Two-Photon Circular Dichroism Spectrometer

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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.