Third Harmonic Microscopy: Dynamic, High-Resolution, Three-Dimensional Imaging Without Bleaching

三次谐波显微镜：动态、高分辨率、三维成像，无需漂白

• 批准号: 9987257
• 负责人: Jeffrey Squier
• 金额: $ 34.48万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9987257&HistoricalAwards=false
• 关键词: Third; Harmonic; Microscopy; Dynamic; Resolution

A high-resolution, real-time, microscope that is based on third-harmonic generation and provides unique possibilities to visualize interfaces in refractive index or third-order nonlinear susceptibility in optically transparent media, and biological systems is being developed. The thin optical sections produced in the third harmonic microscope can be used in conjunction with standard surface rendering techniques to produce three-dimensional images. No fluorophore is necessary to label the specimen as is used in traditional laser fluoresence microscopy, as the signal is generated by endogenous interfaces. Thus, in third harmonic microscopy the images do not fade due to bleaching, and can be viewed for extended periods without loss of intensity or clarity. This imaging technique is therefore particular useful for imaging three-dimensional dynamics in microscopic systems. For dynamic measurements, imaging volumes must be repeatedly addressed in order to develop a reliable time series of transient phenomena. In the past, the effectiveness over which dynamic systems could be measured was limited by the bleaching characteristics of an exogenous label. Third harmonic imaging relies on naturally occurring boundaries within the sample to generate image contrast, and therefore does not fade, and is highly suitable for visualizing dynamic three-dimensional systems.A diode-pumped, femtosecond Nd:glass oscillator optimized for microscopy will be constructed. The laser will produce third harmonic signals that are 16-21 times greater than can be achieved with commercially available lasers. The laser will also be beneficial for 2-photon imaging. Operating at a wavelength of 1.06 micro meter, this light penetrates tissue ~20% deeper than present femtosecond lasers that commonly operate at 800 nm. A novel microscope design will be constructed that can simultaneously capture fluoresence images and third harmonic images with perfect registration. This is important for fully quantifying the image contrast mechanisms in third harmonic microscopy, and enables a quantitative analysis of this new imaging process. The third harmonic microscope will be used in conjunction with an image correlation spectroscopy technique developed specifically to allow rapid measurements of macromolecular aggregation within an intact cellular environment. By combining temporal and spatial autocorrelation analysis of an image time series, it is possible to obtain information on the molecular dynamics and transport properties as well as measuring the state of aggregation of the molecules as a function of time. The combined information can provide insight into the molecular mechanisms that govern many biochemical reactions in living cells. Measurement of molecular interactions and dynamics is integral for a full understanding of how cells build functional macromolecular assemblies as well as how they transduce signals across the plasma membrane following binding of external ligands to receptors localized to the cell surface.

一种基于三次谐波产生的高分辨率、实时显微镜正在开发中，它为光学透明介质和生物系统中的折射率或三阶非线性磁化率界面的可视化提供了独特的可能性。在三次谐波显微镜中产生的薄光学切片可以与标准表面渲染技术结合使用，以产生三维图像。由于信号是由内源性界面产生的，因此不需要像传统激光荧光显微镜那样用荧光团来标记标本。因此，在三次谐波显微镜中，图像不会因漂白而褪色，并且可以在不损失强度或清晰度的情况下长时间观看。因此，这种成像技术对于微观系统中的三维动力学成像特别有用。对于动态测量，成像体积必须反复处理，以便开发可靠的瞬态现象时间序列。在过去，可以测量的动态系统的有效性受到外源标签的漂白特性的限制。三次谐波成像依赖于样本内自然发生的边界来产生图像对比度，因此不会褪色，非常适合于可视化动态三维系统。本文将构建一个用于显微镜优化的二极管泵浦飞秒钕玻璃振荡器。该激光器将产生比商用激光器大16-21倍的三次谐波信号。激光也将有利于双光子成像。工作波长为1.06微米，这种光穿透组织的深度比目前通常工作在800纳米的飞秒激光器深20%。一种新型的显微镜设计将被构建，可以同时捕获荧光图像和三次谐波图像与完美配准。这对于充分量化三次谐波显微镜中的图像对比度机制是重要的，并且可以对这种新的成像过程进行定量分析。三次谐波显微镜将与图像相关光谱技术结合使用，该技术专门用于在完整的细胞环境中快速测量大分子聚集。通过结合图像时间序列的时间和空间自相关分析，可以获得分子动力学和传输特性的信息，以及测量分子的聚集状态作为时间的函数。这些结合起来的信息可以深入了解控制活细胞中许多生化反应的分子机制。分子相互作用和动力学的测量对于充分理解细胞如何构建功能性大分子组装以及它们如何在细胞表面的外部配体与受体结合后将信号传递到质膜上是不可或缺的。