RESUMEN
We present a novel method for the precise tracking of plasmonic gold nanorods (AuNRs) in live cells, enabling a comprehensive understanding of the nanocargo's cellular dynamics. Traditional single particle tracking (SPT) struggles with accurately determining all five spatial parameters (x, y, z, Ï, and θ) in live cells due to various challenges. Our innovation combines electronic tunable lens (ETL) technology with bifocal parallax dark-field (DF) microscopy, allowing continuous adjustment of the imaging focal plane for automatic tracking of both translational and rotational movements of AuNRs. This 5D single-particle orientation and rotational tracking (5D SPORT) method achieves remarkable precision, with 3D localization precisions of 9 (x), 10 (y), and 15 nm (z) and angular resolutions below 2°. To showcase its applicability, we investigated intracellular transport of nanocargos using transferrin-modified AuNRs as the imaging probe. Differentiated transport stages, such as active transport and pause period, were clearly unveiled from the observed dynamics in 5D. This advancement in single particle tracking holds promise for a wide range of applications in biomedical research, particularly when combined with other imaging modalities, such as light sheet fluorescence microscopy.
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.