High performance 937 nm laser allows scientists to see deeper things with lower

Two photon microscopy (2PM) plays a reliable and effective role in non-invasive deep tissue imaging in biomedical research. Since the invention of two-photon microscope at the end of the 20th century, relevant research has been emerging, pushing 2PM from fluorophore to imaging method, and applying it in biochemistry and medicine.

Low repetition rate laser excites a variety of fluorescence signals. Source: Courtesy of T. Qiao (HKU)

How to image deep tissue is always a big challenge for two-photon imaging. Laser sources can potentially address this challenge. However, the traditional Ti: sapphire mode-locked two-photon laser is limited by its high repetition rate and cannot provide high pulse energy for deep tissue imaging at low exposure power. Fiber laser can easily overcome the problem of high repetition rate by adding tens of meters of fiber in the cavity, but in some cases, its gain is low and the signal to noise ratio (SNR) is low.

Recently, according to the Advanced Photonics Nexus, researchers from the Omega Research Group of Kenneth Wong of the University of Hong Kong (HKU) developed a high-performance laser as a new type of light source for multiphoton microscope. They reported a 937 nm laser with a frequency of 1.8 μ M double of all fiber mode-locked laser, with low repetition rate of~9 MHz and high signal-to-noise ratio of 74 dB.

The new 937 nm laser source is realized by self phase modulation in single-mode fiber μ M power increase and pulse width compression. The 937nm laser design is suitable for high-sensitivity deep tissue imaging of various fluorescent proteins. The laser source provides two-photon excitation for a variety of biological tissue types. The penetration depth of mouse brain reaches 620 μ m. The ability of this technique in deep tissue imaging is revealed. The researchers also carried out second harmonic (SHG) imaging, demonstrated unlabeled imaging, and preliminarily verified the potential of this light source in multimodal imaging applications.

Two photon imaging results based on a new 937 nm laser. (a) And (b) two-photon fluorescence images of YFP labeled neurons and fibers in mouse brain slices. (c) Two photon fluorescence images of blood vessels stained with lipophilic tracer at different depths of mouse brain. (d) 3D reconstruction image of mouse brain neurons labeled with EGFP. Source: He, Tang, et al., doi 10.1117/1.APN.1.2.026001

Due to the low repetition frequency and high signal-to-noise ratio of the light source μ For tissue imaging at a depth above m, only 10 mW power is required, while for fiber lasers with a depth significantly lower than 40 mhz, about 200 mW power is required at the same depth. This greatly reduces photobleaching and light damage in imaging, and improves the depth of tissue imaging and the safety of in vivo imaging.

The two-photon excitation efficiency of EGFP and EYFP under 800~1000 nm light.

This work will promote in-depth understanding of the research and biomedical applications of deep tissue imaging. Tian Qiao, a postdoctoral researcher and corresponding author of the University of Hong Kong, commented: "This new type of 937nm laser light source with high signal-to-noise ratio has achieved a good balance between sensitivity, penetration depth and imaging speed in two-photon imaging. Its outstanding performance in two-photon imaging shows that it has exciting potential in biological research, such as deep tissue imaging and multi-mode imaging."

(a) Experimental configuration and frequency doubling setting of all fiber laser source. (b) ASE spectra of TDFs of different lengths (150, 70, 30 and 10 cm, respectively). (c) The ASE spectrum of the 6cm long TDF and the reflectivity of the SESAM are taken as a function of the wavelength.

Source:Deep-tissue two-photon microscopy with a frequency-doubled all-fiber mode-locked laser at 937 nm, Advanced Photonics Nexus (2022). DOI: 10.1117/1.APN.1.2.026001