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
- Light: Science & Applications Vol. 14, Issue 5, Pages: 1265-1277(2025)
- 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
  CLC：
- Received：12 September 2024，
  
  Revised：09 February 2025，
  
  Accepted：12 February 2025，
  
  Published Online：17 March 2025，
  
  Published：31 May 2025
- Accepted：
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Acosta, N. et al. Three-color single-molecule localization microscopy in chromatin. Light: Science & Applications, 14, 1265-1277 (2025).
DOI：

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

Bond, C. et al. Multiplexed DNA-PAINT imaging of the heterogeneity of late endosome/lysosome protein composition. Preprint at bioRxiv https://doi.org/10.1101/2024.03.18.585634 https://doi.org/10.1101/2024.03.18.585634 (2024)..

Hugelier, S. et al. Quantitative single-molecule localization microscopy. Annu. Rev. Biophys. 52 , 139–160 (2023)..

Lelek, M. et al. Single-molecule localization microscopy. Nat. Rev. Methods Prim. 1 , 39 (2021)..

Oddone, A. et al. Super-resolution imaging with stochastic single-molecule localization: concepts, technical developments, and biological applications. Microsc. Res. Tech. 77 , 502–509 (2014)..

Bates, M. et al. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317 , 1749–1753 (2007)..

Ricci, M. A. et al. Super resolution imaging of chromatin in pluripotency, differentiation, and reprogramming. Curr. Opin. Genet. Dev. 46 , 186–193 (2017)..

Heo, S.-J. et al. Aberrant chromatin reorganization in cells from diseased fibrous connective tissue in response to altered chemomechanical cues. Nat. Biomed. Eng. 7 , 177–191 (2023)..

Dhankhar, M. et al. Revealingthe biophysics of lamina-associated domain formation by integrating theoretical modeling and high-resolution imaging. Preprint at bioRxiv https://doi.org/10.1101/2024.06.24.600310 https://doi.org/10.1101/2024.06.24.600310 (2024)..

Zhang, Y. et al. Multicolor super-resolution imaging using spectroscopic single-molecule localization microscopy with optimal spectral dispersion. Appl. Opt. 58 , 2248–2255 (2019)..

Daugird, T. A. et al. Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment. Nat. Commun. 15 , 4178 (2024)..

Kant, A. et al. Active transcription and epigenetic reaction s synergistically regulate meso-scale genomic organization. Nat. Commun. 15 , 4338 (2024)..

Bonev, B. et al. Organization and function of the 3D genome. Nat. Rev. Genet. 17 , 661–678 (2016)..

Jerkovic´, I. et al. Understanding 3D genome organization by multidisciplinary methods. Nat. Rev. Mol. Cell Biol. 22 , 511–528 (2021)..

Zheng, H. et al. The role of 3D genome organization in development and cell differentiation. Nat. Rev. Mol. Cell Biol. 20 , 535–550 (2019)..

Finn, E. H. et al. Extensive heterogeneity and intrinsic variation in spatial genome organization. Cell 176 , 1502–1515.e10 (2019)..

Li, A. et al. Decoding topologically associating domains with ultra-low resolution Hi-C data by graph structural entropy. Nat. Commun. 9 , 3265 (2018)..

Szabo, Q. et al. Principles of genome folding into topologically associating domains. Sci. Adv. 5 , eaaw1668 (2019)..

Mansisidor, A. R. et al. Chromatin accessibility: methods, mechanisms, and biological insights. Nucleus 13 , 236–276 (2022)..

Satam, H. et al. Next-generation sequencing technology: current trends and advancements. Biology 12 , 997 (2023)..

Fudenberg, G. et al. Formation of chromosomal domains by loop extrusion. Cell Rep. 15 , 2038–2049 (2016)..

Nuebler, J. et al. Chromatin organization by an interplay of loop extrusion and compartmental segregation. Proc. Natl. Acad. Sci. USA 115 , E6697–E6706 (2018)..

Davidson, I. F. et al. Genome folding through loop extrusion by SMC complexes. Nat. Rev. Mol. Cell Biol. 22 , 445–464 (2021)..

Schueder, F. et al. Unraveling cellular complexity with transient adapters in highly multiplexed super-resolution imaging. Cell 187 , 1769–1784.e18 (2024)..

Finn, E. H. et al. A high-throughput DNA FISH protocol to visualize genome regions in human cells. STAR Protoc. 2 , 100741 (2021)..

Shim, A. R. et al. Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure. PLoS ONE 19 , e0301000 (2024)..

Khater, I. M. et al. A review of super-resolution single-molecule localization microscopy cluster analysis and quantification. Methods Patterns 1 , 100038 (2020)..

Miron, E. et al. Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin. Sci. Adv. 6 , eaba8811 (2020)..

Ou, H. D. et al. ChromEMT: visualizing 3D chromatin structure and compaction n interphase and mitotic cells. Science (New York, NY) 357 , eaag0025 (2017)..

Li, Y. et al. Analysis of three-dimensional chromatin packing domains by chromatin scanning transmission electron microscopy (ChromSTEM). Sci. Rep. 12 , 12198 (2022)..

Penagos-Puig, A. et al. Heterochromatin as an important driver of genome organization. Front. Cell Dev. Biol. 8 , 579137 (2020)..

Millán-Zambrano, G. et al. Histone post-translational modifications—cause and consequence of genome function. Nat. Rev. Genet. 23 , 563–580 (2022)..

Li, Y. et al. Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function. Sci. Adv. 7 , eabe4310 (2021)..

Almassalha, L. M. et al. Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy. Proc. Natl. Acad. Sci. USA 113 , E6372–E6381 (2016)..

Eid, A. et al. Characterizing chromatin packing scaling in whole nuclei using interferometric microscopy. Opt. Lett. 45 , 4810–4813 (2020)..

Li, W. S. et al. Mature chromatin packing domains persist after RAD21 depletion in 3D. Sci. Adv. 11 , eadp0855 (2025)..

Schnitzbauer, J. et al. Super-resolution microscopy with DNA-PAINT. Nat. Protoc. 12 , 1198–1228 (2017)..

Jing, C. et al. Chemical Tags for Labe ling Proteins Inside Living Cells. Acc. Chem. Res. 44 , 784–792 (2011)..

Lelek, M. et al. Superresolution imaging of HIV in infected cells with FlAsH-PALM. Proc. Natl. Acad. Sci. USA 109 , 8564–8569 (2012)..

Yushchenko, D. A. et al. Tailoring fluorescent labels for far-field nanoscopy. In Far-Field Optical Nanoscopy (eds Tinnefeld, P. et al.) 159–188 (Springer, Berlin, Heidelberg, 2015).

Min, W. et al. Coherent nonlinear optical imaging: beyond fluorescence microscopy. Annu. Rev. Phys. Chem. 62 , 507–530 (2011)..

Walker, R. A. Quantification of immunohistochemistry—issues concerning methods, utility and semiquantitative assessment Ⅰ. Histopathology 49 , 406–410 (2006)..

Dunn, K. W. et al. A practical guide to evaluating colocalization in biological microscopy. Am. J. Physiol.—Cell Physiol. 300 , C723–C742 (2011)..

Castells-Garcia, A. et al. Super resolution microscopy reveals how elongating RNA polymerase Ⅱ and nascent RNA interact with nucleosome clutches. Nucleic Acids Res. 50 , 175–190 (2022)..

Xu, J. et al. Super-resolution imaging of higher-order chromatin structures at different epigenomic states in single mammalian cells. Cell Rep. 24 , 873–882 (2018)..

Xu, J. et al. Imaging higher-order chromatin structures in single cells using stochastic optical reconstruction. Microsc. Bio-Protoc. 9 , e3160 (2019)..

Otterstrom, J. et al. Super-resolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo. Nucleic Acids Res. 47 , 8470–8484 (2019)..

Abdelsayed, V. et al. An optimized buffer for repeatable multicolor STORM. ACS Photonics 9 , 3926–3934 (2022)..

Gogesch, P. et al. The role of Fc receptors on the effectiveness of therapeutic monoclonal antibodies. Int. J. Mol. Sci. 22 , 8947 (2021)..

Ovesný, M. et al. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30 , 2389–2390 (2014)..

Dong, B. et al. Super-resolution spectroscopic microscopy via photon localization. Nat. Commun. 7 , 12290 (2016)..

Weidner, J. et al. Advanced image-free analysis of the nano-organization of chromatin and other biomolecules by single molecule localization microscopy (SMLM). Comput. Struct. Biotechnol. J. 21 , 2018–2034 (2023)..

Nieves, D. J. et al. A framework for evaluating the performance of SMLM cluster analysis algorithms. Nat. Methods 20 , 259–267 (2023)..

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