Surface-Functionalized Halo-Tag Gold Nanoprobes for Live-Cell Long-Term Super-Resolution Imaging of Endoplasmic Reticulum Dynamics.

Super-resolution fluorescence microscopy has emerged as a powerful tool for studying endoplasmic reticulum (ER) dynamics in living cells. However, the lack of high-brightness, high-photostability, and stable labeling probes makes long-term super-resolution imaging of the ER still challenging. Herein, we reported a surface-functionalized Halo-tag gold nanofluorescent probe (GNP-Atto565-fR8-CA) that exhibits excellent brightness, photostability, and biocompatibility. GNP-Atto565-fR8-CA can simultaneously load multiple Atto565 dye molecules, significantly improving its brightness. Modifying the cell-penetrating peptide fR8 enables GNP-Atto565-fR8-CA to be efficiently delivered into the cytoplasm, overcoming the challenge of their easy entrapment in vesicles. Fluorescent labeling of ER proteins via Halo tags enables high specificity and stable labeling of GNP-Atto565-fR8-CA to the ER. The SIM super-resolution imaging results showed that GNP-Atto565-fR8-CA can track and observe the long-term dynamic process of the ER, and can also be used for long-term super-resolution imaging of the dynamic interactions between the ER and other organelles. This work offers a practical tool to study live-cell ER ultrastructure and dynamics.

Sensitivity investigation of a biosensor with resonant coupling of propagating surface plasmons to localized surface plasmons in the near infrared region.

Gold nanoparticles (AuNPs) can be used to improve the performance of propagating surface plasmon resonance (PSPR) refractive index sensors. The resonant coupling effect between PSPR and localized surface plasmon resonance (LSPR) supported by AuNPs on sensitivity remains to be elucidated in terms of evanescent field intensity and distribution. In this study, we directly compare the sensitivity of the PSPR sensor and the resonant coupling mode between the PSPR and LSPR sensors in the wavelength scanning mode. The sensitivity of PSPR can be significantly improved in the near-infrared region excitation wavelength. 1,6-Hexanedithiol was used to achieve a AuNP modified gold film (GF-AuNP). The PSPR excited by the prism coupling mechanism can effectively stimulate LSPR supported by AuNPs in the GF-AuNP, and then resonant coupling is generated. Compared with PSPR, the resonant coupling mode shows a decrease in penetration depth by 28 times and an increase in the surface electric field intensity by 4.6 times in the numerical simulations. The decrease in the penetration depth in the GF-AuNP is made at the expense of bulk sensitivity. The biosensing sensitivity of the GF-AuNP shows up to 7-fold improvement in the carcinoembryonic antigen immunoassay and the GF-AuNP is proven to be a better biosensor. The experimental measurements are in excellent agreement with the theoretical model. This study can be also considered as a guide for the design of plasmonic sensors for detecting multiple substances at different scales, such as cells and proteins.

Superresolution Fluorescence Microscopy of Platelet Subcellular Structures as a Potential Tumor Liquid Biopsy.

Blood-based tumor liquid biopsies are promising as an alternative or complement to tissue biopsies due to their noninvasiveness, convenience, and safety, and there is still a great demand for the discovery of new biomarkers for these biopsies. Here, nanoscale distribution patterns of subcellular structures in platelets, as imaged by structured illumination superresolution fluorescence microscopy, as a new type of potential biomarker for tumor liquid biopsies are presented. A standardized protocol for platelet sample preparation and developed an automated high-throughput image analysis workflow is established. The diagnostic capability based on the statistical analysis of 280 000 superresolution images of individual platelets from a variety of tumor patients, benign mass patients, and healthy volunteers (n = 206) is explored. These results suggest that the nanoscale distribution patterns of α-granules in platelets have the potential to be biomarkers for several cancers, including glioma and cervical, endometrial, and ovarian cancers, facilitating not only diagnosis but also therapeutic monitoring. This study provides a promising novel type of platelet parameter for tumor liquid biopsies at the subcellular level rather than the existing cellular or molecular level and opens up a new avenue for clinical applications of superresolution imaging techniques.