Single-laser-based simultaneous four-wavelength excitation source for femtosecond two-photon fluorescence microscopy
Journal
Biomedical Optics Express
Journal Volume
12
Journal Issue
8
Pages
4661-4679
Date Issued
2021
Author(s)
Abstract
Multicolor labeling of biological samples with large volume is required for omic-level of study such as the construction of nervous system connectome. Among the various imaging method, two photon microscope has multiple advantages over traditional single photon microscope for higher resolution and could image large 3D volumes of tissue samples with superior imaging depth. However, the growing number of fluorophores for labeling underlines the urgent need for an ultrafast laser source with the capability of providing simultaneous plural excitation wavelengths for multiple fluorophores. Here, we propose and demonstrate a single-laser-based four-wavelength excitation source for two-photon fluorescence microscopy. Using a sub-100 fs 1,070-nm Yb:fiber laser to pump an ultrashort nonlinear photonic crystal fiber in the low negative dispersion region, we introduced efficient self-phase modulation and acquired a blue-shifted spectrum dual-peaked at 812 and 960 nm with 28.5% wavelength conversion efficiency. By compressing the blue-shift near-IR spectrum to 33 fs to ensure the temporal overlap of the 812 and 960 nm peaks, the so-called sum frequency effect created the third virtual excitation wavelength effectively at 886 nm. Combined with the 1,070 nm laser source as the fourth excitation wavelength, the all-fiber-format four-wavelength excitation source enabled simultaneous four-color two-photon imaging in Brainbow AAV-labeled (TagBFP, mTFP, EYFP, and mCherry) brain samples. With an increased number of excitation wavelengths and improved excitation efficiency than typical commercial femtosecond lasers, our compact four-wavelength excitation approach can provide a versatile, efficient, and easily accessible solution for multiple-color two-photon fluorescence imaging in the field of neuroscience, biomolecular probing, and clinical applications with at least four spectrally-distinct fluorophores. ? 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Subjects
Blue shift
Conversion efficiency
Efficiency
Femtosecond lasers
Fiber lasers
Fluorescence imaging
Fluorescence microscopy
Fluorophores
Particle beams
Photonic crystal fibers
Photons
Pumping (laser)
Clinical application
Excitation efficiency
Excitation wavelength
Multicolor labeling
Nonlinear photonic crystal fiber
Two-photon fluorescence imaging
Two-photon fluorescence microscopy
Wavelength excitations
Laser excitation
Article
brain
clinical article
connectome
crystal
dispersion
excitation
fluorescence imaging
fluorescence microscopy
human
image quality
local field potential
nervous system
neuroscience
Raman spectrometry
signal noise ratio
single photon emission computed tomography
spectroscopy
SDGs
Publisher
The Optical Society
Type
journal article