Electron microscopy in the twenty-first century

Introduction

Biological electron microscopy (EM) is pivotal in life science research. A measure of its ubiquity is the diversity of its capacity, e.g. three-dimensional structural information about proteins and viruses at Å resolution; three-dimensional reconstructions of cellular organelles and tissues (from a few tens of nanometres to hundreds of micrometres); and two-dimensional and topographic information and elemental microanalysis at subcellular resolution. The capacity of EM can be further enhanced by correlative light and electron microscopy (CLEM) workflows, where light microscopy (LM) events are co-localized with underlying ultrastructure. In its simplest form, LM generates a ‘Google Earth’-like map to identify areas of interest, which are then studied at the ultrastructural level by EM, so overcoming both the physical size limitations imposed on samples by EM and the diffraction limits of resolution imposed by LM ( ). CLEM can also be combined with advanced EM sample preparation and imaging techniques to provide previously unheralded levels of cytoarchitectural insight into biological systems ( ). Pioneering groups now employ correlation in workflows which allow single particle cryo-EM techniques to be applied directly to structures in cells and tissues. Electron tomography is required to first determine the 3D structures of irregular samples such as cells and organelles. Once good tomograms are available, it is possible to extract multiple copies of a structure of interest and then apply single particle methods to obtain averages with greatly improved signal-to-noise ratios.

However, biological EM is not without its challenges. The electron beam is generated under vacuum at pressures and temperatures that are nominally incompatible with liquid water, yet water is the most abundant cellular constituent. Moreover, carbon-based life forms also have poor contrast in the electron microscope because they are composed mainly of light elements. In fact, carbon is so ‘transparent’ in the electron microscope that it is often employed as a film to support biological samples. To overcome these caveats, hydrated ‘live’ tissue is converted to a ‘fixed’ state; conventional protocols employ a series of steps, including chemical fixation, alcohol dehydration and resin infiltration. Heavy metal salts are added for positive staining of fixed and embedded specimens or as negative stains of whole structures that have been deposited on a support film ( ). More recently, cryo-fixation has been adopted to allow the ‘solidification of a biological specimen by cooling with the aim of minimal displacement of its components’ ( ). By using low temperature as a physical fixation strategy, the morphology and dimensions of the living material are retained and soluble cellular components are not displaced, which means that processing artefacts commonly encountered in more conventional room-temperature EM techniques are either reduced or removed. Cryo-EM often allows direct observation of specimens that have not been stained or chemically fixed.

EM in Pathology

Although the use of EM in diagnostic histopathology has declined from the height of its popularity in the 1980s, EM remains an important diagnostic tool in several well-defined areas. Indeed, guidance issued by the UK’s Royal College of Pathologists cites and supports the Association of Clinical Pathologists Best Practice No. 160, which states that ‘Many respondents [to a review of laboratory practice in renal pathology] expressed the opinion that to carry out evaluation of renal biopsy specimens without at least having the availability of electron microscopy is negligent’ ( ). Other specialized areas include the diagnosis of primary ciliary dyskinesia; certain skin/connective tissue disorders, e.g. inherited bullous lesions and Ehlers–Danlos syndrome; and specialized areas of ophthalmic pathology ( ). Despite its continued value and capacity to inform, diagnostic transmission electron microscopy (TEM) has been largely stagnant in the clinical setting; there has been little change in procedures or application for decades. In marked contrast, TEM in the research setting has experienced an explosion in capacity and capability: there is a considerable opportunity for twenty-first century EM to translate to, and to advance, clinical research and diagnostic pathology ( Fig. 1.2.1 , Video 1.2.1 ).