Dual-modulation difference stimulated emission depletion microscopy to suppress the background signal

Stimulated emission depletion(STED)nanoscopy is one of the most well-developed nanoscopy techniques that can provide subdiffraction spatial resolution imaging.Here,we introduce dual-modulation difference STED microscopy(dmdSTED)to suppress the background noise in traditional STED imaging.By applying respective time-domain modulations to the two continuous-wave lasers,signals are distributed discretely in the frequency spectrum and thus are obtained through lock-in demodulation of the corresponding frequencies.The background signals can be selectively eliminated from the effective signal without compromise of temporal resolution.We used nanoparticle,fixed cell,and perovskite coating experiments,as well as theoretical demonstration,to confirm the effectiveness of this method.We highlight dmdSTED as an idea and approach with simple implementation for improving the imaging quality,which substantially enlarges the versatility of STED nanoscopy.

Sub-diffraction dark spot localization microscopy

Single molecular localization microscopy(SMLM)is a useful tool in biological observation with sub-10-nm resolution.However,SMLM is incapable of discerning two molecules within the diffraction-limited region unless with the help of a stochastic on–off switching scheme which yet entails time-consuming processes.Here,we produce a novel kind of focal spot pattern,called sub-diffraction dark spot(SDS),to localize molecules within the sub-diffraction region of interest.In our proposed technique nominated as sub-diffracted dark spot localization microscopy(SDLM),multiple molecules within the diffraction-limited region could be distinguished without the requirement of stochastic fluorescent switches.We have numerically investigated some related impacts of SDLM,such as detection circle diameter,collected photon number,background noise,and SDS size.Simulative localization framework has been implemented on randomly distributed and specifically structured samples.In either two-or three-dimensional case,SDLM is evidenced to have2 nm localization accuracy.