Probably the most important characteristic of fluorescent molecules at low temperatures for the field of BMS-354825 in vivo single molecule spectroscopy are the very narrow absorption and emission spectra . But also in fluorescence microscopy the temperature dependency of the molecules
spectra could be utilized for distinguishing different fluorophore types in cryoFM whose spectra would overlap at ambient temperatures (e.g. improved multi-color measurements). A crucial factor besides the photophysical considerations for cryoFM imaging is the design of the optical system. Most setups developed for cryoFM originated from the field of correlative cryo-microscopy [32••]. The main advantage Epacadostat ic50 of these
implementations is the ability for transferring vitrified samples. Therein, the samples have to be kept below the devitrification temperature of ∼135 K to maintain the structural preservation , thus precooling of the cryo stage is required before insertion of the sample. Additionally, a transfer system allows extraction of the sample for subsequent electron or X-ray microscopy imaging. The typical design of cryo stages for correlative applications consist of an insulated liquid nitrogen cooled chamber, that can be opened for sample exchange, and a long working distance air objective that is kept at ambient temperature either via separation by a glass window or via a temperature
gradient from the sample (Figure 2a,b). Thus the NA of the optical system is limited to <1.0, typically ∼0.8. This restricts the resolution to a range of 400–500 nm. Additionally the number of detectable photons is reduced by almost a factor of 2 compared to high NA oil immersions objectives used for fluorescence microscopy at ambient temperatures. One implementation has shown the principle feasibility of immersing an oil objective with an NA of 1.3 into a cryogen in a liquid nitrogen cooled optical setup . However, neither the resolution nor the imaging quality achieved with this system has been reported find more quantitatively. The application of tomographic imaging to cryoFM allows 3D isotropic resolution [34•]. Another challenge for cryoFM is the stability of a dedicated cryo stage. Small reservoirs of liquid nitrogen, connections to supply hoses and temperature gradients between different components of the cryo stage make these designs very susceptible for having mechanical instabilities while imaging. This can become a problem for precise correlative measurements or in the case of more advanced cryoFM applications (e.g. co-localization studies) or super-resolution techniques. Closed and vacuum insulated systems (Figure 2c), which are mainly used in single molecule spectroscopy, can offer much better temperature and mechanical stabilities [30•].