ULTRAFAST MULTIPHOTON LASER ACTIVATION SYSTEM

本提案的目标是建立一个超快多光子激光激活系统。这个集成的激光仪器系统将被组装起来，以支持一系列创新的生物医学和生物技术研究项目，这些项目是由多光子光化学和光物理实现的。锁模激光器产生的超快（约100飞秒）脉冲允许同时吸收来自明亮脉冲照明的两个或多个光子，用于分子激发，如生物医学研究中使用的多光子激光扫描荧光显微镜。然而，多光子激发的研究正在成为分子生物学和生物医学研究和诊断的有力光化学工具。特别是，将特定基因的DNA与相应的调节蛋白进行多光子光化学交联，可以分析这些基因表达的基本调节因子的分子生物学，并鉴定每种特定的调节酶-DNA配对。所提出的激光系统提供适当的、明亮的、超快的脉冲和紫外（约250）波长的脉冲对，以有效地在体外和体内实现这种交联过程。超快激光系统的高能飞秒脉冲在210纳米到3000纳米的宽范围内提供，以启用或增强整个项目组。超快激光系统的其他用户应用包括：(1)通过多光子光激活开发用于局部微药理学的笼化试剂和笼化神经递质，(2)通过组织中分子的多光子氧化激活研究疾病中的氧化应激效应，(3)生物光损伤机制的研究，(4)几种生物物理技术的发展。(5)研究所有相关的多光子光化学过程的光物理机制。提出的仪器系统促进了几种生物物理技术的发展，特别是多光子光化学方法的发展，用于制造用于生物技术设备的三维复杂微流体系统，光物理学用于增强多光子显微镜的实施和用于生物医学内源性组织荧光的内窥光谱来识别疾病状态。该仪器系统由一个可调谐的钛蓝宝石激光振荡器、再生放大器、两个独立可调谐的光学参数振荡器、用于倍频的谐波发生器、延迟线和相关仪器组成，以满足生物医学用户群体的高脉冲能量和异常宽的调谐范围要求，并为生物医学用户群体安全提供用户友好型仪器。

The objective of this proposal is to create an Ultrafast Multiphoton Laser Activation System. This integrated laser instrument system is to be assembled to support a select set of innovative biomedical and biotechnology research programs that are implemented by multiphoton photochemistry and photophysics. Ultrafast (approximately 100 femtosecond) pulses generated by mode-locked lasers allow simultaneous absorption of two or more photons from bright pulsed illumination for molecular excitation as in the multiphoton laser scanning fluorescence microscopy used in biomedical research in living tissue. However, research on multiphoton excitation is evolving powerful photochemical tools for molecular biology and biomedical research and diagnosis. In particular, multiphoton photochemical crosslinking of the DNA of specific genes to the corresponding regulatory proteins allows assay of the molecular biology of these essential regulators of gene expression and identification of each particular regulatory enzyme-DNA pairing. The proposed laser system provides the appropriate, bright, ultrafast pulses and pulse pairs at ultraviolet (approximately 250) wavelengths to implement efficiently this crosslinking process in vitro and, it is expected, in vivo. The high-energy femtosecond pulses of the Ultrafast Laser System are provided throughout the wide range from 210 nm to 3,000 nm needed to enable or enhance the entire group of projects. Other user's applications of the Ultrafast Laser System include (1) development of caged reagents and caged neurotransmitters for localized micro-pharmacology by multiphoton photoactivation, (2) research on oxidative stress effects in disease by multiphoton oxidant activation of molecules in tissue, (3) investigations of biological photodamage mechanisms, (4) developments of several biophysical technologies, and (5) research on the photophysical mechanisms of all of the relevant multiphoton photochemical processes involved. Several biophysical technology developments are facilitated by the proposed instrument system, in particular, development of a multiphoton photochemical method to fabricate 3-dimensionally complex microfluidic systems for biotechnology devices, and photophysics for enhanced implementation of multiphoton microscopy and for biomedical endoscopic spectroscopy of intrinsic tissue fluorescence to recognize disease states. The instrument system consists of a tunable titanium:sapphire laser oscillator, regenerative amplifier, two independently tunable optical parametric oscillators, harmonic generators for frequency multiplication, delay lines and associated instrumentation in order to encompass the high pulse energy and unusually broad tuning range requirements of the select biomedical user community and to safely provide user friendly instrumentation for the biological user community.