To visualize and analyse molecular structures in their native state and in their cellular context is the overarching aim of in situ structural biology. In the classical structural biology approach purified molecules are investigated isolated from their ‘neighbours’, far from the intricate macromolecular interaction network and thus disentangled from the cellular factory. We have seen stunning results of such isolated molecular structures at atomic or near-atomic resolution obtained by single-particle cryo-electron microscopy (cryo-EM), a direct result of the development and exploitation of direct electron detection devices. However, many supra- and macromolecular assemblies involved in key cellular processes cannot be studied in isolation; their function is so deeply rooted in their cellular context that it is impossible to isolate them without disrupting their structural integrity. Thus the challenge for us now is to apply cryo-EM to protein complexes and other biological objects within their native environment, namely within cells [1,2].

The biggest obstacle in obtaining high-resolution three-dimensional (3D) information on the cellular level is the sample thickness. Resolution scales with thickness in transmission electron microscopy (TEM) and thus minimal invasive preparation methods are needed for several micrometer-sized specimens (e.g. mammalian cells). Cryo-focused ion beam milling or more general FIB micromachining is an alternative to traditional preparation methods. It allows the fabrication of self-supported lamellae, suitable for high-resolution cryo-electron tomography (cryo-ET) studies [3]. These lamellae open up ‘windows’ to the cellular interior, covering tens of square micrometers. They are electron transparent and ‘thin enough’ to obtain molecular resolution information in the cellular context. However, the fidelity with which macromolecules can be visually identified in cryo-electron tomograms is not only related to sample thickness, but on the characteristic ‘material properties’ of the biological samples. Living cells are inherently crowded, but the degree of molecular crowding can vary greatly from one cell type to another (e.g. yeast is denser than algae) and between different regions within a single cell (e.g. the nucleoplasm is denser than the cytoplasm). Thus, a compromise has to be found between what details one would like to see and how big the final tomographic volume should be. Moreover the volume represented in a typical FIB lamella is only a very small fraction of the cell. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. This fact necessitates the introduction of correlative light and electron microscopy (CLEM) in all three dimensions [4]. Together, a sample’s thickness, molecular crowding and the accuracy with which structures can be targeted will ultimately determine the level of detail that can be resolved by cryo-ET.

Here, we present our approaches towards in situ structural biology, – capturing three-dimensional structure within cells at high resolution, unaltered by sample preparation. We will give an overview on recent advances in sample preparation, data collection and data processing, including technology for FIB milling, correlative light and electron microscopy, phase plate imaging and direct electron detection. We demonstrate that these developments can be used in a synergistic manner to produce 3D images of mammalian cells in situ of unprecedented quality, allowing for direct visualization of macromolecular complexes and their spatial coordination in unperturbed eukaryotic cellular environments.

[1] Mahamid J. et al. Science 2016, 351 (6276), 969-972.

[2] Engel BD. et al. PNAS USA 2015, 112 (36): 11264–11269.

[3] Rigort A. and Plitzko JM. Archives of Biochemistry and Biophysics 2015, 581: 122-130.

[4] Arnold J. et al. Biophysical Journal 2016, 110 (4): 860–869.

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