Effects of absorption saturation in colloidal quantum dots as fluorophores for multiphoton fluorescence microscopy

It is shown that colloidal semiconductor nanocrystals (quantum dots), which are promising fluorophores for multiphoton fluorescence microscopy, exhibit a two-photon absorption saturation effect at moderate powers (not exceeding 10 mW) of femtosecond pumping radiation. An analytical expression for the power of two-photon fluorescence of quantum dots as a function of the average pumping power is obtained. With this expression, the deviation of the found dependence from the quadratic law is explained by two factors, i.e., a large two-photon absorption cross section of quantum dots and their slow (compared to the typical pumping pulse-repetition period) relaxation to an unexcited state. Using an LSM 510 Carl Zeiss laser scanning microscope equipped with a Ti:Sa tunable femtosecond laser, a series of model experiments is performed to reveal the saturation effect in a solution of commercially available quantum dots. Good agreement is obtained between the measured dependence of the power of two-photon fluorescence on the average pump power and the theoretical calculation results. It is also experimentally demonstrated that, under fluorescence saturation conditions, the spatial resolution of the method of multiphoton fluorescence microscopy is lost; this effect is analyzed numerically.