Spectral Flow Cytometry: What You Need To Know

1 How To Guide Flow cytometry is a well-established fluorescence-based technology that allows researchers to stain cells with fluorescent probes or dyes to look at antigen expression, DNA content or functional aspects of a cell on a single cell basis. This can be done very quickly, enabling thousands of cells to be analyzed per second. As all fluorochromes will have an emission spectrum spreading over tens or hundreds of nanometers in the visible electromagnetic spectrum, combining fluorochromes can be problematic. Conventional flow cytometers do not measure the entire spectrum of emission but use bandpass filters to select the wavelengths of light around the maximum emission of a fluorochrome. This has two major consequences. Firstly, there will be spectral spillover, or photons ending up in detection channels that we don't want, and secondly, we are not collecting all the emitted light from a fluorochrome, just a selected band. Although spectral analysis has been used in microscopy for a number of years,1 spectral flow cytometry has recently emerged as a powerful technology to address these issues by measuring the whole emission after excitation by each laser in the cytometer. As with all new technologies though, understanding the strengths and possible weaknesses is important for end users. Here, we will look at the most important considerations to run a spectral flow cytometry experiment successfully. How does spectral cytometry work and how is it different to conventional cytometry? Spectral cytometers, just like conventional flow cytometers, will hydrodynamically focus cells to align them to allow single cell measurements. They still use lasers to excite fluorochromes, but they differ in that they will use all lasers to excite all fluorochromes rather than restricting excitation to a single laser. In this way, we can get emission across the entire visible light spectrum. Rather than using relatively wide bandpass filters, spectral cytometers have more, but narrower, detection filters or channels (anywhere from 60–184 depending on the cytometer). In conventional cytometry we are limited by the number of detection channels – we cannot have more fluorochromes than channels available. But with spectral cytometry we will almost always have more channels than fluorochromes in our panel. This has the advantage that one configuration of cytometer will fit most applications, we don’t have to worry about assigning fluorochromes to specific detection channels. Filters and lasers in the cytometer We still need to think about the lasers and filters within the cytometer, so it is still useful to know which lasers we have in our spectral cytometer. For example, it would be hard to use UV-excitable dyes without a UV laser being installed in the cytometer. Not all spectral cytometers will have the same bandpass filters as different manufacturers have designed different configurations. This is no different to conventional cytometry where panels that are designed for one particular cytometer may look slightly different on another. Spectral Flow Cytometry: What You Need To Know Derek Davies SPECTRAL FLOW CYTOMETRY: WHAT YOU NEED TO KNOW 2 How To Guide Dealing with spectral spillover We still have to deal with spectral spillover (overlapping emissions of dyes), but we do this differently. We will still need single color controls (which could be cells or beads) as these will allow us to see the distribution of photons from a fluorochrome into the detection channels on the cytometer. In a conventional cytometer, we use the process of compensation2 to account for spectral overlap. In full spectrum analysis, we do this in a slightly different way using what we generally call spectral unmixing, a form of least squares regression analysis.3 With compensation, we look at x fluorochromes and x detectors, so this is a square matrix that will have one solution. But in unmixing, we have x fluorochromes but x + n detectors, which produces a rectangular matrix to which there may be more than one solution, so we have to use a best fit. However, with the correct controls we can be confident that the unmixing will be a true reflection of the overlap. Why are people moving towards full spectrum analysis? The ability to use more fluorochromes Heterogeneity in populations means that the more analytes we can measure, the more specifically cell populations may be defined. With the introduction of spectral cytometry, we have suddenly moved from being able to measure around 25–30 parameters to around 40–50.4, 5 This does make panel design more difficult but the same rules we have always used still apply. We have to think about the relative brightness of our fluorochromes, our antigen expression levels and antigen expression patterns as well as which markers are expressed on the cells of interest i.e. co-expression. And we always need to think about sources of spillover that may reduce resolution and thus the ability to see a particular population clearly. So, there is still quite a lot of optimization to be done and obviously this is more important and will take longer if we're doing a 50-color experiment than if we are doing a 5-color experiment. The ability to use similar fluorochromes Because we are looking at the whole visible spectrum and exciting fluorochromes with all lasers in the cytometer, there are sufficient differences in the emission spectra for us to be able to use combinations of fluorochromes that would not be possible on a conventional cytometer. The classic example of this is the combination of allophycocyanin (APC) and Alexa Fluor 647. They are both optimally excited by the same laser (red, 640 nm) but they have slightly different peak emissions and if we look at the excitation and emission from the violet (405 nm), blue (488 nm) and yellow (561 nm) lasers, we will see that the spectrum for these two dyes are sufficiently different for us to use them together on a full spectrum flow cytometer. However, we would need to think carefully about which populations to use them for. Ideally, they would be on mutually exclusive markers, for example, in peripheral blood a T-cell marker and a B-cell marker. The ability to account for autofluorescence The final big advantage of full spectrum analysis is that we can use the autofluorescence of our negative cells as a fluorescence parameter in its own right. This can be particularly important in heterogeneous populations where some cell types may have higher autofluorescence than others6 and it is also useful in tissues where cells are naturally highly autofluorescent, for example liver cells, lung cells or solid tumors. SPECTRAL FLOW CYTOMETRY: WHAT YOU NEED TO KNOW 3 How To Guide Practicalities Staining will take longer and with so many antibodies it is certainly worth considering using mastermixes, which will also reduce staining variability. A recent paper showed that overnight staining at lower antibody concentrations may also be worth considering.7 We will still need the appropriate controls, particularly fluorescence minus one (FMO) controls, which allows an appropriate cut-off for positivity to be determined. There are other considerations such as booking an appropriate amount of time on the cytometer, ensuring that sufficient events are acquired, and that files are named and annotated consistently. The MiFlowCyt guidelines can also help when thinking about presentation of data and descriptions of experiments.8 Of course, larger panels will also be more expensive, data files will be larger (which may have storage implications) and there will be some data analysis challenges. Conclusions and the future Should I move to spectral flow cytometry? Well, the general consensus in the cytometry field is that the majority of cytometers within the next few years will be spectral. However, there still remains a place for conventional cytometry, not everybody needs multicolor analysis and sometimes single marker expression is an equally valid experiment. Additionally, as new equipment needs to be acquired to have this capability, there may be funding cycles to be considered. New technology always brings new opportunities, and the rapid development and acceptance of spectral flow cytometry has led to the development of higher multiplexed panels than were previously possible. This will be particularly important in the dissection of complex heterogenous samples. However, if we want to increase the number of measurable analytes, new and novel dye development will be particularly important in the coming years. There are also areas of the spectrum, such as the deep UV and in the infrared regions, that we are currently not using. In addition, there is the intriguing possibility of using just the autofluorescence of cells to determine biological changes. Looking at autofluorescence, alone may be particularly important in cell death assays, cell proliferation and cell signaling. It will be interesting to see what advantages this brings in the coming years. Final thoughts As always, researchers need to weigh up the pros and cons of any technology to be able to make informed decisions as to whether it would give additional insights to their biological question. However, there is little doubt that spectral flow cytometry has the potential to accelerate scientific discovery in the future. Sponsored by SPECTRAL FLOW CYTOMETRY: WHAT YOU NEED TO KNOW 4 How To Guide References 1. Zimmermann T, Rietdorf J, Pepperkok R. Spectral imaging and its applications in live cell microscopy. FEBS Letters 2003; 546. doi:10.1016/S0014-5793(03)00521-0 2. Roederer M. Compensation in flow cytometry. Curr Protoc Cytom. 2002: Chapter 1:Unit 1.14. doi:10.1002/0471142956. cy0114s22. 3. Novo D, Grégori G, Rajwa B. Generalized unmixing model for multispectral flow cytometry utilizing nonsquare compensation matrices. Cytometry. 2013; 83A: 508-520. doi:10.1002/cyto.a.22272 4. Park LM, Lannigan J, Jaimes MC. OMIP-069: Forty-color full spectrum flow cytometry panel for deep immunophenotyping of major cell subsets in human peripheral blood. Cytometry. 2020; 97: 1044-1051. doi:10.1002/cyto.a.24213 5. Konecny AJ, Mage PL, Tyznik AJ, Prlic M, Mair F. OMIP-102: 50-color phenotyping of the human immune system with indepth assessment of T cells and dendritic cells. Cytometry. 2024; 105(6): 430–436. doi:10.1002/cyto.a.24841 6. Bourdely P, Petti L, Khou S, et al. Autofluorescence identifies highly phagocytic tissue-resident macrophages in mouse and human skin and cutaneous squamous cell carcinoma. Front Immunol. 2022;13 doi:10.3389/fimmu.2022.903069 7. Whyte CE, Tumes DJ, Liston A, Burton OT. Do more with less: Improving high parameter cytometry through overnight staining. Curr Protoc. 2022 2: e589. doi:10.1002/cpz1.589 8. Lee JA, Spidlen J, Boyce K, et al. MIFlowCyt: The minimum information about a flow cytometry experiment. Cytometry. 2008; 73(10):926-30. doi:10.1002/cyto.a.20623 About the Author Derek Davies has been involved in Cytometry for over 40 years, firstly as a researcher and then as the head of a large core facility. He has trained thousands of researchers to use analytical flow cytometers and cell sorters as well as advising on experimental design and data analysis.