Spotlight on the Cells’ Ultrastructure

New correlative approach combines superresolution confocal and scanning electron imaging

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Researchers from the Department of Cell Biology, theme Nanomedicine, and the ‘Microscopy Imaging Center’ at the Radboudumc in Nijmegen, Netherlands recently developed and optimized a pipeline for correlative imaging using superresolution (SR) microscopy and scanning electron microscopy (SEM).

ZEISS Airyscan and SEM correlative imaging pipeline
ZEISS Airyscan and SEM correlative imaging pipeline. (1) ITO-coated coverslips are marked on three edges for stage calibration on the ZEISS LSM 880 and Sigma 300. (2) Cells are seeded on the marked coverslips for at least 3 h. (3) Live cells are sonicated to prepare VPMs and immediately fixed. (4) Sample is immunolabeled for the proteins of interest, fixed for the second time and fiducials are added. (5) The sample is imaged with ZEISS Airyscan microscopy. (6) The sample is dehydrated, critical point dried and then sputtered with 5 nm chromium. (7) The sample is imaged with ZEISS SEM microscopy. (8) Fiducials are used to align the LM and SEM image using Matlab.

This state-of-the-art imaging approach allows the correlative visualization of up to three cellular components by SR fluorescence microscopy and the cellular ultrastructure by SEM.

“In other words, one can fluorescently label up to three different proteins and accurately determine their localization with respect to specific cellular ultrastructures”, Ben Joosten, cell biologist and part of the research team at the Radboudumc, explains.

VPM preparation and CPD procedure preserve podosome organization
VPM preparation and CPD procedure preserve podosome organization. (A) DCs were seeded on glass coverslips and after VPM preparation, cells were fixed and stained for actin (cyan), vinculin (green) and zyxin (magenta). After CPD, DCs were imaged by SEM (gray). Shown are representative images of all three channels in three dimensions and the corresponding SEM image. Insets depict two representative podosomes within the cluster. (B) Shown are the SEM-LM overlays for all three channels for the same cells as in (A) Scale bar = 5 μm.

Narrowing the resolution gap

Correlative light and electron microscopy (CLEM) was so far performed using conventional light microscopy (LM) and electron microscopy (EM). Although this offered unique and complementary information from the same cell or tissue sample, the interpretation of those correlative images was challenged by the fact that the lateral resolution of conventional LM (~250 nm) is much worse than the lateral resolution of EM (~2 nm). This is referred to as the “resolution gap”.

The Radboundumc research team
The Radboundumc research team:
Top row from left: Marieke Willemse, Jack Fransen; Bottom row from left: Ben Joosten, Koen van den Dries and Alessandra Cambi

“Our correlative imaging pipeline, called SR-CLEM, narrows this gap as it combines the ultrastructure provided by the ZEISS Sigma SEM with super-resolved fluorescent images acquired with ZEISS LSM 800 with Airyscan (lateral resolution of ~140 nm)”, says Joosten.

He and his colleague Koen van den Dries used SR-CLEM to study the nanoscale architecture of podosomes, small cytoskeletal structures used by leukocytes to transmigrate basement membranes or by osteoclasts to remodel bone tissue. The results of the study are published in Frontiers in Immunology, the most-cited open-access journal in immunology.

“The SR-CLEM approach is particularly interesting for elucidating the organization of complex multimolecular cellular structures as well as for characterizing microorganisms, nanomaterials or nanoparticles and their interaction with cells”, says Jack Fransen, Associate Professor at the Radboudumc.

Read the paper, published in Frontiers in Immunology, here

More information on Correlative Microscopy Solutions from ZEISS

Tags: Confocal Microscopy, Correlative Microscopy, Electron and Ion Microscopy, Super-resolution Microscopy

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