Avalanche Photodiodes (APD)s with specific filters in front of each, to capture the fluorescence data. The optical layout of this system is shown below, taken from the Aurora users guide.

Figure 4:  The Aurora optical layout. 

An example of the number of channels and wavelengths that are measured can be seen in this chart for the blue detector module which has 14 detectors.

Figure 5: Filter configuration for the Blue Detection Module.

One advantage of full spectrum cytometry is capturing the autofluorescence (AF) of cells and using that as a separate spectrum that can be used to remove the AF, allowing for better resolution of the actual signal. As shown in this data from the Sony website where liposomes from cells that expressed GFP were analyzed. In the panel on the left, there appears to be a range of GFP+ expression, including some events along a diagonal. By removing the AF signal, the data is cleaned up and an accurate count of GFP+ liposomes is determined.

Figure 6:  The removal of AF helps to improve the ability to detect fluorescent signals. 

Full-spectrum cytometry also allows for closely related dyes, that would not work together in traditional fluorescent cytometry, to be used in panels on these spectral cytometers.  Take, for example, AlexaFluorⓇ 647 and APC, as shown in this spectral plot and a traditional 660/20 BP filter. It is not possible to use these with a single filter.

Figure 7:  APC (purple) and AlexaFluorⓇ 700 (blue) emission spectra. 

Now, if we look at the same two probes under full-spectrum, in this case, using a 4-laser Aurora, it is clear that these two dyes are different enough and can be separated. 

Figure 8:  APC (purple) and AlexaFluorⓇ 700 (blue) emission spectra. 

As shown from this data from Cytek, cells were gated on CD3+ cells, and CD8 (APC) vs. CD56 (AF6447) are plotted (Test), showing clear separation. For comparison, the control plot shows CD3+ cells were gated, and CD8 (APC) vs. CD56 (PE) is shown, and the results are comparable.

Figure 9:  Results of spectral deconvolution of two overlapping dyes. Data from Cytek.

Full Spectrum Cytometry offers a new mode to measure fluorescence and offers lots of opportunities to expand panels and improve detection, especially in highly autofluorescent cells. Of course, with great power comes great responsibility, and no instrument, no technique can rescue poorly designed experiments. Join us next time for part two of Spectral Cytometry, where we will discuss experimental design. 

To learn more about other important methods 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.

Written By: Tim Bushnell, Ph.D.

The process of fluorescence starts with a photon of light, which causes the excitation of a compound that releases the absorbed energy by emitting a photon of light. This process is very rapid (typically occurring in nanoseconds), and the emitted photon is of higher wavelength (thus lower energy) than the exciting photon. Tools, such as spectral viewers, allow researchers to visualize the spectral profiles of different fluorochromes. Take the fluorescence emission of AlexaFluorⓇ488, which is displayed using the Biolegend spectral viewer.

Figure 1:  Emission profile of AlexaFluorⓇ488. The blue line is the 488 nm excitation line.  

As can be seen, the maximal excitation of this molecule is ~517 nm, but the possible range of emitted photons can reach to over 600 nm.  

In the design of most traditional fluorescence flow cytometry, each detector has a specific filter in front of it that controls the specific wavelength of captured photons. The blue line represents a 520/20 bandpass filter that allows light between 510-530 nm to be measured.  

What if, instead of capturing only 20 nm of light from the emission of this molecule, it was possible to capture the entire spectrum of the emitted fluorescence? If this were possible, more information about each molecule could be captured, allowing for new combinations of fluorochromes that are not traditionally used together. It also would allow us to use more fluorochromes per experiment. 

‘Spectral cytometry’ or better named, Full Spectrum Cytometry, takes advantage of this idea, and has been commercialized by three companies – Sony, Cytek, and Propel, the most recent entry into this field. A quick search on PubMed shows that the first articles on this topic appeared in 1980 and have been on a growth trajectory since then, as shown below. 

Figure 2:  Growth of articles in PubMed with the terms ‘Spectral Flow Cytometry’ or ‘Full Spectrum Cytometry.’

Take a look at the Sony system, which uses a prism-based separation of the incoming light, sending it to a 32 channel PMT.

Figure 3:  The SONY full spectrum cytometry technology. From the Sony webpage.  

The Cytek system, the Aurora, uses multiple Avalanche Photodiodes (APD)s with specific filters in front of each, to capture the fluorescence data. The optical layout of this system is shown below, taken from the Aurora users guide.

Figure 4:  The Aurora optical layout. 

An example of the number of channels and wavelengths that are measured can be seen in this chart for the blue detector module which has 14 detectors.

Figure 5: Filter configuration for the Blue Detection Module.

One advantage of full spectrum cytometry is capturing the autofluorescence (AF) of cells and using that as a separate spectrum that can be used to remove the AF, allowing for better resolution of the actual signal. As shown in this data from the Sony website where liposomes from cells that expressed GFP were analyzed. In the panel on the left, there appears to be a range of GFP+ expression, including some events along a diagonal. By removing the AF signal, the data is cleaned up and an accurate count of GFP+ liposomes is determined.

Figure 6:  The removal of AF helps to improve the ability to detect fluorescent signals. 

Full-spectrum cytometry also allows for closely related dyes, that would not work together in traditional fluorescent cytometry, to be used in panels on these spectral cytometers.  Take, for example, AlexaFluorⓇ 647 and APC, as shown in this spectral plot and a traditional 660/20 BP filter. It is not possible to use these with a single filter.

Figure 7:  APC (purple) and AlexaFluorⓇ 700 (blue) emission spectra. 

Now, if we look at the same two probes under full-spectrum, in this case, using a 4-laser Aurora, it is clear that these two dyes are different enough and can be separated. 

Figure 8:  APC (purple) and AlexaFluorⓇ 700 (blue) emission spectra. 

As shown from this data from Cytek, cells were gated on CD3+ cells, and CD8 (APC) vs. CD56 (AF6447) are plotted (Test), showing clear separation. For comparison, the control plot shows CD3+ cells were gated, and CD8 (APC) vs. CD56 (PE) is shown, and the results are comparable.

Figure 9:  Results of spectral deconvolution of two overlapping dyes. Data from Cytek.

Full Spectrum Cytometry offers a new mode to measure fluorescence and offers lots of opportunities to expand panels and improve detection, especially in highly autofluorescent cells. Of course, with great power comes great responsibility, and no instrument, no technique can rescue poorly designed experiments. Join us next time for part two of Spectral Cytometry, where we will discuss experimental design. 

To learn more about other important methods 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.

Join Expert Cytometry's Mastery Class
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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