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.

  • Article
  • Published:

Imaging intracellular viscosity of a single cell during photoinduced cell death

Nature Chemistry volume 1, pages 69–73 (2009)Cite this article

Abstract

Diffusion-mediated cellular processes, such as metabolism, signalling and transport, depend on the hydrodynamic properties of the intracellular matrix. Photodynamic therapy, used in the treatment of cancer, relies on the generation of short-lived cytotoxic agents within a cell on irradiation of a drug. The efficacy of this treatment depends on the viscosity of the medium through which the cytotoxic agent must diffuse. Here, spectrally resolved fluorescence measurements of a porphyrin-dimer-based molecular rotor are used to quantify intracellular viscosity changes in single cells. We show that there is a dramatic increase in the viscosity of the immediate environment of the rotor on photoinduced cell death. The effect of this viscosity increase is observed directly in the diffusion-dependent kinetics of the photosensitized formation and decay of a key cytotoxic agent, singlet molecular oxygen. Using these tools, we provide insight into the dynamics of diffusion in cells, which is pertinent to drug delivery, cell signalling and intracellular mass transport.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Principles of intracellular viscosity detection and singlet-oxygen generation using a porphyrin dimer.
Figure 2: Emission spectra obtained upon 473 nm excitation of 1 in solution and in cells.
Figure 3: Time-resolved singlet-oxygen phosphorescence traces recorded at 1270 nm from D2O-incubated cells.
Figure 4: Imaging changes in intracellular viscosity using 1 in the ratiometric approach.

Similar content being viewed by others

References

  1. Kao, H. P., Abney, J. R. & Verkman, A. S. Determinants of the translational mobility of a small solute in cell cytoplasm. J. Cell Biol. 120, 175–184 (1993).

    Article  CAS  Google Scholar 

  2. Jones, D. P. Effect of mitochondrial clustering in O2 supply in hepatocytes. Am. J. Physiol. 247, C83–C89 (1984).

    Article  CAS  Google Scholar 

  3. O'Loughlin, M. A., Whillans, D. W. & Hunt, J. W. A fluorescence approach to testing the diffusion of oxygen into mammalian cells. Radiat. Res. 84, 477–495 (1980).

    Article  CAS  Google Scholar 

  4. Uchida, K., Matsuyama, K., Tanaka, K. & Doi, K. Diffusion coefficient for O2 in plasma and mitochondrial membranes of rat cardiomyocytes. Respir. Physiol. 90, 351–362 (1992).

    Article  CAS  Google Scholar 

  5. Hatz, S., Poulsen, L. & Ogilby, P. R. Time-resolved singlet oxygen phosphorescence measurements from photosensitized experiments in single cells: the effect of oxygen diffusion and oxygen concentration. Photochem. Photobiol. 84, 1284–1290 (2008).

    Article  CAS  Google Scholar 

  6. Wojcieszyn, J. W., Schlegel, R. A., Wu, E. S. & Jacobson, K. A. Diffusion of injected macromolecules within the cytoplasm of living cells. Proc. Natl Acad. Sci. USA 78, 4407–4410 (1981).

    Article  CAS  Google Scholar 

  7. Seksek, O., Biwersi, J. & Verkman, A. S. Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus. J. Cell Biol. 138, 131–142 (1997).

    Article  CAS  Google Scholar 

  8. Kuimova, M. K., Yahioglu, G., Levitt, J. A. & Suhling, K. Molecular rotor measures viscosity via fluorescence lifetime imaging. J. Am. Chem. Soc. 130, 6672–6673 (2008).

    Article  CAS  Google Scholar 

  9. Dix, J. A. & Verkman, A. S. Mapping of fluorescence anisotropy in living cells by ratio imaging—Application to cytoplasmic viscosity. Biophys. J. 57, 231–240 (1990).

    Article  CAS  Google Scholar 

  10. Luby-Phelps, K. et al. A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm. Biophys. J. 65, 236–242 (1993).

    Article  CAS  Google Scholar 

  11. Suhling, K. et al. Time-resolved fluorescence anisotropy imaging applied to live cells. Opt. Lett. 29, 584–586 (2004).

    Article  Google Scholar 

  12. Kuimova, M. K., Yahioglu, G. & Ogilby, P. R. Singlet oxygen in a cell: Spatially-dependent lifetimes and quenching rate constants. J. Am. Chem. Soc. 131, 332–340 (2009).

    Article  CAS  Google Scholar 

  13. Haidekker, M. A. & Theodorakis, E. A. Molecular rotors—Fluorescent biosensors for viscosity and flow. Org. Biomol. Chem. 5, 1669–1678 (2007).

    Article  CAS  Google Scholar 

  14. Haidekker, M. A., Brady, T. P., Lichlyter, D. & Theodorakis, E. A. A ratiometric fluorescent viscosity sensor. J. Am. Chem. Soc. 128, 398–399 (2006).

    Article  Google Scholar 

  15. Perrin, F. La fluorescence des solutions: induction moléculaire. Polarisation et durée d'emission. Photochimie. Ann. Phys. (Paris) 12, 169–275 (1929).

    CAS  Google Scholar 

  16. Shinitzky, M., Dianoux, A. C., Gitler, C. & Weber, G. Microviscosity and order in the hydrocarbon region of micelles and membranes determined with fluorescent probes. I. Synthetic micelles. Biochemistry 10, 2106–2113 (1971).

    Article  CAS  Google Scholar 

  17. Collins, H. A. et al. Blood vessel closure using photosensitisers engeneered for two-photon excitation. Nature Photon. 2, 420–424 (2008).

    Article  CAS  Google Scholar 

  18. Balaz, M., Collins, H. A., Dahlstedt, E. & Anderson, H. L. Synthesis of biocompatible conjugated porphyrin dimers for one-photon and two-photon excited photodynamic therapy at NIR wavelengths. Org. Biomol. Chem. 7, 874–888, doi:10.1039/b814789b (2009).

    Article  CAS  Google Scholar 

  19. Kuimova, M. K. et al. Photophysical properties and intracellular imaging of water-soluble porphyrin dimers for two-photon excited photodynamic therapy. Org. Biomol. Chem. 7, 889–896, doi:10.1039/b814791d (2009).

    Article  CAS  Google Scholar 

  20. Dahlstedt, E. et al. One- and two-photon activated phototoxicity of conjugated porphyrin dimers with high two-photon absorption cross-sections. Org. Biomol. Chem. 7, 897–904, doi:10.1039/b814792b (2009).

    Article  CAS  Google Scholar 

  21. Bonnett, R. Chemical Aspects of Photodynamic Therapy (Gordon and Breach, 2000).

    Google Scholar 

  22. Winters, M. U. et al. Photophysics of a butadiyne-linked porphyrin dimer: Influence of conformational flexibility in the ground and first singlet excited state. J. Phys. Chem. C 111, 7192–7199 (2007).

    Article  CAS  Google Scholar 

  23. Förster, T. & Hoffmann, G. Viscosity dependence of fluorescent quantum yields of some dye systems. Z. Phys. Chem. 75, 63–76 (1971).

    Article  Google Scholar 

  24. Borissevitch, I. E., Tominaga, T. T. & Schmitt, C. C. Photophysical studies on the interaction of two water-soluble porphyrins with bovine serum albumin. Effects upon the porphyrin triplet state characteristics. J. Photochem. Photobiol. A 114, 201–207 (1998).

    Article  CAS  Google Scholar 

  25. Lang, K., Mosinger, J. & Wagnerova, D. M. Photophysical properties of porphyrinoid sensitizers non-covalently bound to host molecules; models for photodynamic therapy. Coord. Chem. Rev. 248, 321–350 (2004).

    Article  CAS  Google Scholar 

  26. Zunszain, P. A., Ghuman, J., Komatsu, T., Tsuchida, E. & Curry, S. Crystal structural analysis of human serum albumin complexed with hemin and fatty acid. BMC Struct. Biol. 3: 6 (2003).

    Article  Google Scholar 

  27. Goosey, J. D., Zigler, J. S. & Kinoshita, J. H. Cross-linking of lens crystallins in a photodynamic system—A process mediated by singlet oxygen. Science 208, 1278–1280 (1980).

    Article  CAS  Google Scholar 

  28. Dubbelman, T., Degoeij, A. & Vansteveninck, J. Photodynamic effects of protoporphyrin on human erythrocytes—A nature of cross-linking of membrane proteins. Biochim. Biophys. Acta 511, 141–151 (1978).

    Article  CAS  Google Scholar 

  29. Verweij, H., Dubbelman, T. & Vansteveninck, J. Photodynamic protein cross-linking. Biochim. Biophys. Acta 647, 87–94 (1981).

    Article  CAS  Google Scholar 

  30. Basu, S., Rodionov, V., Terasaki, M. & Campagnola, P. J. Multiphoton-excited microfabrication in live cells via Rose Bengal cross-linking of cytoplasmic proteins. Opt. Lett. 30, 159–161 (2005).

    Article  Google Scholar 

  31. Hatz, S., Lambert, J. D. C. & Ogilby, P. R. Measuring the lifetime of singlet oxygen in a single cell: Addressing the issue of cell viability. Photochem. Photobiol. Sci. 6, 1106–1116 (2007).

    Article  CAS  Google Scholar 

  32. Snyder, J. W., Skovsen, E., Lambert, J. D. C. & Ogilby, P. R. Subcellular, time-resolved studies of singlet oxygen in single cells. J. Am. Chem. Soc. 127, 14558–14559 (2005).

    Article  CAS  Google Scholar 

  33. Battino, R. (ed.) Oxygen and Ozone. Solubility Data Series 7 (Pergamon, 1981).

    Google Scholar 

Download references

Acknowledgements

M.K.K. is grateful to the EPSRC Life Sciences Interface programme for a personal fellowship. We thank STFC for funding access to the Central Laser Facility. This work was supported in part by the Danish National Research Foundation under a block grant for the Center for Oxygen Microscopy and Imaging.

Author information

Authors and Affiliations

  1. Chemistry Department, Imperial College London, Exhibition Road, London, SW7 2AZ, UK

    Marina K. Kuimova

  2. Department of Chemistry, Center for Oxygen Microscopy and Imaging (COMI), University of Aarhus, Århus, DK-8000, Denmark

    Marina K. Kuimova & Peter R. Ogilby

  3. Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Didcot, OX11 0QX, Oxfordshire, UK

    Stanley W. Botchway & Anthony W. Parker

  4. Department of Chemistry, Oxford University, Chemistry Research Laboratory, Oxford, OX1 3TA, UK

    Milan Balaz, Hazel A. Collins & Harry L. Anderson

  5. Department of Physics, King's College London, Strand, London, WC2R 2LS, UK

    Klaus Suhling

Authors
  1. Marina K. Kuimova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stanley W. Botchway
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anthony W. Parker
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Milan Balaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hazel A. Collins
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Harry L. Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Klaus Suhling
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Peter R. Ogilby
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.K.K. designed the research, M.K.K., S.W.B. and A.W.P. measured fluorescence spectra, M.K.K. performed ratiometric imaging and measured singlet-oxygen traces. M.B. and H.A.C. synthesized the porphyrin dimer. All authors discussed the results and contributed to the manuscript.

Corresponding authors

Correspondence to Marina K. Kuimova or Peter R. Ogilby.

Supplementary information

Supplementary information

Supplementary information (PDF 422 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kuimova, M., Botchway, S., Parker, A. et al. Imaging intracellular viscosity of a single cell during photoinduced cell death. Nature Chem 1, 69–73 (2009). https://doi.org/10.1038/nchem.120

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.120

This article is cited by

Search

Advanced 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