Name: A Multilabel Single Molecule Localization Microscopy Protocol for Investigation of Chromatin in the Dense Nuclear Environment (Video) | JoVE Journal
Uploaded: 2026-06-05T13:00:00.000+00:00
Duration: 8 min 49 s
Description: Explore a multilabel single molecule localization microscopy protocol for investigating chromatin in dense nuclear environments using BJ Fibroblast, HeLa, and MCF 10A cells. This computational pipeline enables nanoscale spatial analysis.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by NIH grants U54CA268084, U54CA261694, and R01CA228272, National Science Foundation grants EFMA-1830961 and CBET-2430743, and philanthropic support from Rob and Kristin Goldman, Mr. David Sachs, and the Christina Carinato Charitable Foundation.

<ol><li>Bond, C., Hugelier, S., Xing, J., Sorokina, E. M., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplexed+DNA-PAINT+imaging+of+the+heterogeneity+of+late+endosome%2Flysosome+protein+composition%5BTitle%5D)+AND+%22bioRxiv%3A+The+Preprint+Server+for+Biology%22%5BJournal%5D)">Multiplexed DNA-PAINT imaging of the heterogeneity of late endosome/lysosome protein composition.</a> bioRxiv: The Preprint Server for Biology. , (2024).</li>
<li>Hugelier, S., Colosi, P. L., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+single-molecule+localization+microscopy%5BTitle%5D)+AND+%22Annu.+Rev.+Biophys%22%5BJournal%5D)">Quantitative single-molecule localization microscopy.</a> Annu. Rev. Biophys. 52 (1), 139-160 (2023).</li>
<li>Lelek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+localization+microscopy%5BTitle%5D)+AND+%22Nat.+Rev.+Methods+Primers%22%5BJournal%5D)">Single-molecule localization microscopy.</a> Nat. Rev. Methods Primers. 1, 39(2021).</li>
<li>Oddone, A., Vilanova, I. V., Tam, J., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+imaging+with+stochastic+single-molecule+localization%3A+concepts%2C+technical+developments%2C+and+biological+applications%5BTitle%5D)+AND+%22Microsc+Res+Tech%22%5BJournal%5D)">Super-resolution imaging with stochastic single-molecule localization: concepts, technical developments, and biological applications.</a> Microsc Res Tech. 77 (7), 502-509 (2014).</li>
<li>Bates, M., Huang, B., Dempsey, G. T., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multicolor+super-resolution+imaging+with+photo-switchable+fluorescent+probes%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Multicolor super-resolution imaging with photo-switchable fluorescent probes.</a> Science. 317 (5845), 1749-1753 (2007).</li>
<li>Ricci, M. A., Cosma, M. P., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super+resolution+imaging+of+chromatin+in+pluripotency%2C+differentiation%2C+and+reprogramming%5BTitle%5D)+AND+%22Curr+Opin+Genet+Dev%22%5BJournal%5D)">Super resolution imaging of chromatin in pluripotency, differentiation, and reprogramming.</a> Curr Opin Genet Dev. 46, 186-193 (2017).</li>
<li>Heo, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aberrant+chromatin+reorganization+in+cells+from+diseased+fibrous+connective+tissue+in+response+to+altered+chemomechanical+cues%5BTitle%5D)+AND+%22Nat+Biomed+Eng%22%5BJournal%5D)">Aberrant chromatin reorganization in cells from diseased fibrous connective tissue in response to altered chemomechanical cues.</a> Nat Biomed Eng. 7 (2), 177-191 (2023).</li>
<li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+molecular+characterization+of+the+hippo+signaling+pathway+in+cancer%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Comprehensive molecular characterization of the hippo signaling pathway in cancer.</a> Cell Rep. 25 (5), 1304-1317.e5 (2018).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multicolor+super-resolution+imaging+using+spectroscopic+single-molecule+localization+microscopy+with+optimal+spectral+dispersion%5BTitle%5D)+AND+%22Appl+Opt%22%5BJournal%5D)">Multicolor super-resolution imaging using spectroscopic single-molecule localization microscopy with optimal spectral dispersion.</a> Appl Opt. 58 (9), 2248-2255 (2019).</li>
<li>Daugird, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlative+single+molecule+lattice+light+sheet+imaging+reveals+the+dynamic+relationship+between+nucleosomes+and+the+local+chromatin+environment%5BTitle%5D)+AND+%22Nat+Comm%22%5BJournal%5D)">Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment.</a> Nat Comm. 15 (1), 4178(2024).</li>
<li>Kant, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+transcription+and+epigenetic+reactions+synergistically+regulate+meso-scale+genomic+organization%5BTitle%5D)+AND+%22Nat+Comm%22%5BJournal%5D)">Active transcription and epigenetic reactions synergistically regulate meso-scale genomic organization.</a> Nat Comm. 15 (1), 4338(2024).</li>
<li>Esa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+spectral+precision+distance+microscopy+of+chromatin+nanostructures+after+triple-colour+DNA+labelling%3A+a+study+of+the+BCR+region+on+chromosome+22+and+the+Philadelphia+chromosome%5BTitle%5D)+AND+%22J+Microsc%22%5BJournal%5D)">Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome.</a> J Microsc. 199 (2), 96-105 (2000).</li>
<li>Lelek, M., Di Nunzio, F., Henriques, R., Charneau, P., Arhel, N., Zimmer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Superresolution+imaging+of+HIV+in+infected+cells+with+FlAsH-PALM%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%22%5BJournal%5D)">Superresolution imaging of HIV in infected cells with FlAsH-PALM.</a> Proc Natl Acad Sci. 109 (22), 8564-8569 (2012).</li>
<li>Yushchenko, D. A., Bruchez, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tailoring+fluorescent+labels+for+far-field+nanoscopy%5BTitle%5D)+AND+%22Far-Field+Optical+Nanoscopy%22%5BJournal%5D)">Tailoring fluorescent labels for far-field nanoscopy.</a> Far-Field Optical Nanoscopy. , 159-188 (2015).</li>
<li>Min, W., Freudiger, C. W., Lu, S., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coherent+nonlinear+optical+imaging%3A+beyond+fluorescence+microscopy%5BTitle%5D)+AND+%22Annu+rev+phys+chem%22%5BJournal%5D)">Coherent nonlinear optical imaging: beyond fluorescence microscopy.</a> Annu rev phys chem. 62, 507-530 (2011).</li>
<li>Walker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+immunohistochemistry--issues+concerning+methods%2C+utility+and+semiquantitative+assessment+I%5BTitle%5D)+AND+%22Histopath%22%5BJournal%5D)">Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment I.</a> Histopath. 49 (4), 406-410 (2006).</li>
<li>Dunn, K. W., Kamocka, M. M., McDonald, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+practical+guide+to+evaluating+colocalization+in+biological+microscopy%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">A practical guide to evaluating colocalization in biological microscopy.</a> Am J Physiol Cell Physiol. 300 (4), C723-C742 (2011).</li>
<li>Castells-Garcia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super+resolution+microscopy+reveals+how+elongating+RNA+polymerase+II+and+nascent+RNA+interact+with+nucleosome+clutches%5BTitle%5D)+AND+%22Nucl+Acids+Res%22%5BJournal%5D)">Super resolution microscopy reveals how elongating RNA polymerase II and nascent RNA interact with nucleosome clutches.</a> Nucl Acids Res. 50 (1), 175-190 (2022).</li>
<li>Finn, E. H., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+DNA+FISH+protocol+to+visualize+genome+regions+in+human+cells%5BTitle%5D)+AND+%22STAR+protocols%22%5BJournal%5D)">A high-throughput DNA FISH protocol to visualize genome regions in human cells.</a> STAR protocols. 2 (3), 100741(2021).</li>
<li>Nuebler, J., Fudenberg, G., Imakaev, M., Abdennur, N., Mirny, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+organization+by+an+interplay+of+loop+extrusion+and+compartmental+segregation%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%22%5BJournal%5D)">Chromatin organization by an interplay of loop extrusion and compartmental segregation.</a> Proc Natl Acad Sci. 115 (29), E6697-E6706 (2018).</li>
<li>Davidson, I. F., Peters, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+folding+through+loop+extrusion+by+SMC+complexes%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Genome folding through loop extrusion by SMC complexes.</a> Nat Rev Mol Cell Biol. 22 (7), 445-464 (2021).</li>
<li>Schueder, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unraveling+cellular+complexity+with+transient+adapters+in+highly+multiplexed+super-resolution+imaging%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Unraveling cellular complexity with transient adapters in highly multiplexed super-resolution imaging.</a> Cell. 187 (7), 1769-1784.e18 (2024).</li>
<li>Miron, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+arranges+in+chains+of+mesoscale+domains+with+nanoscale+functional+topography+independent+of+cohesin%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin.</a> Sci Adv. 6 (39), eaba8811(2020).</li>
<li>Ou, H. D., Phan, S., Deerinck, T. J., Thor, A., Ellisman, M. H., O'Shea, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ChromEMT%3A+Visualizing+3D+chromatin+structure+and+compaction+n+interphase+and+mitotic+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">ChromEMT: Visualizing 3D chromatin structure and compaction n interphase and mitotic cells.</a> Science. 357 (6349), eaag0025(2017).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+three-dimensional+chromatin+packing+domains+by+chromatin+scanning+transmission+electron+microscopy+%28ChromSTEM%29%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Analysis of three-dimensional chromatin packing domains by chromatin scanning transmission electron microscopy (ChromSTEM).</a> Sci Rep. 12 (1), 12198(2022).</li>
<li>Penagos-Puig, A., Furlan-Magaril, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterochromatin+as+an+important+driver+of+genome+organization%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">Heterochromatin as an important driver of genome organization.</a> Front Cell Dev Biol. 8, (2020).</li>
<li>Millán-Zambrano, G., Burton, A., Bannister, A. J., Schneider, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+post-translational+modifications+-+cause+and+consequence+of+genome+function%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Histone post-translational modifications - cause and consequence of genome function.</a> Nat Rev Genet. 23 (9), 563-580 (2022).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanoscale+chromatin+imaging+and+analysis+platform+bridges+4D+chromatin+organization+with+molecular+function%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function.</a> Sci Adv. 7 (1), eabe4310(2021).</li>
<li>Li, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mature+chromatin+packing+domains+persist+after+RAD21+depletion+in+3D%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Mature chromatin packing domains persist after RAD21 depletion in 3D.</a> Sci Adv. 11 (4), eadp0855(2025).</li>
<li>Mansisidor, A. R., Risca, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+accessibility%3A+methods%2C+mechanisms%2C+and+biological+insights%5BTitle%5D)+AND+%22Nucleus%22%5BJournal%5D)">Chromatin accessibility: methods, mechanisms, and biological insights.</a> Nucleus. 13 (1), 236-276 (2022).</li>
<li>Li, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decoding+topologically+associating+domains+with+ultra-low+resolution+Hi-C+data+by+graph+structural+entropy%5BTitle%5D)+AND+%22Nat+Comm%22%5BJournal%5D)">Decoding topologically associating domains with ultra-low resolution Hi-C data by graph structural entropy.</a> Nat Comm. 9 (1), 3265(2018).</li>
<li>Szabo, Q., Bantignies, F., Cavalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+of+genome+folding+into+topologically+associating+domains%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Principles of genome folding into topologically associating domains.</a> Sci Adv. 5 (4), eaaw1668(2019).</li>
<li>Satam, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next-generation+sequencing+technology%3A+current+trends+and+advancements%5BTitle%5D)+AND+%22Biology%22%5BJournal%5D)">Next-generation sequencing technology: current trends and advancements.</a> Biology. 12 (7), 997(2023).</li>
<li>Acosta, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-color+single-molecule+localization+microscopy+in+chromatin%5BTitle%5D)+AND+%22Light%3A+Sci+Appl%22%5BJournal%5D)">Three-color single-molecule localization microscopy in chromatin.</a> Light: Sci Appl. 14 (1), 123(2025).</li>
<li>Almassalha, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+conformation%2C+gene+transcription%2C+and+nucleosome+remodeling+as+an+emergent+system%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Chromatin conformation, gene transcription, and nucleosome remodeling as an emergent system.</a> Sci Adv. 11 (2), eadq6652(2025).</li>
<li>Yin, Y., Lee, W. T. C., Rothenberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrafast+data+mining+of+molecular+assemblies+in+multiplexed+high-density+super-resolution+images%5BTitle%5D)+AND+%22Nat+Comm%22%5BJournal%5D)">Ultrafast data mining of molecular assemblies in multiplexed high-density super-resolution images.</a> Nat Comm. 10 (1), 119(2019).</li>
<li>Xu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+imaging+of+higher-order+chromatin+structures+at+different+epigenomic+states+in+single+mammalian+cells%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Super-resolution imaging of higher-order chromatin structures at different epigenomic states in single mammalian cells.</a> Cell Rep. 24 (4), 873-882 (2018).</li>
<li>Xu, J., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+higher-order+chromatin+structures+in+single+cells+using+stochastic+optical+reconstruction+microscopy%5BTitle%5D)+AND+%22Bio-Protocol%22%5BJournal%5D)">Imaging higher-order chromatin structures in single cells using stochastic optical reconstruction microscopy.</a> Bio-Protocol. 9 (3), e3160(2019).</li>
<li>Otterstrom, J., Castells-Garcia, A., Vicario, C., Gomez-Garcia, P. A., Cosma, M. P., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+microscopy+reveals+how+histone+tail+acetylation+affects+DNA+compaction+within+nucleosomes+in+vivo%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Super-resolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo.</a> Nucleic Acids Res. 47 (16), (2019).</li>
<li>Abdelsayed, V., Boukhatem, H., Olivier, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+optimized+buffer+for+repeatable+multicolor+STORM%5BTitle%5D)+AND+%22ACS+Photonics%22%5BJournal%5D)">An optimized buffer for repeatable multicolor STORM.</a> ACS Photonics. 9 (12), 3926-3934 (2022).</li>
<li>Weidner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+image-free+analysis+of+the+nano-organization+of+chromatin+and+other+biomolecules+by+single+molecule+localization+microscopy+%28SMLM%29%5BTitle%5D)+AND+%22Comput+Struct+Biotechnol+J%22%5BJournal%5D)">Advanced image-free analysis of the nano-organization of chromatin and other biomolecules by single molecule localization microscopy (SMLM).</a> Comput Struct Biotechnol J. 21, 2018-2034 (2023).</li>
<li>Nieves, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+framework+for+evaluating+the+performance+of+SMLM+cluster+analysis+algorithms%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">A framework for evaluating the performance of SMLM cluster analysis algorithms.</a> Nat Methods. 20 (2), 259-267 (2023).</li>
<li>Hyun, Y., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+development+of+computational+cluster+analysis+methods+for+single-molecule+localization+microscopy+images%5BTitle%5D)+AND+%22Comput+Struct+Biotechnol+J%22%5BJournal%5D)">Recent development of computational cluster analysis methods for single-molecule localization microscopy images.</a> Comput Struct Biotechnol J. 21, 879-888 (2023).</li>
<li>Markaki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+nuclear+organization+of+transcription+and+DNA+replication%3A+a+topographical+marriage+between+chromatin+domains+and+the+interchromatin+compartment%5BTitle%5D)+AND+%22Cold+Spring+Harb+Symp+Quant+Biol%22%5BJournal%5D)">Functional nuclear organization of transcription and DNA replication: a topographical marriage between chromatin domains and the interchromatin compartment.</a> Cold Spring Harb Symp Quant Biol. 75 (0), 475-492 (2010).</li>
<li>Gelléri, M., Sterr, M., Strickfaden, H., Cremer, C., Cremer, T., Cremer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Space-time+dynamics+of+genome+replication+studied+with+super-resolved+microscopy%5BTitle%5D)+AND+%22Postepy+biochem%22%5BJournal%5D)">Space-time dynamics of genome replication studied with super-resolved microscopy.</a> Postepy biochem. 70 (1), 8-21 (2024).</li>
<li>Cremer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+4D+nucleome%3A+Evidence+for+a+dynamic+nuclear+landscape+based+on+co-aligned+active+and+inactive+nuclear+compartments%5BTitle%5D)+AND+%22FEBS+Letters%22%5BJournal%5D)">The 4D nucleome: Evidence for a dynamic nuclear landscape based on co-aligned active and inactive nuclear compartments.</a> FEBS Letters. 589 (20), 2931-2943 (2015).</li>
</ol>

The authors of this protocol have no disclosures or competing interests.

This three-color SMLM protocol represents a significant advancement in our ability to investigate chromatin organization within the dense nuclear environment. The sequential immunofluorescence labeling approach, combined with point-cloud spatial analysis which uses localization estimates as points as the basis of the analysis, provides researchers with a powerful tool for examining nanoscale relationships between different chromatin modifications and active transcription machinery that were previously undetectable using conventional microscopy techniques.
The protocol&#39;s sequential staining strategy addresses fundamental challenges inherent to multi-target nuclear imaging. Unlike cytoplasmic or membrane-associated structures that are relatively sparse, nuclear chromatin targets exist in extremely high density with overlapping spatial domains. Our modified blocking approach, incorporating serum from secondary antibody host species after each labeling round, effectively prevents cross-reactivity between antibody pairs while maintaining target specificity. The overnight incubations at 4&#176;C ensure complete antibody penetration throughout the nuclear volume, critical for achieving uniform labeling density necessary for quantitative SMLM analysis. This protocol reliably produces images with 15-20 nm resolution across multiple cell lines, making it broadly applicable for chromatin organization studies.
The following are the trouble shooting tips for the imaging session. If the nuclei do not bleach, the most likely cause is insufficient laser intensity at the sample, which may result from a low power setting or an incorrect TIRF angle; in this case, troubleshoot by checking the laser alignment, increasing the laser power, and carefully adjusting the TIRF angle. If blinks in the nucleus are very few but remain constantly bright, this indicates that the nuclei have not bleached sufficiently. If blinks are very sparse and the nuclei are not visible at all, the most likely explanation is unsuccessful staining, and the reagents and antibodies should be checked before repeating the protocol. Conversely, if the number of nuclear blinks is very high (a desirable outcome), a longer bleaching time is required until individual, well-separated single-molecule blinks can be visually resolved. In conventional SMLM experiments, appropriate labeling density can often be estimated using well-defined reference structures such as microtubules; however, chromatin exhibits highly heterogeneous organization and lacks a well-defined ground-truth structure, making such estimation more challenging. Based on empirical optimization and prior experience, we therefore ensure that the effective labeling density reaches approximately 100 localizations per &#181;m&#178; to enable robust reconstruction of chromatin packing domains. In our protocol, we did not use any fiduciary markers but recommend if the users have them. In our experiments, the lateral shifts remain within 0.2 pixels (less than ~5nm), which can be neglected. Therefore, we did not use fiduciary markers.
The choice of H3K9me3, H3K27ac, and RNA polymerase II provides complementary information about chromatin organization at nanoscale level. H3K9me3 serves as an ideal spatial reference because it forms discrete, well-defined clusters that represent constitutive heterochromatin and can be reliably identified through automated clustering algorithms. H3K27ac marks enhancer-associated chromatin that actively participates in gene regulation, while RNA Polymerase II directly indicates sites of active transcription. Together, these three targets allow investigation of how transcriptional machinery and regulatory chromatin modifications organize relative to heterochromatic regions within the nuclear architecture.
The point-cloud analysis framework addresses critical limitations of previous chromatin organization studies by enabling comprehensive spatial analysis in dense nuclear environments. Traditional pairwise comparison methods cannot capture the complex spatial relationships that occur when multiple chromatin modifications coexist within the same nuclear regions. Our approach utilizes joint density analysis to reveal where H3K27ac and RNA polymerase II co-localize relative to H3K9me3 clusters, providing quantitative information that cannot be obtained from separate two-color experiments.
Representative results consistently demonstrate a cohesive organizational model rather than strict chromatin compartmentalization. Both H3K27ac and RNA polymerase II localize preferentially at heterochromatin cluster peripheries across different domain sizes, with quantitative measurements showing positioning within 10 nm of cluster boundaries which supports findings from other groups with similar methods and models of transcription23,28,35,45,46. The joint density analysis reveals that active transcription machinery and enhancer-associated chromatin couple most frequently in regions surrounding heterochromatic clusters. These findings challenge simple phase separation models and support integrated organizational principles where different chromatin modifications maintain close spatial proximity rather than forming distinct separate compartments.
The protocol&#39;s modular design enables investigation of diverse chromatin biology questions through target substitution while maintaining the same analytical framework. Studies of replication timing can substitute for cell cycle proteins such as proliferation cell nuclear antigen (PCNA) and mini chromosome maintenance (MCM), while DNA damage response investigations might target &#947;H2AX and repair factors. Cell cycle studies could examine histone modifications that change throughout division, and differentiation research might focus on epigenetic marks associated with lineage commitment. The sequential labeling approach accommodates any combination of nuclear proteins or chromatin modifications for which reliable antibodies exist, limited primarily by spectral compatibility and cross-reactivity considerations. This flexibility allows researchers to address fundamental questions about chromatin dynamics during various cellular processes while maintaining quantitative spatial analysis capabilities.

The advent of single-molecule localization microscopy (SMLM) has enabled unprecedented exploration of biological structures at the nanometer scale1,2,3,4,5. Beyond single-target imaging, the extension to multi-color SMLM has further advanced the field by allowing simultaneous visualization of multiple molecular species, as well as the spatial and temporal relationships among sub-diffraction structures6,7,8,9,10,11. However, applying multiplexed SMLM to abundantly distributed histone modifications remains challenging because of the dense, polymeric nature of nuclear DNA and the limited accessibility of antibodies within this environment12,13,14,15,16,17,18.
Chromatin exhibits a hierarchical, multiscale organization spanning several orders of magnitude in length, from nanometer-scale nucleosome assemblies to micrometer-scale nuclear architecture. At the largest scales, chromosomes occupy distinct chromosome territories, within which the genome is further partitioned into A/B compartments and topologically associated domains (TADs) that constrain long-range regulatory interactions through mechanisms such as loop extrusion19,20,21,22. At sub-200 nm scales, chromatin is organized as a disordered polymer composed of heterogeneous packing domains (PDs) rather than discrete euchromatin and heterochromatin blocks, with transcriptionally active regions preferentially localizing to PD boundaries23,24,25,26,27,28,29. At the smallest scales (5-20 nm), chromatin consists of irregular nucleosome assemblies and nucleosome clutches, underscoring the absence of a uniform higher-order folding motif and emphasizing the emergent, scale-dependent nature of genome organization24,26,30. With the rapid advancement of sequencing-based approaches such as chromatin immunoprecipitation sequencing and high-throughput chromatin conformation capture19,30,31,32,33, various features of chromatin mesoscale organizational structures have been identified31,32. However, these techniques, in contrast to imaging, fail to capture spatial geometry that is only observed after resolving these structures. Electron microscopy methods such as chromatin electron microscopy (ChromEM24) and chromatin scanning transmission electron microscopy ( ChromSTEM25) have revealed that chromatin is heterogeneous and organized into packing domains at length scales of 50-200 nm25,28,29. While these techniques enable impressive resolution to identify chromatin packing domains, they cannot provide molecularly specific mapping that SMLM offers. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT22) and multiplexed fluorescence in situ hybridization (FISH)19enable high multiplexing; however, DNA-PAINT is strongly affected by elevated background noise arising from random binding events in the oligonucleotide-rich nuclear environment12,34, while traditional heat denaturing based FISH methods require disrupted native chromatin folding. Prior studies have applied super resolution imaging techniques to investigate chromatin at this length scale and have identified a hybrid composition of packing domains, opposing prior phase separation models12,23,34,35. This protocol stems from a previously published paper discussing the biological significance of these findings34. Thus, given its high resolution and multiplexing capabilities, immunostaining-based dSTORM remains the most viable strategy for multi-color chromatin imaging under near-native conditions.
This protocol is not the first to demonstrate labeling of greater than two nuclear targets, with prior studies labeling individual protein complexes or genes12,36. Despite successful labeling of nucleosome post-translational histone modifications, multicolor chromatin SMLM labeling, imaging, and analysis present significant challenges. First, immunostaining in the dense chromatin environment requires optimization of antibody concentration, incubation sequence, and buffer composition to ensure adequate penetration and binding without excessive background. Second, comprehensive analysis of multiple labels is necessary, as the interactions between euchromatin, heterochromatin, and enzymes such as RNA polymerase are likely to extend beyond simple binary exclusions. Thus far, the maximum number of colors demonstrated in chromatin dSTORM imaging remains two18,37,38,39.
Here we present a robust protocol for three-color chromatin SMLM imaging and analysis. Our staining workflow optimizes antibody incubation time and employs improved imaging buffers40for prolonged imaging session for multiple labels. We further describe computational pipelines for two-color distance analysis and three-color joint density analysis, enabling quantitative characterization of relationships between heterochromatin, euchromatin, and transcription machinery. In contrast to earlier two-color chromatin SMLM studies that suggested a separation of heterochromatin and euchromatin, three-color chromatin imaging reveals that the genome is organized into packing domains, with euchromatin and active transcription localized at the periphery of constitutive heterochromatin cores34.
This protocol provides the community with a reproducible framework for performing multi-color chromatin SMLM and establishes analysis strategies suited for multiple functionally conjugated nuclear targets. By bridging methodological gaps, it enables systematic exploration of chromatin domain organization at the supra-nucleosomal level, complementing sequencing and electron microscopy approaches while preserving native nuclear architecture. This article is an extended protocol of a published paper34.

<table><tbody><tr><td>Andor iXon Ultra 888 electron-multiplying CCD&amp;nbsp;</td><td>Andor</td><td>DU-888U3-CSO-#BV&amp;nbsp;</td><td>NA</td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A7030</td><td>Used in blocking and washing buffers. Do not store BSA based blocking buffer for more than 1 month at 4&amp;deg;C</td></tr><tr><td>Changchun New Industries Optoelectronics Tech. Co., Ltd., Model MGL-FN-532 (532 nm)&amp;nbsp;</td><td>PSU-H-LED&amp;nbsp;</td><td>https://www.cnilaser.com/MGL-FN-532.htm</td><td>NA</td></tr><tr><td>Coherent OBIS Laser Box (405 nm, 488 nm, 532 nm, 552 nm, 637 nm)&amp;nbsp;</td><td>Coherent</td><td>1228877&amp;nbsp;</td><td>Lasers collimated with 3&amp;ndash;10 kW/cm&amp;sup3; average power at sample, minimum 10,000 frames collected per wavelength channel with&amp;nbsp;10&amp;ndash;30&amp;nbsp;ms&amp;nbsp;acquisition time.&amp;nbsp;</td></tr><tr><td>DABCO (1,4-Diazabicyclo-(2.2.2)-octane)</td><td>Sigma</td><td>D27802</td><td>NA</td></tr><tr><td>DBPS (1X)</td><td>Thermo Fisher</td><td>14190-136</td><td>NA</td></tr><tr><td>Distilled water</td><td>NA</td><td>NA</td><td>Any Distilled Water is fine&amp;nbsp;</td></tr><tr><td>DTT (Dithiothreitol), 1M</td><td>Sigma</td><td>43816</td><td>NA</td></tr><tr><td>Eight well chambered cover glass</td><td>Cellvis</td><td>C8-1.5H-N</td><td>Can be any glass bottom plate, but volumes must be adjusted depending on vessel.</td></tr><tr><td>Goat anti Mouse AF568</td><td>Thermo Fisher</td><td>A11004</td><td>Stock Concentration: 2 mg/mL&lt;br/&gt; Stability Post Labeling: 2&amp;ndash;3 days</td></tr><tr><td>Goat anti Rabbit AF647</td><td>Thermo Fisher</td><td>A21245</td><td>Stock Concentration: 2 mg/mL&lt;br/&gt; Stability Post Labeling: 4&amp;ndash;5 days</td></tr><tr><td>Goat anti Rat A488</td><td>Thermo Fisher</td><td>A11006</td><td>Stock Concentration: 2 mg/mL&lt;br/&gt; Stability Post Labeling: 2&amp;ndash;3 days</td></tr><tr><td>H3K27ac Primary Antibody</td><td>Thermo Fisher</td><td>MA5-23516</td><td>Stock Concentration: 1.0 mg/mL&amp;nbsp;</td></tr><tr><td>H3K9me3 Primary Antibody&amp;nbsp;</td><td>Abcam</td><td>AB1769156</td><td>Stock Concentration: 1.287 mg/mL&amp;nbsp;</td></tr><tr><td>High Clarity Poly-Propylene Conical Tube 15 mL&amp;nbsp;&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>352096</td><td>Used for Fixative and Quenching solutions&amp;nbsp;</td></tr><tr><td>High Clarity Poly-Propylene Conical Tube 50 mL</td><td>Corning</td><td>352070</td><td>Used for 40 mL working stocks of blocking and washing buffers</td></tr><tr><td>Hydrochloric acid (HCl), 12M</td><td>Sigma</td><td>258148</td><td>NA</td></tr><tr><td>Nikon Eclipse Ti-E with perfect focus system&amp;nbsp;</td><td>NIikon</td><td>TI-DH 611392&amp;nbsp;</td><td>Inverted microscope&amp;nbsp;</td></tr><tr><td>Nikon SR APO TIRF, 100x magnification, 1.49 NA&amp;nbsp;</td><td>Nikon</td><td>&lt;a href=&quot;https://www.microscope.healthcare.nikon.com/products/optics/cfi-apochromat-tirf-series%C2%A0&quot;&gt;https://www.microscope.healthcare.nikon.com/products/optics/cfi-apochromat-tirf-series&amp;nbsp;&lt;/a&gt;</td><td>NA</td></tr><tr><td>Normal goat serum</td><td>Abcam</td><td>AB7481-1002</td><td>Used after first label is done. Should be present in Blocking and Washing Buffers</td></tr><tr><td>Paraformaldehyde 16 %&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Used in fixative solution which should be used up within 2 weeks of making. Keep protected from light at 4&amp;deg;C</td></tr><tr><td>Phosphate buffered saline (PBS) 10X</td><td>Ambion</td><td>AM9625</td><td>NA</td></tr><tr><td>Pipette Tips 1000 uL</td><td>SureOne</td><td>02-707-404</td><td>Any pipette tip is fine as long as it&amp;rsquo;s appropriate for the volume</td></tr><tr><td>RNAPII-PS2</td><td>Abcam</td><td>AB252855</td><td>Stock Concentraion: 0.98 mg/mL</td></tr><tr><td>Sodium Borohydride</td><td>Thermo Fisher</td><td>S678-10</td><td>Use fresh for quenching buffer every single time.&amp;nbsp;</td></tr><tr><td>Sodium hydroxide(NaOH)</td><td>Thermo Fisher&amp;nbsp;</td><td>A16037.36</td><td>NA</td></tr><tr><td>Sodium sulfite</td><td>Sigma</td><td>S0505</td><td>NA</td></tr><tr><td>Triton X-100 10%</td><td>Thermo Fisher&amp;nbsp;</td><td>28314</td><td>NA</td></tr></tbody></table>

a multilabel single molecule localization microscopy protocol for investigation of chromatin in the dense nuclear environment

A Multilabel Single Molecule Localization Microscopy Protocol for Investigation of Chromatin in the Dense Nuclear Environment

Super-resolution microscopy has dramatically advanced our ability to interrogate biological structures beyond the diffraction limit, making it indispensable for studying densely packed nuclear structures such as chromatin, nuclear lamina and nuclear bodies such as nucleoli. Chromatin exhibits multiscale organization-from nanometer-sized nucleosomes to micron-scale domains-necessitating imaging approaches capable of both high resolution and molecular specificity. Single molecule localization microscopy (SMLM), particularly stochastic optical reconstruction microscopy (STORM) , enables precise mapping of epigenetic marks, offering critical insight into chromatin structure and function. However, multi-label imaging in the nuclear environment presents unique challenges, including reduced antibody accessibility, increased non-specific binding, and fluorophore instability. To address these issues, we present a sequential immunolabeling protocol optimized for high-density nuclear environments, enabling robust three-color SMLM with minimal crosstalk and less signal degradation. This method includes optimized buffer formulations, fluorophore selection, and antibody validation strategies to ensure reproducible, high-fidelity labeling across multiple targets. Importantly, we integrate this protocol with a computational analysis pipeline that leverages localizations from one molecular target as spatial anchors (seed points) to quantify inter-target distances, local densities, and multi-label co-affinity. This allows for a detailed spatial analysis of chromatin components at the nanoscale. This protocol serves as a reproducible framework for multi-component imaging and quantitative analysis in dense subcellular environments, offering a powerful tool for researchers investigating complex nuclear architectures like chromatin.

Representative three-color chromatin dSTORM images
The proposed sequential staining protocol has been tested valid for a variety of cell lines, including BJ fibroblast, HCT116, AC16, HeLa, MCF10A, etc. Figure 1 shows the representative images from BJ fibroblast, HeLa and MCF10A cells.
Validation of analysis pipeline using simulated datasets 
The development of a three-color immuno fluorescence protocol necessitated the creation of a specialized computational approach capable of handling the complexity of multi-target nuclear SMLM data (Figure 2A,2B). Given the dense chromatin environment where multiple targets co-exist in proximity, we implemented a point-cloud analysis framework that directly processes localization coordinates rather than reconstructed images. This approach leverages the extensive clustering analysis tools available for point-cloud data40,41,42. We systematically evaluated the analysis pipeline's ability to distinguish biologically meaningful spatial patterns using controlled simulated datasets. Four distribution types were generated to represent distinct chromatin organization scenarios: normal distribution for tightly clustered modifications, uniform distribution for dispersed patterns, toroidal distribution modeling exclusion zones, and random distribution as an organizational control (Figure 2C). These simulated patterns were anchored to genuine heterochromatin cluster positions derived from experimental H3K9me3 data, preserving realistic nuclear spatial constraints. This analysis framework utilizes DBSCAN clustering (epsilon = 50 nm, minimum points = 3) which is one of the many methods for cluster analysis in SMLM data40,41,42. To ensure proper clustering parameters, we have previously described an optimization method tailored for detection of chromatin packing domains34. Please note that when using this as an initial step in the analysis, users will need to optimize the parameters informing the selection through the structure, environment and function of their target. Here the cluster boundaries are determined through convex hull calculations, eliminating assumptions about domain geometry. Our validation demonstrated clear discrimination between all simulated distribution patterns through distinctive distance histogram profiles (Figure 2D). Each spatial organization type produced characteristic signatures when localizations were analyzed relative to heterochromatin centroids. Joint occupancy analysis was tested using dual-marker simulations with controlled spatial relationships (Figure 2E).Spatially segregated markers (normal-toroidal configuration) yielded minimal joint density as expected, resulting in flat distribution profiles (Figure 2E). Overlapping marker patterns (normal-random configuration) produced decreasing joint density with increasing distance from reference points, matching theoretical predictions. These validation results demonstrate our framework's capacity to detect and quantify spatial coupling relationships in complex multi-target datasets. For more information on how these simulated datasets were generated please refer to the full publication34.
Representative results from chromatin organization analysis
Implementation of our validated analysis approach in biological samples reveals characteristic spatial organization patterns achievable through this protocol. Using HeLa cells processed with our three-color staining procedure for H3K9me3, H3K27ac, and RNA Polymerase II, we demonstrate the analytical capabilities enabled by this methodology. H3K9me3 heterochromatin serves as ideal reference system given established evidence for concentric chromatin organization at the 200 nm scale from previous super-resolution investigations23,28,35. Analysis begins with DBSCAN-based identification of heterochromatin domains, which are subsequently categorized by effective radius: small domains (25-40 nm), medium domains (40-80 nm), and large domains (80-253 nm). This size stratification accounts for the documented heterogeneity in DNA packing domain dimensions, with average domains measuring approximately 80 nm in radius25. Distance measurements employ a 1.5x cluster radius search window (Figure 2B top) to capture proximal euchromatin and polymerase signals (Figure 3A,B).
Representative results show both H3K27ac and RNA Polymerase II consistently localizing near heterochromatin boundaries across all domain categories aligning with prior studies28,44 and models for transcription location relative to chromatin domains23,35,45,46. Quantitative distance analysis reveals mean positions very close to domain peripheries: large domains show H3K27ac at -1.0 nm and RNA Polymerase II at 8.4 nm from boundaries, while smaller domains exhibit comparable peripheral associations with minor positional differences. These measurements indicate active chromatin elements concentrate at the interface between repressive and permissive domains rather than being completely excluded. Joint density analysis demonstrates the protocol's capacity to reveal spatial coupling between co-labeled targets (Figure 3C-F). Analysis relative to heterochromatin domains shows peak joint density occurring just outside domain boundaries (r/r₀&gt; 1), indicating preferential H3K27ac-RNA Polymerase II co-localization in peripheral regions (Figure 3G-I). These results showcase how this staining and analysis pipeline can help investigate complex spatial relationships in dense environments.
<img alt="Chromatin immunofluorescence, H3K9me3, H3K27ac, RNAPII markers, microscopy comparison." class="xfigimg" src="/files/ftp_upload/69868/69868fig1.jpg" data-altupdatedat="1782792046000" data-alt="Figure 1"> 
Figure 1: Three color images of the cells used. Three-color images of (A) BJ fibroblast (B) HeLa and (C) MCF10A. <a href="https://www.jove.com/files/ftp_upload/69868/69868fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="DBSCAN clustering diagram, distance analysis, joint density graphs, coordinate data, and proximity study." class="xfigimg" src="/files/ftp_upload/69868/69868fig2.jpg" data-altupdatedat="1782792046000" data-alt="Figure 2"> 
Figure 2: Quantitative analysis framework for spatial organization of targets relative to heterochromatin clusters. (A) Analysis Pipeline for multi-channel SMLM data used in this study. (B) Schematic of distance to periphery calculations for identified heterochromatin clusters and Joint Affinity Counting method. Both methods are used to quantitatively determine the arrangement of two targets relative to the heterochromatin cluster structure. (C) Example scatter distributions around specific heterochromatin clusters used in a study34 to determine distinct biological organizations of targeted structures (clustered inside a domain vs around domain vs not associated and randomly distributed). (D) Distance to periphery histogram for centrally clustered, randomly distributed and toroidally distributed and (E) Joint affinity curves for simulation cases. <a href="https://www.jove.com/files/ftp_upload/69868/69868fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="RNAPII H3K27ac analysis; histograms, microscopy, data charts on gene expression, chromatin study." class="xfigimg" src="/files/ftp_upload/69868/69868fig3.jpg" data-altupdatedat="1782792046000" data-alt="Figure 3"> 
Figure 3: Quantitative spatial relationships of transcription markers around histone domains. (A) Distance to periphery histogram results for exemplary biological data of multi-labeled HeLa cells. For small ( &lt;40 nm), medium (40-80 nm), and large (&gt;120 nm) domains for RNAPII and (B) H3K27ac relative to H3K9me3 clusters. (C-E) Example Images for three label dSTORM image of a HeLa Cells (H3K9me3, H3k27ac, RNAPII2-S2p, with inset and zoomed image for a single domain. (F) Convex hull (red) fitting of domain pictured in E with analysis zone in gray. (G) Affinity plots for RNAPII relative to H3K27ac and H3K27ac (H) relative to RNAPII in the analysis ROI for all domains in the data of medium size in the dataset. (I) Joint density of RNAPII and H3K27ac in analysis ROI for all medium sized domains. <a href="https://www.jove.com/files/ftp_upload/69868/69868fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

We present a three-color chromatin single molecule localization microscopy (SMLM) staining and analysis protocol that enables reproducible mapping of euchromatin, heterochromatin, and RNAP II for spatial analysis. This protocol enables efficient multicolor labeling in dense nuclear environments, including chromatin-associated targets, allowing reliable simultaneous detection.

Super-resolution microscopy has dramatically advanced our ability to interrogate biological structures beyond the diffraction limit, making it ...

Our research examines the epigenetic composition of chromatin packing domains, and how regulatory factors shape their structure and function. This protocol is most useful for investigating spatial relationships between targets and dense cellular environments, such as the nucleus. To begin, weigh bovine serum albumin, so that the final concentration in the required buffer volume for the experiment is 3%Add the bovine serum albumin to a centrifuge tube.Tilt the centrifuge tube to a 45-degree angle to spread out the bovine serum albumin crystals, then add PBS to the tube. This prevents clumping of the crystals. Allow all bovine serum albumin crystals to dissolve naturally at room temperature and avoid shaking or vortexing.Once the crystals are fully dissolved, add Triton X-100 to achieve a final concentration of 0.2%If crystals remain, pipette the solution up and down several times to mix the solution slowly without forming bubbles. Take the live cells from the incubator. Remove the cell culture medium from the dish.Wash the cells by adding enough PBS to cover the cells, and then pipette out the PBS. Next, pipette enough fixative solution to cover the cells and leave the cells to fix for 10 minutes. Using a balance, weigh sodium borohydride to prepare a 0.1%quenching solution.Add the sodium borohydride to a centrifuge tube. Pipette out the fixative solution from the dish. Then, add enough PBS to the dish to cover the cell surface.Place the dish on a shaker for five minutes to wash the cells. Next, add the required volume of PBS to the sodium borohydride in the centrifuge tube. Vortex the tube to mix the quenching solution.Remove the dish from the shaker and discard the PBS from the dish. Add enough quenching solution to cover the surface of the dish. Then, place the dish on a shaker for seven minutes to quench autofluorescence in the cells.Discard the remaining quenching solution in the centrifuge tube into the appropriately-labeled liquid chemical waste container. Remove the dish from the shaker and then remove the quenching solution from the dish. Then, add enough PBS to the dish to cover the surface.Place the dish on a shaker for five minutes to wash the cells. After incubation, remove the PBS and repeat the wash with PBS two additional times. Now, add enough blocking buffer to the dish to cover the surface.Incubate the dish on a shaker for at least one hour to permeabilize the cell membranes and block binding sites. To prepare the primary antibody staining solution, transfer the required volume of the blocking buffer stock to a new centrifuge tube. Add the required volume of primary antibody stock to the blocking buffer to achieve the final concentration specified by the manufacturer.Next, replace the blocking buffer with enough primary antibody staining solution to cover the surface of the dish and incubate on a shaker for one to two hours or for an overnight period. After primary antibody incubation, replace the primary antibody solution with washing buffer. Then, place the dish on a shaker for five minutes to wash the cells.Repeat the washing buffer washed two additional times for a total of three washes. Prepare the secondary antibody staining solution according to the recommended concentration. Calculate the total staining solution volume by adding 0.5 milliliters to the volume required to cover the cells.Transfer the calculated volume from the blocking buffer into a new centrifuge tube to prepare the staining solution. Then, add the appropriate volume of secondary antibody stock to the blocking buffer to obtain the correct final concentration. Wrap the centrifuge tube containing the secondary antibody staining solution with aluminum foil, and mix the solution by pipetting up and down.Add enough secondary antibody staining solution to the dish to cover the surface. Place the dish on a shaker for 40 minutes to attach fluorophores to the labeled cellular targets. Cover the dish with aluminum foil to prevent fluorophore bleaching.After 40 minutes, perform two PBS washes as demonstrated earlier. If storage is preferred, add enough PBS to the dish to cover the cell surface before storage. Wrap the dish with parafilm to prevent liquid evaporation, and then with aluminum foil to prevent fluorophore bleaching.Store the wrapped dish at four degrees Celsius until ready to image. The sequential staining protocol produced representative three-color chromatin dSTORM images for multiple cell lines, including BJ Fibroblast, HeLa, and MCF 10A cells. The analysis pipeline was evaluated using simulated data sets representing normal uniform toroidal and random spatial distributions anchored to heterochromatin cluster positions.Distinct spatial organization patterns produced characteristic distance histogram profiles when localization coordinates were analyzed relative to heterochromatin centroids. Simulated spatially segregated markers in a normal choroidal configuration produced minimal joint density, resulting in a flat distribution profile. Simulated overlapping marker patterns in a normal random configuration produced decreasing joint density with increasing distance from reference points.DB scan-based identification of heterochromatin domains enabled categorization into small, medium, and large domains based on effective radius. Quantitative distance analysis showed that RNA polymerase II and H3K27ac localized near heterochromatin boundaries across small, medium, and large domains. Joint density analysis showed peak H3K27ac and RNA polymerase II colocalization just outside heterochromatin cluster boundaries.Using this protocol, researchers can interrogate the seemingly disordered organization of chromatin to explore the relationship between its structure, regulatory elements, and functional outcomes. Following this procedure, various image-based or point cloud computational analyses can extract quantitative structural features from spatial data, depending on the imaging modality used. Researchers can extend this labeling method by optimizing additional markers and leveraging multi-channel data to enable more advanced and comprehensive analyses.

a-multilabel-single-molecule-localization-microscopy-protocol-for

Research

JoVE Journal

Bioengineering

Single molecule microscopy approch provided novel insights into nuclear transport.

Single-Molecule Imaging of Nuclear Transport

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

CUT&amp;RUN and its variants can be used to determine protein occupancy on chromatin. This protocol describes how to determine protein localization on chromatin using single-cell uliCUT&amp;RUN.

Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

The present protocol describes a method measuring absolute DNA densities within adherent cell nuclei using Voronoi tessellation of single-molecule localization microscopy data, known volume, genome size, and cell cycle stage.

Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

This protocol provides detailed operations on sample preparation, data collection, and analysis of DNA intrinsic fluorescence spectra.

Spectroscopic Super-resolution Imaging of DNA Molecules using Intrinsic Contrast

This protocol presents an efficient workflow for single-molecule localization microscopy imaging of isolated Arabidopsis nuclei, using optimized isolation, fixation, and labeling to achieve reliable nanoscale visualization of chromatin and RNA Polymerase II. This approach can be readily adapted to other chromatin-associated proteins or histone modifications for high-resolution imaging.

A Streamlined Protocol for Single-Molecule Localization Microscopy in Arabidopsis Nuclei

Explore a multilabel single molecule localization microscopy protocol for investigating chromatin in dense nuclear environments using BJ Fibroblast, HeLa, and MCF 10A cells. This computational pipeline enables nanoscale spatial analysis.

A Multilabel Single Molecule Localization Microscopy Protocol for Investigation of Chromatin in the Dense Nuclear Environment

Explore More Videos