Today let's look at web-tools for understanding fluorophore signatures. These spectral viewer tools can be very useful for designing flow cytometry panels because they allow you to visualise where you're going to get spectral overlap and therefore spreading in the panel. Some of these tools also provide a readout of the proposed panel's complexity, which is meant to measure how well the unmixing will work. Larger complexity values mean you're more likely to have distortion of the negative events because you're trying to separate very similar signatures.
As an aside, some of these tools have dedicated panel design functions beyond comparing spectra of fluorophores. That's a whole other can of worms that we can get into another time.
Fluorofinder
Let's start with FluoroFinder. I believe this is the most complete of all the web-tools. The spectral viewer contains fluorophores from all vendors and has tools for the Aurora, the ID7000 and the Bigfoot. It does not currently have any tools for the Discover S8. The fluorophore database is very complete, containing Qdots, BV/BUVs, Star Brights, Spark dyes, Real dyes, Brilliant Blues, Biotium CF dyes, Alexa Fluors. At the moment, it's missing a couple of the newest Real dyes from BD as well as some of BioLegend's latest offerings.
There are some glitches, as you'll see below. Also, I personally dislike that I have to make an account, and that this account needs to be linked to a facility to see the available machines. If your facility hasn't told FluoroFinder which machines you have access to, it's a bit more difficult to use the tool.
Being able to compare the spectra of fluorophores from different vendors is super helpful. For instance, we can pull up CF514 and Spark Blue 550 plus CF405L and Spark Violet 538, dyes from Biotium and BioLegend, respectively. Funnily, we only get two traces.
This is because these pairs are identical. They have different brand names, but are, presumably, the same molecule under the hood. Looking at the similarity matrix tells us these have a similarity score of 1, i.e., 100%. Knowing this, we could consider conjugating our own antibodies to the CF dyes if they weren't available to purchase as pre-made conjugates.
Here's another example looking at Cytek's cFluors. Notice anything?
If we read the small print on the Cytek cFluor reagents, we can discover more rebranding of fluorophores with cFluor names. This is useful to know for panel design and if you're planning to do any assays where organic fluorophores might be damaged (phospho-flow, for example).
Also, here is where we start to see glitches in FluoroFinder. I tried pretty hard to set the colours of those traces to be distinct. They're supposed to look like this:
And the similarity/complexity index calculations make very little sense. It seems that if you add and subtract fluorophores, the calculation gets confused and quickly escalates the number (at one point, it say 3e16). The same combination gives a complexity index of 106 on Cytek Cloud whereas the example above comes to 47. That's not to say the two tools should produce exactly the same numbers, but ideally they should be internally consistent.
Sony
Sony also offers a spectral viewer tool, but it's just FluoroFinder loading on Sony's webpage in a smaller window. It's a bit harder to see, but you don't have to create an account, which is a plus. It's also a bit slow at times, presumably because it's porting back and forth to FluoroFinder. To use it, select your version of the ID7000 and add in the fluorophores.
Cytek
Cytek provides a spectral viewer tool for all versions of the Aurora. There are actually two versions; an open, legacy version that isn't being updated anymore, and Cytek Cloud, which contains most of the latest fluorophores (missing a few StarBrights, Real dyes and Fire dyes). You need a free account to use the Cloud. The Cloud contains a very useful feature estimating the reduction in stain index (separation) you will see between pairs of fluorophores. This is much more reflective of how the dyes will behave in your panel than the similarity index.
BioLegend
BioLegend offers spectral viewer tools for both the Aurora and the ID7000. They contain all the latest fluorophores from BioLegend, plus a decent selection of competitor dyes. Just deselect any lasers you lack on your machine to use the tools.
Bio-Rad
Bio-Rad's spectral analyser is general purpose, serving conventional flow, spectral flow, microscopy and western blotting. Select Spectral Analyzer, then your version of the Aurora. It's odd that it only offers support for the Aurora because, like Sony, this appears to be running off FluoroFinder.
BD
BD's web-tool is the only one I'm aware of that offers support for the FACSDiscover S8. It only covers BD instruments, though. The tool provides spectra for all the BD dyes that have been officially released. It lacks all the unique fluorophores from BioLegend and Bio-Rad, and doesn't have Qdots either. Select the Fluorochomes tab to enter dyes.
ThermoFisher Thermo's SpectraViewer works best for conventional flow or microscopy. You can add laser lines and filters to build a virtual version of your machine. There's a nice guide on how to use it here.
Beckman Coulter
This tool is also geared towards conventional flow. Limited to Beckman Coulter instruments.
Miltenyi Biotec
Another spectrum viewer that works for conventional flow. It has the MACS brand instruments, and a few others that are harder to find such as the ZE5. It's powered by EasyPanel and contains a good range of fluorophores.
If I've missed any that you use or would recommend, let me know and I'll check them out.
Comentários