Development and Application of Two- Photon Excitation Stimulated Emission Depletion Microscopy for Superresolution Fluorescence Imaging in Thick Tissue The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Takasaki, 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. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:11051181 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Development and Application of Two-photon Excitation Stimulated Emission Depletion Microscopy for Superresolution Fluorescence Imaging in Thick Tissue A dissertation presented by Kevin T. Takasaki to The Committee on Higher Degrees in Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biophysics Harvard University Cambridge, Massachusetts May 2013 © 2013 Kevin T. Takasaki All rights reserved. Dissertation Advisor: Prof. Bernardo L. Sabatini Kevin T. Takasaki Development and Application of Two-photon Excitation Stimulated Emission Depletion Microscopy for Superresolution Fluorescence Imaging in Thick Tissue Abstract Two-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. iii Table of Contents 1. Introduction ................................................................................................................. 1 1.1. Motivation ............................................................................................................ 1 1.2. Fluorescence microscopy in neurobiology ........................................................... 1 1.3. Superresolution fluorescence microscopy ............................................................ 3 1.4. Description of thesis ............................................................................................. 3 2. Concepts and theory .................................................................................................... 4 2.1. Fluorescence microscopy ..................................................................................... 4 2.1.1. Flourescence ................................................................................................. 4 2.1.2. Two-photon excitation laser-scanning microscopy ...................................... 5 2.1.3. Resolution limit of conventional fluorescence microscopy .......................... 6 2.1.4. Resolution enhancement by deconvolution .................................................. 7 2.1.5. Linear deconvolution .................................................................................... 8 2.2. Stimulated emission depletion microscopy ........................................................ 10 2.2.1. Stimulated emission .................................................................................... 10 2.2.2. Stimulated emission depletion .................................................................... 11 2.2.3. Resolution enhancement in STED microscopy .......................................... 12 2.2.4. Theory of STED ring formation ................................................................. 13 3. Two-photon excitation STED microscopy with continuous wave (CW) STED ...... 18 3.1. Introduction ........................................................................................................ 18 3.2. Design and operation of the CW STED2P microscope ..................................... 18 3.2.1. Description of the microscope .................................................................... 18 3.2.2. Co-alignment of excitation and STED lasers.............................................. 21 3.2.3. Two-photon excitation and CW STED of fluorescent dyes ....................... 21 3.2.4. CW STED2P imaging of fluorescent beads................................................ 23 3.3. CW STED2P imaging in brain tissue ................................................................. 26 3.3.1. Consideration of refractive index mismatch ............................................... 26 3.3.2. Sample preparation, materials, and methods .............................................. 27 3.3.3. Enhanced resolution of dendritic spines ..................................................... 28 3.3.4. Effect of lateral resolution enhancement on 3D reconstruction .................. 31 3.3.5. Use of CW STED2P for morphological analysis ....................................... 33 3.3.6. Consideration of phototoxicity of STED imaging in tissue ........................ 35 3.4. Discussion .......................................................................................................... 37 4. Two-photon excitation STED microscopy with pulsed STED ................................. 39 4.1. Introduction ........................................................................................................ 39 4.2. Design and operation of the pSTED2P microscope ........................................... 40 4.2.1. Description of the microscope .................................................................... 40 4.2.2. Co-alignment of excitation and STED laser pulses .................................... 44 4.2.3. Demonstration and characterization of pulsed STED ................................. 44 4.2.4. Interaction of excitation and STED laser polarization ................................ 46 4.2.5. pSTED2P imaging of 40 nm fluorescent beads .......................................... 48 4.3. pSTED2P imaging in brain tissue ...................................................................... 50 4.3.1. Sample preparation, materials, and methods .............................................. 50 4.3.2. Comparison of CW and pulsed STED resolution ....................................... 51 4.3.3. Effect of depth on the resolution of STED imaging in tissue ..................... 52 iv 4.3.4. Effect of depth on the efficiency of STED ................................................. 54 4.3.5. Imaging of dendritic spines with 60 nm resolution .................................... 57 4.3.6. Accurate imaging and measurement of dendritic spine morphology ......... 59 4.4. Discussion .......................................................................................................... 62 5. Application of pSTED2P to studying the biophysics of dendritic spines ................. 64 5.1. Introduction ........................................................................................................ 64 5.2. Dendritic spines as biochemical compartments ................................................. 64 5.2.1. Diffusive compartmentalization in dendritic spines ................................... 64 5.2.2. Morphological determinants of diffusional transfer in dendritic spines ..... 66 5.3. Dendritic spines as electrical compartments ...................................................... 69 5.3.1. Electrical compartmentalization in dendritic spines ................................... 69 5.3.2. Morphological determinants of electrical compartmentalization of spines 70 5.4. Discussion .......................................................................................................... 73 6. Summary and future directions ................................................................................. 74 6.1. Thesis summary.................................................................................................. 74 6.2. Future directions ................................................................................................. 74 6.2.1. Improving depth penetration of two-photon excitation STED microscopy 74 6.2.2. Identification and implementation of alternative fluorophores .................. 75 6.2.3. Biophysical, structural, and functional studies of nanoscale systems ........ 76 v Acknowledgements This dissertation would not have existed without the support of many people, of whom only an incomplete list could be rendered here. I can only be grateful and humbled by the recognition of having received so much, and so much more than deserved. It is a privilege to be able to thank my advisor, Professor Bernardo Sabatini, for providing the inspiration and support that made this research possible, as well as for invaluable years of guidance and mentoring. It is likewise a privilege to thank my coworkers and friends in the Sabatini