Super Resolution Pump-Probe Microscopy for Biomedical Imaging

描述(申请人提供)：该项目将开发一种超分辨率泵浦-探测调制(PPM)显微镜来成像非荧光分子。影像已成为生物医学研究和临床预后治疗的重要工具。光学成像系统，特别是荧光显微镜，在生物医学研究中得到了广泛的应用。最近，受激发射耗尽(STED)显微镜利用点扩散函数(PSF)工程技术突破了衍射极限，达到了纳米级的分辨率。所有这些超分辨技术都是基于分子荧光的，大多数应用都需要将外源荧光团标记到靶分子上。然而，标记过程会改变分子的活性或定位。最近，泵浦-探测显微镜通过探测两个飞秒/皮秒激光脉冲与非荧光分子相互作用时不同分子能级之间的能量跃迁，展示了它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，这两种方式都使用两束激光来激发分子，然后探测或耗尽分子。这一方案的创新之处在于开发了一种泵浦-探测调制(PPM)显微镜，它将STED显微镜中使用的PSF工程方法与泵浦-探测方法相结合，实现了对非荧光分子进行纳米分辨率成像的目标。这项拨款申请有两个具体目标：(1)开发一种基于受激发射和基态耗尽的泵浦-探测调制显微镜，可以超分辨率成像金纳米棒和Atto612Q分子；(2)用双光子激发PPM显微镜成像活的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.