Photoactivated Localization Microscopy (PALM) can determine the position of a fluorophore with an error that is approximately equal to the point spread function (PSF) of the fluorescence microscope divided by the square root of the number of photons. This technique relies on capturing only one fluorophore within a given region, by using a photo-activatable fluorescent protein (PA-FP). One activates the PA-FP population weakly so that only one or two molecules are activated within an area equal to the PSF, maps them until the activated proteins bleach, and then repeats the cycle.

We are currently developing a cryogenic-PALM system intended to keep the specimen at temperatures below -140˚C. There are two advantages to this technique: (1) the bleaching rate is reduced so that one collects more photons and hence achieves greater localization precision, and (2) plunge-freezing (for thin samples) or high-pressure freezing (for thicker samples) combined with a sub -140˚C cold stage can preserve the specimen in an essentially native state without the need for fixatives.

Using a custom-built PALM microscope we have been able to show that PA-FPs can be activated at temperatures below -140˚C. We also find that the bleaching rate relative to the rate of fluorescence appears to be reduced by at least an order of magnitude. Currently, we are developing a cold stage that will be able to accommodate a high numerical aperture objective, with the potential to achieving 1-2nm molecular localization.
Cryogenic Photoactivated Light Microscopy