 Super-resolution microscopy

Super-resolution microscopy recovers spatial detail beyond the conventional diffraction limit by acquiring information beyond a single ordinary image. Stimulated emission depletion microscopy confines detectable fluorescence with a depletion beam; photoactivated localization microscopy separates emitters in time and estimates each emitter's position before combining the estimates.

The distinction between finding one isolated emitter and resolving many adjacent emitters matters. An isolated point spread function can be localized more precisely than its width if enough photons are collected, but overlapping unknown emitters cannot simply be treated as isolated ones. Label density, photon shot noise and specimen motion therefore constrain the final optical resolution, even when the reconstructed image uses very small pixels.

Super-resolution means resolution beyond the diffraction limit.

First you shine a lot of light which saturates most fluorophores, leaving very few active.

They you can observe fluorophores firing one by one. Their exact position is a bit stochastic and beyond the diffraction limit, but so long as there aren't to many in close proximity, you can wait for it to fire a bunch of times, and the center of the Gaussian is the actual location.

From this we see that super-resolution microscopy is basically a space-time tradeoff: the more time we wait, the better spacial resolution we get. But we can't do it if things are moving too fast in the sample.

Tradeoff with cryoEM: you get to see things moving in live cell. Electron microscopy fully kills cells, so you have no chance of seeing anything that moves ever.

Caveats: