Three-dimensional super-resolution protein localization correlated with vitrified cellular context

Abstract

We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

“…Previous work describing such structures has identified them as autolysosomes, which are autophagosomes after fusion with lysosomes (Klionsky et al, 2014). In the context of INS-1E cells, these structures are likely involved in degrading vesicle content including insulin and may therefore act as a regulatory control for insulin secretion, though they were not clearly surrounded by two membranes as expected for autophagosomes (Goginashvili et al, 2015, Marsh et al, 2007, Liu et al, 2015). The lysed vesicles and probable autolysosomes may have formed here simply because of the unnaturally high levels of CgA expression following transfection.…”

“…To facilitate image correlation, we added 500 nm blue fluorospheres to the samples before plunge-freezing [as in (Bykov et al, 2016, Schorb and Briggs, 2014, Chang et al, 2014, Liu et al, 2015, Schellenberger et al, 2014)]. Significantly, these fluorospheres were visible in both cryo-LM and cryo-EM modalities.…”

“…Unfortunately, because the sample has to be kept frozen, long-working-distance air-objective lenses with low numerical apertures are used instead of oil-immersion lenses. Therefore, to increase the resolution of light microscopy under cryogenic conditions, several “super-resolution” cryo-CLEM studies have now been performed (Chang et al, 2014, Liu et al, 2015, Kaufmann et al, 2014). …”

“…Cryo-CLEM experiments on mammalian cells have been previously performed using organic fluorescent dyes or proteins tagged with fluorophores (Kaufmann et al, 2014, Liu et al, 2015, Schorb and Briggs, 2014, Schorb et al, 2016, Bykov et al, 2016, Schellenberger et al, 2014, Jun et al, 2011). Though organic dyes are very bright, it can be challenging to use these compounds to efficiently label specific proteins inside cells.…”

Structure and Epigenetic Regulation of Chromatin Fibers

“…Well-characterized chromatin fibers reconstituted in vitro, including compacted 30-nm fibers and open nucleosomal arrays, would be perfect structural references for analyzing the 3D organization of chromatin fibers in situ in these studies. The combination of cryo-EM with super-resolution fluorescence imaging techniques has been recently developed to visualize and quantify the ultrastructure of cryopreserved cells (Chang et al 2014;Liu et al 2015). The combination of genomic approaches (such as micro-C and RICC-seq) and CRISPR (clustered regularly interspaced short palindromic repeat)-based imaging techniques may enable us to probe the ultrastructure and 3D organization of chromatin fiber at defined genomic regions in the intact nuclei.…”