Recently, a new technique called MINFLUX was promoted and attained ~1-nanometer precision. However, MINFLUX is incapable of discerning two molecules within the diffraction-limited region unless with the help of on-off switching scheme of SMLM which yet entails time-consuming processes. Here, we produce a novel kind of focal spot pattern, called sub-diffraction dark spot, 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 fluorescent switches. We have numerically presented the SDLM modality and some impacts, like intensity, are investigated. Simulative localization framework has been implemented on randomly-distributed and specifically-structured samples. SDLM is evidenced to have high localization accuracy and stability in densely-packed fluorescent solution.
Super-resolution optical microscopy has enabled the observation of ultra-fine structures and features beyond the diffraction limit, among which nonlinear absorption has been a useful tool to investigate physical and biological characteristics in sub-100 nanometer range. Saturation competition microscopy (SAC), based on nonlinear absorption principle, has been demonstrated to obtain high resolution in either fluorescence or non-fluorescence imaging of biological applications. Furthermore, fluorescent nanodiamonds (FNDs) have been widely researched as nitrogenvacancy centers (NVCs) in FNDs are important medium in quantum entanglement. Here, we report on the characterization of NVCs using pulse SAC (pSAC) microscopy. Resolution of λ/6 has been reached and the experimental results shows that it has better signal-to-background ratio (SBR) with lower illumination intensity in contrast to stimulated emission depletion microscopy (STED).