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）使能技术和特定的挑战主题，06-NS-103：2009年ARRA下的神经科学突破技术，美国国立卫生研究院神经疾病和中风研究所（NINDS）下的健康和科学研究挑战赠款（RFA-OD-09- 003）。 双光子激光扫描显微镜（2 PLSM）允许前所未有的荧光成像神经组织内的神经元结构和功能。 然而，由于在2 PLSM中深层组织穿透和激发所需的近红外（NIR）光子，与传统的共焦显微镜相比，这种方法的分辨率较差。 在这里，我们建议开发超分辨率2 PLSM荧光成像来分析脑组织深处的小神经结构。 利用近红外激光进行双光子激发和单光子激发发射耗尽（STED），实现了超越经典阿贝衍射极限的深部组织超分辨成像。 使用原型显微镜的初步数据展示了3倍超分辨率成像到~100微米的深度。 随着进一步的改进，结合2 PLSM/ STED超分辨率显微镜将实现比传统2 PLSM分辨率提高约10倍，允许活组织内的纳米级成像。 这种改进的分辨率允许监测精细尺度的突触和神经元结构的可塑性在原位。 鉴于神经结构的扰动在人类神经精神疾病模型中很常见，因此必须开发更好的方法，如2 PLSM/STED，以评估完整脑组织中的这些缺陷。 由于光传播到大脑深处的能力很差，因此很难对大脑深处进行高分辨率成像。 我们建议设计和实现一个超分辨率显微镜，允许在脑组织内的分辨率超过衍射极限成像。

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.