Non-degenerate multiphoton microscopy for deep brain imaging

 描述（由申请人提供）：本提案的总体目标是推动多光子显微镜的深度渗透，其目标是需要大规模记录皮质活动的神经科学应用，其中不能放松高分辨率要求（单微米级）。为了实现深度高分辨率成像，同时保持足够的测量信噪比以用于活动成像（例如，用于检测单个尖峰诱导的钙瞬变），我们将开发一种非常规的“非简并”2-光子显微镜，该显微镜利用了最近的实践证明，即使用长波长光（~1700 nm）用于深穿透3-光子显微镜的优点，但避开了3-光子吸收（3 PA）的低概率。我们的交付成果--与其他地方旨在大体积采样的工程努力相辅相成--将对我们重建空间分布的神经元回路活动的能力产生变革性影响，为目前不可行的生物学假设的测试提供前所未有的机会。新技术的种子是一种众所周知的现象，其中荧光团分子对第二光子的吸收通过由第一光子的吸收引起的中间状态而增强。这保证了在给定波长的正确组合的情况下激发效率的增加。对于我们的深度穿透目标，IR光束将向皮质组织内的焦点体积递送高光子通量。第二较高能量光子束将具有较低强度。因此，虽然更高能量的光子束将在脑组织中经历更高的散射，但是该光束的通量要求将被放宽（与传统的双光子显微镜相比），有助于实现深度成像。重要的是，通过增加IR光束的强度，同时降低较短波长的IR光束的强度， 通过使用波长光束，我们将减少大脑表面上不必要的“散焦”激发。这是因为较短波长光束在表面处将不具有足够的光子密度，而IR光束将位于可见发射荧光团的简并2光子吸收（2 PA）范围之外。最后，我们将实施一种创新的自适应光学策略，以纠正光束传输更高能量光子所经历的相位失真。具体而言，我们使用IR光束作为参考点（“引导星星”），并调整第二光束的相位以达到最大重叠，该IR光束可以很好地聚焦在组织内部深处。总体而言，我们预期在皮质组织内实现约1.6 mm的穿透，同时避免过多的激光功率并在简并激发模式下保留单独IR光束的2 PA激发体积特性。我们的最终交付成果将是一个原型设备，其概念验证证明了其使用合成钙指示剂和遗传编码的钙敏感荧光蛋白进行体内脑活动成像的性能。该项目将在3年内为学术界与工业界的伙伴关系提案奠定基础，以全面开发和部署新技术作为商业产品。

 DESCRIPTION (provided by applicant): The overarching goal of this proposal is to push the depth penetration of multiphoton microscopy targeting neuroscience applications in need of large-scale recording of cortical activity where high resolution requirement (in the order of singl microns) cannot be relaxed. To achieve deep high-resolution imaging while retaining sufficient signal-to-noise ratio of the measurements for imaging of activity (e.g., for detection of single spikes induced calcium transients), we will develop an unconventional "non-degenerate" 2-photon microscopy capitalizing on the recent practical demonstration of the advantage of using long wavelength light (~1700 nm) for deep penetration 3-photon microscopy but circumventing the low probability of 3-photon absorption (3PA). Our deliverables - complementary to engineering efforts elsewhere aimed at large-volume sampling - would have a transformative impact on our ability to reconstruct spatially distributed neuronal circuit activity providing unprecedented opportunities for tests of biological hypotheses that are currently unfeasible. The seed of the new technology is a well-known phenomenon where absorption of the second photon by the fluorophore molecule is enhanced through an intermediate state induced by absorption of the first photon. This warrants an increase in the excitation efficiency given the right combination of the wavelengths. For our goal of deep penetration, the IR beam will deliver high photon flux to the focal volume inside the cortical tissue. The second higher energy photon beam will have lower intensity. Thus, while the higher energy photon beam would experience higher scattering in the brain tissue, the flux requirement for this beam will be relaxed (compared to that in the conventional 2-photon microscopy) helping to achieve deep imaging. Importantly, by increasing the intensity of the IR beam while lowering the intensity of the shorter wavelength beam, we will decrease the unwanted "out-of-focus" excitation on the brain surface. This is because the shorter wavelength beam will not have enough photon density at the surface while the IR beam will lie outside the degenerate 2- photon absorption (2PA) range for visible emission fluorophores. Finally, we will implement an innovative Adaptive Optics strategy to correct the phase distortions that will be experienced by the beam delivering higher energy photons. Specifically, we use the IR beam, which can be focused well deep inside the tissue, as a reference point ("guiding star") and adjusting the phase of the second beam to reach the maximum overlap. Overall, we expect to achieve ~1.6 mm penetration inside the cortical tissue while avoiding excessive laser power and retaining the excitation volume characteristic for 2PA of the IR beam alone in the degenerate excitation mode. Our endpoint deliverable will be a prototype device with the proof-of-concept demonstration of its performance for imaging of brain activity in vivo using synthetic calcium indicators and genetically encoded calcium-sensitive fluorescent proteins. This project will lay the groundwork for an academic-industry partnership proposal in 3 years to fully develop and deploy the new technology as a commercial product.