Cryostats that offer a very high imaging stability usually do not have the possibility of a transfer system for imaging vitrified samples [37 and 38••]. The integrations of objectives and optical imaging paths in the column of a transmission electron microscope [8] or X-ray microscope [17], which were already equipped with sample transfer systems, represent approaches of a thermally stable fluorescence cryo-imaging system. They are beneficial for correlative cryo-microscopy from a sample handling point of view, but the NA of the optical imaging system is further reduced by spatial restrictions inside the column, limiting the resolution even more

than compared to setups for cryoFM with objectives outside the cryo chamber. Currently, the major drawback in cryoFM is the relatively low resolution. The development of a dedicated cryo immersion objective to reach an NA above 1.0 and thereby click here a resolution comparable to applications at ambient temperatures is one of the most important requirements. This will be dependent on how well an objective can be designed and built for operation under cryo conditions without creating strong aberrations due to different thermal expansion coefficients of the different elements in the objective. In parallel, super-resolution methods might be adapted learn more to cryo conditions to overcome

the diffraction limit in cryoFM. Here, the mechanical stability of the system will be

of greatest importance as the image acquisition takes substantially longer than for basic fluorescence imaging. Recently, the feasibility Sulfite dehydrogenase of reaching a stability with a sample drift in the range of 100 nm per hour has been reported [30•]. The foundation of most super-resolution methods, which have been developed for fluorescence microscopy at ambient temperatures, is the photo-switching of fluorophores [39] used for labeling the structures or proteins of interest. As discussed above, various studies have been performed to investigate photo-switching of fluorescent proteins and organic dye molecules at low temperatures. Methods based on single molecule localization [40] are dependent on the time the fluorescent molecules remain in the bright and the dark state. It has been shown that single molecule localization accuracy in the subnanometer range can be achieved using photo-switching of isolated organic dye molecules with relatively long life-times of the bright state in conjunction with suppressed photo-bleaching in cryo conditions [30•]. However, only if the life-time of the dark state is much longer than the life-time of the bright state, densely located single molecule signals can be separated from each other for a precise position determination, necessary for super-resolution imaging.