Contrast Agents for Correlative Light and Electron Microscopy

The Gibbs Lab Researches Contrast Agents for CLEM

Background on Correlative Light and Electron Microscopy

Electron microscopy (EM) is an important tool for cell biology as it provides near angstrom resolution of cells enabling direct visualization of ultrastructure, membrane shapes, and organelle architecture. Light microscopy (LM), especially fluorescence microscopy, has also proven to be an invaluable biological imaging tool as numerous different biomolecules can be tagged and imaged specifically in a single sample. Both LM and EM have limitations, namely resolution for LM and labeling of multiple biomolecules within a single sample for EM. However, combination of these two imaging techniques provides highly complimentary data and is realized through the correlative light and electron microscopy (CLEM) technique.

Challenges with CLEM

Although CLEM instruments are available, the necessary dual sample labeling with heavy metals for EM contrast and fluorophores for LM is a continued challenge as heavy metal labeling is known to quench fluorescence signal. CLEM is generally performed by either dual labeling the sample with heavy metals and fluorophores and imaging on an instrument with dual capability or performing fluorescence microscopy, followed by heavy metal labeling and EM. The advantage to simultaneous fluorescence microscopy and EM is the ability to collect well correlated imaging datasets, however heavy metal fluorophore quenching and limited sample labeling are continued difficulties. The advantage to performing fluorescence imaging followed by EM labeling and imaging is that all fluorescence imaging techniques can be utilized and heavy metal stains do not compromise fluorescence signal. However, even with advanced CLEM technology imaging field of view correlation is challenging due to changes in sample size and shape during EM staining.

CLEM in the Gibbs Lab

The Gibbs Lab is characterizing fluorophore behavior in the presence of EM sample staining reagents. Representative fluorophores with varied chemical scaffolds are being utilized to model the behavior of each fluorophore type in the presence of the stains and solvents necessary for optimal EM imaging. OHSU has a renowned EM facility termed the OHSU FEI Living Lab that houses transmission electron microscopy (TEM), dual beam scanning electron microscopy (SEM), and CLEM tools that are being utilized in the current work. The overall goal of this project is to improve fluorescence staining for CLEM imaging to enable simultaneous EM and LM to understand cellular ultrastructure in the context of individual biomolecules.