Multi-photon fluorescence microscopy imaging of mitochondria in living cells excited by Yb-doped femtosecond fiber laser utilizing two- and three-photon competitive absorption

Abstract

By employing an Yb-doped femtosecond fiber laser oscillating at 1030 nm, multi-photon fluorescence microscopy imaging was demonstrated, utilizing competitive two- and three-photon absorption processes for excitation of the fluorescence probes. Competitive two- and three-photon absorptions occurred for all six of the fluorescence probes employed. The mitochondria of human embryonic kidney 293 cells were stained with the probes, and multi-photon fluorescence images were obtained in all cases. The brightest multi-photon fluorescence image was obtained when employing the probe compound possessing the longest cut-off wavelength. This can be attributed to the larger contribution of two-photon absorption to the competitive excitation process. Thus, by utilizing competitive two- and three-photon absorption processes, multi-photon fluorescence images can be acquired using Yb-doped femtosecond fiber laser excitation with a variety of probes.