Three-color single-molecule localization microscopy in chromatin

Nicolas Acosta; Ruyi Gong; Yuanzhe Su; Jane Frederick; Karla I. Medina; Wing Shun Li; Kiana Mohammadian; Luay Almassalha; Geng Wang; Vadim Backman

doi:10.1038/s41377-025-01786-1

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Three-color single-molecule localization microscopy in chromatin

Original Articles

- Three-color single-molecule localization microscopy in chromatin
- Three-color single-molecule localization microscopy in chromatin
- LSA 2025年14卷第5期页码：1265-1277
- Affiliations：
  
  1.Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA
  2.Center for Physical Genomics and Engineering, Northwestern University, Evanston, IL 60208, USA
  3.IBIS Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, IL 60208, USA
  4.Applied Physics Program, Northwestern University, Evanston, IL 60208, USA
  5.Department of Gastroenterology and Hepatology, Northwestern Memorial Hospital, Chicago, IL 60611, USA
- Author bio：
  
  Geng Wang (wanggeng@northwestern.edu)
  Vadim Backman (v-backman@northwestern.edu)
- Funds：
- DOI：10.1038/s41377-025-01786-1
  中图分类号：
- 收稿日期：2024-09-12，
  
  修回日期：2025-02-09，
  
  录用日期：2025-02-12，
  
  网络出版日期：2025-03-17，
  
  纸质出版日期：2025-05-31
- Accepted：
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Three-color single-molecule localization microscopy in chromatin[J]. LSA, 2025,14(5):1265-1277.

Acosta, N. et al. Three-color single-molecule localization microscopy in chromatin. Light: Science & Applications, 14, 1265-1277 (2025).
Three-color single-molecule localization microscopy in chromatin[J]. LSA, 2025,14(5):1265-1277. DOI： 10.1038/s41377-025-01786-1.

Acosta, N. et al. Three-color single-molecule localization microscopy in chromatin. Light: Science & Applications, 14, 1265-1277 (2025). DOI： 10.1038/s41377-025-01786-1.

摘要

Abstract

Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales

from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods

such as STORM

are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However

these efforts are challenged by the high-density nuclear environment

which can affect antibody binding affinities

diffusivity and non-specific interactions. Optimizing buffer conditions

fluorophore stability

and antibody specificity is crucial for achieving effective antibody conjugates. Here

we demonstrate a sequential immunolabeling protocol that reliably enables three-color studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm

which utilizes localizations from one target as seed points for distance

density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories

but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.

关键词

Keywords

references

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