Cytometry and Antibody Technology

Spectral Flow Cytometry

 How is Spectral Flow Cytometry Different from Traditional Flow Cytometry?

First let’s go through some terminology because there isn’t exactly a consensus. For a long time traditional flow cytometers (also called conventional or classical – pick your favorite term) have been configured so that one detector is assigned to a single fluorophore. Spectral flow cytometry is a newer technology where all of the detectors in the instrument are used to examine all of the fluorophores in the panel. There is one manufacturer that uses the term “full spectrum flow cytometry”, which is just another term for spectral flow cytometry.

Another difference is that traditional flow cytometers use compensation to distinguish fluorophores, whereas spectral flow cytometers use spectral unmixing. Both compensation and spectral unmixing are mathematical methods that are applied to the data.

In the CAT Facility there are several cytometers that are configured as spectral flow cytometers. An overview of the CAT Facility instruments can be found below (note: the Bigfoot can easily switch between traditional compensation and spectral unmixing):

Spectral Analyzers
Spectral Cell Sorters

Who Should Use a Spectral Flow Cytometer?

Many researchers become interested in spectral flow cytometry because they need to run a very large panel. Spectral cytometers are definitely an excellent choice for large panels (15+ markers)! However, these cytometers are fantastic with any panel size – it doesn’t matter if there are 4 or 34 markers. Anyone who is anticipating running a large panel at some point in their project is encouraged to run smaller panels on a spectral cytometer before working up to the large panel.

Spectral cytometers are also capable of separating highly overlapping fluorophores – this is what makes the large panels possible. That also means that any projects that require the use of these difficult fluorophore combinations will benefit from the spectral technology. For example, GFP and YFP are much easier to separate on a spectral cytometer than a traditional cytometer.

Feel free to contact the staff to discuss your project and determine which cytometer is the best fit for your needs!

Considerations for Setting Up an Experiment on a Spectral Flow Cytometer

The first thing to know about running a large panel is that it will take time to set up. It’s impossible to accurately predict exactly how long it will take, but a one month optimization time for a large panel is a very rough starting point – many factors, including panel size, could increase or decrease that time.

For further details on a typical spectral experiment setup workflow, see the Spectral Experiment Design resource.

Preparing samples for a spectral cytometer is very comparable to preparing samples for a traditional cytometer. The main difference to keep in mind is that that spectral cytometers gather information from the entire fluorophore emission spectra with high sensitivity. This increased sensitivity comes with many benefits (increased panel size, separation of GFP and YFP, etc,), but also means that spectral cytometers are especially sensitive to issues in the single stain controls used to calculate spectral unmixing. In fact, the most common cause of bad data is that poor quality controls were used to calculate unmixing. The good news is that anyone who already follows best practices for compensation controls for traditional cytometry will be able to easily prepare high quality spectral unmixing controls. But anyone who is used to taking some shortcuts in creating single stained controls may find that those shortcuts are incredibly detrimental to the data quality. The CAT Facility provides extensive training to ensure that users are equipped with the necessary information for creating proper controls and all other aspects of a spectral experiment.

In terms of panel design, the same strategies can be used for both traditional and spectral panels. Instead, it’s important to consider the configuration of the cytometer and plan the panel for each configuration. Because there are often major differences between manufacturers (detector types, number of detectors, detector weighting in unmixing algorithm, etc) it is unreasonable to expect that a panel will behave exactly the same way on cytometers from different manufacturers. A panel designed for a Fortessa will likely look different on an Aurora, and a panel that works beautifully on the Aurora will look different on the Bigfoot. A period of panel optimization should be anticipated when transferring a panel from one cytometer to another.

Exciting Features

Separate Highly Overlapping Fluorophores

One example of highly overlapping fluorophores is APC and Alexa Fluor 647. Distinguishing these two fluorophores would not be possible on a traditional flow cytometer. On a spectral cytometer the signatures of APC and Alexa Fluor 647 are similar, but the overall signatures are distinct. Therefore both can be used in the same panel. This allows researchers to significantly increase the number of markers in their panels. Users in the CAT Facility typically run panels around 20-30 markers, but at the moment 40 to 45 markers is possible.


Increased Resolution with Autofluorescence Extraction

All cells have intrinsic autofluorescence (figure panel A) and the intensity and signature of the autofluorescence can between cell types or even be affected by various factors and treatments (i.e. fixation). If a sample has a particularly high amount of autofluorescence, it may be useful to apply a feature called autofluorescence extraction in addition to the spectral unmixing (figure panel B,C). With the autofluorescence extraction feature turned on, the autofluorescence signature will be treated almost as if it was a fluorophore, so the unmixed data file will contain all of the typical fluorophore parameters as well as an additional parameter for autofluorescence. By moving the autofluorescence signal from the fluorophore parameters to its own dedicated parameter, the resulting fluorophore parameters may have better separation between the positive and negative populations.


Spectral Flow Cytometers at the CAT Facility

The CAT Facility has instruments from two manufacturers: the Cytek Aurora is a benchtop analyzer and the ThermoFisher Bigfoot is a cell sorter. More information on these instruments as well as resources for running spectral experiments can be found in the links below.


Spectral Analyzers
Aurora Blue, Aurora Red


Droplet-Based Sorter
Traditional and spectral capabilities