Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale

Abstract

We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.

Keywords: CLEM; cryoEM; cryogenic electron tomography; electron microscopy; fluorescence microscopy; super-resolution microscopy.

Figure 1

Examples from recent srCryoCLEM studies. In these overlays, the grayscale images are 2D slices from CET reconstructions, and the SR information, shown in a color overlay, identifies specifically labeled proteins. In panels a and b, there are additional colored features of the manual annotation of structures such as granules, protein fibrils, and membranes directly visible in the CET. (a) Two examples of srCryoCLEM imaging of the fusion construct VipA-PA-GFP (red/yellow) that binds the tubular structure (blue) in the bacterium Myxococcus xanthus. The insets below show the electron density associated with the blue tubular structures (16). Panel a adapted from Reference 16 with permission; copyright 2014 Springer Nature. (b, left) srCryoCLEM image of TOM20-Dronpa (color map) on top of CET data of a mitochondrion from HEK293 cells. (Right) TOM20-Dronpa localizations (green spheres) in 3D decorating the manually annotated mitochondrial membrane (purple) and cristae (blue) (17). Panel b adapted from Reference 17/CC-BY 4.0. (c, left) srCryoCLEM of the fusion construct rsEGFP2-MAP2 in U2OS cells. The color map approximates the density of rsEGFP2-MAP2 localizations. (Right) The same CET data displayed to the left absent fluorescence information showing the underlying microtubules (18). Panel c adapted from Reference 18/CC-BY 4.0. (d, left) DL overlay of a Caulobacter crescentus cell expressing the fusion construct PAmKate-McpA in which the color displays the pixelated fluorescence intensity. (Right) Single-molecule localizations of PAmKate-McpA (red circles) in which the precision of the localization is shown as the diameter of the circle (19). Panel d adapted from Reference 19/wCC-BY 4.0. Abbreviations: CET, cryogenic electron tomography; DL, diffraction limit; SR, super resolution; srCryoCLEM, super-resolution cryogenic correlative light and electron microscopy.

Figure 2

Cryogenic electron tomography (CET) sample preparation and data collection methods. (a) Transmission electron microscopy grids for cryogenic electron microscopy consist of a metallic mesh covered with a thin substrate layer. (b) Plunge freezing and high-pressure freezing are the most common methods used to rapidly freeze the sample. These methods preserve the sample in its native hydrated state encased in amorphous ice. (c) Thick samples cannot be directly analyzed due to the limited penetration of the electron beam. (Left) Focused ion beam milling uses a beam of gallium ions to ablate the sample to produce a thin lamella. (Right) Vitreous ice sectioning uses a diamond blade to cut serial thin sections. (d) CET data consists of multiple projections of the sample acquired by rotating the sample relative to the transmitted electron beam. These projections are then computationally reconstructed to produce a 3D volume that can be annotated to visualize features of interest. Data in panel d adapted with permission from Reference ; copyright 2014 Springer Nature.

Figure 3

Different forms of super-resolution microscopy that have been demonstrated under cryogenic conditions. Abbreviations: ISM, image-scanning microscopy; PALM, photoactivated localization microscopy; PSF, point-spread function; RESOLFT, reversibly saturable optical fluorescence transition; SIM, structured illumination microscopy; SMACM, single-molecule active control microscopy.

Figure 4

Experimental workflow for the conduction of a super-resolution cryogenic correlative light and electron microscopy (srCryoCLEM) experiment. Black arrows show possible paths through the workflow, and colored arrows show workflows that have been used by specific researchers.

Figure 5

Changes in the photophysical and photochemical landscape under cryogenic temperatures. (a) Fluorescent labels such as (left) a fluorescent protein or (right) a small molecule dye held in a solid host such as vitreous ice have limited conformational space to explore, and excitation/emission dipoles are held rigidly (double black arrow), leading to polarized excitation and emission. The solid host also limits the diffusion of molecular oxygen, which plays a key role in both photobleaching and triplet-state quenching. (b) Cryogenic temperatures limit thermal energy, reducing the ability to overcome thermal barriers (black arrows) in photoswitching reaction pathways on the excited- and ground-state manifolds. Optical electronic transitions are shown by colored arrows: blue for activation, green for fluorescence excitation, and red for fluorescence emission. (c) Extreme cryogenic temperatures, <4 K, can lead to narrow single-molecule absorption line widths. The local environment for each absorber is different and can shift these absorption bands, leading to each absorber (black profiles) having its own unique resonance wavelength that can be specifically excited with a narrowband tunable laser (green) to produce fluorescence. Sparse excitation using the narrow homogeneous line widths has been demonstrated as a viable active control mechanism for super resolution using organic dyes in crystalline hosts but has not been explored as a control mechanism for biological environments.