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

一个高分辨率，实时，显微镜，是基于三次谐波产生，并提供独特的可能性，可视化界面的折射率或三阶非线性极化率在光学透明介质，和生物系统正在开发。在三次谐波显微镜中产生的薄光学切片可以与标准表面绘制技术结合使用以产生三维图像。没有荧光团是必要的标记样品，因为在传统的激光荧光显微镜中使用的信号是由内源性界面产生的。因此，在三次谐波显微镜中，图像不会由于漂白而褪色，并且可以在不损失强度或清晰度的情况下长时间观察。因此，这种成像技术对于在微观系统中成像三维动力学是特别有用的。对于动态测量，必须重复处理成像体积，以便形成瞬态现象的可靠时间序列。在过去，可以测量动态系统的有效性受到外源性标签的漂白特性的限制。三次谐波成像依赖于样品内部自然产生的边界来产生图像对比度，因此不会褪色，非常适合动态三维系统的可视化。该激光器将产生比商业上可获得的激光器大16-21倍的三次谐波信号。该激光器也将有利于双光子成像。这种光以1.06微米的波长工作，比目前通常以800 nm工作的飞秒激光器穿透组织深约20%。一种新型的显微镜设计将被构造，可以同时捕获荧光图像和第三谐波图像与完美的注册。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，并且能够定量分析这种新的成像过程。三次谐波显微镜将与专门开发的图像相关光谱技术结合使用，以允许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，可以获得关于分子动力学和传输特性的信息，以及测量作为时间的函数的分子聚集状态。这些综合信息可以提供对控制活细胞中许多生化反应的分子机制的深入了解。分子相互作用和动力学的测量对于全面理解细胞如何构建功能性大分子组装体以及它们如何在外部配体与定位于细胞表面的受体结合后跨越质膜传递信号是不可或缺的。

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.