Three-color single-molecule localization microscopy in chromatin DOI Creative Commons
Nicolas Acosta, Ruyi Gong, Yuanzhe Su

et al.

Light Science & Applications, Journal Year: 2025, Volume and Issue: 14(1)

Published: March 17, 2025

Abstract Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments domains at different length scales, from nanometers micrometers. Single molecule localization (SMLM) methods, such as STORM, are essential studying chromatin supra-nucleosome level due their target epigenetic marks that determine organization. Multi-label imaging necessary unpack its complexity. However, these efforts challenged by high-density nuclear environment, which can affect antibody binding affinities, diffusivity non-specific interactions. Optimizing buffer conditions, fluorophore stability, specificity crucial achieving effective conjugates. Here, we demonstrate sequential immunolabeling protocol reliably enables three-color studies within dense environment. This couples multiplexed datasets robust analysis algorithm, utilizes localizations one seed points distance, density multi-label joint affinity measurements explore all three targets. Applying this algorithm analyze distance reveals heterochromatin euchromatin not-distinct territories, but transcription couple periphery heterochromatic clusters. work step in molecular environment capacity investigation multi-component systems enhanced accuracy.

Language: Английский

Three-color single-molecule localization microscopy in chromatin DOI Creative Commons
Nicolas Acosta, Ruyi Gong, Yuanzhe Su

et al.

Light Science & Applications, Journal Year: 2025, Volume and Issue: 14(1)

Published: March 17, 2025

Abstract Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments domains at different length scales, from nanometers micrometers. Single molecule localization (SMLM) methods, such as STORM, are essential studying chromatin supra-nucleosome level due their target epigenetic marks that determine organization. Multi-label imaging necessary unpack its complexity. However, these efforts challenged by high-density nuclear environment, which can affect antibody binding affinities, diffusivity non-specific interactions. Optimizing buffer conditions, fluorophore stability, specificity crucial achieving effective conjugates. Here, we demonstrate sequential immunolabeling protocol reliably enables three-color studies within dense environment. This couples multiplexed datasets robust analysis algorithm, utilizes localizations one seed points distance, density multi-label joint affinity measurements explore all three targets. Applying this algorithm analyze distance reveals heterochromatin euchromatin not-distinct territories, but transcription couple periphery heterochromatic clusters. work step in molecular environment capacity investigation multi-component systems enhanced accuracy.

Language: Английский

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