Determining two-photon absorption cross sections via nonresonant multiphoton photoacoustic spectroscopy

Abstract

Multiphoton excitation of exogenous dyes and endogenous biochemical species has been used extensively for tissue diagnosis by fluorescence spectroscopy. Unfortunately, the majority of endogenous biochemical chromophores have low quantum yields, less than 0.2, therefore determining two-photon cross sections of weakly luminescencing molecules is difficult using two-photon fluorescence spectroscopy. Accurate determination of two-photon cross sections of these biochemicals could provide insight into fluorescence signal reduction caused by the absorption of excitation energy by non-target intracellular species. Non-resonant multiphoton photoacoustic spectroscopy (NMPPAS) is a novel technique we have developed for condensed matter measurements that has the potential for accurately determining two-photon absorption cross-sections of chemicals with small or non-existant fluorescence quantum yields. In this technique, near infrared light is used to generate an ultrasonic signal following a non-resonant two-photon excitation process. This ultrasonic wave is directly related to the non-radative relaxation of the chromophore of interest and is measured using a contact piezoelectric ultrasonic transducer. The signal from the ultrasonic transducer can then be used to calculate two-photon absorption cross sections. This paper will describe the validation of this technique by measuring the two-photon absorption cross- sections of well characterized chromophores such as rhodamine B and coumarin 1 in solution as well as riboflavin in a gelatin tissue phantom.