Stochastic Micro-Pattern for Automated Correlative Fluorescence - Scanning Electron Microscopy

Abstract

Studies of cellular surface features gain from correlative approaches, where live cell information acquired by fluorescence light microscopy is complemented by ultrastructural information from scanning electron micrographs. Current approaches to spatially align fluorescence images with scanning electron micrographs are technically challenging and often cost or time-intensive. Relying exclusively on open-source software and equipment available in a standard lab, we have developed a method for rapid, software-assisted alignment of fluorescence images with the corresponding scanning electron micrographs via a stochastic gold micro-pattern. Here, we provide detailed instructions for micro-pattern production and image processing, troubleshooting for critical intermediate steps, and examples of membrane ultra-structures aligned with the fluorescence signal of proteins enriched at such sites. Together, the presented method for correlative fluorescence – scanning electron microscopy is versatile, robust and easily integrated into existing workflows, permitting image alignment with accuracy comparable to existing approaches with negligible investment of time or capital.

Receptor‐Free Signaling at Curved Cellular Membranes

“…[19,49,100,101] Crucial insights on endocytosis have been gained via TIRF microscopy, where the evanescent field was used to delineate spatio-temporal enrichment of individual proteins at the basal membrane. Here, protein enrichment to transient or long-lived membrane deformations can be quantified in living and fixed cells using various fluorescenceand/or electron-based approaches.…”

“…[100,101] • Advantages: Can visualize the localization of curvature-sensitive proteins (i.e., fluorescence) as well as membrane topography (i.e., scanning electron microscopy) under physiological conditions. [100,101] • Advantages: Can visualize the localization of curvature-sensitive proteins (i.e., fluorescence) as well as membrane topography (i.e., scanning electron microscopy) under physiological conditions.…”

“…Here, protein enrichment to transient or long-lived membrane deformations can be quantified in living and fixed cells using various fluorescenceand/or electron-based approaches. [19,49,100,101] Crucial insights on endocytosis have been gained via TIRF microscopy, where the evanescent field was used to delineate spatio-temporal enrichment of individual proteins at the basal membrane. [30] Still, as curvature-sensing proteins at high concentration may induce ectopic (artificial) membrane deformations, [21,108] endogenous protein levels should be considered when interpreting such measurements.…”

“…Correlative light-electron microscopy • Principle: Combination of data obtained via fluorescence image and scanning electron micrograph. [100,101] • Advantages: Can visualize the localization of curvature-sensitive proteins (i.e., fluorescence) as well as membrane topography (i.e., scanning electron microscopy) under physiological conditions.…”

Parallel Acquisition of Plasma Membrane Ultrastructure and Cytosolic Protein Localisation in Cultured Cells via Correlated Immunogold SEM

“…Electron microscopy (EM), in contrast, allows detailed acquisition of a specimen with a resolution of <1 nm, but lacks information on protein localisation. To reliably extract ultrastructural information and compare it to that of proteins enriched at such sites, hybrid approaches that have correlated light and electron microscopy (CLEM) have proven useful [ 1 , 2 , 3 ]. Here, information from fluorescence-based images and electron micrographs are separately acquired and subsequently merged.…”