Two-Photon Circular Dichroism Spectrometer

Tinoco（1975）和Power（1975）首先从理论上讨论了双光子圆二色性（TPCD），即两个左圆偏振光子与两个右圆偏振光子的微分吸收。虽然相关的TP-ORD效应已被证实，但该技术在理论上与拉曼光学活性相似，尚未得到实验证明（Gedanken & Tamir, 1987）。我们提出了一种基于钛蓝宝石飞秒激光技术的TPCD光谱仪，采用非常灵敏的瞬态热光栅检测方法。我们主要感兴趣的领域是蛋白质的二级结构。鉴于这种蛋白质缺乏荧光，这种应用要求直接吸收或基于热的方法来检测TPCD信号。TPCD作为一种与传统的圆二色性（CD）互补的方法，在测定蛋白质二级结构和检测细微构象变化方面具有几个潜在的优势。这些是(1)提供了一个新的线性独立的数据集，可能允许对二级结构进行独特的确定；(2)TPCD光谱从300到600 nm覆盖了对应于单光子能量到150到300 nm的电子激发态，从而将激发范围扩展到更高的能量，并覆盖了a-螺旋的预期更高的激子成分。(3)三个独立的TPCD光谱（对于相同频率的双光子和随机取向的样品）证明了独立的信息。该光谱仪基于高重复频率（1khz）飞秒激光技术。双光子CD信号的检测将基于微分瞬态热光栅方法。这种方法最近被证明为CD测量提供了一种非常灵敏的方法，以前也被证明是一种非常灵敏的方法，用于进行两p光子吸收测量。我们的建议是结合这两种能力来提供新的双光子CD信息。基于先前双光子测量的详细信噪比计算和期望微分双光子截面的可靠估计表明，该方法将获得良好的信噪比。

Two-photon circular dichroism (TPCD), the differential absorption of two left circularly polarized photons vs. two right circularly polarized photons, was first discussed theoretically by Tinoco (1975) and by Power (1975). This technique, which is theoretically similar to Raman optical activity, has not yet been demonstrated experimentally although the related TP-ORD effect has been demonstrated (Gedanken & Tamir, 1987). We propose to construct a TPCD spectrometer based on recently developed commercially available Ti:sapphire femtosecond laser technology using the very sensitive transient thermal grating detection method. Our primary area of interest is the secondary structure of proteins. Given the lack of fluorescence of such proteins, this application dictates a direct absorption or thermally based methodology for detection of the TPCD signal. TPCD offers several potential advantages as a method complimentary to conventional circular dichroism (CD) for the determination of protein secondary structure and detection of subtle conformational changes. These are (1 ) a new, linearly independent data set is provided potentially permitting a unique determination of secondary structure, (2) the TPCD spectrum extending from 300 to 600 nm covers electronic excited states corresponding in one-photon energy to 150 to 300 nm thus extending the range of the excitations to higher energy and covering the expected higher exciton components of the a-helix, (3) there are three independent TPCD spectra (for two-photons of the same frequency and randomly oriented samples) proving independent information. The spectrometer to be constructed is based on high-repetition rate (1 kHz) femtosecond laser technology. The detection of the two-photon CD signal will be based on the differential transient thermal grating method. This method has been recently shown to provide a very sensitive method for CD measurements and has previously been shown to be a very sensitive method for performing two-p hoton absorption measurements. Our proposal is to combine these two capabilities to provide new two-photon CD information. A detailed signal to noise calculation based on prior two-photon measurements using this grating method and a reliable estimate of the expected differential two-photon cross section shows that excellent signal to noise will be achieved.