Fluorescent in situ hybridization of synaptic proteins imaged with super-resolution STED microscopy

Super-resolution fluorescence microscopy is still a developing field. One of the limitations has been that standard labeling assays, which had been developed for conventional imaging, must be adjusted and optimized for each super-resolution method. These methods are more sensitive to noise, and require more intense labeling than conventional microscopy, which is not always trivial to achieve. Here, we describe the use of stimulation-emission depletion (STED) microscopy to locate messenger RNAs (mRNAs) in single neurons with high spatial precision. We address several technical difficulties we encountered in using fluorescent in situ hybridization (FISH) for STED imaging. We optimized the experimental protocol to detect mRNAs and proteins simultaneously, by performing FISH and immunostaining on the same samples. We tested our imaging approach in primary hippocampal neurons, studying the mRNAs of three important presynaptic proteins (synaptobrevin, synaptotagmin, and synaptophysin). Our approach allowed us to relate changes in mRNA levels and localization to neuronal physiology, under different activity regimes and also during neuronal development. We conclude that FISH can be performed efficiently using super-resolution techniques. This should contribute significantly to the clarification of the molecular mechanisms that govern mRNA distribution and dynamics within cells.