Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events

Abstract

Summary Biological studies have relied on two complementary microscope technologies – light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale to look for unique or rare events, and it also provides an opportunity for live imaging, whereas the forte of electron microscopy is the high resolution. Traditionally light and electron microscopy observations are carried out in different populations of cells/tissues and a ‘correlative’ inference is drawn. The advent of true correlative light‐electron microscopy has allowed high‐resolution imaging by electron microscopy of the same structure observed by light microscopy, and in advanced cases by video microscopy. Thus a rare event captured by low‐resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy. Here, the potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.

Strukturierte Beleuchtung in der Hochauflösungsmikroskopie

“…Aber auch dann wird sich Hochauflösung in der Weitfeldmikroskopie nicht atomaren Grenzen nähern können. Deshalb gibt es schon jetzt Anstrengungen in der korrelativen Mikroskopie, Hochauflösungsbilder mit elektronenmikroskopischen Aufnahmen zu überlagern, um so die Position eines Proteins in einer noch hö-her aufgelösten Zellstruktur zu bestimmen [24]. Auf diesem Gebiet werden vor allem hohe Anstrengungen nötig sein, Fixierungsverfahren zu entwickeln, die beiden Technologien gerecht werden.…”

Multi-dimensional correlative imaging of subcellular events: combining the strengths of light and electron microscopy

“…3). A classical example of this approach was the study of intracellular transport routes between the Golgi apparatus and the cell membrane (Mironov and Beznoussenko 2009;Mironov et al 2000). Finally, another labeling approach, based on correlative enzyme-cytochemistry, uses the localization of reaction products of NADH oxidase by cerium-based cytochemistry (Ellis 2008).…”

“…Currently, the areas to watch out for are 'smart' probes that yield signals at high spatial resolutions and the development of new standardized sample preparation methods for producing accurate, artifact-free, and reproducible CM data. There is also consensus that much can be expected from dedicated cryo-sample preparation (McDonald 2009) and from in situ methods for sample manipulation (Hekking et al 2009;Mironov and Beznoussenko 2009). Nowadays, several companies offer solutions for rapid transfer of vitrified samples across microscopy platforms or stages that allow precise relocation of the sample between LM and EM platforms.…”

“…Source: 'ISI Web of Knowledge-Web of Science' on the choice of sample preparation method(s). Various (noninvasive) sample preparation approaches have successfully enabled CM of intact structures, such as whole-mount cells; however, the majority of approaches deal with (invasive) preand post-processing preparation procedures, such as permeabilization, detergent-extraction, sectioning, and the like (Mironov and Beznoussenko 2009). Finally, these various classes can be divided further into subclasses determined mainly by sample fixation (e.g., chemical versus physical fixation) (McDonald 2009;Subramaniam 2005) or labeling (e.g., fluorescent and/or electron-dense tags; see next section) (Giepmans 2008;Sosinsky et al 2007).…”

Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples

“…Cryo-fixation with immunogold labelling was used for subcellular localisation of H + /K + -ATPase in gastric parietal cells [98], immunolocalisation of collagen in human articular cartilage [99] and of Connexin43 proteins in rat myocardium [100]. It is worth mentioning that cryo-fixation in combination with ET [16,66,101] and CLEM [102][103][104] gives a chance to understand not only cell ultrastructure but also dynamics of the cellular processes.…”