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, China2Chinese University of Hong Kong, Faculty of Medicine, Department of Psychiatry, Hong Kong, China3Chinese University of Hong Kong, Faculty of Medicine, Department of Medicine and Therapeutics, Hong Kong, China4Chinese University of Hong Kong, Prince of Wales Hospital, Li Ka Shing Institute of Health Sciences, Hong Kong, China5Fudan University, Shanghai Medical College, Institute for Translational Brain Research, Shanghai, China6University of Hong Kong, School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, Hong Kong, China7University of Hong Kong, State Key Laboratory of Brain and Cognitive Sciences, Hong Kong, China8South China University of Technology, School of Physics and Optoelectronics, Guangzhou, China9Advanced Biomedical Instrumentation Centre, Hong Kong, Chinashow less
DOI: 10.1117/1.APN.1.2.026001
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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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Fig. 1. Two-photon excitation efficiency of the EGFP and EYFP under the illumination from 800 to 1000 nm.
Fig. 2. (a) Experimental configuration of the all-fiber laser source and the frequency-doubling setup. (b) ASE spectra of the TDFs with different lengths (150, 70, 30, and 10 cm, respectively). (c) ASE spectrum of the 6-cm-long TDF and the reflectance of the SESAM as a function of the wavelength.
Fig. 3. (a) Spectrum of the laser oscillator in the mode-locked state. (b) Pulse train and (c) RF spectrum of the output signal from the laser oscillator. The inset of (c) is the RF spectrum within a 200-MHz span. (d) Average output power as a function of the pump power in the amplifier. (e) Spectrum and (f) pulse width of the amplified signal. (g) Beam profile of the frequency-doubled laser centered at 937 nm. (h) Spectrum and (i) pulse width of the frequency-doubled laser after the PPLN crystal. “a. u.”: arbitrary units.
Fig. 4. (a) Schematic diagram of the 2PM experimental setup with both epi and transmitted detections. GM, galvanometric mirror; L, lens; M, mirror; Obj., objective; Con., condenser; DM, dichroic mirror; F, filters; PMT, photomultiplier tube. (b) Lateral and (d) axial PSFs of the transmitted-detection scheme. (c) The lateral resolution and (e) DOF of the transmitted-detection scheme. (f) Lateral and (h) axial PSFs of the epi-detection scheme. (g) The lateral resolution and (i) DOF of the epi-detection scheme. “a. u.”: arbitrary units.
Fig. 5. (a)–(d) Two-photon fluorescence images of YFP-labeled neurons and fibers in a mouse brain slice. (e)–(g) Two-photon fluorescence images of Alexa Fluor 488- and Alexa Fluor 568-stained mouse kidney slice. Scale bar: . FOV: . Frame rate: 0.37 Hz (mouse brain) and 0.18 Hz (mouse kidney). Power after obj.: 18 mW (mouse brain) and 13 mW (mouse kidney).
Fig. 6. (a) Two-photon fluorescence images of the DiI-stained vasculatures at different depths of the mouse brain. The depth value labeled with “*” denotes an axially projected image: 210*, 160 to ; 335*, 300 to ; 445*, 430 to ; 505*, 480 to . The corresponding intensity profile of the blood vessel at the depth of (b) and (c) . “a. u.”: arbitrary units. (d), (e) Vasculatures of different structures. Scale bar: . FOV: . Frame rate: 0.37 Hz. Power after obj.: 12 mW.
Fig. 7. (a) 3D reconstruction of the 2PM images of the EGFP-labeled mouse brain neurons. (b)–(d) Typical images of the neurons located at different depths. (e) 3D images of the GFP-labeled mouse brain vasculature. (f) Typical image of the blood vessel. Scalar bar: .
Fig. 8. SHG images of the mouse [(a), (b)] skull, [(c), (d)] leg, and [(e), (f)] tail. FOV: . Frame rate: 0.19 Hz. Power after obj.: 10 mW. Scale bar: .