However, the spatial resolution of conventional optical microscopy is limited to ~200 nm due to the optical diffraction limitation 3, 4, 5, 6. Optical microscopy imaging is very dependable and, as such, is the most widely used analysis method in biomedical and molecular biology research 1, 2. This novel 3D fluorescence-free SRM technique was successfully applied to resolve the positions of various nanoparticles on glass and gold nanospots ( in vitro) as well as in a living single cell ( in vivo) with subdiffraction limited resolution in 3D.
Compared with the commonly used least-square method, the least-cubic method was more useful for finding the center in asymmetric cases (i.e., nanorods) with high precision and accuracy. Final, 3D super-resolution microscopy (SRM) images were obtained by resolving 3D coordinates and their Cramér-Rao lower bound-based localization precisions in an image space (530 nm × 530 nm × 300 nm) with a specific voxel size (2.5 nm × 2.5 nm × 5 nm). The 3D coordinates of individual GNP, SNP, and GNR nanoparticles ( x, y, z) were resolved by fitting the data with 3D point spread functions using a least-cubic algorithm and collation. Single-particle images were then compared with simulation data. Various plasmonic nanoparticles on a glass slide (i.e., gold nanoparticles, GNPs silver nanoparticles, SNPs and gold nanorods, GNRs) were imaged and sliced in the z-direction to a thickness of 10 nm. Augmented three-dimensional (3D) subdiffraction-limited resolution of fluorescence-free single-nanoparticles was achieved with wavelength-dependent enhanced dark-field (EDF) illumination and a least-cubic algorithm.