Multiplexed Nanoscopy via Buffer Exchange.

Understanding cellular functions, particularly in their intricate complexity, can greatly benefit from the spatial mapping of diverse molecules through multitarget single-molecule localization microscopy (SMLM). Existing methodologies, primarily restricting the encoding dimensions to color and lifetime or requiring cyclic staining, often involve broad chromatic detection, specialized optical configurations, or sophisticated labeling techniques. Here, we propose a simple approach called buffer-exchange stochastic optical reconstruction microscopy (beSTORM), which introduces an additional dimension to differentiate between single molecules irrespective of their spectral properties. This method leverages the distinguishable photoblinking responses to distinct buffer conditions, offering a straightforward yet effective means of fluorophore discrimination. Through buffer exchanges, beSTORM achieves multitarget SMLM imaging with minimal crosstalk. Direct integration with expansion microscopy (ExM) demonstrates its capability to resolve up to six proteins at the molecular level within a single emission color without chromatic aberration. Overall, beSTORM presents a highly compatible imaging platform, promising significant advancements in highly multiplexed nanoscopy for exploring multiple targets in biological systems with nanoscale precision.

Single-molecule localization microscopy reveals the ultrastructural constitution of distal appendages in expanded mammalian centrioles.

Distal appendages (DAPs) are vital in cilia formation, mediating vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Although numerous DAP proteins arranging a nine-fold symmetry have been studied using superresolution microscopy analyses, the extensive ultrastructural understanding of the DAP structure developing from the centriole wall remains elusive owing to insufficient resolution. Here, we proposed a pragmatic imaging strategy for two-color single-molecule localization microscopy of expanded mammalian DAP. Importantly, our imaging workflow enables us to push the resolution limit of a light microscope well close to a molecular level, thus achieving an unprecedented mapping resolution inside intact cells. Upon this workflow, we unravel the ultra-resolved higher-order protein complexes of the DAP and its associated proteins. Intriguingly, our images show that C2CD3, microtubule triplet, MNR, CEP90, OFD1, and ODF2 jointly constitute a unique molecular configuration at the DAP base. Moreover, our finding suggests that ODF2 plays an auxiliary role in coordinating and maintaining DAP nine-fold symmetry. Together, we develop an organelle-based drift correction protocol and a two-color solution with minimum crosstalk, allowing a robust localization microscopy imaging of expanded DAP structures deep into the gel-specimen composites.

Comparison of Emergency Room Visits and Rehospitalization for Bleeding Complications following Transurethral Procedures for the Treatment of Benign Prostatic Hyperplasia: A Population-Based Retrospective Cohort Study.

BACKGROUND: The postoperative bleeding complications associated with laser surgery of the prostate and transurethral resection of the prostate (TURP) were compared. METHODS: We used the Taiwan National Health Insurance Research Database to conduct an observational population-based cohort study. All eligible patients who received transurethral procedures between January 2015 and September 2018 were enrolled. Patients who received laser surgery or TURP were matched at a ratio of 1:1 by using propensity score matching, and the association of these procedures with bleeding events was evaluated. RESULTS: A total of 3302 patients who underwent elective transurethral procedures were included. The multivariable Cox regression analysis revealed that diode laser enucleation of the prostate (DiLEP) resulted in significantly higher emergency room risks within 90 days after surgery due to clot retention than the Monopolar transurethral resection of the prostate (M-TURP) (Hazard Ratio: 1.52; 95% Confidence Interval [CI], 1.06-2.16, p = 0.022). Moreover, GreenLight photovaporization of the prostate (PVP) (0.61; 95% CI, 0.38-1.00 p = 0.050) and thulium laser vaporesection of the prostate (ThuVARP) (0.67; 95% CI, 0.47-0.95, p = 0.024) resulted in significantly fewer rehospitalization due to clot retention than did M-TURP. No significant increase in blood clots were observed in patients using comedications and those with different demographic characteristics and comorbidities. CONCLUSIONS: Among the investigated six transurethral procedures for Benign prostatic hyperplasia, PVP and ThuVARP were safer than M-TURP because bleeding events and clot retention were less likely to occur, even in patients receiving anticoagulant or antiplatelet therapy. However, DiLEP and holmium laser enucleation of the prostate (HoLEP) did not result in fewer bleeding events than M-TURP.

Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages.

Subdistal appendages (sDAPs) are centriolar elements that are observed proximal to the distal appendages (DAPs) in vertebrates. Despite the obvious presence of sDAPs, structural and functional understanding of them remains elusive. Here, by combining super-resolved localization analysis and CRISPR-Cas9 genetic perturbation, we find that although DAPs and sDAPs are primarily responsible for distinct functions in ciliogenesis and microtubule anchoring, respectively, the presence of one element actually affects the positioning of the other. Specifically, we find dual layers of both ODF2 and CEP89, where their localizations are differentially regulated by DAP and sDAP integrity. DAP depletion relaxes longitudinal occupancy of sDAP protein ninein to cover the DAP region, implying a role of DAPs in sDAP positioning. Removing sDAPs alter the distal border of centrosomal γ-tubulins, illustrating a new role of sDAPs. Together, our results provide an architectural framework for sDAPs that sheds light on functional understanding, surprisingly revealing coupling between DAPs and sDAPs.