Probing diffusion and photochemical properties through localized photobleaching

Abstract

Two-photon excitation can excite physiological ultraviolet (UV) chromophores at visible wavelengths and holds significance for applications such as non-linear microscopy, micro-pharmacology, localized heating and tissue ablation. However, little quantitative data is available on the absolute two-photon absorption cross sections ((sigma)₂) of these molecules. Their low two-photon absorbance and limited solubility implies that the sensitivity required for such measurements is much higher than that available from standard techniques. We employ the recently developed generalized z-scan technique to measure the absolute value of the (sigma)₂ of tryptophan, the most ubiquitous of the physiological near-UV chromophores, and obtain the value of 32.0 +/- 1.2 mGM (1 GM equals 10^-50 cm⁴sec/photon/molecule) at 532 nm. This is the first determination of the absolute (sigma)₂ of any biological UV chromophore, and can be used to calibrate the previously reported relative two-photon excitation spectra of a number of such molecules.