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 p...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 1.8μm 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 620μm 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 530μm,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.展开更多
基金The National Natural Science Foundation of China(Nos.51772159,51472125)the Natural Science Foundation of Zhejiang Province(No.LZ17E020001)K.C.Wong Magna Fund in Ningbo University
基金Research Grants Council of the Hong Kong Special Administrative Region of China(HKU C7074-21GF,HKU 17205321,HKU 17200219,HKU 17209018,CityU T42-103/16-N)and Health@InnoHK program of the Innovation and Technology Commission of the Hong Kong SAR Government.
文摘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 1.8μm 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 620μm 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 530μm,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.
