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
Gold nanoparticles (AuNPs) have become increasingly useful in recent years for their roles in nanomedicine, cellular biology, energy storage and conversion, photocatalysis, and more. At the single-particle level, AuNPs have heterogeneous physical and chemical properties which are not resolvable in ensemble measurements. In the present study, we developed an ultrahigh-throughput spectroscopy and microscopy imaging system for characterization of AuNPs at the single-particle level using phasor analysis. The developed method enables quantification of spectra and spatial information on large numbers of AuNPs with a single snapshot of an image (1024 × 1024 pixels) at high temporal resolution (26 fps) and localization precision (sub-5 nm). We characterized the localized surface plasmonic resonance (SPR) scattering spectra of gold nanospheres (AuNSs) of four different sizes (40-100 nm). Comparing to the conventional optical grating method which suffers low efficiency in characterization due to spectral interference caused by nearby nanoparticles, the phasor approach enables high-throughput analysis of single-particle SPR properties in high particle density. Up to 10-fold greater efficiency of single-particle spectro-microscopy analysis using the spectra phasor approach when compared to a conventional optical grating method was demonstrated.
RESUMEN
The development of nanomaterials such as two-dimensional (2D) layered materials advanced applications in many fields, including biosensors format based on field-effect transistors. The unique physical and chemical properties of 2D layered materials enable the detection limit of biomolecules as low as â¼1 pg/mL. The majority of 2D layered materials contain different structural features and defects introduced in chemical synthesis and fabrication processing. These structural features have different physicochemical properties, causing heterogeneous adsorption of bioreceptors like antibodies, enzymes, etc. Understanding the correlation between the adsorption of bioreceptors and properties of structural features is essential for building highly efficient, sensitive biosensors based on 2D layered materials. Here, we utilize a single-molecule localization-based super-resolved fluorescence imaging method to unveil the inhomogeneous adsorption of antibody fragments on 2D layered molybdenum disulfide (MoS2). The surface coverage of antibody fragments on MoS2 thin flakes is quantitatively measured and compared at different structural features and different layer thicknesses. The methodology in the current work can be extended to study bioreceptor adsorption on other types of 2D layered materials and pave a way to improve biosensors' sensitivity based on defect engineering 2D layered materials.
