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

用于活细胞非侵入性成像的相干反斯托克斯拉曼多重显微镜

• 批准号: BB/D001013/1
• 负责人: Paola Borri
• 金额: $ 34.87万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD001013%2F1
• 关键词: Coherent; Anti; Stokes; Raman; multiplex

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.

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，用于活细胞的实时研究。这种仪器将允许在生理条件下用化学对比剂对细胞和亚细胞结构进行非侵入性显微镜检查，而不需要染色或表达标记的蛋白质，以及在完整组织中具有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，并且仍然是在活细胞和组织内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，除非染色，否则在光学显微镜下很难区分。荧光显微镜，使用染料标记的抗体或蛋白质与荧光标签的融合，提供了一种高度灵敏和特异的生物分子可视化方法。然而，当用于活细胞中的真实的时间观察时，这些修饰的生物分子提出了它们的行为是真实的还是人为的问题。此外，所有荧光探针都易于发生光漂白，即在用光激发后荧光强度的不可逆退化。光学显微镜的另一个主要困难发生在试图以高轴向分辨率和长穿透深度对完整组织内的活细胞进行成像时。这种新型显微镜的关键思想是通过光与化学键中特定共振振动的散射（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反斯托克斯拉曼散射（汽车）时，这种现象可以被相干增强。由于汽车非线性地依赖于激发光强度，因此仅在小的聚焦体积中实现用于汽车生成的足够强度，其中激发光子被集中，从而导致类似于多光子荧光显微镜的固有高分辨率和切片能力。汽车显微镜在细胞生物学成像中的应用最近已经被一些小组探索，但仍处于相当早期的阶段。需要大量的开发工作来将该技术改进到成熟状态。我们提出了一种新的多重汽车显微镜的建设，能够检测几种化学物质在平行与改进的化学选择性和灵敏度相比，在文献中报道到现在。为了开发生物背景下的仪器，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，即使用最先进的荧光显微镜也难以接近。这些问题是：（a）致病酵母白假丝酵母（Candida albicans）在细胞程序性死亡（凋亡）过程中线粒体结构的变化和模式酵母酿酒酵母（Saccharomyces cerevisiae）在呼吸振荡过程中线粒体结构的变化;（B）小鼠乳腺细胞终芽的生长、发育和凋亡;（c）黏菌盘基网柄菌（Dictyosteelium discoideum）发育过程中细胞含水量和膜动力学的动态变化。除了生物科学学院的合作者外，来自世界各地的物理和生物学科的研究人员可能会从这项工作的成果中受益。这种新型显微镜技术的使用也可能与医疗应用有关，以改善疾病的诊断和治疗。此外，拟议的研究包含实现经济设计的多重汽车显微镜，其可能的广泛应用，使显微镜制造商可能会对这个项目感兴趣。

项目成果

Coherent anti-Stokes Raman micro-spectroscopy using spectral focusing: theory and experiment

• DOI: 10.1002/jrs.2264
• 发表时间: 2009-07-01
• 期刊: JOURNAL OF RAMAN SPECTROSCOPY
• 影响因子: 2.5
• 作者: Langbein, Wolfgang;Rocha-Mendoza, Israel;Borri, Paola
• 通讯作者: Borri, Paola

Coherent anti-Stokes Raman microspectroscopy using spectral focusing with glass dispersion

• DOI: 10.1063/1.3028346
• 发表时间: 2008-11-17
• 期刊: APPLIED PHYSICS LETTERS
• 影响因子: 4
• 作者: Rocha-Mendoza, Israel;Langbein, Wolfgang;Borri, Paola
• 通讯作者: Borri, Paola

CARS microscopy using linearly chirped ultrafast laser pulses

使用线性啁啾超快激光脉冲的 CARS 显微镜

• DOI: 10.1117/12.809411
• 发表时间: 2009
• 作者: Rocha-Mendoza I

Differential CARS microscopy with linearly chirped femtosecond laser pulses

• DOI: 10.1117/12.873872
• 发表时间: 2011-02
• 作者: W. Langbein;I. Rocha-Mendoza;F. Masia;C. Di Napoli;I. Pope;P. Watson;P. Borri

Differential coherent anti-Stokes Raman scattering microscopy with linearly chirped femtosecond laser pulses.

• DOI: 10.1364/ol.34.002258
• 发表时间: 2009-08
• 期刊: Optics letters
• 影响因子: 3.6
• 作者: I. Rocha-Mendoza;W. Langbein;P. Watson;P. Borri