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Two-photon microscopy (2PM) is a valuable tool in biomedical research, as it allows for noninvasive imaging deep into tissues. However, the high repetition frequency of traditional Ti:sapphire mode-locked lasers limits imaging depth at low exposure power, and fiber lasers, which overcome high repetition rates, can still suffer from low gains and low signal-to-noise (SNR) ratio. Researchers from the University of Hong Kong have now developed a new frequency-doubled all-fiber mode-locked laser at 937 nm with a low repetition rate and a high SNR, which enables penetration deeper into tissues while requiring a fraction of the power of other fiber lasers.

New 937 nm Laser Enables Deeper Imaging with 2-photon Microscopy

The novel laser source relies on self-phase modulation in a single-mode fiber to simultaneously boost its 1.8 μm power and compress the pulse width. The system achieves a low repetition rate of ~9 MHz and a high SNR of 74 dB and does not require any optical filters or specialty fibers. Due to its low repetition frequency and high SNR, the laser source requires only 10 mW to image tissue at depths of over 600 µm; by comparison, a 40 Mhz fiber laser requires approximately 200 mW at similar depth. This low power requirement greatly reduces photobleaching and photodamage during imaging, which improves deep tissue imaging as well as live cell imaging. 


The researchers demonstrated 2PM excitations on samples including mouse brains, blood vessels and mouse kidney. The laser achieved a penetration depth of 620 μm into mouse brain tissue, and also enabled high-resolution and contrast images of blood vessels up to ~530 μm deep. Additionally, the team conducted second-harmonic generation (SHG) imaging of intact mouse skull, leg and tail using the laser source, demonstrating label-free imaging and initially validating the system’s potential for multimodal imaging applications. This research was published in Advanced Photonics Nexus. 


Two-photon imaging results, based on the novel 937-nm laser. (a) and (b) Two-photon fluorescence images of YFP-labeled neurons and fibers in a mouse brain slice. (c) Two photon fluorescence images of the lipophilic tracer-stained vasculatures at different depths of the mouse brain. (d) 3D reconstruction of the images of EGFP-labeled mouse brain neurons.


“This novel high-SNR 937-nm laser source achieves a good balance between sensitivity, penetration depth, and imaging speed for two-photon imaging,” said corresponding author Tian Qiao. “Its great performance in two-photon imaging indicates its exciting potential for biological investigations, such as in vivo deep-tissue imaging and multimode imaging.” 


In addition to its high performance, the laser is also compact in size, and the use of telecommunication-grade fibers, rather than specialty fibers, minimizes its cost, the authors wrote.