Plasma Membrane-Promoted Aggregation of BODIPY Dimers Enables Live-Cell Super-Resolution Imaging

Abstract

Molecular aggregation profoundly alters the optical properties of fluorophores, yet its behavior in dynamic biological environments remains insufficiently understood and rarely harnessed functionally. Here, we report a membrane-controlled aggregation strategy based on plasma membrane-targeted BODIPY dimers engineered to undergo tunable H - and J-aggregation. By modulating the linker length, we establish a clear structure-aggregation relationship in which dimers form intramolecular H-aggregates in polar media, while two-dimensional confinement within lipid bilayers promotes intermolecular aggregation with red-shifted emission. Quantitative studies in model membranes reveal a threshold-dependent transition toward aggregate formation above a probe/lipid ratio of 1/100, highlighting the lipid bilayer as an active supramolecular platform that enhances probe-probe encounters and favors emissive excitonic states. In live cells, this membrane-promoted J-aggregation generates spontaneous blinking behavior that enables dual-channel single-molecule localization microscopy without specialized imaging buffers. Mechanistic investigations using HaloTag constructs confirm that while intramolecular aggregation can occur, membrane-driven intermolecular collisions strongly amplify J-aggregate formation. These findings demonstrate that biological membranes can serve as dynamic two-dimensional reactors for excitonic coupling, and establish membrane-induced J-aggregation of small-molecule fluorophores as a functional and generalizable principle for bioimaging.

References

[1] M. Collot, S. Pfister, and A. S. Klymchenko, “Advanced Functional Fluorescent Probes for Cell Plasma Membranes,” Current Opinion in Chemical Biology 69 (2022): 102161.

[2] G. Jacquemet, H. Hamidi, and J. Ivaska, “Filopodia in Cell Adhesion, 3D Migration and Cancer Cell Invasion,” Current Opinion in Cell Biology 36 (2015): 23-31.

[3] M. Bénard, C. Chamot, D. Schapman, et al., “Combining Sophisticated Fast FLIM, Confocal Msicroscopy, and STED Nanoscopy for Live-Cell Imaging of Tunneling Nanotubes,” Life Science Alliance 7 (2024): e202302398.

[4] M. Collot, E. Boutant, M. Lehmann, and A. S. Klymchenko, “BODIPY With Tuned Amphiphilicity as a Fluorogenic Plasma Membrane Probe,” Bioconjugate Chemistry 30 (2019): 192-199.

[5] S. Pfister, V. Le Berruyer, K. Fam, and M. Collot, “A Photoactivatable Plasma Membrane Probe Based on a Self-Triggered Photooxidation Cascade for Live Cell Super-Resolution Microscopy,” Angewandte Chemie International Edition 64 (2025): e202425276.

[6] M. Collot, P. Ashokkumar, H. Anton, et al., “MemBright: A Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience,” Cell Chemical Biology 26 (2019): 600-614.e7.

[7] M. Lorizate, O. Terrones, J. A. Nieto-Garai, et al., “Super-Resolution Microscopy Using a Bioorthogonal-Based Cholesterol Probe Provides Unprecedented Capabilities for Imaging Nanoscale Lipid Heterogeneity in Living Cells,” Small Methods 5 (2021): 2100430.

[8] A. D. Thompson, M. H. Omar, F. Rivera-Molina, Z. Xi, A. J. Koleske, and D. K. Toomre, “Long-Term Live-Cell STED Nanoscopy of Primary and Cultured Cells With the Plasma Membrane HIDE Probe DiI-SiR,” Angewandte Chemie International Edition 56 (2017): 10408-10412.

[9] M. Collot, E. Boutant, K. T. Fam, L. Danglot, and A. S. Klymchenko, “Molecular Tuning of Styryl Dyes Leads to Versatile and Efficient Plasma Membrane Probes for Cell and Tissue Imaging,” Bioconjugate Chemistry 31 (2020): 875-883.

[10] R. Yan, K. Chen, and K. Xu, “Probing Nanoscale Diffusional Heterogeneities in Cellular Membranes Through Multidimensional Single-Molecule and Super-Resolution Microscopy,” Journal of the American Chemical Society 142 (2020): 18866-18873.

[11] D. I. Danylchuk, S. Moon, K. Xu, and A. S. Klymchenko, “Switchable Solvatochromic Probes for Live-Cell Super-Resolution Imaging of Plasma Membrane Organization,” Angewandte Chemie International Edition 58 (2019): 14920-14924.

[12] S.-H. Shim, C. Xia, G. Zhong, et al., “Super-Resolution Fluorescence Imaging of Organelles in Live Cells With Photoswitchable Membrane Probes,” Proceedings of the National Academy of Sciences of the United States of America 109 (2012): 13978-13983.

[13] V. Breton, P. Nazac, D. Boulet, and L. Danglot, “Molecular Mapping of Neuronal Architecture Using STORM Microscopy and New Fluorescent Probes for SMLM Imaging,” Normal Pressure Hydrocephalus 11 (2024): 014414.

[14] A. Kusumi, T. A. Tsunoyama, K. M. Hirosawa, R. S. Kasai, and T. K. Fujiwara, “Tracking Single Molecules at Work in Living Cells,” Nature Chemical Biology 10 (2014): 524-532.

[15] L. Cognet, C. Leduc, and B. Lounis, “Advances in Live-Cell Single-Particle Tracking and Dynamic Super-Resolution Imaging,” Current Opinion in Chemical Biology 20 (2014): 78-85.

[16] D. Albrecht, C. M. Winterflood, M. Sadeghi, T. Tschager, F. Noé, and H. Ewers, “Nanoscopic Compartmentalization of Membrane Protein Motion at the Axon Initial Segment,” Journal of Cell Biology 215 (2016): 37-46.

[17] C. K. Spahn, M. Glaesmann, J. B. Grimm, A. X. Ayala, L. D. Lavis, and M. Heilemann, “A Toolbox for Multiplexed Super-Resolution Imaging of the E. coli Nucleoid and Membrane Using Novel PAINT Labels,” Scientific Reports 8 (2018): 14768.

[18] I. O. Aparin, R. Yan, R. Pelletier, et al., “Fluorogenic Dimers as Bright Switchable Probes for Enhanced Super-Resolution Imaging of Cell Membranes,” Journal of the American Chemical Society 144 (2022): 18043-18053.

[19] Q. Qi, Y. Liu, V. Puranik, et al., “Photoswitchable Fluorescent Hydrazone for Super-Resolution Cell Membrane Imaging,” Journal of the American Chemical Society 147 (2025): 16404-16411.

[20] P. J. Macdonald, S. Gayda, R. A. Haack, Q. Ruan, R. J. Himmelsbach, and S. Y. Tetin, “Rhodamine-Derived Fluorescent Dye With Inherent Blinking Behavior for Super-Resolution Imaging,” Analytical Chemistry 90 (2018): 9165-9173.

[21] Z. Liu, Y. Zheng, T. Xie, et al., “Clickable Rhodamine Spirolactam Based Spontaneously Blinking Probe for Super-Resolution Imaging,” Chinese Chemical Letters 32 (2021): 3862-3864.

[22] N. Lardon, L. Wang, A. Tschanz, et al., “Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy,” Journal of the American Chemical Society 143 (2021): 14592-14600.

[23] Q. Qiao, W. Liu, J. Chen, et al., “An Acid-Regulated Self-Blinking Fluorescent Probe for Resolving Whole-Cell Lysosomes With Long-Term Nanoscopy,” Angewandte Chemie International Edition 61 (2022): e202202961.

[24] Y. Zheng, Z. Ye, X. Zhang, and Y. Xiao, “Recruiting Rate Determines the Blinking Propensity of Rhodamine Fluorophores for Super-Resolution Imaging,” Journal of the American Chemical Society 145 (2023): 5125-5133.

[25] S. Pfister, S. Walter, A. Perrier, and M. Collot, “Spontaneously Blinking Spiroamide Rhodamines for Live SMLM Imaging of the Plasma Membrane,” Chemical Communications 61 (2025): 6170-6173.

[26] L. Saladin, V. Breton, V. Le Berruyer, et al., “Targeted Photoconvertible BODIPYs Based on Directed Photooxidation-Induced Conversion for Applications in Photoconversion and Live Super-Resolution Imaging,” Journal of the American Chemical Society 146 (2024): 17456-17473.

[27] L. Saladin, V. Le Berruyer, M. Bonnevial, P. Didier, and M. Collot, “Targeted Photoactivatable Green-Emitting BODIPY Based on Directed Photooxidation-Induced Activation and Its Application to Live Dynamic Super-Resolution Microscopy,” Chemistry—A European Journal 30 (2024): e202403409.

[28] P. Ashokkumar, M. Collot, and A. S. Klymchenko, “Fluorogenic Squaraine Dendrimers for Background-Free Imaging of Integrin Receptors in Cancer Cells,” Chemistry—A European Journal 27 (2021): 6795-6803.

[29] K. T. Fam, L. Saladin, A. S. Klymchenko, and M. Collot, “Confronting Molecular Rotors and Self-Quenched Dimers as Fluorogenic BODIPY Systems to Probe Biotin Receptors in Cancer Cells,” Chemical Communications 57 (2021): 4807-4810.

[30] I. A. Karpenko, M. Collot, L. Richert, et al., “Fluorogenic Squaraine Dimers With Polarity-Sensitive Folding as Bright Far-Red Probes for Background-Free Bioimaging,” Journal of the American Chemical Society 137 (2015): 405-412.

[31] K. T. Fam, M. Collot, and A. S. Klymchenko, “Probing Biotin Receptors in Cancer Cells With Rationally Designed Fluorogenic Squaraine Dimers,” Chemical Science 11 (2020): 8240-8248.

[32] L. Esteoulle, F. Daubeuf, M. Collot, et al., “A Near-Infrared Fluorogenic Dimer Enables Background-Free Imaging of Endogenous GPCRs in Living Mice,” Chemical Science 11 (2020): 6824-6829.

[33] O. Florès, Y. Berthomé, L. Weiss, et al., “Click-Functionalized Cyanine Fluorogenic Dimers for Improved Detection of GPCRs: Application to Imaging of ApelinR in Living Cells,” Chemistry—A European Journal 31 (2025): e202500379.

[34] F. Bouhedda, K. T. Fam, M. Collot, et al., “A Dimerization-Based Fluorogenic Dye-Aptamer Module for RNA Imaging in Live Cells,” Nature Chemical Biology 16 (2020): 69-76.

[35] D. Kim, U. Lee, J. Bouffard, and Y. Kim, “Glycosaminoglycan-Induced Emissive J-Aggregate Formation in a Meso-Ester BODIPY Dye,” Advanced Optical Materials 8 (2020): 1902161.

[36] K. Li, X. Duan, Z. Jiang, et al., “J-Aggregates of Meso-[2.2]Paracyclophanyl-BODIPY Dye for NIR-II Imaging,” Nature Communications 12 (2021): 2376.

[37] J. Heo, D. P. Murale, H. Y. Yoon, et al., “Recent Trends in Molecular Aggregates: An Exploration of Biomedicine,” Aggregate 3 (2022): e159.

[38] F. Würthner, T. E. Kaiser, and C. R. J.-A. Saha-Möller, “J-Aggregates: From Serendipitous Discovery to Supramolecular Engineering of Functional Dye Materials,” Angewandte Chemie International Edition 50 (2011): 3376-3410.

[39] W. Chen, C.-A. Cheng, E. D. Cosco, et al., “Shortwave Infrared Imaging With J-Aggregates Stabilized in Hollow Mesoporous Silica Nanoparticles,” Journal of the American Chemical Society 141 (2019): 12475-12480.

[40] P. Sun, Q. Wu, X. Sun, et al., “J-Aggregate Squaraine Nanoparticles With Bright NIR-II Fluorescence for Imaging Guided Photothermal Therapy,” Chemical Communications 54 (2018): 13395-13398.

[41] M. H. Y. Cheng, K. M. Harmatys, D. M. Charron, J. Chen, and G. Zheng, “Stable J-Aggregation of an Aza-BODIPY-Lipid in a Liposome for Optical Cancer Imaging,” Angewandte Chemie 131 (2019): 13528-13533.

[42] S. Adhikari, J. Moscatelli, E. M. Smith, C. Banerjee, and E. M. Puchner, “Single-Molecule Localization Microscopy and Tracking With Red-Shifted States of Conventional BODIPY Conjugates in Living Cells,” Nature Communications 10 (2019): 3400.

[43] P. Rybczynski, A. Smolarkiewicz-Wyczachowski, J. Piskorz, et al., “Photochemical Properties and Stability of BODIPY Dyes,” International Journal of Molecular Sciences 22 (2021): 6735.

[44] F. Bergström, I. Mikhalyov, P. Hägglöf, R. Wortmann, T. Ny, and L. B.-Å. Johansson, “Dimers of Dipyrrometheneboron Difluoride (BOD-IPY) With Light Spectroscopic Applications in Chemistry and Biology,” Journal of the American Chemical Society 124 (2002): 196-204.

[45] T. T. Vu, M. Dvorko, E. Y. Schmidt, et al., “Understanding the Spectroscopic Properties and Aggregation Process of a New Emitting Boron Dipyrromethene (BODIPY),” Journal of Physical Chemistry C 117 (2013): 5373-5385.

[46] A. B. Descalzo, P. Ashokkumar, Z. Shen, and K. Rurack, “On the Aggregation Behaviour and Spectroscopic Properties of Alkylated and Annelated Boron-Dipyrromethene (BODIPY) Dyes in Aqueous Solution,” ChemPhotoChem 4 (2020): 120-131.

[47] S. Michelis, L. Danglot, R. Vauchelles, A. S. Klymchenko, and M. Collot, “Imaging and Measuring Vesicular Acidification With a Plasma Membrane-Targeted Ratiometric PH Probe,” Analytical Chemistry 94 (2022): 5996-6003.

[48] G. Gramse, A. Dols-Perez, M. A. Edwards, L. Fumagalli, and G. Gomila, “Nanoscale Measurement of the Dielectric Constant of Supported Lipid Bilayers in Aqueous Solutions With Electrostatic Force Microscopy,” Biophysical Journal 104 (2013): 1257-1262.

[49] L. J. Patalag, L. P. Ho, P. G. Jones, and D. B. Werz, “Ethylene-Bridged Oligo-BODIPYs: Access to Intramolecular J-Aggregates and Superfluorophores,” Journal of the American Chemical Society 139 (2017): 15104-15113.

[50] N. M. Gretskaya and I. I. Mikhalyov, “Some Patterns in Dimer II Formation in BODIPY-FL-Labeled Lipids,” Russian Journal of Bioorganic Chemistry 35 (2009): 759-765.

[51] L. Saladin, V. Le Berruyer, M. Bonnevial, P. Didier, and M. Collot, “Targeted Photoactivatable Green-Emitting BODIPY Based on Directed Photooxidation-Induced Activation and Its Application to Live Dynamic Super-Resolution Microscopy,” Chemistry—A European Journal 30 (2024): e202403409.

[52] A. Kamkaew, S. H. Lim, H. B. Lee, L. V. Kiew, L. Y. Chung, and K. Burgess, “BODIPY Dyes in Photodynamic Therapy,” Chemical Society Reviews 42 (2012): 77-88.

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