New technologies for in vivo spectral resolved high speed multiphoton microscopsy

体内光谱分辨高速多光子显微镜新技术

• 批准号: 9021702
• 负责人: Elly Nedivi
• 金额: $ 19.5万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9021702
• 关键词: Algorithms; Biological; Brain; Cell Culture Techniques; Cell Nucleus; Cell physiology; Cells; Cellular Structures; Color; Complex; Confocal Microscopy; Event; Fourier Transform; Goals; Health; Image; Image Analysis; Imagery; Inhibitory Synapse; Label; Maps; Methods; Microscope; Microscopy; Monitor; Morphology; Neurobiology; Neurons; Noise; Proteins; Resolution; Scanning; Signal Transduction; Speed; Structure; Subcellular structure; Synapses; System; Technology; Testing; Time; Tissues; Vesicle; base; design; detector; hippocampal pyramidal neuron; in vivo; in vivo imaging; instrument; instrumentation; microscopic imaging; multi-photon; neocortical; new technology; next generation; novel; protein protein interaction; protein transport; submicron; temporal measurement

 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.

 描述（由申请人提供）：光谱分辨成像在许多生物学研究中普遍存在，从绘制突触动力学图谱到监测细胞内信号传导，以及研究蛋白质-蛋白质相互作用。独立监测突触、细胞核、蛋白质运输囊泡和各种其他多组分复合物等细胞结构的寿命和动态的能力对于揭示其细胞功能及其组装和分解至关重要。虽然使用共焦显微镜对同一细胞中 3-4 种不同蛋白质进行光谱分辨可视化是很常见的 在固定脑切片或细胞培养中，体内动态多蛋白成像仍然是一个挑战，但许多细胞内和细胞间相互作用依赖于完整组织的背景。我们的目标是开发和实施与高通量多光子显微镜兼容的光谱解析技术，以实现多组分亚细胞结构的大体积体内成像。在前两个目标中，我们建议测试两种用于大体积、高速成像的新型光谱测量方法，它们各有优缺点，可以根据不同的成像需求进行定制。第三个目标是，我们将开发一种基于小波的高效泊松去噪光谱解混合算法，该算法可以通过允许对信噪比低得多的图像进行精确分析来潜在地增强这两种方法。目标 1：设计色散光谱仪 (DS)，利用多阳极 PMT (MAPMT) 在多焦点多光子显微镜 (MMM) 系统中实现高光谱成像。目标 2：设计傅里叶变换光谱仪 (FTS)，以实现 MMM 和宽视场多光子显微镜 (WFMM) 系统中的高光谱成像。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。