Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches

Abstract

Much of our understanding of the spatial organization of and interactions between cellular organelles and macromolecular complexes have been the result of imaging studies utilizing either light- or electron-based microscopic analyses. These classical approaches, whilst insightful, are nonetheless limited by either restrictions in resolution or by the sheer complexity of generating multi-dimensional data. Recent advances in the use and application of X-rays to acquire micro- and nano-tomographic datasets offer an alternative methodology to visualize cellular architecture at the nanoscale. These new approaches allow for the sub-cellular analyses of unstained vitrified cells, three-dimensional localization of specific protein targets, and have served as an essential tool in bridging light and electron correlative microscopy experiments. Here, we review the theory, instrumentation details, acquisition principles and applications of both soft X-ray tomography and X-ray microscopy, and how the use of these techniques offers a succinct means of analyzing three-dimensional cellular architecture. We discuss some of the recent work that has taken advantage of these approaches, and detail how they have become integral in correlative microscopy workflows.

Deconstructing 3D Structured Materials by Modern Ultramicrotomy for Multimodal Imaging and Volume Analysis across Length Scales

“…It is presently one of the hot topics in structural cell biology. [29] However, resolution of these non-invasive techniques is limited, and detailed, high-resolution chemical information at the nanometer scale in 3D is simply not achievable so far. [3] To overcome the limitation of spatial resolution in 3D, invasive techniques such as focused-ion-beam milling [8] as well as plasma [30] and laser milling [31] have been developed and in recent years variants of ultramicrotomy sectioning have been advanced for biological samples.…”

Deletion of taf1 and taf5 in zebrafish capitulate cardiac and craniofacial abnormalities associated with TAFopathies through perturbations in metabolism

“…X-ray microscopy was performed using a Zeiss Xradia Versa 520 XRM microscope at WUCCI ( Bayguinov et al, 2020 ). Zebrafish larvae were fixed in 4% paraformaldehyde, then transferred to Lugol's Iodine for 3 weeks, after which samples were imaged.…”

Analysis of cell–nanoparticle interactions and imaging of in vitro labeled cells showing barcorded endosomes using fluorescent thiol-organosilica nanoparticles surface-functionalized with polyethyleneimine

“…Intraendosomal sorting could be expected to play important roles, such as an enhancement of the signaling response to molecules in the endosome and improvement of the efficiency of antigen presentation. To clarify these hypotheses, ultraprecise and comprehensive morphological studies of cellular endocytosis using higher resolution and multimodal approaches on a nanoscale, such as correlative light and electron microscopy (CLEM) with 3-D reconstruction, [41][42][43] are required as important approaches and are under investigation.…”