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

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.

The SARS-CoV-2 nucleocapsid protein associates with the replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

“…The fixed immunostained samples were expanded following a published procedure ( 42 ) and imaged either on a confocal or on a light sheet microscope as previously reported ( 43 ). Briefly, immunostained cells were incubated with 0.25% glutaraldehyde in PBS for 15 minutes, washed 3 times with PBS and then incubated with monomer solution (1xPBS, 2M NaCl, 2.5% acrylamide, 0.15% N,N’-methylenebisacrylamide, 8.625% sodium acrylate) for ~2 m at RT.…”

Towards a Quantitative Single Particle Characterization by Super Resolution Microscopy: From Virus Structures to Antivirals Design

“…On the other hand, Gag itself accumulates in budding areas forming three different patterns: small clusters (40-50 nm), medium clusters (140 nm) and patchy aggregates of bigger sizes (Figures 5B,C), characterized by dSTORM, PALM (Blanchard et al, 2020). (B) Expansion/light sheet of cells infected with live attenuated influenza NPs (Mascheroni et al, 2020). A Welch t-test was performed, where n = 10 and **p < 0.01.…”

“…On the other hand, to study the influenza replication one could focus on the nucleoproteins (NPs) since they are interacting with the vRNA of the virus and are involved in the viral replication ( Portela and Digard, 2002 ; Krug and Fodor, 2013 ). For this purpose, Mascheroni et al (2020) used a non-conventional super-resolution approach combining expansion microscopy and light sheet microscopy. Expansion microscopy ( Chen et al, 2015 ) is a technique that uses mechanical forces to physically expand the cell in order to enlarge the structures that are closer to the diffraction limit, which, in combination with light sheet microscopy, could allow the identification of NPs located in ∼250 nm vesicular structures surrounding the nucleus of the cell.…”

“…Expansion microscopy ( Chen et al, 2015 ) is a technique that uses mechanical forces to physically expand the cell in order to enlarge the structures that are closer to the diffraction limit, which, in combination with light sheet microscopy, could allow the identification of NPs located in ∼250 nm vesicular structures surrounding the nucleus of the cell. It was presumed that the ribonucleoprotein-genome (RNP) complex starts to assemble inside the membranes of the endosome machinery in the cytoplasm ( Figure 4B ) after being released from the nucleus in a process that involved enlargement of the nucleopores via caspases, characterized by SIM ( Mühlbauer et al, 2015 ; Mascheroni et al, 2020 ).…”

“…Scale bar 200 nm ( Blanchard et al, 2020 ). (B) Expansion/light sheet of cells infected with live attenuated influenza NPs ( Mascheroni et al, 2020 ). A Welch t-test was performed, where n = 10 and ** p < 0.01.…”