Correlative microscopy methods that maximize specimen fidelity and data completeness, and improve molecular localization capabilities

Abstract

Correlative microscopy techniques interrogate biological systems more thoroughly than is possible using a single modality. This is particularly true if disparate data types can be acquired from the same specimen. Recently, there has been significant progress towards combining the structural information obtained from soft X-ray tomography (SXT) with molecular localization data. Here we will compare methods for determining the position of molecules in a cell viewed by SXT, including direct visualization using electron dense labels, and by indirect methods, such as fluorescence microscopy and high numerical aperture cryo-light microscopy. We will also discuss available options for preserving the in vivo structure and organization of the specimen during multi-modal data collection, and how some simple specimen mounting concepts can ensure maximal data completeness in correlative imaging experiments.

Keywords: CFM; Cellular imaging; Cryo-Flourescence microscopy; FM; Hybrid methods; LAC; Localization; PSF; SXT; Tomography; YFP; cryogenic fluorescence microscopy; fluorescence microscopy; linear absorption coefficient; point spread function; soft X-ray tomography; yellow fluorescent protein.