The next step in understanding cellular regulation is how interconnectivity between different types of organelles is important for function ( Gatta and Levine, 2016). Complications in organelle performance are associated with many pathologies ranging from infection, neurodegeneration, and cancer ( Wu et al., 2018 Ballabio and Bonifacino, 2019 Ferguson, 2019 Sironi et al., 2020). 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.Įukaryotic cells are compartmentalized in organelles delimited by distinctive intracellular membranes, each with special biochemical functions yet functioning together to maintain cellular homeostasis. 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. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM.
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.
Gridded coverslips registration#
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. However, the application of volume-CLEM is hampered by limitations in throughput and 3D correlation efficiency. 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. Therefore, directly linking molecular to nanoscale ultrastructural information is crucial in understanding cellular physiology. The key advantage is that the film is easily cut and punched to allow EM processing.Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. The material of CMC correlative microscopy coverslips is compatible with cell culture growth and exhibit good adhesion for cell cultures. CMC correlative microscopy coverslips are not designed to replace the optically superior quartz or glass coverslips as the film itself has a structure which can be visible in certain illumination conditions. They are specifically designed to allow identification and location of particular areas of interest with bright field or fluorescence microscopes and then processing for electron microscopy. CMC correlative Microscopy Coverslips are made using a polyester-based film with unique reticule patterns.
0 kommentar(er)
