Super Resolution Pump-Probe Microscopy for Biomedical Imaging

描述（由申请人提供）：本项目将开发一种超分辨率泵浦-探测调制（PPM）显微镜，以成像非荧光分子。影像学已成为生物医学研究和临床预后与治疗的重要工具。光学成像系统，特别是荧光显微镜，已广泛用于生物医学研究。最近，受激发射耗尽（STED）显微镜已达到纳米分辨率使用点扩展函数（PSF）工程技术，以打破衍射极限。所有这些超分辨率技术都是基于分子荧光，大多数应用需要将外源荧光团标记到靶分子上。然而，标记过程可以改变分子的活性或定位。最近，泵浦-探测显微镜证明了其通过检测当两个飞秒/皮秒激光脉冲与分子相互作用时不同分子能级之间的能量跃迁来成像非荧光分子的能力。泵浦-探测显微镜和STED显微镜的共同特点是两种模式都使用两束激光来激发分子，然后探测或耗尽分子。该方案的创新之处在于开发了一种泵浦-探测调制（PPM）显微镜，该显微镜结合了STED显微镜中使用的PSF工程方法和泵浦-探测方法，以实现以纳米分辨率成像非荧光分子的目标。该资助申请有两个具体目标：（1）开发基于受激发射和基态耗尽的泵浦-探测调制显微镜，可以以超分辨率成像金纳米棒和Atto612Q分子;（2）用双光子激发PPM显微镜成像活体果蝇脑组织中的多巴胺。

DESCRIPTION (provided by applicant): This project will develop a super resolution pump-probe modulation (PPM) microscope to image non-fluorescent molecules. Imaging has become an important tool in biomedical research and clinical prognosis and treatment. Optical imaging systems, particularly fluorescence microscopy, have been extensively used in biomedical research. Recently, stimulated emission depletion (STED) microscopy has achieved nanometer resolution using point-spread function (PSF) engineering techniques to break the diffraction limit. All these super resolution techniques are based on molecular fluorescence with most applications requiring tagging exogenous fluorophores onto targeted molecules. However, the labeling process can alter the activity or localization of the molecules. Recently pump-probe microscopy demonstrated its ability to image non-fluorescent molecules by detecting the energy transitions between different molecular energy levels when two femtosecond/picosecond laser pulses are interacting with the molecules. The common feature of the pump-probe microscope and the STED microscope is that both modalities use two laser beams to excite and then to probe or deplete the molecules. The innovation of this proposal is developing a pump-probe modulation (PPM) microscope that combines the PSF engineering method used in STED microscopy and the pump-probe method to achieve the goal of imaging non-fluorescent molecules with nanometer resolution. There are two specific aims in this grant application: (1) Develop a pump-probe modulation microscope based on stimulated emission and ground state depletion that can image gold nanorods and Atto612Q molecules with super resolution; and (2) Image dopamine in live Drosophila brain tissue with two-photon excitation PPM microscopy.