Efficient and Accurate Synapse Detection With Selective Structured Illumination Microscopy on the Putative Regions of Interest of Ultrathin Serial Sections.

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.

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy.

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.