Abstract
Correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) system has been fast becoming a powerful technique with the advantage to allow the fluorescent labeling and direct visualization of the close-to-physiologic ultrastructure in cells at the same time, offering unique insights into the ultrastructure with specific cellular function. There have been various engineered ways to achieve cryo-CLEM including the commercial FEI iCorr system that integrates fluorescence microscope into the column of transmission electron microscope. In this study, we applied the approach of the cryo-CLEM-based iCorr to image the syntaphilin-immobilized neuronal mitochondria in situ to test the performance of the FEI iCorr system and determine its correlation accuracy. Our study revealed the various morphologies of syntaphilin-immobilized neuronal mitochondria that interact with microtubules and suggested that the cryo-CLEM procedure by the FEI iCorr system is suitable with a half micron-meter correlation accuracy to study the cellular organelles that have a discrete distribution and large size, e.g. mitochondrion, Golgi complex, lysosome, etc.
Highlights
Fluorescence light microscopy (FM) offers large-scale, time-resolved, and dynamic visualization of positions of interest (POIs) in cells and provides a variety of information in cellular processes
Besides chemical fixation, cryo-vitrification technique has provided a good immobilization of cellular ultrastructures in their native state, which can be imaged by cryo-electron
After transfection with the plasmids of Dendra2-SNPH and TagRFP-mito, the cotransfected cells showed that all axonal mitochondria became immobilized as previously reported (Kang et al 2008), and the fluorescence signals from SNPH and mitochondria were significantly colocalized (Fig. 1B)
Summary
Fluorescence light microscopy (FM) offers large-scale, time-resolved, and dynamic visualization of positions of interest (POIs) in cells and provides a variety of information in cellular processes. FM could only provide localization information of labeled molecules, but with a lack of structural context in the cells. Electron microscopy (EM) has been applied into the study of cellular ultrastructures for many years and can provide detailed structural information of cells in nanometer-scale resolution. Besides chemical fixation, cryo-vitrification technique has provided a good immobilization of cellular ultrastructures in their native state, which can be imaged by cryo-electron. This article is published with open access at Springerlink.com
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