Fig. 3

a A presentative diagram of imaging zebrafish embryo by Second Harmonic Generation Microscopy (SHG) and Third Harmonic Generation (THG): A titanium-sapphire laser generates pulses of 200 fs at 1200 nm. The laser beam passes on galvanometric mirrors (GM) to reach the sample through the objective lens. A dichroic mirror reflects the scattered signals from the sample, and the other dichroic mirror separates them into two channels (THG) and (SHG/2PF). The signals focused on the photomultiplier tube detectors (PMT), one for THG, and the other for SHG and 2PF. b A scheme of the light spectrum in SHG and THG microscopy. The excitation wavelength is 1200 nm, while the emission spectra of THG at 400 nm, SHG at 600 nm, and Two-Photon 2PF from 600 to 800 nm with a peak at ~ 650 nm generated by the sample