Improvement in Resolution of Multiphoton Scanning Structured Illumination Microscopy via Harmonics

We describe a multiphoton(mP)-structured illumination microscopy(SIM)technique,which demonstrates substantial improvement in image resolution compared with linear SIM due to the nonlinear response of fluorescence.This nonlinear response is caused by the effect of nonsinusoidal structured illumination created by scanning a sinusoidally modulated illumination to excite an mP fluorescence signal.The harmonics of the structured fluorescence illumination are utilised to improve resolution.We present an mP-SIM theory for reconstructing the super-resolution image of the system.Theoretically,the resolution of our m P-SIM is unlimited if all the high-order harmonics of the nonlinear response of fluorescence are considered.Experimentally,we demonstrate an 86 nm lateral resolution for two-photon(2P)-SIM and a 72 nm lateral resolution for second-harmonic-generation(SHG)-SIM.We further demonstrate their application by imaging cells stained with F-actin and collagen fibres in mouse-tail tendon.Our method can be directly used in commercial mP microscopes and requires no specific fluorophores or high-intensity laser.

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

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.