Chapter 4 - Correlative light–electron microscopy

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Abstract

Recent advances in combining light and electron microscopy imaging techniques provide the means to correlate dynamic biological processes with the underlying structural correlates in situ. In this chapter, we provide snapshots of current advances targeting quantitative correlation of the dynamic state of a biological pathway with high-resolution structural information in the same window of time and space.

Introduction

Complexity in cell biological processes has traditionally been tackled by a reductionist approach where the corresponding system is broken down into small isolated pieces so that the most relevant parts of the system and interactions among them can be identified and studied. The reductionist agenda assumes that knowing the behavior of the participating molecules is sufficient for providing a mechanistic description of the behavior of the system. Indeed, a large body of work carried out over the past two decades has determined, at atomic resolution, minimal structural domains of protein components that in turn suggested pathways of assembly and organization. Although these high-resolution structural approaches provide critical information about individual molecules, allowing interpolation into the inner workings of molecular assemblies, as the development of integrated system models requires in-depth understanding of all system components; this knowledge becomes truly meaningful only if it can be related to the components working as an ensemble within a living cell. In this chapter, we describe how a newly developing hybrid approach that aims at seamlessly tying light and electron microcopy imaging techniques (correlative light and electron microscopy, cLEM; van Driel et al., 2009) is being harnessed to allow structural integration of these molecular models at the organelle and cellular levels.

Structural biology research is increasingly focusing on interpreting dynamic biological processes and pathways by mining structural variations originating from the micro-, meso-, and macroscales. Toward this goal, spatial and functional correlation of data derived from live-cell imaging (via light microscopy, LM) with information derived from high-resolution transmission electron cryo-microscopy (cryo-EM) and cryo-tomography (cryo-ET) is indispensable. Live-cell imaging approaches, which combine LM with genetically or specifically combined fluorophores, can track dynamically a set of proteins via multiplexing approaches to provide the means of following simultaneously multiple processes to derive the hierarchy and kinetics of the relations between activities. Cryo-EM or cryo-ET provides the ability to determine, in a fully hydrated state and in situ, the three-dimensional (3D) structures of the underlying large, dynamic macromolecular assemblies that through structural adaptations govern these processes. Thus, cLEM is establishing approaches and technologies to systematically and quantitatively determine structure–function correlates in a physiologically relevant environment.

Section snippets

Correlative Light and Electron Microscopy (cLEM) Implementation

cLEM implementations can be divided into two tiers in terms of the level of correlative details. The first tier consists of the identification of regions of interest without correlative knowledge of the components, and the second tier includes direct localization of fluorescent tags at the electron microscopy (EM) level which allows one to one correspondence between the fluorophore and the assembly under investigation.

Future Perspectives

Detection at the EM level requires electron dense markers such as gold clusters or quantum dots. In contrast to fluorescence tags, which can be genetically engineered into the cells under study, these EM markers need to be introduced into the cell by other means. In the context of cryo-cLEM, this fact constitutes an obstacle because the introduction of EM tags may interfere with the native state of the cell and/or distort its ultrastucture. We and others have begun to develop a new methodology

Outlook

The proof of principal for possible adaption of high-resolution oil-immersion LM microscopes with frozen hydrated samples (Le Gros et al., 2009) with advances in generating fluorescent protein fusions (Werner et al., 2009), advances of computer-controlled microscopes, digital cameras, and aberration-reducing electron-optics will allow cryo-cLEM to seamlessly integrate data that can span four orders of magnitude (nanometers to tens of microns) in length scale to allow establishing unified models

Acknowledgments

Drs. Hanein and Volkmann thank Karen L. Anderson for the cell preparations and cLEM imaging and Dr. Davaler Anjum for the tomography data collection and image processing presented in Figs. 1 and 2. The funding sources for Drs. Dorit Hanein and Niels Volkmann that supported this study are the National Institutes of Health Cell Migration Consortium, Grant Number U54 GM064346 and NIGMS, Grant Number P01 GM066311.

References (28)

  • L.F. van Driel et al.

    Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells

    Eur. J. Cell Biol.

    (2009)
  • N. Volkmann

    A novel three-dimensional variant of the watershed transform for segmentation of electron density maps

    J. Struct. Biol.

    (2002)
  • A. Al-Amoudi et al.

    Cryo-electron microscopy of vitreous sections

    EMBO J.

    (2004)
  • E. Betzig et al.

    Imaging intracellular fluorescent proteins at nanometer resolution

    Science

    (2006)
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