RESUMEN
Single-molecule localization microscopy (SMLM), a type of super-resolution fluorescence microscopy, has become a strong technique in the toolbox of chemists, biologists, physicists, and engineers in recent years for its unique ability to resolve characteristic features at the nanoscopic level. It drastically improves the resolution of optical microscopes beyond the diffraction limit, with which previously unresolvable structures can now be studied. Spectrally resolved super-resolution fluorescence microscopy via multiplexing of different fluorophores is one of the greatest advancements among SMLM techniques. However, current spectrally resolved SMLM (SR-SMLM) methodologies present low spatial resolution due to loss of photons, low throughput due to spectral interferences, or require complex optical systems. Here, we overcome these drawbacks by developing a SR-SMLM methodology using a color glass filter. It enables high throughput and improved photon usage for hyperspectral imaging at the nanoscopic level. Our methodology can readily distinguish fluorophores of close spectral emission and achieves sub-10 nm localization and sub-5 nm spectral precisions.
RESUMEN
Understanding the host-guest interactions in porous materials is of great importance in the field of separation science. Probing it at the single-molecule level uncovers the inter- and intraparticle inhomogeneity and establishes structure-property relationships for guiding the design of porous materials for better separation performance. In this work, we investigated the dynamics of host-guest interactions in core-shell mesoporous silica particles under in situ conditions by using a fluorogenic reaction-initiated single-molecule tracking (riSMT) approach. Taking advantage of the low fluorescence background, three-dimensional (3D) tracking of the dynamics of the molecules inside the mesoporous silica pore was achieved with high spatial precision. Compared to the commonly used two-dimensional (2D) tracking method, the 3D tracking results show that the diffusion coefficients of the molecules are three times larger on average. Using riSMT, we quantitatively analyzed the mass transfer of probe molecules in the mesoporous silica pore, including the fraction of adsorption versus diffusion, diffusion coefficients, and residence time. Large interparticle inhomogeneity was revealed and is expected to contribute to the peak broadening for separation application at the ensemble level. We further investigated the impact of electrostatic interaction on the mass transfer of molecules in the mesoporous silica pore and discovered that the primary effect is on the fraction rather than their diffusion rates of resorufin molecules undergoing diffusion.
