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

H Moritomo,S Onishi,Y Makino,Y Suzuki,J Kawamata,K Matsumoto

doi:10.1063/5.0004953

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.

Highlights

Compared with the femtosecond Ti:sapphire laser, which is a common light source of multi-photon microscopes, the Ybdoped fs fiber laser has several advantages, such as stability against changes of the surrounding environment, e.g., humidity or temperature, and the small size of its oscillator.1–4 using a fs fiber laser as the excitation light source of a two-photon fluorescence microscope enables two-photon imaging even under severe conditions, such as in an operating room of a hospital or an animal research laboratory, where a Ti:sapphire laser may not be stable
The lower the order of photon absorption, the higher the multi-photon absorption efficiency that was observed. This trend indicated that brighter multi-photon fluorescence microscopy images would be obtained when competitive scitation.org/journal/adv two- and three-photon absorptions were utilized as the excitation processes, compared with the case of three-photon absorption
Judging from the higher efficiency of competitive twoand three-photon absorptions than that of pure three-photon absorption, it is expected that the excitation power needed for obtaining multi-photon fluorescence microscope images of human embryonic kidney 293 (HEK293) cells should be smaller when staining with BP, NP, PYJ, and ACJ than in the case of FLJ and NPJ

Summary

Introduction

Compared with the femtosecond (fs) Ti:sapphire laser, which is a common light source of multi-photon microscopes, the Ybdoped fs fiber laser has several advantages, such as stability against changes of the surrounding environment, e.g., humidity or temperature, and the small size of its oscillator.1–4 using a fs fiber laser as the excitation light source of a two-photon fluorescence microscope enables two-photon imaging even under severe conditions, such as in an operating room of a hospital or an animal research laboratory, where a Ti:sapphire laser may not be stable. 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.

Results

Conclusion