RESUMO
Critical determinants of synaptic functions include subcellular locations, input sources, and specific molecular characteristics. However, there is not yet a reliable and efficient method that can detect synapses. Electron microscopy is a gold-standard method to detect synapses due to its exceedingly high spatial resolution. However, it requires laborious and time-consuming sample preparation and lengthy imaging time with limited labeling methods. Recent advances in various fluorescence microscopy methods have highlighted fluorescence microscopy as a substitute for electron microscopy in reliable synapse detection in a large volume of neural circuits. In particular, array tomography has been verified as a useful tool for neural circuit reconstruction. To further improve array tomography, we developed a novel imaging method, called "structured illumination microscopy on the putative region of interest on ultrathin sections", which enables efficient and accurate detection of synapses-of-interest. Briefly, based on low-magnification conventional fluorescence microscopy images, synapse candidacy was determined. Subsequently, the coordinates of the regions with candidate synapses were imaged using super-resolution structured illumination microscopy. Using this system, synapses from the high-order thalamic nucleus, the posterior medial nucleus in the barrel cortex were rapidly and accurately imaged.
RESUMO
We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging.