Coherent Anti-Stokes Raman multiplex microscopy for non-invasive imaging of living cells

我们的目标是开发和展示一种新一代的显微镜，具有更高的灵敏度和化学特异性，可用于活细胞的实时研究。这种仪器将允许在具有化学对比的生理条件下对细胞和亚细胞结构进行非侵入性的显微镜检查，而不需要标记蛋白的染色或表达，以及在完整组织中高穿透深度的三维成像能力。光学显微镜是推动细胞生物学进步的不可或缺的工具，而且仍然是在活细胞和组织中获得空间和时间分辨率的唯一实用手段。然而，大多数细胞成分没有颜色，在光学显微镜下很难区分，除非它们被染色。荧光显微镜，使用染料标记的抗体或蛋白质与荧光标记的融合，提供了一种高度敏感和特异的生物分子可视化方法。然而，当用于活细胞的实时观察时，这些修饰的生物分子提出了问题，它们的行为是真实的还是人为的。此外，所有的荧光探针都容易发生光漂白，即光激发后荧光强度的不可逆降解。光学显微镜的另一个主要困难是试图以高轴向分辨率和长穿透深度成像完整组织中的活细胞。这种新型显微镜的关键思想是通过光的散射和化学键中特定的振动共振(拉曼散射)来获得图像对比度。当使用两个短激光脉冲共振激发振动并产生所谓的相干安替斯托克斯拉曼散射(CARS)时，这一现象可以被相干增强。由于CARS与激发光强的非线性相关，只有在激发光子集中的小焦区才能获得足够的CARS产生强度，从而获得了类似于多光子荧光显微镜的固有的高分辨率和切片能力。最近，几个小组探索了CARS显微镜在细胞生物学成像中的应用，但仍处于相当早期的阶段。要将这项技术改进到成熟状态，需要大量的开发工作。我们建议构建一种新的多重CARS显微镜，与目前文献报道的相比，该显微镜能够并行地检测几种化学物种，并且提高了化学选择性和灵敏度。为了开发用于生物学背景的仪器，我们将与生物科学学院的生物学家合作，他们将提供生物样本和有趣的问题，即使使用最先进的荧光显微镜也很难解决这些问题。这些问题是：a)致病白色念珠菌在细胞程序性死亡(凋亡)过程中线粒体结构的变化，以及模型酵母在呼吸振荡过程中线粒体结构的变化，b)小鼠乳腺细胞的末端芽生长、发育和凋亡，c)盘基网柄菌发育过程中细胞水分含量和膜动力学的动态变化。除了生物科学学院的合作者外，来自世界各地物理和生物学科的研究人员可能会从这项工作的结果中受益。这种新的显微技术的使用也可能在医学应用中具有相关性，以改进疾病的诊断和治疗。此外，本研究还为多用途汽车显微镜的广泛应用提供了一种经济的设计方案，从而使显微镜制造商对该项目很感兴趣。

Our objective is to develop and demonstrate a new microscope generation with improved sensitivity and chemical specificity for real-time studies on living cells. Such an instrument will allow non-invasive, microscopic examination of cells and subcellular structures under physiological conditions with chemical contrast without the need to stain or express tagged proteins, together with three-dimensional imaging capability of high penetration depth in intact tissues. Optical microscopy is an indispensable tool that is driving progress in cell biology, and is still the only practical means of obtaining spatial and temporal resolution within living cells and tissues. However, most cellular constituents have no colour and they are hard to distinguish under a light microscope unless they are stained. Fluorescence microscopy, using antibodies labelled with dyes or fusion of proteins with fluorescent tags has provided a highly sensitive and specific method of visualizing biomolecules. However, when used for real time observations in living cells, these modified biomolecules raise questions if their behaviour is real or artefactual. Furthermore, all fluorescent probes are prone to photo-bleaching that is an irreversible degradation of the fluorescence intensity after excitation with light. Another major difficulty in light microscopy occurs when attempting to image living cells within intact tissues with high axial resolution and long penetration depth. The key idea of this new microscope is to obtain the image contrast via scattering of light with the specific resonances of vibration in chemical bonds (Raman scattering). This phenomenon can be coherently enhanced when using two short laser pulses to resonantly excite the vibrations and generate the so-called Coherent Antistokes Raman Scattering (CARS). Since CARS depends nonlinearly on the exciting light intensity, sufficient intensities for CARS generation are achieved only in the small focal volume where the exciting photons are concentrated resulting in an intrinsic high resolution and sectioning capability similar to multi-photon fluorescence microscopy. The application of CARS microscopy for imaging in cell biology has been recently explored by a few groups but is still at a rather early stage. Significant development effort is needed to improve this technique to a mature state. We propose the construction of a new multiplex CARS microscope able to detect several chemical species in parallel with improved chemical selectivity and sensitivity as compared to what reported in literature up to now. To develop the instrument for the biological context we will collaborate with biologists within the School of Biosciences, who will provide biological samples and interesting problems that are difficult to approach even with state-of-the-art fluorescence microscopy. These problems are: a) the change in structure of mitochondria in the pathogenic yeast Candida albicans during programmed cell death (apoptosis) and in the model yeast Saccharomyces cerevisiae during respiratory oscillations, b) the end-bud growth, development and apoptosis of cells in mouse mammary glands, c) the dynamic changes in cell water content and membrane dynamics during development of the slime mold Dictyostelium discoideum. Besides the collaborators within the School of Biosciences, researchers from both physics and biological disciplines worldwide might benefit from the outcome of this work. The usage of this novel microscopy technique is also likely to be of relevance in medical applications, to improve the diagnostic and treatment of diseases. Additionally, the proposed research contains the realization of an economic design of the multiplex CARS microscope for its possible widespread application, so that microscope manufacturers are likely to be interested in this project.