Supraresolution 2-photon microscopy in brain tissue

描述（由申请人提供）：本申请涉及广泛的挑战领域（06）使能技术和具体挑战主题，06- ns -103: 2009年ARRA下的神经科学突破技术，国家神经疾病和中风研究所（NINDS）下的NIH健康与科学研究挑战拨款（RFA-OD-09- 003）。双光子激光扫描显微镜（2PLSM）允许前所未有的荧光成像神经元结构和功能深入神经组织。然而，由于在2PLSM中需要近红外（NIR）光子来穿透和激发深层组织，与传统的共聚焦显微镜相比，这种方法的分辨率较差。在这里，我们建议发展超溶液2PLSM荧光成像来分析脑组织深处的小神经结构。通过使用近红外激光器进行2光子激发和1光子激发发射耗尽（STED），实现了经典阿贝衍射极限以外的深层组织超溶液成像。使用原型显微镜的初步数据显示，3倍超溶液成像深度为~100微米。随着进一步的改进，结合2PLSM/ STED超溶液显微镜的分辨率将比传统的2PLSM提高约10倍，允许在活组织内进行纳米级成像。这种改进的分辨率允许在原位监测精细尺度的突触和神经元结构可塑性。鉴于神经结构的扰动在人类神经精神疾病模型中很常见，必须开发更好的方法，如2PLSM/STED来评估完整脑组织中的这些缺陷。由于光线传播到大脑组织的能力差，在大脑深处进行高分辨率成像是困难的。我们建议设计并实现一种超溶液显微镜，可以在超过衍射极限的分辨率下对脑组织进行成像。

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.