IDBR: A Focus-Engineered Coherent Anti-Stokes Raman Scattering (FE-CARS) Microscope for Biological Imaging

该奖项是为了表彰通过聚焦工程技术改进相干反斯托克斯拉曼散射（CARS）显微镜的对比度。相干抗斯托克斯拉曼散射（CARS）显微镜已经迅速上升为具有化学选择性的细胞和组织成像的非侵入性工具。基于其独特的对脂质、蛋白质和DNA的振动选择性，car在生物学问题上的潜在应用是广泛的。然而，由于无所不在的非共振背景和一些分子群的弱振动响应的存在，到目前为止，应用仅限于脂质成像。为了开拓CARS显微镜在生物学和生物医学领域的新应用，背景抑制和对比度改善方法势在必行。这项工作介绍了一种新的方法，以CARS显微镜，提供即时的对比度提高。该方法是基于控制从焦体发射的CARS波的空间干扰。发射控制是通过使用空间光调制器（SLM）对入射激光脉冲进行空间相位整形或焦点工程来实现的。使用适当的相位掩模可以控制不需要的背景贡献的破坏性干扰，而建设性干扰则会突出振动共振物体。一种聚焦工程（FE-） CARS显微镜将被开发，具有以下里程碑：1)通过计算机控制入射横向光束轮廓的相位整形来表征和优化FE-CARS显微镜的对比度改善；2)将FE-CARS与双光子激发荧光（TPEF）和聚焦工程二次谐波（SE-SHG）相结合，用于组织和细胞的多模态成像。FE-CARS多模态成像将测试a)脂质沉积和钙羟基磷灰石晶体；B)在甘油或二甲基亚砜的影响下胶原纤维的结构变化。预计该显微镜的发展将对生物成像研究产生深远的影响。该仪器将用于培养非线性显微镜领域的研究生、本科生和大学预科生。该仪器将在几个外展项目中使用。在这些项目中，PI组织了大学生物科学学院的少数民族科学项目和为期4周的高中生暑期学校。

This award is for developing improved contrast in Coherent Anti-Stokes Raman Scattering [CARS] Microscopy through Focus-Engineering [FE-CARS]. Coherent anti-Stokes Raman scattering (CARS) microscopy has rapidly ascended as a noninvasive tool for imaging cells and tissues with chemical selectivity. Based on its unique vibrational selectivity to lipids, proteins, and DNA, the full range of potential CARS applications to biological problems is vast. Nonetheless, due to the presence of a ubiquitous nonresonant background and the weak vibrational response of several molecular groups, applications have been limited to imaging of lipids so far. To open up a new range of applications for CARS microscopy in biology and biomedicine, background suppression and contrast improvement methods are imperative. This work introduces a new approach to CARS microscopy, which provides immediate contrast improvement. The approach is based on controlling the spatial interference of the CARS waves emanating from the focal volume. Emission control is achieved by spatial phase shaping, or focal engineering, of the incident laser pulses using a spatial light modulator (SLM). The use of appropriate phase masks allows controlled destructive interference of unwanted background contributions, while constructive interference accentuates vibrationally resonant objects. A focus-engineered (FE-) CARS microscope will be developed, with the following milestones: 1) Characterizing and optimizing contrast improvement with FE-CARS through computer-controlled phase shaping of the incident transverse beam profiles; 2) Combining FE-CARS with two-photon excited fluorescence (TPEF) and focus-engineered second harmonic generation (SE-SHG) for multimodal imaging of tissues and cells. FE-CARS multimodal imaging will be tested on a) lipid depositions and calcium hydroxyapapatite crystals; b) structural changes to collagen fibers under the influence of glycerol or dimethyl-sulfoxide. The proposed microscope development is anticipated to have far reaching implications for biological imaging studies. The proposed instrument will be used for training graduate students, undergraduate students and pre-college students in the field of nonlinear microscopy. The instrument will be used in several outreach programs. Among these programs, the PI organized the Minority Science Program of the School of Biological Sciences at the university and a 4-week Summer School for high school students.