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Electron microscopy of cardiac 3D nanodynamics: form, function, future

Abstract

The 3D nanostructure of the heart, its dynamic deformation during cycles of contraction and relaxation, and the effects of this deformation on cell function remain largely uncharted territory. Over the past decade, the first inroads have been made towards 3D reconstruction of heart cells, with a native resolution of around 1 nm3, and of individual molecules relevant to heart function at a near-atomic scale. These advances have provided access to a new generation of data and have driven the development of increasingly smart, artificial intelligence-based, deep-learning image-analysis algorithms. By high-pressure freezing of cardiomyocytes with millisecond accuracy after initiation of an action potential, pseudodynamic snapshots of contraction-induced deformation of intracellular organelles can now be captured. In combination with functional studies, such as fluorescence imaging, exciting insights into cardiac autoregulatory processes at nano-to-micro scales are starting to emerge. In this Review, we discuss the progress in this fascinating new field to highlight the fundamental scientific insight that has emerged, based on technological breakthroughs in biological sample preparation, 3D imaging and data analysis; to illustrate the potential clinical relevance of understanding 3D cardiac nanodynamics; and to predict further progress that we can reasonably expect to see over the next 10 years.

Key points

  • Electron microscopy (EM) methods are currently the only means of obtaining (sub-)nanometre-scale information on most biological structures.

  • After decades of dwindling interest, seminal developments in sample preparation, imaging and analysis have led to a renaissance of EM.

  • However, data acquisition and processing remain time-consuming and laborious, and the inability to observe dynamic events in live cells limits the uptake and utility of EM.

  • Recent developments promise the advent of temporally resolved, structure–function-correlative, large-volume, 3D EM.

  • Initial applications of these new developments show EM to be a powerful driver of modern fundamental and translational heart research.

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Fig. 1: Imaging modalities for the investigation of 3D cardiac structure and function across scales.
Fig. 2: Comparison of ‘conventional’ (chemical fixation/dehydration) and ‘native’ (HPF) sample preservation.
Fig. 3: Advantages of 3D analysis.

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Acknowledgements

The authors thank the staff at the Electron Microscopy Core Facility EMBL Heidelberg for many years of on-site support and advice, as well as A. Vlachos, J. Madl and J. O’Reilly, all at the University of Freiburg, for helpful comments on the manuscript. E.A.R.-Z. is a German Research Foundation Emmy Noether Fellow (DFG #396913060). The authors are members of the German Research Foundation Collaborative Research Centre SFB1425 (DFG #422681845).

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Correspondence to Eva A. Rog-Zielinska.

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Nature Reviews Cardiology thanks Pradeep Luther, Montserrat Samsó and Christian Soeller for their contribution to the peer review of this work.

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Related links

IMOD: https://bio3d.colorado.edu/imod/

Glossary

Electron microscopy

(EM). A microscopy method in which the sample is exposed to a beam of accelerated electrons, while images are obtained either by trans-illumination of samples with spatially variable permeability for electrons, or reconstructed from electrons reflected by the sample surface in scanning mode; this allows in-plane linear resolutions of <1 nm (102–103 times higher than conventional photon-based microscopy).

Electron tomography

(ET). A 3D EM method in which a sample (typically 200–300 nm thick) is tilted relative to the imaging plane of the electron beam, and individual transmitted images are captured (usually between −60o and +60o tilt levels, and often along two mutually perpendicular tilt axes); using these images, the sample volume can be reconstructed with a native voxel size <1 nm3.

Cryo-EM

A method of imaging samples while frozen and hydrated; by omitting fixatives, solvent substitution, resin embedding and heavy metal staining, researchers can visualize native nanostructural details down to the level of single molecules.

Serial block-face or focused ion beam SEM

3D EM methods in which, between individual runs of SEM image acquisition, a thin layer of the sample surface is removed, either mechanically or using a focused ion beam; the sample volume is reconstructed from voxels whose resolution is limited by the surface removal technique, usually yielding voxels of ≥10 nm3.

Anisotropic

The property of an object, here the unitary 3D imaging readouts (voxels), that does not have edge lengths of equal size; usually, the edge lengths in the imaging plane are identical, while that between imaging planes is larger (the voxel shape is a square cuboid).

Isotropic

The property of an object, here the unitary 3D imaging readouts (voxels), that has edge lengths of equal size (the voxel shape is a cube).

Single-particle analysis

(SPA). A variant of cryo-EM that uses post-acquisition methods to three-dimensionally reconstruct individual molecules (such as proteins) by combining multiple images (usually thousands) of a population of molecules at random angular orientations; this approach allows researchers to achieve near-atomic-scale structural resolution.

Correlative light and electron microscopy

(CLEM). An approach in which a single sample is first imaged using fluorescence microscopy (to visualize the presence or dynamics of suitable reporters in live or fixed cells) and then, aided by meticulous transfer of coordinates, re-imaged using EM; correlation of the resulting data sets allows researchers to interrelate nanoscale to mesoscale structural and functional information on cells and subcellular structures.

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Kohl, P., Greiner, J. & Rog-Zielinska, E.A. Electron microscopy of cardiac 3D nanodynamics: form, function, future. Nat Rev Cardiol 19, 607–619 (2022). https://doi.org/10.1038/s41569-022-00677-x

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