Portable and wavelength-tunable two-photon microscopy

描述(由申请人提供)：近年来，在细胞和分子水平上实现对生物系统的详细了解的努力导致了对成像和显微镜技术进步的越来越多的要求。与标准共焦显微镜相比，双光子激发具有分辨率更高、背景噪声更少、组织穿透更深、光损伤更小等显著优点。然而，到目前为止，一直需要高峰值功率的钛宝石激光器作为激发源。钛宝石激光器及其泵浦系统的高成本和笨重的体积，以及飞秒脉冲从源到显微镜的光学耦合的困难，严重限制了双光子显微镜的实际应用。在过去的十年里，光纤通信的快速发展使基于光纤的光学设备变得广泛可用和负担得起。一台小课本大小的飞秒光纤激光器可以输出8kW峰值功率的光脉冲。 在这个项目中，我们将展示一种用于双光子显微镜的波长可调的近红外光源，称为TP-FLEX。该系统基于超快光纤激光器、孤子可调谐光纤波长移位器和荧光显微镜的集成。除了更简单的激发光脉冲，TP-FLEX提供的快速波长可调谐性还将显著增强双光子显微镜的能力。生物样品的多色标记与波长可调的激发相结合，将通过探测器和激发源之间的时域同步实现快速多色成像。此外，TP-FLEX可调谐激光系统将紧凑，波长移动光纤将用于光纤激光器和显微镜之间的灵活互连，允许光源的便携性。该系统的成功演示将增加多光子显微镜在生物、化学和分析应用领域的可及性。这里提出的跨学科研究项目汇集了三名拥有光纤设备和系统、生物物理学和光谱学以及细胞生物学和显微镜专业知识的教职员工。这一综合努力对于开发先进生物成像的创新方法至关重要。

DESCRIPTION (provided by applicant): In recent years, efforts to achieve a detailed understanding of biological systems at the cellular and molecular levels have led to increasing requirements for advances in imaging and microscopy. Compared to standard confocal microscopy, two-photon excitation offers distinct advantages including better resolution, less background noise, deeper tissue penetration and less photo-damage. However, a high peak-power Ti: Sapphire laser has until now been required as the excitation source. The high cost and cumbersome size of the Ti:Sapphire laser and its pumping system, along with the difficulty of optical coupling of femtosecond pulses from the source to the microscope, severely limit practical applications for two-photon microscopy. During the last decede, rapid advances in fiber-optic communications have made fiber-based optical devices widely available and affordable. A femtosecond fiber laser the size of a small textbook can deliver >8kW peak power of optical pulses. In this project, we will demonstrate a wavelength-tunable near-infrared light source, called TP-FLEX, for two-photon microscopy. The system is based on the integration of an ultrafast fiber laser, a soliton-based tunable fiber-optic wavelength shifter and a fluorescence microscope. In addition to a substantially simpler generation of excitation optical pulses, rapid wavelength tunability provided by TP-FLEX will significantly enhance the capability of two-photon microscopy. Multi-color labeling of biological samples combined with the ability of wavelength-tunable excitation will enable rapid multi-color imaging through time-domain synchronization between the detector and the excitation source. Moreover, the TP-FLEX tunable laser system will be compact and the wavelength shifting fiber will serve to flexibly interconnect between the fiber laser and the microscope, allowing portability of the light source. The successful demonstration of this system will result in increased accessibility of multi-photon microscopy for a wide range of biological, chemical, and analytical applications. The interdisciplinary research project proposed here brings together three faculty members with expertise in fiber-optic devices and systems, biophysics and spectroscopy and cell biology and microscopy. This integrated effort is critical for the development of an innovative methodology for advanced biological imaging.