With correlative light and electron microscopy (CLEM) we target specifically labeled sparse or transient structures and determine the molecular structure in three dimensions inside cells, by combining live fluorescent microscopy with cryo fluorescent microscopy and cryo electron microscopy. We make use of correlative microscopy to investigate cell biological structures within a biomedical context
Light microscopy (LM) and electron microscopy (EM) are powerful techniques to image cells and tissues. Both techniques have their specific advantages and limitations. EM can image fixed samples at a resolution better than 5 nm while with fluorescent LM images fluorophores without the surrounding context with a resolution in the order of several 100 nm. By combining the two techniques in a correlative light and electron microscopy (CLEM) setup the advantages of both techniques can be combined. A major requirement for CLEM is that the sample preparation is suitable for both imaging conditions.
Cryo-immobilization by plunge-freezing is a well-known fixation method for EM after which the sample can be directly imaged at liquid nitrogen temperatures (cryo-EM). We also made use of cryo-fixation for LM imaging (cryo-LM) giving the advantage that no chemical fixation is needed and that many fluorescent dyes are compatible with cryo-fixation.
One of our processed cellular cryo-electron tomography images is shown above. The image shows a surface rendering of cellular interior by cryo-ET. Actin (orange), intermediate filaments (red), Vesicles (yellow), microtubules (green) and storage granules (purple) are depicted against a slice through the tomogram (grey).
We implemented a two-step cryo-CLEM approach in which the cryo-fixed sample is subsequently imaged by cryo-LM and cryo-EM (
Koning et al., 2014a). The FM is used to target specifically labeled sparse or transient cellular structures, which then are imaged by cryo electron tomography to generate a 3D structure. The images are subsequently combined into a single file for visualization ( Koning et al., 2013). We were able to target specific subcellular membranes inside Streptomyces bacteria and determine a three-dimensional map of these intricate structures.
The image shows an example of Cryo-CLEM on Streptomyces. It is an overlay of a fluorescence light microscopy image (purple), a cryo-electron microscopy image (grey) and tomographic surface rendering (green).
This cryo-CLEM approach is capable to image (cryo-)fixed structures only. However, cells are dynamic, changing and moving structures. With the goal to expand CLEM with live imaging capabilities, we integrated a live fluorescent microscope with the plunge-freezing device we often apply for cryo-fixation into a specialized apparatus we named Microscopy and Vitrification Integrated System (MAVIS). MAVIS enables live tracking of specific cells and fixation of cells at a desired time point (
Koning et al., 2014b).
The image shows the MAVIS, a Microscopy and Vitrification device. It illustrates a prototype of a fluorescence microscope build upon a vitrification device for the preparation of biological samples for cryo-CLEM.