Supraresolution 2-photon microscopy in brain tissue

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DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-NS-103: Breakthrough technologies for neuroscience under the ARRA of 2009, NIH Challenge Grants in Health and Science Research (RFA-OD-09- 003) under the National Institute of Neurological Disorders and Strokes (NINDS). Two-photon laser scanning microscopy (2PLSM) has allowed unprecedented fluorescent imaging of neuronal structure and function deep within neural tissue. However, due to the near infrared (NIR) photons necessary for deep tissue penetration and excitation in 2PLSM, the resolution of this approach is poor compared to that of conventional confocal microscopy. Here we propose to develop supraresolution 2PLSM fluorescence imaging to analyze small neural structures deep within brain tissue. Deep tissue supraresolution imaging beyond the classical Abbe diffraction limit is accomplished by using NIR lasers for 2-photon excitation and 1-photon stimulation emission depletion (STED). Preliminary data using a prototype microscope demonstrates 3-fold supraresolution imaging to depths of ~100 microns. With further refinements, combined 2PLSM/ STED supraresolution microscopy will achieve ~10 fold improvement in resolution over conventional 2PLSM, allowing nanoscale imaging within live tissue. This improved resolution permits monitoring of fine-scale synapse and neuronal structural plasticity in situ. Given that perturbations of neural structure are common in models of human neuropsychiatric diseases, better methods such as 2PLSM/STED must be developed to evaluate these defects in intact brain tissue. High resolution imaging deep within the brain is difficult due to the poor ability of light to propagate into this tissue. We propose to design and implement a supraresolution microscope that allows imaging within brain tissue at resolution beyond the diffraction limit.