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: 30μm. FOV: 150×150μm. 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 260μm; 335*, 300 to 370μm; 445*, 430 to 460μm; 505*, 480 to 530μm. The corresponding intensity profile of the blood vessel at the depth of (b) 580μm and (c) 445μm. “a. u.”: arbitrary units. (d), (e) Vasculatures of different structures. Scale bar: 30μm. FOV: 225×225μm. 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: 50μm.

Fig. 8. SHG images of the mouse [(a), (b)] skull, [(c), (d)] leg, and [(e), (f)] tail. FOV: 225×225μm. Frame rate: 0.19 Hz. Power after obj.: 10 mW. Scale bar: 50μm.