Correlative Organelle Microscopy: Fluorescence Guided Volume Electron Microscopy of Intracellular Processes

Abstract

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Therefore, directly linking molecular to nanoscale ultrastructural information is crucial in understanding cellular physiology. Volume or three-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. However, the application of volume-CLEM is hampered by limitations in throughput and 3D correlation efficiency. In order to address these limitations, we describe a novel pipeline for volume-CLEM that provides high-precision (<100 nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) datasets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated into a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume EM and obviates the need for post-correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between the endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D.

Bimodal endocytic probe for three-dimensional correlative light and electron microscopy

“…By using the 3D distribution of endocytosed fBSA-Au, we can select ROIs by cryo-FM, use this fluorescence to identify where to make lamella in the cryo-FIB-SEM, and locate the targeted organelles within the lamella during subsequent imaging with cryo-ET. fBSA-Au can be a highly suitable fiducial marker, especially in integrated FM in FIB-SEM systems, where the ROI (e.g., lamella) identified by FM can be directly prepared for cryo-ET ( Smeets, 2020 ; Timmermans et al., 2016 ; Loginov et al, 2022 ). An additional benefit of endocytosed nanogold fiducials for cryo-ET is accurate tracking during tilt series acquisition and improved tilt-series alignment for image reconstruction, as reported by others ( Berger et al., 2021 ).…”

CLEMSite, a software for automated phenotypic screens using light microscopy and FIB-SEM

“…At the software level, that would require improving the targeting accuracy using smaller grids and extending the maps to 3D coordinates. 3D registration against a light microscopy Z-stack would considerably help to constrain the field of view during acquisition (Loginov et al, 2022), thus reducing the imaging time and keeping the field-of-view position during tracking. At the instrument level, this would require, first, stabilizing the ion beam before the critical region is acquired, to compensate for the change between high currents for milling and fine currents for sectioning; second, to make sure that the fine current beam hits exactly the front face of the milled cross-section and then prevent milling artifacts; finally, the second direction is to increase the number of samples acquired per session.…”

Bimodal Endocytic Probe for Three-Dimensional Correlative Light and Electron Microscopy

“…By using the 3D distribution of endocytosed fBSA-Au, we can select ROIs by cryo-FM, use this fluorescence to identify where to make lamella in the cryo-FIB-SEM, and locate the targeted organelles within the lamella during subsequent imaging with cryo-ET. fBSA-Au can be a highly suitable fiducial marker, especially in integrated FM in FIB-SEM systems, where the ROI (e.g., lamella) identified by FM can be directly prepared for cryo-ET (Smeets, 2020;Timmermans et al, 2016;Loginov et al, 2022). An additional benefit of endocytosed nanogold fiducials for cryo-ET is accurate tracking during tilt series acquisition and improved tilt-series alignment for image reconstruction, as reported by others (Berger et al, 2021).…”