Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue and super-resolution microscopy

Abstract

Fluorescence microscopy is a vital tool in biomedical research but faces considerable challenges in achieving uniform or bright labeling. For instance, fluorescent proteins are limited to model organisms, and antibody conjugates can be inconsistent and difficult to use with thick specimens. To partly address these challenges, we developed a labeling protocol that can rapidly visualize many well-contrasted key features and landmarks on biological specimens in both thin and thick tissues or cultured cells. This approach uses established reactive fluorophores to label a variety of biological specimens for cleared-tissue microscopy or expansion super-resolution microscopy and is termed FLARE (fluorescent labeling of abundant reactive entities). These fluorophores target chemical groups and reveal their distribution on the specimens; amine-reactive fluorophores such as hydroxysuccinimidyl esters target accessible amines on proteins, while hydrazide fluorophores target oxidized carbohydrates. The resulting stains provide signals analogous to traditional general histology stains such as H&E or periodic acid–Schiff but use fluorescent probes that are compatible with volumetric imaging. In general, the stains for FLARE are performed in the order of carbohydrates, amine and DNA, and the incubation time for the stains varies from 1 h to 1 d depending on the combination of stains and the type and thickness of the biological specimens. FLARE is powerful, robust and easy to implement in laboratories that already routinely do fluorescence microscopy.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Overview of FLARE for fluorescent labeling of biological samples.
Fig. 2: A summary of suggested workflows when combining FLARE with DNA FISH and immunofluorescence for clearing or expansion.
Fig. 3: Comparison of fixation conditions for expanded RPE cells stained by FLARE.
Fig. 4: FLARE staining of 5-µm-thick FFPE human tissue microarrays.
Fig. 5: Compatibility of FLARE with diverse procedures.
Fig. 6: Gelation chamber setup.
Fig. 7: A step-by-step illustration of the FLARE procedure for the expanded sample.
Fig. 8: FLARE labeling of ~1-mm-thick kidney tissue.
Fig. 9: A step-by-step illustration of the FLARE procedure for the optically cleared sample.
Fig. 10: Demonstration of expansion uniformity using a MAP-based protocol.
Fig. 11: Proper sample orientation during staining and image acquisition.
Fig. 12: Demonstration of the inner filter effect for densely labeled samples.

Data availability

Data other than those presented in the paper and in the authors’ previous paper3 are available from the corresponding author upon request.

Code availability

The custom Wolfram Mathematica scripts used for stitching tiling images (Fig. 4a,h) are available as Supplementary Code.

References

  1. Bradbury, A. & Plückthun, A. Reproducibility: standardize antibodies used in research. Nature 518, 27–29 (2015).

    CAS  PubMed  Article  Google Scholar 

  2. Schnell, U., Dijk, F., Sjollema, K. A. & Giepmans, B. N. G. Immunolabeling artifacts and the need for live-cell imaging. Nat. Methods 9, 152–158 (2012).

    CAS  PubMed  Article  Google Scholar 

  3. Mao, C. et al. Feature-rich covalent stains for super-resolution and cleared tissue fluorescence microscopy. Sci. Adv. 6, eaba4542 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Titford, M. Progress in the development of microscopical techniques for diagnostic pathology. J. Histotechnol. 32, 9–19 (2009).

    CAS  Article  Google Scholar 

  5. Sigal, Y. M., Zhou, R. & Zhuang, X. Visualizing and discovering cellular structures with super-resolution microscopy. Science 361, 880–887 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. Schermelleh, L. et al. Super-resolution microscopy demystified. Nat. Cell Biol. 21, 72–84 (2019).

    CAS  PubMed  Article  Google Scholar 

  7. Sahl, S. J., Hell, S. W. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat. Rev. Mol. Cell Biol. 18, 685–701 (2017).

    CAS  PubMed  Article  Google Scholar 

  8. Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy. Science 347, 543–548 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Chozinski, T. J. et al. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat. Methods 13, 485–488 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Tillberg, P. W. et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat. Biotechnol. 34, 987–992 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Ku, T. et al. Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nat. Biotechnol. 34, 973–981 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Richardson, D. S. & Lichtman, J. W. Clarifying tissue clearing. Cell 162, 246–257 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T. & Ueda, H. R. Chemical principles in tissue clearing and staining protocols for whole-body cell profiling. Annu. Rev. Cell Dev. Biol. 32, 713–741 (2016).

    CAS  PubMed  Article  Google Scholar 

  14. Glaser, A. K. et al. Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues. Nat. Commun. 10, 2781 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  15. Klingberg, A. et al. Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy. J. Am. Soc. Nephrol. 28, 453–459 (2017).

    Article  Google Scholar 

  16. Masselink, W. et al. Broad applicability of a streamlined ethyl cinnamate-based clearing procedure. Development 146, dev166884 (2019).

    PubMed  Article  Google Scholar 

  17. Beliveau, B. J. et al. Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc. Natl Acad. Sci. USA 109, 21301–21306 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Chen, Y. et al. Rapid pathology of lumpectomy margins with open-top light-sheet (OTLS) microscopy. Biomed. Opt. Express 10, 1257 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Xie, W. et al. Microscopy with ultraviolet surface excitation for wide-area pathology of breast surgical margins. J. Biomed. Opt. 24, 12 (2019).

    Article  Google Scholar 

  20. Yu, C.-C. et al. Expansion microscopy of C. elegans. eLife 9, e46249 (2020).

    CAS  PubMed  Article  Google Scholar 

  21. M’Saad, O. & Bewersdorf, J. Light microscopy of proteins in their ultrastructural context. Nat. Commun. 11, 3850 (2020).

    PubMed  PubMed Central  Article  Google Scholar 

  22. Damstra, H. G. J. et al. Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx). Preprint at bioRxiv https://doi.org/10.1101/2021.02.03.428837 (2021).

  23. Sim, J. et al. Whole-ExM: expansion microscopy imaging of all anatomical structures of whole larval zebrafish. Preprint at bioRxiv https://doi.org/10.1101/2021.05.18.443629 (2021).

  24. Elfer, K. N. et al. DRAQ5 and eosin (‘D&E’) as an analog to hematoxylin and eosin for rapid fluorescence histology of fresh tissues. PLoS ONE 11, e0165530 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  25. Serafin, R., Xie, W., Glaser, A. K. & Liu, J. T. C. FalseColor-Python: a rapid intensity-leveling and digital-staining package for fluorescence-based slide-free digital pathology. PLoS ONE 15, e0233198 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Yun, D. H. et al. Ultrafast immunostaining of organ-scale tissues for scalable proteomic phenotyping. Preprint at bioRxiv https://doi.org/10.1101/660373 (2019).

  27. Ku, T. et al. Elasticizing tissues for reversible shape transformation and accelerated molecular labeling. Nat. Methods 17, 609–613 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Susaki, E. A. et al. Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues. Nat. Commun. 11, 1982 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. Zhao, S. et al. Cellular and molecular probing of intact human organs. Cell 180, 796–812.e19 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Bertiaux, E. et al. Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid. PLOS Biol. 19, e3001020 (2021).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Bancroft’s Theory and Practice of Histological Techniques 7th edn (eds Suvarna, S. K. et al.) (Elsevier Churchill Livingstone, 2013).

  32. Apgar et al. Fluorescence microscopy of rat embryo sections stained with haematoxylin-eosin and Masson’s trichrome method: fluorescence microscopy using haematoxylin-eosin and Masson’s trichrome. J. Microsc. 191, 20–27 (1998).

    CAS  PubMed  Article  Google Scholar 

  33. Giacomelli, M. G. et al. Virtual hematoxylin and eosin transillumination microscopy using epi-fluorescence imaging. PLoS ONE 11, e0159337 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  34. Glaser, A. K. et al. Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nat. Biomed. Eng. 1, 0084 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  35. Kasten, F. H., Burton, V. & Glover, P. Fluorescent Schiff-type reagents for cytochemical detection of polyaldehyde moieties in sections and smears. Nature 184, 1797–1798 (1959).

    CAS  PubMed  Article  Google Scholar 

  36. Li, Z. et al. Application of periodic acid–Schiff fluorescence emission for immunohistochemistry of living mouse renal glomeruli by an ‘in vivo cryotechnique’. Arch. Histol. Cytol. 69, 147–161 (2006).

    CAS  PubMed  Article  Google Scholar 

  37. Shi, X. et al. Label-retention expansion microscopy. J. Cell Biol. 220, e202105067 (2021).

  38. Karagiannis, E. D. et al. expansion microscopy of lipid membranes. Preprint at bioRxiv http://biorxiv.org/lookup/doi/10.1101/829903 (2019).

  39. Wen, G. et al. Evaluation of direct grafting strategies via trivalent anchoring for enabling lipid membrane and cytoskeleton staining in expansion microscopy. ACS Nano 14, 7860–7867 (2020).

    CAS  PubMed  Article  Google Scholar 

  40. Götz, R. et al. Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy. Nat. Commun. 11, 6173 (2020).

    PubMed  PubMed Central  Article  Google Scholar 

  41. Chang, J.-B. et al. Iterative expansion microscopy. Nat. Methods 14, 593–599 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Ross, M. H. Histology: A Text and Atlas with Correlated Cell and Molecular Biology (Lippincott Williams & Wilkins, 2006).

  43. Bucur, O. et al. Nanoscale imaging of clinical specimens using conventional and rapid-expansion pathology. Nat. Protoc. https://doi.org/10.1038/s41596-020-0300-1 (2020).

  44. Unnersjö-Jess, D. et al. Confocal super-resolution imaging of the glomeruluar filtration barrier enables by tissue expansion. Kidney Int. https://doi.org/10.1016/j.kint.2017.09.019 (2017).

  45. Hermanson, G. Bioconjugate Techniques 2nd edn (Elsevier, 2008).

  46. Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J. & Waggoner, A. S. Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 4, 105–111 (1993).

    CAS  PubMed  Article  Google Scholar 

  47. Murray, E. et al. Simple, scalable proteomic imaging for high-dimensional profiling of intact systems. Cell 163, 1500–1514 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Chozinski, T. J. et al. Volumetric, nanoscale optical imaging of mouse and human kidney via expansion microscopy. Sci. Rep. https://doi.org/10.1038/s41598-018-28694-2 (2018).

  49. Power, R. M. & Huisken, J. A guide to light-sheet fluorescence microscopy for multiscale imaging. Nat. Methods 14, 360–373 (2017).

    CAS  PubMed  Article  Google Scholar 

  50. Santi, P. A. Light sheet fluorescence microscopy: a review. J. Histochem. Cytochem. 59, 129–138 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Liu, J. T. C. et al. Harnessing non-destructive 3D pathology. Nat. Biomed. Eng. https://doi.org/10.1038/s41551-020-00681-x (2018).

  52. Jiang, N. et al. Superresolution imaging of Drosophila tissues using expansion microscopy. Mol. Biol. Cell 29, 1413–1421 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. Richter, K. N. et al. Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy. EMBO J. https://doi.org/10.15252/embj.201695709 (2017).

Download references

Acknowledgements

This work was supported by the University of Washington, NIDDK Diabetic Complications Consortium grants DK076169 and DK115255 (J.C.V.), NIH grants R01 MH115767 (J.C.V.), R01 CA244170 (J.T.C.L.), R01 EB031002 (J.T.C.L.) and K99 CA240681 (A.K.G.), and DoD PCRP grant W81XWH-18-10358 (J.T.C.L.). NW BioTrust, a core service for patient consenting, and NWBioSpecimen, a core service for procurement and annotation of research biospecimens, are supported by National Cancer Institute grant P30 CA015704 (G. Gilliland, principal investigator (PI)), Institute of Translational Health Sciences grant UL1 TR000423 (M. Disis, PI), the University of Washington School of Medicine and Department of Pathology, and Fred Hutchinson Cancer Research Center.

Author information

Authors and Affiliations

Authors

Contributions

C.M., M.Y.L., A.K.G., M.A.W., J.T.C.L. and J.C.V. designed the methodology. C.M., M.Y.L., A.K.G., M.A.W. and A.R.H performed the experiments and analysis. C.M., M.Y.L., A.K.G., M.A.W., J.T.C.L. and J.C.V. wrote the paper, and all authors commented on the manuscript. J.C.V. supervised the project.

Corresponding author

Correspondence to Joshua C. Vaughan.

Ethics declarations

Competing interests

J.T.C.L and A.K.G are co-founders and hold equity in Lightspeed Microscopy Inc., of which J.T.C.L is also a board member. The other authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Mao, C. et al. Sci. Adv. 6, eaba4542 (2020): https://doi.org/10.1126/sciadv.aba4542

Chen, Y. et al. Biomed. Opt. Express 10, 1257 (2019): https://doi.org/10.1364/BOE.10.001257

Supplementary information

Supplementary information

Supplementary Method 1, Supplementary Figs. 1–4, Supplementary Tables 1 and 2 and the first frame of Supplementary Video 1.

Reporting Summary

Supplementary Video 1

Open-top light-sheet imaging of a ~1-mm-thick mouse kidney section. (Adapted with permission from ref. 3, AAAS, under a Creative Commons license CC BY-NC 4.0.)

Supplementary Code 1

Custom Wolfram Mathematica scripts used for stitching tiling images.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lee, M.Y., Mao, C., Glaser, A.K. et al. Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue and super-resolution microscopy. Nat Protoc 17, 819–846 (2022). https://doi.org/10.1038/s41596-021-00667-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-021-00667-2

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing