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 has been the result of imaging studies utilizing either light- or electron-based microscopic analyses. These classical approaches, while insightful, are nonetheless limited either by restrictions in resolution or by the sheer complexity of generating multidimensional data. Recent advances in the use and application of X-rays to acquire micro- and nanotomographic data sets offer an alternative methodology to visualize cellular architecture at the nanoscale. These new approaches allow for the subcellular analyses of unstained vitrified cells and 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.