High‐Resolution Microscopy for Structural Studies of Biological Systems in Cells


Atomic resolution structures of proteins are essential for understanding the biological roles played by these molecules. However, in order to understand fully how the molecules function in living systems, other techniques must be applied that provide insights into how they interact in cells and tissues. No single technique can provide all the information needed, but a multi‐technique approach can be applied to provide information on different aspects of molecular structure and interaction in cells and tissues. Methods that can be applied include a range of fluorescence‐based microscopy techniques, including ‘super‐resolution’ microscopy, which provides subdiffraction limit images. When accompanied by other techniques such as single particle tracking, electron microscopy and molecular dynamics simulation, these methods can provide new insights into the functioning of complex biological processes.

Key Concepts

  • Linking structure and function requires methods that can provide structural information on molecules and molecular complexes in cells.
  • ‘Super‐resolution’ optical methods that can image below the diffraction limit allow us to fill in the resolution gap between conventional optical and electron microscopy.
  • Single‐molecule techniques allow us to investigate individual interactions without the requirement to synchronise cellular processes.
  • Often no single technique can provide all the information needed so an approach combining different imaging methods is often required.
  • Molecular dynamics simulations can help in the interpretation of in cellulo structural data.

Keywords: macromolecular structure; super‐resolution microscopy; single‐molecule methods; cryo‐electron microscopy; FRET‐FLIM; molecular dynamics simulation; epidermal growth factor receptor; multimodal imaging

Figure 1. Techniques available for structural studies. Upper: Some of the more common techniques, together with their resolution ranges (crystal structure is the Drosophila EGFR ectodomain). Lower: Aspects of structure that can be investigated using these methods. Reproduced with permission from Alvarado et al., . © Springer Nature.
Figure 2. Application of super‐resolution microscopy to study receptor clustering in cells. (a) dSTORM image of HER4 in CHO cells, labelled with affimer‐Alexa 647 (bar 2 µm). (b) HER4 cluster radius determined from data in a by Bayesian analysis. (c) Number of HER4 molecules per cluster determined from the radii shown in b. (a–c) Source: Tiede, https://elifesciences.org/articles/24903. Licensed under CC by 4.0. (d) Analysis of HER3 molecules per cluster from STORM image of receptors labelled with affibody‐Alexa 647 in SKBR3 cells. The data show the effect of the ligand neuregulin (NRG) and the tyrosine kinase inhibitors Lapatinib and Bosutinib on receptor clustering. Source: Claus, https://elifesciences.org/articles/32271. Licensed under CC by 4.0.
Figure 3. FLImP to determine the oligomer architecture of EGFR. (a) FLImP distribution of separations of EGF bound to EGFR in CHO cells. (b) Full‐length structural model of an EGFR tetramer as a dimer of active dimers assembled by face‐to‐face interactions. Source: Needham, https://www.nature.com/articles/ncomms13307. Licensed under CC by 4.0.
Figure 4. EGFR in CHO cells imaged by transmission electron microscopy. (a) Cells were labelled with EGF that had been conjugated with 5 nm gold nanoparticles, fixed with paraformaldehyde and glutaraldehyde and stained with 1% reduced Osmium, 1% tannic acid and 1% sodium sulphate. The samples were dehydrated in serial Ethanol, incubated in a 50/50 solution of the embedding medium Embed 812 in propylene oxide for 60 min, then infiltrated with pure Embed 812 twice for 90 min and hardened overnight at 60 °C. Blocks were cut into 70 nm thick sections using a Leica UC6 ultramicrotome and mounted on formvar‐coated copper slot grids (Synaptek). Arrows show uncoated vesicles, and the circle shows a clathrin‐coated vesicle. (b) EGFR labelled with EGF‐Au in CHO cell sections post‐stained on‐grid with lead citrate for 5 s for added contrast of membrane structures. (c) Expanded view of the area bounded by the red box in b. Two labelled EGFR are visible with a separation of ∼12 nm, consistent with the EGFR dimer when taking into account the dimensions of the ligand. Imaging was performed on a FEI Tecnai G2 Spirit BioTWIN TEM microscope with Gatan Orius CCD camera at 120 kV. Bar for all panels 100 nm.
Figure 5. Single particle tracking to investigate EGFR mobility in cells. (a) Single particle tracks of EGFR in live CHO cells. The magenta lines show tracks accumulated over approximately 12 s. For clarity, the underlying image is shown in a negative format so the darker spots are fluorescent EGFR visible in the selected frame. (b) Mean squared displacement (MSD) versus time plots for PIP2 (blue), EGFR (green) and EGFR in the presence of the cholesterol‐depleting treatment methyl‐β‐cyclodextrin (red). (c) FLImP data showing EGF separations on EGFR in CHO cells exposed to methyl‐β‐cyclodextrin. Source: Needham, https://www.nature.com/articles/ncomms13307. Licensed under CC by 4.0.
Figure 6. Use of FRET‐FLIM to investigate EGFR conformation in cells. (a) Typical FRET‐FLIM image of EGFR in cells (bar 50 µm). (b) FRET efficiency used to calculate DOCA from EGF to the plasma membrane; data in red are from receptors treated with mAb 2E9 to block low‐affinity binding, data in blue are in the absence of 2E9 and represent the entire EGFR population. (c) Extended dimer model of EGFR; EGF‐membrane distance is consistent with the DOCA measurement for low‐affinity receptors. (d) Model of EGFR relaxed on the plasma membrane; EGF‐membrane distance is consistent with the DOCA measurement for high‐affinity receptors. Reproduced with permission from Tynan et al., . © American Society for Microbiology.


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Further Reading

Becker W (2012) Fluorescence lifetime imaging‐‐techniques and applications. Journal of Microscopy 247 (2): 119–136.

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Shashkova S and Leake MC (2017) Single‐molecule fluorescence microscopy review: shedding new light on old problems. Bioscience Reports 37. DOI: 10.1042/BSR20170031.

Shen H, Tauzin LJ, Baiyasi R, et al. (2017) Single particle tracking: from theory to biophysical applications. Chemical Reviews 117 (11): 7331–7376.

Sydor AM, Czymmek KJ, Puchner EM and Mennella V (2015) Super‐resolution microscopy: from single molecules to supramolecular assemblies. Trends in Cell Biology 25 (12): 730–748.

Yu J (2016) Single‐molecule studies in live cells. Annual Review of Physical Chemistry 67: 565–585.

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Clarke, David T, Zanetti‐Domingues, Laura C, and Martin‐Fernandez, Marisa L(Dec 2018) High‐Resolution Microscopy for Structural Studies of Biological Systems in Cells. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0027945]