Figure 2.

Schematic overview of different stand-alone cryo stage and cryostat designs for cryoFM. (a) Cryo stage design for an inverted microscope setup. Cooling is achieved by pumping liquid nitrogen (LN2) through the cryo stage. Imaging is performed through a long working distance air objective which is separated from the cryo environment by a glass window and kept at ambient temperature (for details see [45]). (b) Cryo stage for an upright microscope configuration [10] with an autonomous LN2 cooling mechanism. The objective is dipping into the cold nitrogen atmosphere inside the cryo stage. An air objective with a long working distance is required to avoid heat transfer to the sample. Additional cooling of the objective allows reducing the working distance and thus an objective with a larger NA can be used to increase resolution [36]. Full integration of the objective into the cryo stage and immersion imaging under cryo conditions have been shown with a design of overall similar principle [9]. (c) Vacuum insulated cryostat. Temperature stability and range (also liquid helium (LHE) cooling possible) is increased compared to cryo stages as shown in (a) and (b), but the implementation of a sample transfer mechanism is very complicated. Higher NA air objectives can be used if placed inside the cryostat, but the NA is limited to <1.0 due to the incompatibility of cryo immersion with vacuum [37,38^••].