Where in the cell is my protein?

Abstract

The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253-278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.How well can we align the fluorescence map to the cryo-EM map? 9Concluding remarks and outstanding questions 10

Super-resolution fluorescence imaging of cryosamples does not limit achievable resolution in cryoEM

“…In this report, we have presented data that addresses the question of whether exposure to intense light sources damages cryoEM samples. This is an important open question for the prospect of combining (super-resolution) fluorescence microscopy with cryoEM -a novel methodology that has been gaining popularity in recent years 17,18 With apoferritin as a representative non-photosensitive protein in the presence of the photosensitizing fluorophore rsEGFP2, we expect that our findings are also valid for cellular samples containing fluorescent fusion proteins. Given that there are no observable differences in apoferritin at <2.0 Å, we expect it to be rare for fluorescence imaging of cryosamples to lead to any observable differences in in situ cryo-electron tomography datasets, where resolution often does not reach this level.…”

Bridging length scales from molecules to the whole organism by cryoCLEM and cryoET

“…However, this is challenging because of the risk of devitrication caused by the high intensity illumination necessary for these techniques. 32,33 Additional positional accuracy can also be achieved if the label itself is sufficiently electron dense to be observed directly within the cryoEM image. For this purpose, nanogold, quantum dots, or DNA origami scaffolds may be suitable.…”

“…This is therefore sufficient to ensure the correct mapping of locations on the EM grid for cryoET data collection of the uorescent target. 32,33 An important experimental design consideration in cryoCLEM experiments is the availability and type of uorescent label, which is not trivial because this must be compatible with maintaining the viability of cells or tissues in an otherwise functional or physiologically 'normal' state. Thus, depending on the location of the target, biochemical or immunological labels must overcome physico-chemical barriers, including slow diffusion rates within tissues, lipid membranes and the crowded environment of cell cytoplasm.…”

“…This is therefore sufficient to ensure the correct mapping of locations on the EM grid for cryoET data collection of the fluorescent target. 32,33…”

Cryo-plasma FIB/SEM volume imaging of biological specimens

“…The process of thinning (milling) requires a focused ion beam; the current state of the art uses Ga + ion beams. For lamella preparation, ensuring the ROI is contained within the lamella commonly employs correlative light microscopy (cryo-CLEM) relying on fluorescent marker (usually a tagged protein) prior to, and during milling (DeRosier, 2021; Klein et al, 2021; Mahamid et al, 2015). However, fluorescence microscopy under cryogenic conditions suffers from poor axial resolution (> 300 nm), reducing its effectiveness as a targeting tool for lamella preparation.…”