Correlative GFP-immunoelectron microscopy in yeast

Correlative light and electron microscopy represents the ultimate goal for the visualization of cell biological processes. In theory, it is possible to combine the strengths of both methods, that is, the live-cell imaging of the movement of GFP-tagged proteins captured by fluorescence microscopy with an image of the fine structural context surrounding the tagged protein imaged and localized by immunoelectron microscopy. In practice, inherent technical limitations of the two individual methods and their combination make the technique very complex to handle. Here, we present a high-pressure freezing and freeze-substitution protocol which fulfills the key criterion for correlative microscopy, namely, the ability to achieve excellent visibility of fine structures without disrupting the antigens recognized by the immunolabeling protocol. This is achieved by a fixative-free freeze-substitution and low-temperature embedding.