ADAPT: Adaptive and predictive arbitrary point scanning two photon tomography

多光子显微镜目前在活体成像方面是其他光学方法中无与伦比的，因为它允许对标记的结构和蛋白质进行成像，同时提供具有亚细胞轴向分辨率的大光学穿透深度，保持相对较低的光漂白水平。这些优势，加上激发基因编码的钙和电压指示器的可能性，使多光子显微镜成为啮齿动物神经元研究的首选方法，以破译大脑功能和了解神经元疾病。高速下的光学采样对于准确捕捉神经活动是至关重要的，因为动作电位的特征时间尺度是几毫秒的量级，而钙瞬变在100毫秒范围内相关。目前的成像技术没有将快速记录速率与体积采样充分结合起来，而体积采样对于完全理解固有的三维神经元电路同样重要。通过这个编织项目，我们的目标是推进和定制利用衍射激发技术进行的光谱-时间激光成像，该技术由一个合作伙伴先前介绍，以应对神经光子学的挑战：i)获取分层神经元结构(皮质、肠)在其功能过程的固有时间尺度(20赫兹容积率)的体积成像；ii)开发专用近红外源进行成像，并受益于成熟的GCaMP荧光蛋白质家族；iii)分析和优化信号产量和样品损伤，引入快速自适应扫描和采样协议；iv)通过在组织深度嵌入非线性受信标记物来校正波前扭曲和解释标本移动。为了实现这些目标，Adapt的研究伙伴与激光物理学家和光谱时相多光子成像的发明者Sebastian Karpf(GER)合作，建立了940 nm的体积成像系统，该系统经过完美调整，可以在20赫兹的体积成像中对GFP样分子成像；纳米光子学和非线性光学专家Luigi Bonacina(CH)利用源的主动调制和定制的三维扫描解决方案来改进、评估和将破坏性技术转化为自适应和有针对性的采样。此外，蓝天实验室将建立一个专门的倍频探测通道，从非线性导星收集信息，以抵消传播引起的波前扭曲，并考虑样本运动(心跳、呼吸)。最后，神经生理学专家Pieter Vanden Berghe(BE)将与LB一起研究高速、高级扫描协议带来的优势，以优化和研究与最先进的飞秒系统相比的信号产量和样本损伤。PV还将使用GCaMP将Adapt技术应用于体积神经元活动成像，以突破当前神经元活体成像的界限。

Multiphoton microscopy is today unrivalled among other optical approaches in intravital imaging, as it allows imaging of labelled structures and proteins while providing large optical penetration depth with sub-cellular axial resolution, keeping comparatively low photobleaching levels. These advantages, along with the possibility of exciting genetically encoded calcium and voltage indicators, have made multiphoton microscopy the method of choice for neuronal studies in rodents to decipher brain function and understand neuronal diseases. Optical sampling at high speeds is of paramount importance for accurately capturing neural activity, as the characteristic timescales for action potentials are of the order of a few milliseconds, while Ca transients correlate in the 100 ms range. Current imaging technologies do not sufficiently combine fast recording rates with volumetric sampling, which is equally important to fully understand the inherently three-dimensional neuronal circuitry. With this WEAVE project, we aim to advance and tailor the spectro-temporal laser imaging by diffractive excitation technique previously introduced by one partner to the challenges of neurophotonics by: i) acquiring volumetric imaging of layered neuronal structures (cortex, intestine) at the inherent timescales of their functional processes (20Hz volume rate); ii) developing a dedicated near infrared source to image and benefit from the matured GCaMP family of fluorescence proteins; iii) analyzing and optimizing signal yield and sample damage introducing rapid adaptive scanning and sampling protocols; iv) correcting wavefront distortions and accounting for specimen movements by employing nonlinear fiduciary markers embedded in tissue depth. To achieve these goals, the ADAPT research partners have joined in a synergetic WEAVE consortium with laser physicist and inventor of spectro-temporal Multiphoton imaging Sebastian Karpf (GER) to build a volumetric imaging system at 940 nm that is perfectly tuned to image GFP-like molecules at 20 Hz volumetric imaging; nanophotonics and nonlinear optics expert Luigi Bonacina (CH) to advance, assess, and translate the disruptive technology to adaptive and targeted sampling exploiting the active modulation of the source and bespoke solutions for three-dimensional scanning. Further, LB will set-up a dedicated SHG detection channel to collect information from a nonlinear guide star to counteract propagation-induced wavefront distortions and account for sample movements (heartbeat, breathing). Finally, neurophysiology expert Pieter Vanden Berghe (BE) will study together with LB the advantages brought forth by the high-speed, advanced scanning protocols to optimize and investigate signal yield and sample damage as compared to state-of-the-art femtosecond systems. PV will also apply the ADAPT technology to volumetric neuronal activity imaging using GCaMP to push the current boundaries of neuronal intravital imaging.