HoT Auto-Blinking Probes Enable Real-Time, Super-Resolution Chromatin Imaging in Live Cells and Tissues-Wang et al
Description
Single-molecule localization microscopy (SMLM) enables visualization of chromatin architecture at nanoscale resolution. However, high-performance DNA probes suitable for SMLM in both live cells and tissues remain limited. We developed HoT (Hoechst-6-TAMRA (6-Carboxytetramethylrhodamine) derivative) probes—rhodamine-based derivatives conjugated to a Hoechst moiety—through structural fine-tuning of rhodamine spirocyclization. HoTs are self-assembling, auto-blinking probes with excellent photostability and high temporal resolution. They are permeable to live cells and enable long-term, real-time chromatin nanoscopic imaging in live and fixed cells, and in tissue sections. In live cells, we identified nanoscale features in the 3D organization of chromatin and quantified DNA fiber kinetics at high resolution. We quantified DNA compaction in single cells within retinal and colon cancer sections. OligoSTORM-labeled gene loci can be visualized and measured within their HoT-labeled chromatin footprints. Our work provides powerful tools for investigating chromatin structure and functions in living cells and tissues, with applications ranging from cancer diagnosis to retinal regeneration.
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The dataset was generated using high-resolution fluorescence microscopy to visualize chromatin structure. Imaging was performed using Single-Molecule Localization Microscopy (SMLM) and MINFLUX . Samples, including live and fixed cells as well as tissue sections (e.g., from retina and colon cancer), were labeled with HoT (Hoechst-6-TAMRA derivative) DNA probes. These probes are self-blinking fluorescent markers developed through structural optimization of rhodamine spirocyclization. The data includes SR images capturing the nanoscale 2D, 3D organization of chromatin and the quantification of DNA compaction in tissue sections. Additionally, the dataset contains correlated imaging data where specific gene loci, labeled via OligoSTORM were visualized within their broader HoT-labeled chromatin context. The primary reagents used were the HoT probes, and the data acquisition relied on SMLM and MINFLUX hardware and software.