Heat denaturation enables multicolor X10-STED microscopy

Expansion microscopy (ExM) improves imaging quality by physically enlarging the biological specimens. In principle, combining a large expansion factor with optical super-resolution should provide extremely high imaging precision. However, large expansion factors imply that the expanded specimens are dim and are therefore poorly suited for optical super-resolution. To solve this problem, we present a protocol that ensures the expansion of the samples up to 10-fold, in a single expansion step, through high-temperature homogenization (X10ht). The resulting gels exhibit a higher fluorescence intensity than gels homogenized using enzymatic digestion (based on proteinase K). This enables the sample analysis by multicolor stimulated emission depletion (STED) microscopy, for a final resolution of 6-8 nm in neuronal cell cultures or isolated vesicles. X10ht also enables the expansion of 100-200 mu m thick brain samples, up to 6-fold. The better epitope preservation also enables the use of nanobodies as labeling probes and the implementation of post-expansion signal amplification. We conclude that X10ht is a promising tool for nanoscale resolution in biological samples.

Automated summary

Expansion microscopy (ExM) is improved by high-temperature homogenization (X10ht), which allows for the expansion of samples up to 10-fold in a single step. This method enables the use of multicolor stimulated emission depletion (STED) microscopy for high-resolution imaging and improves epitope preservation for labeling probes and signal amplification. X10ht holds promise for nanoscale resolution in biological samples.