• Advanced Photonics Nexus
  • Vol. 1, Issue 2, 026001 (2022)
Hongsen He1、†, Huajun Tang1, Meng Zhou1, Hei Ming Lai2、3、4, Tian Qiao1、*, Yu-xuan Ren5, Cora S. W. Lai6、7, Ho Ko2、3、4, Xiaoming Wei8, Zhongmin Yang8, Kevin K. Tsia1、9, and Kenneth K. Y. Wong1、9、*
Author Affiliations
  • 1University of Hong Kong, Department of Electrical and Electronic Engineering, Hong Kong, China
  • 2Chinese University of Hong Kong, Faculty of Medicine, Department of Psychiatry, Hong Kong, China
  • 3Chinese University of Hong Kong, Faculty of Medicine, Department of Medicine and Therapeutics, Hong Kong, China
  • 4Chinese University of Hong Kong, Prince of Wales Hospital, Li Ka Shing Institute of Health Sciences, Hong Kong, China
  • 5Fudan University, Shanghai Medical College, Institute for Translational Brain Research, Shanghai, China
  • 6University of Hong Kong, School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, Hong Kong, China
  • 7University of Hong Kong, State Key Laboratory of Brain and Cognitive Sciences, Hong Kong, China
  • 8South China University of Technology, School of Physics and Optoelectronics, Guangzhou, China
  • 9Advanced Biomedical Instrumentation Centre, Hong Kong, China
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    DOI: 10.1117/1.APN.1.2.026001 Cite this Article Set citation alerts
    Hongsen He, Huajun Tang, Meng Zhou, Hei Ming Lai, Tian Qiao, Yu-xuan Ren, Cora S. W. Lai, Ho Ko, Xiaoming Wei, Zhongmin Yang, Kevin K. Tsia, Kenneth K. Y. Wong. Deep-tissue two-photon microscopy with a frequency-doubled all-fiber mode-locked laser at 937 nm[J]. Advanced Photonics Nexus, 2022, 1(2): 026001 Copy Citation Text show less

    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.

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    Supplementary Materials
    Hongsen He, Huajun Tang, Meng Zhou, Hei Ming Lai, Tian Qiao, Yu-xuan Ren, Cora S. W. Lai, Ho Ko, Xiaoming Wei, Zhongmin Yang, Kevin K. Tsia, Kenneth K. Y. Wong. Deep-tissue two-photon microscopy with a frequency-doubled all-fiber mode-locked laser at 937 nm[J]. Advanced Photonics Nexus, 2022, 1(2): 026001
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