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首页> 外文会议>Conference on adaptive optics and wavefront control for biological systems III >Nonlinear Adaptive Optics: Aberration Correction in Three Photon Fluorescence Microscopy for Brain Imaging
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Nonlinear Adaptive Optics: Aberration Correction in Three Photon Fluorescence Microscopy for Brain Imaging

机译：非线性自适应光学：三个光子荧光显微镜中的像差校正脑成像

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Multiphoton fluorescence microscopy is a well-established technique for deep-tissue imaging with subcellular resolution. Three-photon microscopy (3PM) when combined with long wavelength excitation was shown to allow deeper imaging than two-photon microscopy (2PM) in biological tissues, such as mouse brain, because out-of-focus background light can be further reduced due to the higher order nonlinear excitation. As was demonstrated in 2PM systems, imaging depth and resolution can be improved by aberration correction using adaptive optics (AO) techniques which are based on shaping the scanning beam using a spatial light modulator (SLM). In this way, it is possible to compensate for tissue low order aberration and to some extent, to compensate for tissue scattering. Here, we present a 3PM AO microscopy system for brain imaging. Soliton self-frequency shift is used to create a femtosecond source at 1675 nm and a microelectromechanical (MEMS) SLM serves as the wavefront shaping device. We perturb the 1020 segment SLM using a modified nonlinear version of three-point phase shifting interferometry. The nonlinearity of the fluorescence signal used for feedback ensures that the signal is increasing when the spot size decreases, allowing compensation of phase errors in an iterative optimization process without direct phase measurement. We compare the performance for different orders of nonlinear feedback, showing an exponential growth in signal improvement as the nonlinear order increases. We demonstrate the impact of the method by applying the 3PM AO system for in-vivo mouse brain imaging, showing improvement in signal at 1-mm depth inside the brain.
机译：多光子荧光显微镜是具有亚细胞分辨率的深组织成像的良好技术。当具有长波长激发结合时的三光子显微镜（下午3pm）允许在生物组织（例如小鼠脑中）中的两光子显微镜（2pm）更深入的成像，因为由于焦点背景光可能进一步降低高阶非线性激励。如在下午2PM系统中所示，通过使用自适应光学（AO）技术的像差校正可以基于使用空间光调制器（SLM）来通过像差校正来改善成像深度和分辨率。以这种方式，可以补偿组织低阶像差和一定程度，以补偿组织散射。在这里，我们提出了3磅的AO显微镜系统进行脑成像。孤子自频移位用于在1675nm处创建飞秒源，微机电（MEMS）SLM用作波前整形装置。我们使用修改的非线性版本的三点相移干扰测量法扰乱了1020段SLM。用于反馈的荧光信号的非线性确保当光斑尺寸减小时信号在增加，允许在不直接相位测量的情况下在迭代优化过程中进行相位误差的补偿。我们比较不同的非线性反馈令的性能，显示由于非线性顺序增加的信号改善中的指数增长。我们通过对体内鼠标脑成像应用3PM AO系统来证明该方法的影响，显示大脑内1毫米深度的信号的改善。

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