FIGURE 1.

From a living fluorescent biological sample to an embedded specimen for light and electron microscopy, pitfalls and trade-offs. (A) Living matter is essentially composed of water and proteins, lipids and carbohydrates. Protein functions are related to their structural shape, supported by several scaffolding proteins. (B) During chemical fixation the sample is immobilized by cross-linking. The fixative, mostly aldehyde compounds, creates bonds between proteins, lipids and carbohydrates. Diffusion and fixation efficiency is affected by the sample’s density and the pH of local sub-compartments. (C) In the following dehydration in increasing concentrations of solvent, unbound fixative is progressively eluted together with the water bound in the sample. (D) The solvent is then progressively replaced by resin and polymerization is initiated (by heat or UV polymerization). Depending on the type of resin, chemical bonds can be formed between sample and resin, which provide complementary fixation. (E) Physical fixation, on the other hand, instantaneously and homogeneously immobilizes the entire sample, going from a hydrated living state (H₂OL) to a cryo-immobilized state (H₂OS) in a matter of milliseconds. Ice crystal growth resulting in damage of the ultrastructure is prevented and the water molecules remain in place. (F) The vitrified specimen is dehydrated in solvent at −90°C. The solvent substitutes the solid water, molecule by molecule. If chemical fixatives are added, these are simultaneously diffused into the specimen. These fixatives become active only above −60°C. (G) Graphical legend. (H) The CLEM in-resin fluorescence paradigm. Moving from the living, fluorescent sample to the ideal fixed electron microscopy material inevitably causes fluorescent protein quenching. The ideal IRF protocol is a compromise between acceptable fluorescence loss to identify the protein of interest after resin embedding, and adequate ultrastructure preservation and imaging contrast to achieve exploitable EM images.