Grant: Research Tools and Instruments

Fluorescence microscopy has made tremendous contributions to the studies of organelles dynamics in living cells but is restricted by the resolution of diffraction-limit light microscopy (~200 nm). Super-resolution microscopy, such as stimulated emission depletion (STED) microscopy, has overcome the diffraction barrier, achieving a lateral resolution of ~50 nm. STED applies a donut-shaped depletion laser to confocal excitation and resolution is determined by the ratio between the maximal intensity in the depletion beam and the intensity at which the probability of fluorescence emission is reduced by half. While high intensity depletion beam enhance resolution, they often result in photobleaching and phototoxicity for live cell imaging. STED microscopy has contributed enormously to the research programs of the applicants and many other labs at UBC, enabling the novel characterization of endoplasmic reticulum (ER) nanodomains (Nabi), ER-mitochondria contact sites (Nabi, Hamarneh) and ER reorganization upon Zika virus infection (Nabi, Jean, Hamarneh). Vogl used STED to study disruption of cortactin or oxysterol binding protein-related protein 9 at membrane contact sites at tubulobulbar complex (TBC) in testes. Craig used expansion microscopy (ExM) and STED super-resolution microscopy to study synaptic connections in neuropsychiatric disorders. NSERC RTI funding has allowed us to upgrade our Leica TCS SP8 STED 3X microscope with a 775 nm STED laser, an 86X/1.2 water STED white objective, and fluorescence lifetime analysis with tauSTED, reducing depletion laser intensity required, enhancing resolution, and enabling gentler live-cell STED imaging. However, the detector configuration and setup of our tauSTED system limits multichannel imaging. tauSTED time-gating STED (gSTED) applications only work with HyD detectors and with only two HyD detectors, our STED system can only acquire simultaneous imaging for up to two channels based on between-line acquisition, in which one line of the first channel is followed by the acquisition of one line of the second channel, thus enabling quasi-simultaneous two-colour imaging. We request funding for new HyD-SMD (single molecule detection) detectors to enable: 1) simultaneous 4-color live cell imaging; 2) stable 3-4 channel 3D tauSTED or gSTED imaging (between line or between-frame to replace current between-stack imaging); 3) Enhanced tauSTED imaging with the new HyD-SMD detectors that acquire 1 photon per pulse. We also request software upgrades and a workstation to make the STED system and off-line data analysis more accessible to multiple users and acquire tauXtend, an improved Leica deconvolution software module that uses lifetime data. A new tauSTED super-resolution microscope cost ~$2 million. In contrast, tauSTED upgrade of our Leica TCS SP8 STED 3X microscope for <$150,000 represents a highly cost- effective approach to enhance our super--resolution capabilities to best--of--class.