Development of fast multi-photon microscope for kHz-imaging of in vivo neuronal network activity

双光子显微镜（TPM）可以对大脑进行高分辨率的3D成像。TPM已经被广泛用于阿尔茨海默病和痴呆症等退行性疾病的研究。然而，为了研究整个神经网络的神经活动和疾病相关的修改，必须以高帧速率获得图像。突触信息传输发生在毫秒和亚毫秒的时间尺度上，因此需要kHz的帧速率。加州洛杉矶大学（UCLA）的Bahram Jalali教授小组已经用单光子显微镜（FIRE显微镜）实现了这一目标。该DFG研究提案的目的是开发一种快速TPM系统，从而在高分辨率下实现更高的组织成像深度。通过采用快速激光与时间编码成像技术相结合，应达到100 MHz的像素速率和1.5 kHz的帧速率（256x256像素）。为了实现这一目标，将使用先进的调制技术与高灵敏度光电倍增管（PMT）对双光子信号的检测相结合来产生信号。光电倍增管具有亚纳秒的时间分辨率，因此也适用于并行荧光寿命成像（FLIM）。这也将导致所提出的系统的FLIM成像速度的急剧增加。总的来说，所设想的多模态显微镜将产生超过10倍的速度增强相比，传统的TPM系统，并因此适合于在轴突潜在的尖峰事件固有的时间分辨率在体内的神经网络的成像中的应用。

Two-Photon microscopy (TPM) enables high-resolution, 3-D imaging of the brain. TPM is already widely used for the study of degenerative diseases like Alzheimer´s and dementia. However, in order to study neural activity and disease-related modifications of whole neural networks, the images must be obtained at high frame rates. The synaptic information transport occurs at the millisecond and sub-millisecond timescale, so kHz frame-rates are desired. This was already achieved with one-photon microscopy (FIRE microscope) in the group of Prof. Bahram Jalali at the University of California Los Angeles (UCLA). The aim of this DFG research proposal is the development of a fast TPM system, thus enabling much higher imaging depth in tissue at high resolution. By employing fast lasers in combination with a time-encoded imaging technique, pixel rates of 100 MHz and frame rates of 1.5 kHz at 256x256 pixels shall be reached. In order to accomplish this, the signals will be generated using advanced modulation techniques paired with the detection of the two-photon signals by highly sensitive photomultiplier tubes (PMT). PMTs have time-resolutions of sub-ns and are thus also suited for fluorescence lifetime imaging (FLIM) in parallel. This would also lead to a dramatic increase of FLIM imaging speeds by the proposed system. Over all, the envisaged multi-modal microscope would yield a speed enhancement of more than a factor of 10 as compared to conventional TPM systems and is thus suited for application in imaging of neural networks in vivo at time-resolutions intrinsic to axon potential spiking events.