Correlative stochastic optical reconstruction microscopy and electron microscopy.

Doory Kim,Thomas J Deerinck,Mark H Ellisman,Yaron M Sigal,Hazen P Babcock,Xiaowei Zhuang,Michael A Fox

doi:10.1371/journal.pone.0124581

Abstract

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets.

Highlights

Fluorescence light microscopy (LM) and electron microscopy (EM) are two of the most widely used imaging modalities for probing cellular structures
Most of sample preparations for either stochastic optical reconstruction microscopy (STORM) or transmission EM (TEM) were not changed from the standard protocols used for each imaging modality
As the fluorescence signal acquisition precedes the addition of any EM fixative or stains, the optimal fixative and stain could be used for EM, and neither STORM nor EM images were substantially compromised

Summary

Introduction

Fluorescence light microscopy (LM) and electron microscopy (EM) are two of the most widely used imaging modalities for probing cellular structures. These modalities have distinct strengths and weaknesses that complement each other. Multi-color imaging using spectrally distinct fluorescent labels allows several molecular targets to be imaged simultaneously and their interactions to be directly probed. Various super-resolution fluorescence imaging techniques have been developed to substantially surpass the diffraction limit, allowing molecular structures in cells to be imaged with nanometer-scale resolution[1,2,3]. Transmission and scanning EM methods provide higher image resolution than light microscopy, including super-resolution fluorescence

Methods

Results

Conclusion