Sabatini, Bernardo LuisTakasaki, Kevin Takao2013-09-182013-09-182013Takasaki, Kevin Takao. 2013. Development and Application of Two-Photon Excitation Stimulated Emission Depletion Microscopy for Superresolution Fluorescence Imaging in Thick Tissue. Doctoral dissertation, Harvard University.http://dissertations.umi.com/gsas.harvard:10976http://nrs.harvard.edu/urn-3:HUL.InstRepos:11051181Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of 1-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular features of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality which overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. In this thesis, I describe the development of 2PLSM combined with STED microscopy for superresolution fluorescence imaging of neurons embedded in thick tissue. Furthermore, I describe the application of this method to studying the biophysics connecting synaptic structure and function in dendritic spines.en-USBiophysicsNeurosciencesOpticsmicroscopysuperresolutionsynapseThesis or Dissertation2013-09-18