An Easy Path for Correlative Electron and Super-Resolution Light Microscopy

Dorothea Pinotsi,Silvia Campioni,Gabor Csucs,Simona Rodighiero

doi:10.1038/s41598-019-52047-2

Abstract

A number of new Correlative Light and Electron Microscopy approaches have been developed over the past years, offering the opportunity to combine the specificity and bio-compatibility of light microscopy with the high resolution achieved in electron microscopy. More recently, these approaches have taken one step further and also super-resolution light microscopy was combined with transmission or scanning electron microscopy. This combination usually requires moving the specimen between different imaging systems, an expensive set-up and relatively complicated imaging workflows. Here we present a way to overcome these difficulties by exploiting a commercially available wide-field fluorescence microscope integrated in the specimen chamber of a Scanning Electron Microscope (SEM) to perform correlative LM/EM studies. Super-resolution light microscopy was achieved by using a recently developed algorithm - the Super-Resolution Radial Fluctuations (SRRF) - to improve the resolution of diffraction limited fluorescent images. With this combination of hardware/software it is possible to obtain correlative super-resolution light and scanning electron microscopy images in an easy and fast way. The imaging workflow is described and demonstrated on fluorescently labelled amyloid fibrils, fibrillar protein aggregates linked to the onset of multiple neurodegenerative diseases, revealing information about their polymorphism.

Highlights

The development of correlative Light and Electron Microscopy (LM and EM) methods has contributed to significant advances in the understanding of biological, chemical and physical processes[1,2,3,4,5]
The simplicity of the method we describe is based on: (a) the commercial availability of a wide-field fluorescence microscope that can be mounted inside an Scanning Electron Microscope (SEM) chamber and (b) the application of a recently developed algorithm called Super Resolution Radial Fluctuations (SRRF) for simple and cost-effective super-resolution light microscopy[16]
As an example application demonstrating the value of our approach, we studied the morphology of dual-color labeled amyloid fibrils formed by the human α-Synuclein (α-Syn) protein, whose aggregation is related to the onset of Parkinson’s disease[17,18]

Summary

Introduction

The development of correlative Light and Electron Microscopy (LM and EM) methods has contributed to significant advances in the understanding of biological, chemical and physical processes[1,2,3,4,5]. It is clear that a synergic combination of super-resolution LM combined with various forms of EM could lead to unprecedented information on the process under study Often such approaches are based on protocols which are complex and very difficult to implement and require transferring of the sample between different setups, the use of patterned substrates[10,11], as well as rigorous image analysis algorithms for image alignment and correlation[12,13,14,15]. The simplicity of the method we describe is based on: (a) the commercial availability of a wide-field fluorescence microscope that can be mounted inside an SEM chamber and (b) the application of a recently developed algorithm called Super Resolution Radial Fluctuations (SRRF) for simple and cost-effective super-resolution light microscopy[16] This method can be performed with a standard wide-field fluorescent microscope and solely uses an ImageJ plugin for the image analysis (requiring a time-lapse acquisition of the field of view of interest). The obtained results show that with our approach we are able to distinguish the “seed” units from the elongating monomers incorporated in the fibers, and to identify details on the filaments that are involved in the bundles

Methods

Results

Conclusion