新型多光子荧光显微成像原理与技术研究
文献类型：学位论文
| 作者 | 乔玲玲 |
| 学位类别 | 博士 |
| 答辩日期 | 2011 |
| 授予单位 | 中国科学院上海光学精密机械研究所 |
| 导师 | 程亚 |
| 关键词 | 双光子荧光 分辨率 成像深度 飞秒激光直写 微光学 微流体 |
| 其他题名 | The study of principles and techniques of novel multiphoton fluorescence microscopy in bio-imaging |
| 中文摘要 | 随着人们对生命科学认知的需求，多光子荧光显微术已经成为一种重要的生物成像技术。多光子激发需要同时吸收多个光子来完成电子跃迁，是一个非线性光学过程，因此它的产生需要很高的激发光强（＞109 w/cm2）。近年来出现的超短脉冲激光器，如皮秒激光器、飞秒激光器具有较高的峰值强度和较低的平均功率，是多光子荧光显微术的理想光源。多光子激发仅发生在几何焦点附近光强较高的区域（焦点外的光强较低不足以激发多光子荧光），因此多光子荧光显微术天然地具有三维层析能力，同时也大大减弱了焦点之外区域荧光分子的光漂白和光损伤。此外，多光子荧光显微术通常以近红外光作为激发光源，与可见光相比，近红外光在生物组织中的散射和吸收小，因此具有显著增强的穿透能力。 尽管多光子荧光显微术具有上述优点，但它仍然存在一些局限性。例如，在对高散射生物组织成像时，激发光强随深度的增加呈指数衰减，为了在更深处维持同样的荧光信号强度，需要按指数不断增加激发光强度。深度越深，所需要的激发光强就越高。当激发光强超过一定的阈值时，一方面会导致焦点之外区域背景荧光噪声的产生从而降低信噪比，另一方面也加剧了样品表面荧光分子的光漂白和光损伤。因此，通过增加光强并不能无限制地提高成像深度，焦点外的背景荧光噪声从根本上限制了成像深度的进一步提高。如何突破现有多光子成像技术在层析深度方面的不足是该研究领域的一大难点，本文主要致力于寻求一些全新的成像方案来解决上述问题。 此外，光流控芯片因具有低消耗、高效率和高灵敏度等传统生化分析系统无可比拟的优点，目前已经在生物医学领域掀起了一场重大革命。利用光流器件实现显微镜系统中的一些复杂功能，将非常有益于成像系统的微型化和集成化，毫无疑问会推动更多的生物医学应用。为此，本论文也特别演示了微光学、微流体元件在显微成像系统中的应用。 本文主要有以下几个创新点： 1首次将飞秒激光光学参量放大器（OPA）作为激发光源，应用于双光子荧光显微成像系统。实验中对比了飞秒振荡器 (800 nm)、飞秒放大器(800 nm)以及OPA (1240 nm)三种激发光源在散射体中的成像深度, 结果表明使用长波长激发光源能够大大地提高成像深度。该实验证实了OPA作为非线性荧光显微术的激发光源的可行性。 2首次提出一种同轴双色双光子荧光显微成像方案。本方案采用外环内圆的入射模式，两入射光束仅在焦点区域实现空间重叠，可有效避免焦点外的背景荧光。利用蒙特卡罗模拟证明：与普通的双光子成像相比，该方案在对高散射生物组织成像时具有更高的信噪比，能够有效提高层析深度。该方案是一种有望突破现有双光子成像层析深度的全新技术方案。 3首次利用飞秒激光再生放大器作为激发光源，从实验上观测到了吲哚（氨基酸重要组分）的双色双光子荧光信号。该实验证实了飞秒放大器作为双色双光子荧光显微成像术的激发光源的可行性。 4首次将飞秒激光直写的微透镜作为成像物镜，用于双光子荧光成像系统，并对生物组织成像。实验中测量的横向和纵向分辨率分别为1.6 µm和11.1 µm，此外，该实验还展示了微透镜对植物叶子组织的成像质量与5倍商用物镜相当。此实验初步演示了飞秒激光微纳加工技术制备的微透镜在小型化非线性荧光显微镜（如双光子内窥镜）上具有潜在的应用价值。 5利用飞秒激光直写技术将微光学透镜和微流体通道集成在石英玻璃芯片中，并演示了微透镜对微通道中的物体具有5倍放大镜的功能。该集成器件在芯片实验室中具有潜在的应用价值。 |
| 英文摘要 | Multiphoton fluorescence microscopy has become one of the core technologies in bio-imaging applications. It is based on the nearly simultaneous absorption of two (or more) low energy photons promoting an electronic transition. The multiphoton fluorescence excitation is a nonlinear optical process available only at high excitation intensity (>109 w/cm2). Ultrashort pulse lasers with pulse durations ranging from picosecond (ps) to femtosecond (fs) are ideal sources for multiphoton excitation because of their high peak intensities and low average powers. Multiphoton fluorescence microscopy intrinsically provides three-dimensional (3D) sectioning ability because the excitation is confined to the high-intensity region at the focus and there is virtually no out-of-focus fluorescence generation (the laser intensity out of the focal area is just not high enough for multiphoton process). For the same reason, the out-of-focus photobleaching and phototoxicity are also effectively eliminated. In addition, the infrared (IR) wavelength mostly used in multiphoton excitation helps reduce scattering in imaging of thick bio-tissues, enabling imaging deeply into the sample. Despite the above-mentioned benefits, multiphoton excitation does have its limitations. When imaging into the highly scattering bio-tissues, the high scattering loss makes it necessary to increase the excitation intensity so as to maintain an adequate peak intensity at the focus for multiphoton fluorescence excitation. The deeper the image plane, the higher the required intensity of the excitation beam. When the intensity is raised beyond a certain range, the fluorescence can already be efficiently excited in the out-of-focus area, causing strong noise and reducing the signal-noise-ratio (SNR). Moreover, it can induce out-of-focus photobleaching and phototoxicity. Therefore, the imaging depth cannot be increased infinitely by increasing excitation intensity and is fundamentally limited by the onset of out-of-focus fluorescence generation near the top of the sample. At present, it is a challenge to break the fundamental imaging-depth limit in multiphoton fluorescence micrscopy. The research in this thesis aims to deal with the challenging issues by developing some novel multiphoton excitation schemes. Additionally, compared with traditional systems, optofluidics allows for performing biochemical analysis with low consumption, high speed of analysis and high sensitivity, and has created a revolution in biomedical sciences. In general, the optofluidics can perform some multifunctionalities in microscopes. There is no doubt that the employment of optofluidics will benefit the miniaturization of imaging systems and then propel a range of biomedical applications. Therefore, this thesis has also demonstrated the applications of microoptics and microfluidics in microscope imaging systems. The main progresses of this thesis are shown as follows: First, OPA is employed in the two-photon fluorescence imaging system as the excitation source. The maximum imaging depth in highly scattering medium are compared at three different laser sources, including femtosecond oscillator, femtosecond amplifier and OPA. The results show that the imaging depth can be significantly improved by using long wavelength. This experiment establishes the feasibility of using amplified 1240-nm excitation source in nonlinear optical microscopy. Second, the concentric two-color two-photon (C2C2P) fluorescence excitation scheme is proposed to overcome the background excitation. In this proposed method, the two excitation beams are separated in space before reaching their common focal spot. Monte Carlo simulation shows that, in comparison with the one-color two-photon excitation scheme, the C2C2P fluorescence microscopy provides a significantly greater penetration depth for imaging into a highly scattering bio-tissues. The C2C2P excitation is a promising scheme to break the fundamental imaging-depth limit in two-photon fluorescence micrscopy. Third, two-color two-photon (2C2P) excitation of indole is observed upon simultaneous illumination at 400 and 800 nm femtosecond pulses from a Ti: Sapphire regenerative amplifier. This experiment demonstrates the feasibility of using a femtosecond amplifier as the excitation source in 2C2P microscopy. Fourth, a micro-lens fabricated by laser micromachining is employed for two-photon fluorescence imaging of biological tissues as an imaging objective. The lateral and axial resolutions of this microlens are measured to be 1.6 µm and 11.1 µm, respectively. Moreover, this experiment has demonstrated that the TPFM images of a plant leaf tissue obtained with this microlens are comparable to that achieved with a 5× objective lens. This work represents a critical step towards the application of micro-optical components fabricated by femtosecond laser micromachining in miniaturized nonlinear fluorescence microscope. Fifth, a microlens and a microfluidic channel are integrated into a fused silica glass chip using femtosecond laser micromachining. Moreover, this experiment has also demonstrated that the fabricated microlens exhibits good imaging performance with 5× magnification, showing great potential in the future lab-on-a-chip applications. |
| 语种 | 中文 |
| 源URL | [http://ir.siom.ac.cn/handle/181231/15674]  |
| 专题 | 上海光学精密机械研究所_学位论文 |
| 推荐引用方式 GB/T 7714 | 乔玲玲. 新型多光子荧光显微成像原理与技术研究[D]. 中国科学院上海光学精密机械研究所. 2011. |
入库方式： OAI收割
来源：上海光学精密机械研究所
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