The spatial resolution of cryo fluorescence microscopy (cryo-FLM) is more limited compared to the resolution that fluorescence microscopy (FLM) at ambient temperatures can achieve. This is because cryogenic conditions inhibit the use of immersion objectives. In light microscopy, the high numerical aperture (NA) of immersion objectives determines the resolution achievable, as calculated by Abbe's diffraction limit formula. With an oil immersion objective (NA 1.4 or higher) it is possible to achieve the lateral resolution of around 200 nm1. In contrast, the air objectives that are used in cryo fluorescence microscopy are limited to NA<1 and are therefore only able to reach resolutions of 400 to 500 nm.
In addition to the limitation in NA, cryogenic conditions change many aspects of the fluorophores. This leads to some benefits as well as some challenges for cryo-FLM.
A benefit is that some fluorophores emit more photons at cryogenic temperatures, making each fluorophore brighter. However, this upside is cancelled out by the fact that about 80% of fluorophores are excited into a so-called triplet state, from where they do not emit any fluorescence. This means that only 1 in 5 fluorophores is actually contributing to the signal.
Another change is that the half-life of fluorophores is increased dramatically. This means one can use longer exposure times to get more signal without bleaching the fluorophores. However, this also means that molecules in the cell that contribute to autofluorescence have a longer half-life as well. This translates into very high background autofluorescence in certain cell types.
The last change that should be taken into account is that the excitation and emission spectra of the fluorophores can change at cryogenic temperatures. Being aware of these changes and taking them into account can save you a lot of headaches later down the line. At Delmic we optimize our integrated fluorescence solutions for work under cryogenic conditions.
1 Abbe, E. Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv für Mikroskopische Anatomie 9, 413–418 (1873)
Watch the video of our applications specialist talking about cryo-FLM