We report on third-harmonic generation (THG) of biomolecular solutions at the fluid/glass interface as a means to probe resonant contributions to their nonlinear absorption spectra that could serve as contrast mechanisms for functional imaging. Our source was 100 fs laser pulses whose center wavelength varied from 760 to 1000 nm. We find evidence of a two-photon resonance in the ratio of third-order susceptibilities, sample ͑3͒ ͑3͒ / glass ͑3͒ , for aqueous solutions of Rhodamine B, Fura-2, and hemoglobin and a three-photon resonance in sample ͑3͒ ͑3͒ / glass ͑3͒ for solutions of bovine serum albumin. Consistent with past work, we find evidence of a one-photon resonance of sample ͑3͒ ͑3͒ / glass ͑3͒ for water, while confirming a lack of resonant enhancement for benzene. At physiological concentrations, hemoglobin in different ligand-binding states could be distinguished on the basis of features of its THG spectrum.
Porphyrin molecules have a highly conjugated cyclic structure and are theorized to have unusually large two-photon absorptivities (sigmaTPA), i.e., sigmaTPA approximately 10(2) GM. The authors tested this claim. Ultrafast two-photon absorption (TPA) spectroscopy was performed on solutions of hemoglobin, which contains a naturally occurring metaloporphyrin. They used a pump-probe technique to directly detect the change in transmission induced by TPA over the wavelength range of lambda0=780-880 nm. As controls, they measured the TPA of the dyes rhodamine 6G and B; their measurements both verify and extend previously reported values. In new results, hemoglobin was found to have a peak two-photon absorptivity of sigmaTPA approximately 150 GM at lambda0=825 nm, near a resonance of the Soret band. This value supports theoretical expectations. They also found a significant difference in the TPA of carboxyhemoglobin versus oxyhemoglobin, e.g., sigmaTPA=61 GM versus sigmaTPA=18 GM, respectively, at lambda0=850 nm, which shows that the ligand affects the electronic states involved in TPA.
The recently developed technique of ultrafast third harmonic generation (THG) micro-spectroscopy is discussed. The approach is easily adapted to a standard laser scanning microscope and allows for two and three photon resonances to be identified in non-fluorescent unlabeled samples. This work provides nonlinear microscopists with a tool for further understanding the contrast and damage mechanisms they will encounter under nonlinear excitation. Here, we use THG micro-spectroscopy to investigate the nonlinear optical properties of hemoglobin over the spectral range of 770-1000 nm with 100 fs long nJ energy laser pulses. We demonstrate the ability of this approach to distinguish different ligand binding states in physiological solutions of human hemoglobin.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.