The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

What photon from yonder fluorochrome breaks?  It is … umm… hmmm. Let me see. Excitation off a 561 nm laser, with an emission maximum of 692 nm.

I’m sure if Shakespeare was a flow cytometrist, he might have written that very scene. But the play is lost in time. However, since the protagonist had difficulty determining what fluorochrome was emitting photons, let’s consider how this could be figured out.

In my opinion, one of the handiest flow cytometry tools is the spectral viewer. This tool helps visualize the excitation and emission profile of different fluorochromes, as well as allowing you to add both excitation lasers and emission filters. Additionally, with a myriad of new fluorochromes on the market, being able to visualize the spectral profiles in conjunction with more traditional fluorochromes is useful when deciding whether to expand a panel or switch fluorochromes.

So, why might you want to switch fluorochromes? There are many reasons to consider switching. First and foremost is the desire to take advantage of newly released reagents.

There have been a lot of changes in the flow cytometry field since I last wrote about this topic. From companies being acquired to the release of new spectral viewers to the need for both spectra and spectral analyzers.

So, before we dive into the power of these tools, peruse this list (presented in alphabetical order). 

What Can A Spectral Viewer Tell You?

Figure 1 shows the typical spectrum for a fluorochrome – in this case FITC. The output of the FITC spectrum is shown in panel A. The dashed lines represent the excitation profile for the fluorochrome, while the solid, filled line shows the emission profile. By moving the cursor over the excitation or emission profile, the percent of max excitation or emission is displayed (Fig.1B). Finally, it is possible to place an excitation laser and emission filter on the graph. This allows you to better visualize where there may be potential issues when more than one fluorochrome is shown.  

Another nice feature of spectral viewers is that by selecting different excitation wavelengths, some of these tools will show the output normalized to the excitation of the given fluorochrome by the given wavelength. As show in Figure 2A, when the 488 nm laser is selected, the emission profiles are shown. In the table below, the spectra is a column with %Max, indicating the percent of maximal excitation. The table next to that shows the percent emission being captured by the selected filter. In Figure 2B, the excitation laser is changed to 488 nm. The resulting spectra are shown based on the excitation percentage for each fluorochrome. Notice PerCP-Cy5.5 is higher than the other spectra. By looking at the %Max, we find that PerCP-Cy5.5 has about a 40% with 405 nm light. The consequence of this becomes apparent during panel design.

In Figure 3A, the excitation laser is 405 nm and the spectra for BV711 and PerCP-Cy5.5 are plotted. If you look at the table, you can see that 41.3% of the PerCP-CY5.5 leaks into the 710/50 filter used to measure BV711. Thus, if both these fluorochromes are in a panel, it’s important to ensure they’re on mutually exclusive antigens. That way, the spillover from PerCP-CY5.5 does not impact the signal of cells labeled with BV711.

By looking at the excitation profile of the fluorochrome of interest, we can also gain insight into its characteristics. Figure 3B illustrates this using BV711. Notice the small excitation around 680-690 nm. This second excitation peak is what one would expect if the dye of interest was a tandem dye, which BV711 is.

How Do Spectral Viewers Help Full Spectrum Flow?

The traditional spectral viewers are focused on the premise of measuring one fluorochrome in a single detector. With full spectrum flow cytometry becoming the new standard in measuring fluorescence, these systems operate on the premise of measuring the whole spectrum across multiple excitation lines. Thus, a new tool was developed – one that displays the full spectrum of a given fluorochrome. The output of this type of spectral viewer is shown below in Figure 4 using FITC, BV711 and PerCP-CY5.5.

This viewer is specific for the Cytek Aurora, and in this case, the 5-laser system. Across the bottom are the different detectors for each excitation laser (i.e., UV1-UV16 represent the 16 detectors off the UV laser excitation and so on). Thus, it’s possible to look at the spectra across the excitation lasers at once. A nice feature is that it’s possible to highlight a specific fluorochrome, as shown in Figure 4B. In this case, BV711 was selected revealing 5 different peaks. The right panel shows that PerCP-5.5 also has 5 peaks, but sufficiently different from BV711, making it easier to use these two fluorochromes together on a full spectrum system rather than a traditional fluorescent flow cytometer.

It’s also possible to explore in silico combinations of fluorochromes as shown in Figure 4C which plots EGFP, FITC and YFP. In general, these can’t be easily used on a traditional fluorescent flow cytometer, but with spectral cytometry, it’s possible to use them together – although optimization may prove difficult.

Conclusion

Spectral viewers are an essential tool in the flow cytometrist’s bag of tricks. With new fluorochromes coming onto the market, it’s useful to compare the fluorochromes being used in a panel to see if these new ones may offer some advantages. Of course, you may have to bounce around to different viewers depending on the fluorochrome.

So, bookmark these links (or just this article), play with them to understand what the output reveals, and be ready to use them when the time comes to design a panel.

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