New technologies for in vivo spectral resolved high speed multiphoton microscopsy

 描述（由申请人提供）：光谱分辨成像在许多生物学研究中是普遍存在的，从映射突触动力学到监测细胞内信号传导和研究蛋白质-蛋白质相互作用。独立监测细胞结构（如突触、细胞核、蛋白质运输囊泡和各种其他多组分复合物）的寿命和动力学的能力对于揭示它们的细胞功能以及它们的组装和拆卸至关重要。虽然使用共聚焦显微镜在同一细胞中的3-4种不同蛋白质的光谱分辨可视化是相当常规的， 在固定的脑切片或细胞培养物中，体内动态多蛋白质成像仍然是一个挑战，然而许多细胞内和细胞间的相互作用依赖于完整组织的环境。我们的目标是开发和实施光谱分辨技术，与高通量多光子显微镜兼容，允许大容量，多组分亚细胞结构的体内成像。在前两个目标中，我们提出测试两种新的光谱方法，用于大容量，高速成像，具有各自的优点和缺点，可以定制以满足不同的成像需求。在第三个目标中，我们将开发一个高效的基于小波的泊松去噪光谱解混算法，它可以通过允许对SNR低得多的图像进行准确分析来增强这两种方法。目标1：设计一个色散光谱仪（DS），使高光谱成像的多焦多光子显微镜（MMM）系统利用多阳极光电倍增管（MAPMT）。目标2：设计一个傅里叶变换光谱仪（FTS），使MMM和宽场多光子显微镜（WFMM）系统的高光谱成像。目标3：开发一种形态学引导的泊松去噪最大似然（MLE）谱分解算法，以降低原始图像的SNR要求。

 DESCRIPTION (provided by applicant): Spectrally-resolved imaging is ubiquitous in numerous biological studies ranging from mapping synapse dynamics, to monitoring of intracellular signaling, and studying protein-protein interactions. The ability to independently monitor the lifetime and dynamics of cellular structures, such as the synapse, the nucleus, protein trafficking vesicles, and various other multicomponent complexes is critical to revealing their cellular function as well as their assembly and disassembly. While spectrally resolved visualization of 3-4 different proteins in the same cell is quite routine using confocal microscopy in fixed brain sections or in cell culture, dynamic multi-protein imaging in vivo remains a challenge, yet many intra- and inter- cellular interactions are dependent on the context of an intact tissue. Our goal is to develop and implement spectrally resolved technologies that are compatible with high throughput multiphoton microscopy to allow large volume, in vivo imaging of multicomponent subcellular structures. In the first two aims we propose testing two novel spectrometric approaches for large volume, high-speed imaging, with respective strengths and weaknesses, that can be tailored to tackle different imaging needs. In the third aim, we will develop a highly efficient wavelet based Poisson denoised spectral un-mixing algorithm that can potentially enhance both approaches by allowing accurate analysis of images with far lower SNR. Aim 1: Design a dispersive spectrometer (DS) to enable hyperspectral imaging in a multifocal multiphoton microscope (MMM) system utilizing multianode PMTs (MAPMT). Aim 2: Design a Fourier transform spectrometer (FTS) to enable hyperspectral imaging in MMM and wide-field multiphoton microscopy (WFMM) systems. Aim 3: Develop a morphology-guided, Poisson-denoised maximum likelihood (MLE) spectral decomposition algorithm to reduce SNR requirement of raw images.