Ultra-deep tissue imaging by super-nonlinear fluorescence microscopy

超非线性荧光显微镜超深层组织成像

• 批准号: 8769558
• 负责人: Wei Min
• 金额: $ 24万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8769558
• 关键词: Acute; Address; Alzheimer' s Disease; Applications Grants; Area; Biological; Biomedical Research; Brain; Brain imaging; Chemicals; Dependence; Drops; Dyes; Embryology; Fluorescence; Fluorescence Microscopy; Future; Generations; Goals; Huntington Disease; Image; Imaging Device; Imaging Techniques; In Vitro; Lasers; Life; Light; Medical; Microscopy; Modality; Molecular; Monitor; Mus; Neurons; Neurosciences; Organism; Penetration; Process; Proteins; Resolution; Sampling; Science; Signal Transduction; Slice; Staging; Techniques; Time; Tissues; Work; brain tissue; fluorophore; imaging probe; in vivo; innovation; light microscopy; nervous system disorder; novel; oncology; optical imaging; public health relevance; small molecule; synthetic protein; tissue phantom; two-photon

DESCRIPTION (provided by applicant): It is highly desirable to be able to probe biological activities with subcellular resolution deep inside live organisms. To this end, light microscopy is the most powerful and versatile modality. In particular, by employing a spatially confined excitation via a nonlinear transition, two-photon excited fluorescence microscopy has become indispensable for imaging scattering samples such as brain. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast, S/B, defines a fundamental imaging-depth limit (about 1 mm for mouse brain tissues), which cannot be overcome by increasing excitation intensity. Thus, how to image deeper than the fundamental imaging- depth limit poses a grand challenge for many biomedical studies including neuroscience, embryology and oncology. Novel optical imaging techniques that accomplish this goal would undoubtedly open up new avenues, transforming our ability to monitor living systems. We propose to address this challenge by exploring a unique probe-centered strategy as opposed to the popular wave-centered approaches. We realize that these exists a special class of imaging probes that can occupy metastable on- and off- states which can be manipulated by external light at proper wavelengths. By harnessing this unique class of photoswitchable probes, we propose to develop a novel platform of super-nonlinear (higher than quadratic dependence of its signal on the laser intensity) fluorescence microscopy which should be able to promote the S/B contrast and extend the fundamental imaging-depth limit of two-photon microscopy. Specifically, we will focus on two seemingly opposite but actually related techniques which couple photo-switchable probes with two-photon microscopy, namely, multiphoton activation and imaging (MPAI) and multiphoton deactivation and imaging (MPDI). Our preliminary results have demonstrated the validity of both MPAI and MPDI on three-dimensional tissue phantoms. Moreover, the 4th order super-nonlinear dependence of the signal on laser intensity was also verified experimentally. Hence, we aim to further develop and perfect the technique to the stage where it can be applied to imaging various scattering samples, particularly brain tissues, with a much better S/B contrast and depth penetration. Specifically, we plan to (1) systematically evaluate the emerging generation of photoswitchable probes (including fluorescent proteins and synthetic dyes); (2) apply the most promising probes into brain tissue slice imaging and, (3) ultimately be able to perform in vivo deep brain MPAI or MPDI with 2.4 times deeper than what two-photon microscopy can ever achieve. The proposed technical innovation has the potential to change the future landscape of in vivo light microscopy, take bio-imaging into new areas of biomedicine that have been previously uncharted.

描述（由申请人提供）：非常希望能够以亚细胞分辨率探测活体内部深处的生物活性。为此，光学显微镜 最强大、最通用的方式。特别是，通过非线性跃迁采用空间限制的激发，双光子激发荧光显微镜已成为对大脑等散射样品进行成像必不可少的。然而，由于散射损耗，入射激光功率随着成像深度呈指数下降，离焦荧光最终会压倒焦内信号。由此产生的成像对比度 S/B 损失定义了基本的成像深度限制（对于小鼠脑组织约为 1 毫米），该限制无法通过增加激发强度来克服。因此，如何比基本成像深度限制更深地成像对包括神经科学、胚胎学和肿瘤学在内的许多生物医学研究提出了巨大的挑战。实现这一目标的新颖光学成像技术无疑将开辟新的途径，改变我们监测生命系统的能力。 我们建议通过探索独特的以探针为中心的策略来应对这一挑战，而不是流行的以波为中心的方法。我们意识到存在一类特殊的成像探针，它们可以占据亚稳态的开和关状态，可以通过适当波长的外部光进行操纵。通过利用这种独特的光开关探针，我们建议开发一种新型的超非线性（其信号对激光强度的二次依赖性高于）荧光显微镜平台，该平台应该能够提高 S/B 对比度并扩展基本的双光子显微镜的成像深度限制。具体来说，我们将重点关注两种看似相反但实际上相关的技术，将光切换探针与双光子显微镜结合起来，即多光子激活和成像（MPAI）和多光子失活和成像（MPDI）。 我们的初步结果证明了 MPAI 和 MPDI 在三维组织模型上的有效性。此外，信号对激光强度的四阶超非线性依赖性也得到了实验验证。因此，我们的目标是进一步开发和完善该技术，使其能够应用于对各种散射样本（特别是脑组织）进行成像，并具有更好的 S/B 对比度和深度穿透能力。具体来说，我们计划（1）系统评估新兴一代光开关探针（包括荧光蛋白和合成染料）； (2) 将最有前途的探针应用于脑组织切片成像，(3) 最终能够进行体内深部脑 MPAI 或 MPDI，深度是双光子显微镜所能达到的 2.4 倍。所提出的技术创新有可能改变体内光学显微镜的未来格局，将生物成像带入以前未知的生物医学新领域。