Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm

TLDR

The authors measured two‑photon excitation spectra of 11 fluorophores from 690 to 1050 nm with 100‑fs Ti:sapphire pulses and derived absolute cross sections for Rhodamine B and Fluorescein by comparison to one‑photon fluorescence. The results show identical one‑photon and two‑photon emission spectra, wavelength‑independent TPE emission, fluorescence intensity proportional to the square of excitation intensity within ±4% below saturation, and absolute cross sections for the remaining nine fluorophores.

Abstract

Measurements of two-photon fluorescence excitation (TPE) spectra are presented for 11 common molecular fluorophores in the excitation wavelength range 690 nm < λ < 1050 nm. Results of excitation by ∼100-fs pulses of a mode-locked Ti:sapphire laser are corroborated by single-mode cw Ti:sapphire excitation data in the range 710 nm < λ < 840 nm. Absolute values of the TPE cross section for Rhodamine B and Fluorescein are obtained by comparison with one-photon-excited fluorescence, assuming equal emission quantum efficiencies. TPE action cross sections for the other nine fluorophores are also determined. No differences between one-photon- and two-photon-excited fluorescence emission spectra are found. TPE emission spectra are independent of excitation wavelength. With both pulsed and cw excitation the fluorescence emission intensities are strictly proportional to the square of the excitation intensity to within ±4% for excitation intensities sufficiently below excited-state saturation.