Research Starter Grant: Three-Dimensional Polarization Second-Harmonic Generation Microscopy

伊利诺伊大学厄巴纳-香槟分校的Toussaint博士获得了一笔赠款，用于开发一种三维偏振二次谐波显微镜。这种方法是传统荧光显微镜的一种替代方法，使用非线性光学散射来完全消除对外部探针的任何依赖，而是使用被研究样本的化学性质作为造影剂，从而实现真正的非侵入性成像。这里需要注意的是，倍频显微镜适用于一类特殊但极其相关的生物系统--那些显示出有序的分子空间排列(非中心对称)的系统。在这种情况下，这样的系统在与强激光脉冲相互作用时，自然产生频率是原始激光脉冲两倍的光。这种倍频光被用来为样品成像，其方式与双光子荧光显微镜中使用的荧光大致相同。然而，该技术的应用已经被限制为仅利用产生倍频的原始光的电场矢量(偏振)的两个自由度。在这种情况下，极化被用来产生关于样品的分子对称性的信息。所提出的技术将利用光的全3D偏振态，以便首次产生三维偏振倍频。三维偏振倍频显微镜将通过将矢量束与标准偏振光学相结合来创建。具体地说，径向偏振矢量光束在显微镜S焦点处产生增强的偏振，而横向偏振分量是通过操纵商业偏振光学获得的。这一新仪器将促进生物发现方面的进展，并为生物科学和工程学方面的本科生和研究生提供独特的培训和教育机会。通过跨学科的研究和开发，这些学科的学生将对设计和开发以及细胞生物学的基本原理有深入的了解。

A grant is awarded to Dr. Toussaint of the University of Illinois at Urbana-Champaign to develop a three-dimensional polarization second harmonic generation microscope. This approach is an alternative approach to traditional fluorescence microscopy using nonlinear optical scattering to completely obviate any reliance on exogenous probes, and rather uses the chemical properties of the specimen under study as the contrast agent, thereby allowing for truly noninvasive imaging. The caveat here is that SHG microscopy works for a special, but extremely relevant, class of biological systems?those that exhibit an ordered spatial arrangement of molecules (noncentrosymmetry). In this case, such systems upon interacting with an intense laser pulse naturally produce light at twice the frequency of the original laser pulse. This SHG light is what is used to image specimens in much the same way as fluorescence is used in two-photon fluorescence microscopy. However, utilization of this technique has been limited to exploiting only two degrees-of-freedom of the electric field vector (polarization) of the original light generating the SHG. In this case, the polarization is used to yield information on the molecular symmetries of specimens. The proposed technique will utilize the full 3D state of polarization of the light in order to generate, for the first time, three-dimensional polarization SHG. Three-dimensional polarization SHG microscopy will be created by integrating vector beams with standard polarization optics. Specifically, a radially polarized vector beam produces an enhanced polarization at a microscope?s focus, while the transverse polarization components are obtained from manipulation of commercial polarization optics. This new instrument will allow for advances in biological discovery as well as provide unique training and educational opportunities to undergraduate and graduate students in the biological sciences as well as engineering. Through interdisciplinary research and development students across these disciplines will gain insights into the fundamentals of design and development as well as cell biology.