Fluorescence cryo-microscopy: current challenges and prospects

Abstract

Studying biological structures with fine details does not only require a microscope with high resolution, but also a sample preparation process that preserves the structures in a near-native state. Live-cell imaging is restricted mostly to the field of light microscopy. For studies requiring much higher resolution, fast freezing techniques (vitrification) are successfully used to immobilize the sample in a near-native state for imaging with electron and X-ray cryo-microscopy. Fluorescence cryo-microscopy combines imaging of vitrified samples with the advantages of fluorescence labeling of biological structures. Technical considerations as well as the behavior of fluorophores at low temperatures have to be taken into account for developing or adapting super-resolution methods under cryo conditions to exploit the full potential of this technique.

Figure 1

Resolution scale. Colored bars represent the resolution achieved with the according microscopy techniques in biological samples. Opaque color corresponds to a range routinely accessible, best values that have been achieved but are not routine are indicated by increased transparency. Super-resolution fluorescence microscopy already reaches a similar range as X-ray microscopy for chemically fixed samples, but remains very challenging for live-cell imaging. CryoFM, which also provides the possibility of imaging biological structures in a near-native state, is currently limited in resolution even more than conventional fluorescence microscopy. The development of cryo immersion objectives and the adaptation of super-resolution methods for cryo conditions will lead to a dramatic increase of the resolution in cryoFM (indicated by dashed lines).

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^••].