Single-laser-based simultaneous four-wavelength excitation source for femtosecond two-photon fluorescence microscopy

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.

Nyquist-exceeding high voxel rate acquisition in mesoscopic multiphoton microscopy for full-field submicron resolution resolvability

“…The Fidelity-2 fiber laser produced <60 fs pulses at a center-wavelength of around 1,070 nm, which was optically coupled to a 7 mm-long photonic crystal fiber to introduce a negative dispersion for self-phase modulation-enabled spectral broadening ( Hsiao et al., 2021 ), so as to obtain the lower excitation wavelengths required for the Alexa Fluor 546 and Thy1-GFP related experiments. A half-wave plate was added prior to the fiber-coupling system to enable spectrum-shaping by means of polarization rotation ( Apolonski et al., 2002 ).…”

Multi‐Photon Microscopy

“…For optimal excitation of three spectrally diverse fluorescent proteins (CFP, YFP, and RFP) it is necessary—owing to unequal absorption coefficients and fluorophore quantum yields, and because scattering‐induced attenuation with depth is color dependent—to either tune the wavelength of the pulsed laser source or simultaneously produce at least two spectrally distinct pulsed excitations (Mahou et al., 2012). Another option is to employ a single laser producing four wavelengths (Hsiao, Huang, Borah, Chen, & Sun, 2021) for multi‐photon microscopy. Several improvements to the initial Brainbow transgenic lines have been made (Cai, Cohen, Luo, Lichtman, & Sanes, 2013) to facilitate imaging and analysis of the mouse connectome.…”

Advances in Ultrafast Fiber Lasers for Multiphoton Microscopy in Neuroscience

“…Similarly, innovative fiber amplifiers concentrate on chirped pulse amplification and direct amplification approaches for increasing the energy of ultra-short laser pulses. Eventually, the average power of the laser pulses increased to over 1 Watts [18] at MHz repetition rates, which is essential for achieving multiphoton effects such as two-and three-photon fluorescence, as well as label-free imaging in the brain [19].…”