What would a perfect fluorescent label look like?
This, as it turns out, is less of an abstract question and you might first think. In fact, if we think about it in terms of one primary channel for fluorescence detection and every other channel being one in which a fluorescent label contributes spread becomes a pretty easy definition —
The perfect label would emit so cleanly, it would only hit the desired detector and have fluorescence nowhere else.
I know what you’re thinking — in spectral cytometry, we no longer have one-to-one mapping of fluorescent labels with detectors. That’s absolutely correct, but if we had truly perfect labels we could leverage every single detector of any cytometer to ask one question per detector per cell. Spectral is a response to not having perfect labels — leveraging the signature of a dye to great effect for phenotyping. However, there is no magic wand that removes spillover spread
Spillover spread, which we see manifest as a “trumpet” effect in our data, is measurement error caused through photon counting, and, getting back to our concept of a “perfect” label, more spreading error occurs when more fluorophores are detected by an individual detector.
In fact, we can go some steps further as we design our perfect label:
- It should not be cross-excited at all and should emit, cleanly into 1 detector, not spilling into any other detectors
- It should only be as bright as it needs to be
The second requirement is truly interesting — and is one of the main reasons we developed the first digital fluorescent labels with variable brightness levels for 2 of our labels — NovaBlue 610 and NovaBlue 660 — with many more to come!
NovaFluors help us attack and manage the problem of spillover spread like no other technology to date with clean fluorescence, and the ability to choose fluorophore intensity.
So, in the end you can either take comfort in Salvador Dali’s admonition that “Have no fear of perfection – you’ll never reach it.” or you can get close to spectral clarity and perfection by trying out a NovaFluor CD4 Kit today.
For more light reading on this topic, we highly recommend Laura Johnston’s excellent guide to spillover.