Fluorescence CLEM in biology: historic developments and current super‐resolution applications

Abstract

Correlative light and electron microscopy (CLEM) is a powerful imaging approach that allows the direct correlation of information obtained on a light and an electron microscope. There is a growing interest in the application of CLEM in biology, mainly attributable to technical advances in field of fluorescence microscopy in the past two decades. In this review, we summarize the important developments in CLEM for biological applications, focusing on the combination of fluorescence microscopy and electron microscopy. We first provide a brief overview of the early days of fluorescence CLEM usage starting with the initial rise in the late 1970s and the subsequent optimization of CLEM workflows during the following two decades. Next, we describe how the engineering of fluorescent proteins and the development of super‐resolution fluorescence microscopy have significantly renewed the interest in CLEM resulting in the present application of fluorescence CLEM in many different areas of cellular and molecular biology. Lastly, we present the promises and challenges for the future of fluorescence CLEM discussing novel workflows, probe development and quantification possibilities.

Serial Block Face‐Scanning Electron Microscopy as a Burgeoning Technology

“…Combining SBF-SEM with other imaging techniques, such as fluorescence microscopy, can provide complementary information for a more comprehensive diagnosis. [8,17,60] New imaging modalities technologies are constantly evolving and offer a multitude of potential throughputs which may increase the applicability of SBF-SEM. [8,17,60] Developing robust correlative workflows that integrate data from multiple imaging modalities may enhance the clinical utility of SBF-SEM.…”

“…[8,17,60] New imaging modalities technologies are constantly evolving and offer a multitude of potential throughputs which may increase the applicability of SBF-SEM. [8,17,60] Developing robust correlative workflows that integrate data from multiple imaging modalities may enhance the clinical utility of SBF-SEM. [8] By addressing the challenges of sample preparation, imaging speed, data analysis, and integration with other diagnostic modalities, SBF-SEM has the potential to become a more actively utilized tool in pathology and real-time diagnosis in hospital settings.…”

Diversity of cells and signals in the cardiovascular system

“…Also, of note here are expansion microscopy methods (Wassie et al., 2019; Gallagher & Zhao, 2021) enabling subdiffraction resolutions to be attained using simple hardware and emerging techniques such as minFlux (Gwosch et al., 2020) and minSTED (Weber et al., 2021), which are achieving near‐atomic scale resolutions comparable to electron microscopy. The impressive power of these light microscopy techniques to reveal the molecular workings of cells is further enhanced by the addition of ultrastructural context through correlative light and electron microscopy (CLEM) techniques (Dillard et al., 2018; Mezache et al., 2019; Heiligenstein & Lucas, 2022; Jeong & Kim, 2022; van den Dries et al., 2022). A more detailed review of current super‐resolution methods is provided in the excellent recent review by Jacquemet et al.…”

Microspectroscopy: functional imaging of biological systems

“…Here, the gap between in vivo light microscopy and electron microscopy is closed. Cambi and co-workers present a historical overview of CLEM combined with fluorescence microscopy [3]. In particular, they describe how the engineering of fluorescent proteins and the development of super-resolution fluorescence microscopy have significantly renewed the interest in CLEM resulting in the present application of fluorescence CLEM in many different areas of cellular and molecular biology.…”