Correlative cryo-fluorescence and cryo-scanning electron microscopy as a straightforward tool to study host-pathogen interactions.

Martin Strnad,Jana Elsterová,Jana Nebesářová,Ryan O M Rego,Jana Schrenková,Marie Vancová,Libor Grubhoffer

doi:10.1038/srep18029

Abstract

Correlative light and electron microscopy is an imaging technique that enables identification and targeting of fluorescently tagged structures with subsequent imaging at near-to-nanometer resolution. We established a novel correlative cryo-fluorescence microscopy and cryo-scanning electron microscopy workflow, which enables imaging of the studied object of interest very close to its natural state, devoid of artifacts caused for instance by slow chemical fixation. This system was tested by investigating the interaction of the zoonotic bacterium Borrelia burgdorferi with two mammalian cell lines of neural origin in order to broaden our knowledge about the cell-association mechanisms that precedes the entry of the bacteria into the cell. This method appears to be an unprecedentedly fast (<3 hours), straightforward, and reliable solution to study the finer details of pathogen-host cell interactions and provides important insights into the complex and dynamic relationship between a pathogen and a host.

Highlights

A fundamental leap towards direct, fully artifact-free CLEM has been made possible by the introduction of a cryo-fluorescence microscope[8,9]
With regard to fluorescent tags, cryo-CLEM is suitable for correlating membrane permeable dyes and gene fusion proteins such as green fluorescent protein (GFP)[15]
The goal of this study is to provide a very fast microscopy procedure (Fig. 1) that is highly convenient for high-resolution studies of pathogen-host cell interactions in a native state

Summary

Introduction

A fundamental leap towards direct, fully artifact-free CLEM has been made possible by the introduction of a cryo-fluorescence microscope[8,9]. Unlike procedures where the object of interest is studied by FM before vitrification, the all-cryo-workflow (cryo-CLEM) obviates all structural or positional changes that may occur during the period between fluorescence observation and cryo-immobilization, or during the transfer of the sample to the electron microscope[11]. The goal of this study is to provide a very fast microscopy procedure (Fig. 1) that is highly convenient for high-resolution studies of pathogen-host cell interactions in a native state. Using this novel correlative cryo-FM and cryo-SEM approach, we sought to further define the cell binding properties of B. burgdorferi and the suggested propensity of B. burgdorferi towards invasion of nonphagocytic cells

Objectives

Methods

Results

Conclusion