The CCiTUB Electron Cryomicroscopy Unit, with more than 15 years of experience in cryo-methods, has adapted its Cryoimmobilization, Freeze substitution and inclusion protocols in acrylic resins so that users can carry out Correlative Light and Electron Microscopy studies (CLEM). Cryoimmobilization by High Pressure Freezing (HPF) consists of fixing the sample by combining a pressure rise (2,100 bar) and an ultrafast temperature drop (minimum 10,000 Celsius degrees per second), achieving pass the water from the sample directly to the state of vitreous ice, avoiding the state of crystalline ice and, therefore, maintaining the biological structure in a close-to-native state. In addition, by means of cryo-methods, samples are processed at low temperatures, limiting the use of chemical agents and thus minimizing the structural artifacts derived from conventional methods.
Over the last decade, CLEM has become a widely used technique for exploring biological and biomedical samples at different scales, as both types of microscopy offer very different but at the same time complementary resolutions. While Optical Microscopy can be applied directly to the acquisition of images of cell and tissue samples in vivo, Electron Microscopy requires fixed samples, either by conventional methods or by cryo-methods. The main advantage of cryo-methods is that they provide a high degree of preservation of all cellular components, as well as target epitopes of antibodies and fluorescent proteins, while acrylic resins allow access of antibodies to their target epitopes. In this way, an optimal cellular context is created for the application of CLEM techniques, both for samples that already incorporate fluorescence and for samples to which double immunolabeling must be added: for fluorescence (fluorophore-conjugated antibodies) and for electron microscopy (colloidal gold-conjugated antibodies).
Once the samples for CLEM have been prepared, they are first observed under a fluorescence microscope, where the areas of interest are selected and acquired. Subsequently, the same samples are observed under the transmission electron microscope, where the same previously photographed areas are searched and images of their ultrastructure are acquired. Finally, images of the same area are obtained at two complementary resolutions, which can be overlaid and thus determine in detail the infrastructure of the fluorescent areas.
In CCiTUB users can perform CLEM experiments, obtaining fluorescence microscopy images in the Advanced Optical Microscopy Unit and subsequently transmission electron microscopy images in the TEM-SEM Unit and Electron Cryomicroscopy Unit. In this way the user can obtain a much higher volume of information than would be available using only one of the techniques.