Superresolution (SR) light microscopy and electron microscopy (EM) have together revolutionized our knowledge of biological networks, offering an exceptionally precise correlation of fluorescent proteins with cellular structure. One of the most crucial elements in any SR correlative imaging methodology are the probes. However, different characteristics are demanded of probes for different imaging procedures, and mutual incompatibility is a genuine possibility.
Fluorescent proteins typically necessitate the existence of water vapor, and are not fluorescent in high vacuum. Conversely, electron imaging calls for high vacuum. Moreover, the addition of a heavy metal stain for EM contrast typically destroys fluorescence.
Correlative Light and Electron Microscopy
Standard correlative light and electron microscopy (CLEM) is consequently carried out by preliminarily imaging the sample under the required conditions for light microscopy, and subsequently introducing the EM stain and successive electron imaging. However, this multiplies the risk of sample contamination when alternating between the respective imaging technologies. Furthermore, the fact that each process is recorded on microscopes, with vastly different resolutions and fields of view, can cause further complications.
Alternative Superresolution Imaging Technique
This study suggests an alternative superresolution imaging technique, administered within a system encompassing an optical platform incorporated with a scanning electron microscope. The SR-iCLEM (superresolution integrated CLEM) technique permits the collection of coterminous correlative images in one coherent network.
Research  indicates how fluorescence from the conventionally used probes GFP and YFP is preserved in resin. This study has demonstrated that GFP and YFP blink when fixed in acrylic resin, and imaged in ambient conditions. Hela cells infected with YFP-A3 vaccinia, or transfected with GFP-C1, were high pressure frozen, freeze substituted and set in HM20 resin as already indicated . 200 nm segments were incised and the cells of interest were situated on the SR-iCLEM system in wide field (WF) mode at 200 Pa partial pressure of water, which has hitherto been proven to be the optimal pressure for WF imaging in vacuo .
Figure 1. (a) SR-EM overlay showing localization of YFP-A3 vaccinia virus within a HeLa cell. (b),(c) Enlargements of one of the boxed areas shown in A. Scale bars: 10 μm (a), 1 μm (b), 1 μm (c) N: Nucleus
Blinking of the fluorophores was instigated by intensifying the laser power to a density of 330 W/cm2, and series of roughly 30000 images were gathered at the nominated site. The system was subsequently pumped down to high vacuum and EM images were obtained by implementing a backscattered electron detector in the SEM. These SR images were reconstituted using Thunder-STORM.
This series of WF-SR-EM could be replicated at various cells across the section or at the cell across successive sections to reconstruct the 3D volume. Overlays of the WF-EM and SR-EM images produced by this imaging sequence supply data on the fluorescently labeled cellular structures.
YFP and GFP were proven to blink in vacuo when embedded in acrylic resin. Ultrathin segments of HeLa cells infected with YFP-A3 vaccinia virus were imaged utilizing this methodology and the images reconstituted, engendering a superresolution localization accuracy of 85 nm, as calculated using Fourier Ring Correlation .
The intensification of resolution between the SR and WF functions can be perceived from the WF-EM and SR-EM overlays in Fig. 1. Although the fluorescence could be attributed to singular virus particles, based on the WF images, the SR reconstruction provides robust evidence that it derives from the core of the virus. Along the same lines, the SR reconstruction shows the GFP signal to be localized in the membrane subdomains (Fig. 2).
It is crucial to point out that the allocation of the SR signal to particular organelles could not be conclusively made on the evidence of SR imaging alone, and EM data was necessitated for reference. In addition, the simultaneous EM imaging could potentially yield ‘ground truth’ data for evaluating the SR reconstruction algorithms and parameters.
The impact of the pressure of water vapor on blinking was examined and 200 Pa was identified as a ‘sweet spot’ for both fluorescence intensity and blinking of GFP in IRF sections. Further research demonstrated that, GFP blinking could be regulated by alternating across pressures.
The effect of electron contrast on blinking was also studied and high fluorescence intensity was found to correspond with greater blinking properties, whereas high EM contrast resulted in decreased blinking.
Figure 2. (a) SR-EM overlay showing localization of GFP-C1 within a HeLa cell. (b),(c) Enlargements of one of the boxed areas shown in A, showing the mitochondria (M), endoplasmic reticulum (arrows), and a putative autophagosome (*) . Scale bars: 5 μm (a), 1 μm (b), 1 μm (c)
 C. J. Peddie et al., Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells, Ultramicroscopy 143, 3–14 (2014)
 E. Brama et al., Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope, J. Chem. Biol. 8, 179–188 (2015)
 R. P. Nieuwenhuizen et al., Measuring image resolution in optical nanoscopy, Nat. Methods 10, 557–562 (2013)
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