Full spectrum flow cytometry enables high-throughput characterisation of 40+ parameters at the single cell level.
While conventional flow cytometers measure peak fluorophore emission for each dye used, full spectrum flow cytometers measure the entire emission spectrum of each dye, so no specific filter is dedicated to any given signal. Instead, the instrument measures the entire emission spectrum of each fluorophore per laser and stiches them together into a unique spectral fingerprint. To delineate fluorescent signals, conventional flow cytometers use compensation and calculate a spillover matrix, whereas spectral flow cytometers use spectral unmixing, a mathematical algorithm that requires the use of reference spectra to calculate individual spectral fingerprints and put them into their own individual parameters. Meaning two fluorophores with identical peaks emission signals to be used in combination, as long as there are slight differences somewhere along the spectral fingerprint.
Full spectrum plot of Brilliant Violet 750 fluorophore. Each spectral signature is made of the collection of light in each detector and per each detector the median fluorescence intensity (MFI) is shown. The color-coding corresponds to a top-down view of contour levels of a regular counts histogram. The red is the area of the most ca4counts and there is a gaussian distribution of events showing in cooler colors.
One of the extraordinary powers of spectral cytometry is the ability to capture the autofluorescence (AF) signature of different cells types or entities within a sample and to use these as unique markers for identification, and/or for specific removal during unmixing.
AF is an intrinsic characteristic of cells caused by the presence of fluorescent biological compounds within the cell; these can include lipofuscin, riboflavin and flavin coenzymes, structural proteins, metabolites and cellular organelles. In flow cytometry AF creates background that can impair the detection of dim markers emitting light at the same wavelengths andcan attribute to false positives if not accounted for. Full spectrum flow cytometry makes it easy to identify and account for AF in any tissue type. It is also possible to use AF to identify and quantify unlabelled populations based on their autofluorescent signatures (e.g., different phytoplankton populations).
Full spectrum flow cytometry enables high-throughput characterisation of 40+ parameters at the single cell level, which gives researchers greatly increased flexibility in panel design and allowing for the generation of high-dimensional immunophenotyping panels of a size only before seen with mass cytometry. The Cytek Aurora is a full spectrum flow cytometer which can resolve 40+ colours in a single panel. There are four Auroras housed at MIMR, two with a 5-laser configuration (UV, Violet, Blue, Yellow-Green and Red), and two with a 4-laser configuration (Violet, Blue, Yellow-Green and Red). Three of our four Auroras are equipped with plate loaders to allow for high-throughput sample acquisition.
The Hugh Green Cytometry Centre are leaders in the field of full spectrum flow cytometry and have published several papers in well-known journals. We were the first group to compare full spectrum flow cytometry data with mass cytometry (CyTOF) data, and we found that the quality of the full spectrum data was optimal for high-dimensional data analysis algorithms. The following figure shows how data collected on these two technology platforms compare using UMAP, a dimensionality reduction technique that reduces high-dimensional data visualisation to two dimensions.
Full spectrum plot of Brilliant Violet 750 fluorophore. The spectral signature is made from the light collected per detector in the system, they are grouped by laser and stitched together, the median fluorescence intensity (MdnFI) is shown per detector. The colour-coding corresponds to a top-down view of contour levels of a regular counts histogram. The red is the area of peak counts and there is a gaussian distribution of events shown in cooler colours.
We have also published two protocols in Current Protocols of Cytometry to help the community successfully use full spectrum flow cytometry. One paper covers full spectrum flow cytometry panel design and the other one full spectrum flow cytometry panel optimisation.