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对生物系统进行详细了解的努力导致了对成像和显微镜技术进步的要求越来越高。与标准共聚焦显微镜相比，双光子激发具有明显的优势，包括更好的分辨率，更少的背景噪声，更深的组织穿透和更少的光损伤。然而，到目前为止，需要一个高峰值功率的Ti：蓝宝石激光器作为激发源。钛蓝宝石激光器及其泵浦系统的高成本和笨重的尺寸，以及飞秒脉冲从光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。在过去的十年中，光纤通信的快速发展使得基于光纤的光学设备广泛使用并且价格低廉。一个小型教科书大小的飞秒光纤激光器可以提供80千瓦的光脉冲峰值功率。

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.