Combining sample expansion and light sheet microscopy for the volumetric imaging of virus-infected cells with super-resolution.

Luca Mascheroni,Oliver Dibben,Edward Ward,Clemens F Kaminski,Katharina M Scherer,James D Manton

doi:10.1364/boe.399404

Abstract

Expansion microscopy is a sample preparation technique that enables the optical imaging of biological specimens at super-resolution owing to their physical magnification, which is achieved through water-absorbing polymers. The technique uses readily available chemicals and does not require sophisticated equipment, thus offering super-resolution to laboratories that are not microscopy-specialised. Here we present a protocol combining sample expansion with light sheet microscopy to generate high-contrast, high-resolution 3D reconstructions of whole virus-infected cells. The results are superior to those achievable with comparable imaging modalities and reveal details of the infection cycle that are not discernible before expansion. An image resolution of approximately 95 nm could be achieved in samples labelled in 3 colours. We resolve that the viral nucleoprotein is accumulated at the membrane of vesicular structures within the cell cytoplasm and how these vesicles are positioned relative to cellular structures. We provide detailed guidance and a video protocol for the optimal application of the method and demonstrate its potential to study virus-host cell interactions.

Highlights

Expansion microscopy is a technique that relies on the physical magnification of biological samples in order to visualise details that are spaced more closely than the diffraction limit of light (~300 nm).[1 2]
We study the suitability of expansion microscopy and light sheet microscopy for the imaging of virus-infected samples using human A549 cells infected with live attenuated influenza vaccine (LAIV), a modified low-virulence variant of the influenza A virus that is the basis of flu vaccine formulations and sold under the names Fluenz (USA and Canada) and FluMist (Europe).[16 17]
By combining expansion microscopy with light sheet microscopy, we demonstrate how high-contrast 3D models of whole LAIV-infected cells can be reconstructed at superresolution in three colour channels, highlighting the viral nucleoprotein located in cytosolic vesicles

Summary

Introduction

Expansion microscopy is a technique that relies on the physical magnification of biological samples in order to visualise details that are spaced more closely than the diffraction limit of light (~300 nm).[1 2] The physical expansion is achieved by embedding fixed specimens in a polymer matrix that can absorb water, forming a socalled hydrogel. This approach generates volumetrically isotropic expansion and allows the bypassing of the diffraction barrier of light microscopy without any need for sophisticated instruments, enabling laboratories that possess standard fluorescence microscopes to image their samples at super-resolution. The expansion process dilutes the fluorophore concentration, decreasing the fluorescence intensity up to a hundredfold, which hinders the imaging of expanded samples with microscopes incapable of collecting a large number of photons or those requiring very high fluorescent signals

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