Multi-Photon Super-Linear Image Scanning Microscopy Using Upconversion Nanoparticles

Abstract

Super-resolution fluorescence microscopy is of great interest in life science studies for visualizing subcellular structures at the nanometer scale. Among various kinds of super-resolution approaches, image scanning microscopy (ISM) offers a doubled resolution enhancement in a simple and straightforward manner, based on the commonly used confocal microscopes. ISM is also suitable to be integrated with multi-photon microscopy techniques, such as two-photon excitation and second-harmonic generation imaging, for deep tissue imaging, but it remains the twofold limited resolution enhancement and requires expensive femtosecond lasers. Here, the super-linear ISM (SL-ISM) pushes the resolution enhancement beyond the factor of two is presented and experimentally demonstrated, with a single low-power, continuous-wave, and near-infrared laser, by harnessing the emission nonlinearity within the multiphoton excitation process of lanthanide-doped upconversion nanoparticles (UCNPs). Based on a modified confocal microscope, a resolution of ≈120 nm, 1/8th of the excitation wavelength is achieved. Furthermore, a parallel detection strategy of SL-ISM with the multifocal structured excitation pattern is demonstrated, to speed up the acquisition frame rate. This method suggests a new perspective for super-resolution imaging or sensing, multi-photon imaging, and deep-tissue imaging with simple, low-cost, and straightforward implementations. Show more