Deep-tissue two-photon microscopy with a frequency-doubled all-fiber mode-locked laser at 937 nm

Abstract

In two-photon microscopy, low illumination powers on samples and a high signal-to-noise ratio (SNR) of the excitation laser are highly desired for alleviating the problems of photobleaching and phototoxicity, as well as providing clean backgrounds for images. However, the high-repetition-rate Ti: sapphire laser and the low-SNR Raman-shift lasers fall short of meeting these demands, especially when used for deep penetrations. Here, we demonstrate a 937-nm laser frequency-doubled from an all-fiber mode-locked laser at 1.8 μm with a low repetition rate of ∼9 MHz and a high SNR of 74 dB. We showcase two-photon excitations with low illumination powers on multiple types of biological tissues, including fluorescence imaging of mouse brain neurons labeled with green and yellow fluorescence proteins (GFP and YFP), DiI-stained and GFP-labeled blood vessels, Alexa Fluor 488/568-stained mouse kidney, and second-harmonic-generation imaging of the mouse skull, leg, and tail. We achieve a penetration depth in mouse brain tissues up to 620 μm with an illumination power as low as ∼10 mW, and, even for the DiI dye with an extremely low excitation efficiency of 3.3%, the penetration depth is still up to 530 μm, indicating that the low-repetition-rate source works efficiently for a wide range of dyes with a fixed excitation wavelength. The low-repetition-rate and high-SNR excitation source holds great potential for biological investigations, such as in vivo deep-tissue imaging.

New approaches for low phototoxicity imaging of living cells and tissues

“…A typical 40‐MHz fiber laser requires 20 times more energy to penetrate tissue to a similar depth. [ 31 ]…”

635 nm femtosecond fiber laser oscillator and amplifier

“…The emergence of ultrafast laser pulse generation, a milestone in laser science, has triggered incredible progress across a wide array of disciplines, encompassing industrial applications, energy technologies, life sciences, and beyond 1 , 2 . Cutting-edge innovations, such as robust laser ignition systems, 3 vibrant laser 3D printing, 4 mind-boggling attosecond science, 5 and high-resolution microscopic imaging, 6 are effectively powered by ultrafast lasers. Among the various laser platforms that have been developed, fiber oscillators, esteemed for their compact design, outstanding performance, and cost-effectiveness, have become one of the mainstream technologies for generating femtosecond laser pulses 7 …”

High-power Raman soliton generation tunable from 1.76 to 1.84 µm in all-fiber polarization-maintaining erbium-doped amplifier

“…He et al demonstrated a high-power all-fiber mode-locked laser at 1870 nm. The maximum output power of femtosecond laser is 341 mW at a pump power of 4.5 W [9]. However, to realize the short-wavelength operation of the TDF laser, short length of active fiber and high excitation densities need to be used to reduce the reabsorption in TDF, which result in significant power loss.…”