We propose new probes for correlative super-resolution fluorescence and electron microscopy that use biorthogonal reactions - Click, SNAP and HALO tags - to label targets in living cells. To minimize quenching of the fluorophores by the gold particles, the small Undecagold (11 gold atoms) gold cluster label will be used: this has minimal absorption at wavelengths above 500 nm and therefore minimal overlap with emission of longer-wavelength fluorophores. Undecagold will be conjugated to ATTO647N and SiR (siliconized rhodamine), longer-wavelength fluorophores which are optimized for super-resolution imaging, and to Click and HALO tag ligands for bioorthogonal reactivity in living cells as well as for general labeling use. We hypothesize that this probe configuration will provide much brighter fluorescence signals and will enable live cell and super-resolution imaging, which often photon are limiting. These probes will combine high resolution, dense and potentially quantitative labeling with a much smaller probe size than antibodies, thus facilitating cellular delivery, diffusion and targeting in living cells. Preparations will be optimized to provide retenton of 80% native fluorescence by quantum yield, with average of 1.0 or more fluorophores per gold label and 1:1 Undecagold : targeting group; retention of native Click and HALO reactivity will be demonstrated by conjugation to test proteins bearing the conjugate tags, followed chromatographic separation, spectroscopic characterization and in vitro blot and light microscopy labeling. The new probes will then be validated in correlative super-resolution fluorescence and EM labeling experiments: SIM/STED and EM studies to (a) localize G-protein coupled receptors (GPCRs; e.g. smoothened); and (b) visualize the structure of the ciliary pocket by correlative fluorescence and electron microscopy. Fluorescence brightness and quantum yield will be compared with those of combined fluorescent and gold immunoprobes fluorescence SIM and STED signals will be correlated with EM localization of targets.
A new class of combined fluorescent and gold labeling reagents will be developed that will enable labeling for both super- resolution fluorescence microscopy and electron microscopy in a single labeling procedure; furthermore, these new probes will use biorthogonal reactions for labeling, and thus may be used in living cells. These will be the first combined fluorescent and gold labeling reagents. Synthetic strategies and choice of labeling components will be optimized to minimize fluorescence quenching and deliver brighter fluorescent signals, using smaller probes capable of high density labeling and fast tissue penetration. Deliverables will include research tools to leverage the power of correlated electron microscopy to provide a morphological context and macromolecular localization for super-resolution fluorescence microscopy, to bring a new level of resolution to the study of biological processes at high resolution in both prepared specimens and in living cells. In addition to the specific application proposed for validation, our approach is intended to provide an enabling technology that will stimulate the development of complementary tools for other large-scale projects such as the BRAIN initiative to map the structure, function, connectivity and plasticity of neural circuits. Such systems pose great challenges for conventional microscopic methods because information is required on both the distribution of targets within entire systems, and the precise localization of specific functional components at nanometer-scale resolution.
