In vivo three- and four-photon fluorescence microscopy using a 1.8 µm femtosecond fiber laser system.

Abstract

Multiphoton microscopy has enabled us to image cellular dynamics in vivo. However, the excitation wavelength for imaging with commercially available lasers is mostly limited between 0.65-1.04 µm. Here we develop a femtosecond fiber laser system that produces ∼150 fs pulses at 1.8 µm. Our system starts from an erbium-doped silica fiber laser, and its wavelength is converted to 1.8 µm using a Raman shift fiber. The 1.8 µm pulses are amplified with a two-stage Tm:ZBLAN fiber amplifier. The final pulse energy is ∼1 µJ, sufficient for in vivo imaging. We successfully observe TurboFP635-expressing cortical neurons at a depth of 0.7 mm from the brain surface by three-photon excitation and Clover-expressing astrocytes at a depth of 0.15 mm by four-photon excitation.