Deep-tissue two-photon microscopy with a frequency-doubled all-fiber mode-locked laser at 937 nm

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.