Label-free 3D-CLEM Using Endogenous Tissue Landmarks

Manja Luckner,Steffen Burgold,Severin Filser,Maximilian Scheungrab,Yilmaz Niyaz,Eric Hummel,Gerhard Wanner,Jochen Herms

doi:10.1016/j.isci.2018.07.012

Abstract

SummaryEmerging 3D correlative light and electron microscopy approaches enable studying neuronal structure-function relations at unprecedented depth and precision. However, established protocols for the correlation of light and electron micrographs rely on the introduction of artificial fiducial markers, such as polymer beads or near-infrared brandings, which might obscure or even damage the structure under investigation. Here, we report a general applicable “flat embedding” preparation, enabling high-precision overlay of light and scanning electron micrographs, using exclusively endogenous landmarks in the brain: blood vessels, nuclei, and myelinated axons. Furthermore, we demonstrate feasibility of the workflow by combining in vivo 2-photon microscopy and focused ion beam scanning electron microscopy to dissect the role of astrocytic coverage in the persistence of dendritic spines.

Highlights

Studying biological key events within complex model systems relies on dynamic and functional imaging at optimum spatial and temporal resolution
The region of interest (ROI) can be marked by photo-oxidation of fluorophores (Grabenbauer et al, 2005), by affinity labeling with peroxidases (Knott et al, 2009) or by the use of exogenous fiducial markers like polymer beads (Kukulski et al, 2012), quantum dots (Masich et al, 2006), or nearinfrared branding (NIRB) (Bishop et al, 2011)
We show that blood vessels, nuclei, and myelinated axons can be used for precise correlation of Light microscopy (LM) and electron microscopy (EM) images with micrometer accuracy, allowing retrieval of structures as small as single synapses

Summary

Introduction

Studying biological key events within complex model systems relies on dynamic and functional imaging at optimum spatial and temporal resolution. The ROIs can be marked by photo-oxidation of fluorophores (Grabenbauer et al, 2005), by affinity labeling with peroxidases (Knott et al, 2009) or by the use of exogenous fiducial markers like polymer beads (Kukulski et al, 2012), quantum dots (Masich et al, 2006), or nearinfrared branding (NIRB) (Bishop et al, 2011) Useful, these approaches require processing of the tissue samples and thereby might obscure the target structure or even deteriorate their ultrastructure. There are some protocols to reduce resin embedding, these methods comprise several delicate preparation steps or specialized equipment (Kizilyaprak et al, 2014; Belu et al, 2016; Lucas et al, 2017; Schieber et al, 2017) These issues were addressed by developing a ‘‘flat embedding’’ preparation to enable direct LM visualization of endogenous fiducial markers, present throughout the brain parenchyma. The feasibility of the protocol was confirmed by revealing the intimate interplay of perisynaptic astrocytic

Methods

Results

Discussion

Conclusion