New correlative approach combines superresolution confocal and scanning electron imaging
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).
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.
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”.
“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.