super-resolution-imaging-shared-management-protocol-for-confocal

Super-Resolution Imaging and Shared Management: A Protocol for Confocal Microscopy with Multiplex Detection

Super-resolution microscopy (SRM) overcomes the diffraction limit of conventional light microscopy, enabling nanoscale visualization of subcellular ...

a-multicolor-3d-storm-high-resolution-visualization-protocol-for

A Multicolor 3D-STORM High-Resolution Visualization Protocol for Collagen Mineralization in Self-Assembled Recombinant Type I Collagen Fibrils

This protocol describes a multicolor three-dimensional stochastic optical reconstruction microscopy (3D-STORM) method for nanoscale visualization of ...

Zhejiang University School of Medicine

Polycomb group (PcG) proteins maintain cell identity by repressing gene expression during development. Surprisingly, emerging studies have recently reported that a number of PcG proteins directly activate gene expression during cell fate determination process. However, the mechanisms by which they direct gene activation in pluripotency remain poorly understood. Here, we show that Phc1, a subunit of canonical polycomb repressive complex 1 (cPRC1), can exert its function in pluripotency maintenance via a PRC1-independent activation of Nanog. Ablation of Phc1 reduces the expression of Nanog and overexpression of Nanog partially rescues impaired pluripotency caused by Phc1 depletion. We find that Phc1 interacts with Nanog and activates Nanog transcription by stabilizing the genome-wide chromatin interactions of the Nanog locus. This adds to the already known canonical function of PRC1 in pluripotency maintenance via a PRC1-dependent repression of differentiation genes. Overall, our study reveals a function of Phc1 to activate Nanog transcription through regulating chromatin architecture and proposes a paradigm for PcG proteins to maintain pluripotency.

PHC1 maintains pluripotency by organizing genome-wide chromatin interactions of the Nanog locus.

Super-Resolution Radial Fluctuation (SRRF) enables live-cell super-resolution imaging, but requires careful parameter selection. Here, we quantify the impact of NanoJ-SRRF parameters on microtubule imaging using FWHM and SQUIRREL-based error mapping. Ring radius proved most critical, with values >1.0 degrading resolution and fidelity. Radiality magnification and axes in ring had minimal impact. Advanced parameters revealed pitfalls: "remove positivity constraint" degraded resolution by 43%, while gradient weighting catastrophically reduced fidelity (RSP = 0.204 ± 0.116). Temporal Radiality Average (TRA) outperformed Temporal Radiality Auto-Correlations (TRAC), milimizing artifacts. This study establishes the first evidence-based guidelines for live-cell tubulin imaging: ring radius ≤1.0, TRA mode prioritization, and avoidance of gradient weighting. Integrating FWHM and SQUIRREL offers a robust opitimization framework for cytoskeletal dynamics.

Parameter optimization of NanoJ-SRRF for live-cell microtubule imaging.

Super-resolution fluorescence microscopy (SRM) has enabled visualization of nanoscale cellular structures, but systematic evaluation of resolution assessment methods across diverse biological structures and SRM modalities remains lacking. Here, we comparatively assessed three resolution metrics-Full Width at Half Maximum (FWHM), decorrelation analysis, and Fourier Ring Correlation (FRC)-across two SRM techniques (Super-resolution Radial Fluctuation, SRRF; Stimulated Emission Depletion, STED) using key subcellular structures: microtubules (filaments), mitochondria (membranes), and nuclear pore protein Nup98 (single particles) in HeLa/U2OS cells. Our results showed decorrelation analysis provided robust resolution estimates across all structures and modalities (confocal/SRRF/STED), exhibiting superior performance for dense nuclear pore complexes where FWHM failed due to overlapping point spread functions. FWHM was effective for discrete structures (microtubules/mitochondria) but unsuitable for densely packed features. NanoJ-SQUIRREL's integrated FRC analysis successfully quantified SRRF resolution for all structures, with resolution-scaled Pearson coefficient (RSP > 0.90) confirming minimal reconstruction artifacts. STED achieved significantly higher resolution than SRRF, but severe photobleaching prevented FRC application. Our study demonstrates decorrelation analysis offers universal robustness across structures and modalities, FWHM is suitable for discrete structures, and NanoJ-SQUIRREL provides standardized artifact validation and FRC-based resolution quantification for computational super-resolution. This study establishes a framework for context-appropriate resolution assessment in cell biology, emphasizing integration of structural features and technical principles.

ArticleTotal : 2	Year
Publication title	2026
Publication title	2026

Article	Year
PHC1 maintains pluripotency by organizing genome-wide chromatin interactions of the Nanog locus. Nature communications\| PubMed ID: 33990559	2021
Parameter optimization of NanoJ-SRRF for live-cell microtubule imaging. BioTechniques\| PubMed ID: 40693644	2025
Resolution assessment of super-resolution microscopy imaging: structural and technical dependencies for cell biology. Cytotechnology\| PubMed ID: 40894291	2025

Qianbing Zhao

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