Lysosomes are multifunctional, subcellular organelles with roles in plasma membrane repair, autophagy, pathogen degradation and nutrient sensing. Dysfunctional lysosomes underlie Alzheimer’s disease, Parkinson’s disease and rare lysosomal storage diseases, but their contributions to these pathophysiologies are unclear. Live imaging has revealed lysosome subpopulations with different physical characteristics including dynamics, morphology or cellular localization. Here, we chemically resolve lysosome subpopulations using a DNA-based combination reporter that quantitatively images pH and chloride simultaneously in the same lysosome while retaining single-lysosome information in live cells. We call this technology two-ion measurement or 2-IM. 2-IM of lysosomes in primary skin fibroblasts derived from healthy individuals shows two main lysosome populations, one of which is absent in primary cells derived from patients with Niemann–Pick disease. When patient cells are treated with relevant therapeutics, the second population re-emerges. Chemically resolving lysosomes by 2-IM could enable decoding the mechanistic underpinnings of lysosomal diseases, monitoring disease progression or evaluating therapeutic efficacy.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Data availability

The data that support the plots within this paper and the findings of this study are available from the corresponding author upon reasonable request.

Additional information

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


  1. 1.

    Settembre, C., Fraldi, A., Medina, D. L. & Ballabio, A. Signals from the lysosome: a control centre for cellular clearance and energy metabolism. Nat. Rev. Mol. Cell Biol. 14, 283–296 (2013).

  2. 2.

    Fraldi, A., Klein, A. D., Medina, D. L. & Settembre, C. Brain disorders due to lysosomal dysfunction. Annu. Rev. Neurosci. 39, 277–295 (2016).

  3. 3.

    Pu, J., Guardia, C. M., Keren-Kaplan, T. & Bonifacino, J. S. Mechanisms and functions of lysosome positioning. J. Cell Sci. 129, 4329–4339 (2016).

  4. 4.

    Wasmeier, C., Hume, A. N., Bolasco, G. & Seabra, M. C. Melanosomes at a glance. J. Cell Sci. 121, 3995–3999 (2008).

  5. 5.

    Faurschou, M. & Borregaard, N. Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 5, 1317–1327 (2003).

  6. 6.

    Blott, E. J. & Griffiths, G. M. Secretory lysosomes. Nat. Rev. Mol. Cell Biol. 3, 122–131 (2002).

  7. 7.

    Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010).

  8. 8.

    Pertoft, H., Wärmegård, B. & Höök, M. Heterogeneity of lysosomes originating from rat liver parenchymal cells. Metabolic relationship of subpopulations separated by density-gradient centrifugation. Biochem. J. 174, 309–317 (1978).

  9. 9.

    Li, X. et al. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation. Nat. Cell Biol. 18, 404–417 (2016).

  10. 10.

    Matteoni, R. & Kreis, T. E. Translocation and clustering of endosomes and lysosomes depends on microtubules. J. Cell Biol. 105, 1253–1265 (1987).

  11. 11.

    Pu, J. et al. BORC, a multisubunit complex that regulates lysosome positioning. Dev. Cell 33, 176–188 (2015).

  12. 12.

    Kornak, U. et al. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104, 205–215 (2001).

  13. 13.

    Ba, Q., Raghavan, G., Kiselyov, K. & Yang, G. Whole-cell scale dynamic organization of lysosomes revealed by spatial statistical analysis. Cell Rep. 23, 3591–3606 (2018).

  14. 14.

    Seiji, M., Shimao, K., Birbeck, M. S. C. & Fitzpatrick, T. B. Subcellular localization of melanin biosynthests. Ann. NY Acad. Sci. 100, 497–533 (2006).

  15. 15.

    Raposo, G., Tenza, D., Murphy, D. M., Berson, J. F. & Marks, M. S. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells. J. Cell Biol. 152, 809–824 (2001).

  16. 16.

    Chakraborty, K., Leung, K. & Krishnan, Y. High lumenal chloride in the lysosome is critical for lysosome function. eLife 6, e28862 (2017).

  17. 17.

    Saha, S., Prakash, V., Halder, S., Chakraborty, K. & Krishnan, Y. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells. Nat. Nanotech. 10, 645–651 (2015).

  18. 18.

    Van Dyke, R. W. Proton pump-generated electrochemical gradients in rat liver multivesicular bodies. Quantitation and effects of chloride. J. Biol. Chem. 263, 2603–2611 (1988).

  19. 19.

    Luzio, J. P., Pryor, P. R. & Bright, N. A. Lysosomes: fusion and function. Nat. Rev. Mol. Cell Biol. 8, 622–632 (2007).

  20. 20.

    Weinert, S. et al. Lysosomal pathology and osteopetrosis upon loss of H+-driven lysosomal Cl accumulation. Science 328, 1401–1403 (2010).

  21. 21.

    Guzman, R. E., Grieschat, M., Fahlke, C. & Alekov, A. K. ClC-3 is an intracellular chloride/proton exchanger with large voltage-dependent nonlinear capacitance. ACS Chem. Neurosci. 4, 994–1003 (2013).

  22. 22.

    Biwersi, J., Tulk, B. & Verkman, A. S. Long-wavelength chloride-sensitive fluorescent indicators. Anal. Biochem. 219, 139–143 (1994).

  23. 23.

    Arosio, D. et al. Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nat. Methods 7, 516–518 (2010).

  24. 24.

    Kuner, T. & Augustine, G. J. A genetically encoded ratiometric indicator for chloride. Neuron 27, 447–459 (2000).

  25. 25.

    Surana, S., Shenoy, A. R. & Krishnan, Y. Designing DNA nanodevices for compatibility with the immune system of higher organisms. Nat. Nanotech. 10, 741–747 (2015).

  26. 26.

    Modi, S., Nizak, C., Surana, S., Halder, S. & Krishnan, Y. Two DNA nanomachines map pH changes along intersecting endocytic pathways inside the same cell. Nat. Nanotech. 8, 459–467 (2013).

  27. 27.

    Chu, T. C. et al. Aptamer:toxin conjugates that specifically target prostate tumor cells. Cancer Res. 66, 5989–5992 (2006).

  28. 28.

    Pinheiro, A. V., Han, D., Shih, W. M. & Yan, H. Challenges and opportunities for structural DNA nanotechnology. Nat. Nanotech. 6, 763–772 (2011).

  29. 29.

    Bhatia, D. et al. Quantum dot-loaded monofunctionalized DNA icosahedra for single-particle tracking of endocytic pathways. Nat. Nanotech. 11, 1112–1119 (2016).

  30. 30.

    Prakash, V., Saha, S., Chakraborty, K. & Krishnan, Y. Rational design of a quantitative, pH-insensitive, nucleic acid based fluorescent chloride reporter. Chem. Sci. 7, 1946–1953 (2016).

  31. 31.

    Modi, S., Halder, S., Nizak, C. & Krishnan, Y. Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways. Nanoscale 6, 1144–1152 (2014).

  32. 32.

    Modi, S. et al. A DNA nanomachine that maps spatial and temporal pH changes inside living cells. Nat. Nanotech. 4, 325–330 (2009).

  33. 33.

    Surana, S., Bhat, J. M., Koushika, S. P. & Krishnan, Y. An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism. Nat. Commun. 2, 340 (2011).

  34. 34.

    Halder, S. & Krishnan, Y. Design of ultrasensitive DNA-based fluorescent pH sensitive nanodevices. Nanoscale 7, 10008–10012 (2015).

  35. 35.

    Gough, P. J. & Gordon, S. The role of scavenger receptors in the innate immune system. Microbes Infect. 2, 305–311 (2000).

  36. 36.

    Bhatia, D., Surana, S., Chakraborty, S., Koushika, S. P. & Krishnan, Y. A synthetic icosahedral DNA-based host–cargo complex for functional in vivo imaging. Nat. Commun. 2, 339 (2011).

  37. 37.

    Chakraborty, K., Veetil, A. T., Jaffrey, S. R. & Krishnan, Y. Nucleic acid-based nanodevices in biological imaging. Annu. Rev. Biochem. 85, 349–373 (2016).

  38. 38.

    Veetil, A. T. et al. Cell-targetable DNA nanocapsules for spatiotemporal release of caged bioactive small molecules. Nat. Nanotech. 12, 1183–1189 (2017).

  39. 39.

    Surana, S., Bhatia, D. & Krishnan, Y. A method to study in vivo stability of DNA nanostructures. Methods 64, 94–100 (2013).

  40. 40.

    Schuchman, E. H. & Desnick, R. J. Types A and B Niemann–Pick disease. Mol. Genet. Metab. 120, 27–33 (2017).

  41. 41.

    Vanier, M. T. & Millat, G. Niemann–Pick disease type C. Clin. Genet. 64, 269–281 (2003).

  42. 42.

    Desnick, R. J. & Schuchman, E. H. Enzyme replacement and enhancement therapies: lessons from lysosomal disorders. Nat. Rev. Genet. 3, 954–966 (2002).

  43. 43.

    Ory, D. S. et al. Intrathecal 2-hydroxypropyl-β-cyclodextrin decreases neurological disease progression in Niemann-Pick disease, type C1: a non-randomised, open-label, phase 1–2 trial. Lancet 390, 1758–1768 (2017).

  44. 44.

    Johnson, D. E., Ostrowski, P., Jaumouillé, V. & Grinstein, S. The position of lysosomes within the cell determines their luminal pH. J. Cell Biol. 212, 677–692 (2016).

  45. 45.

    Kornhuber, J. et al. Identification of new functional inhibitors of acid sphingomyelinase using a structure–property–activity relation model. J. Med. Chem. 51, 219–237 (2008).

  46. 46.

    Cenedella, R. J. Cholesterol synthesis inhibitor U18666A and the role of sterol metabolism and trafficking in numerous pathophysiological processes. Lipids 44, 477–487 (2009).

  47. 47.

    He, X. et al. Characterization of human acid sphingomyelinase purified from the media of overexpressing Chinese hamster ovary cells. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1432, 251–264 (1999).

  48. 48.

    Devany, J., Chakraborty, K. & Krishnan, Y. Subcellular nanorheology reveals lysosomal viscosity as a reporter for lysosomal storage diseases. Nano Lett. 18, 1351–1359 (2018).

  49. 49.

    Aue, W. P., Bartholdi, E. & Ernst, R. R. Two‐dimensional spectroscopy. Application to nuclear magnetic resonance. J. Chem. Phys. 64, 2229–2246 (1976).

  50. 50.

    Boaz, H. & Rollefson, G. K. The quenching of fluorescence. Deviations from the Stern–Volmer law. J. Am. Chem. Soc. 72, 3435–3443 (1950).

Download references


This work was supported by the University of Chicago Women’s Board, Pilot and Feasibility award from an NIDDK center grant P30DK42086 to the University of Chicago Digestive Diseases Research Core Center, MRSEC grant no. DMR-1420709, Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust, C-084, ANL-UChicago collaborative grant and University of Chicago start-up funds to Y.K. Y.K. is a Brain Research Foundation Fellow.

Author information

Author notes

  1. These authors contributed equally: KaHo Leung, Kasturi Chakraborty.


  1. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    • KaHo Leung
    • , Kasturi Chakraborty
    • , Anand Saminathan
    •  & Yamuna Krishnan
  2. Grossman Institute of Neuroscience, Quantitative Biology and Human Behavior, The University of Chicago, Chicago, IL, USA

    • KaHo Leung
    • , Kasturi Chakraborty
    • , Anand Saminathan
    •  & Yamuna Krishnan


  1. Search for KaHo Leung in:

  2. Search for Kasturi Chakraborty in:

  3. Search for Anand Saminathan in:

  4. Search for Yamuna Krishnan in:


K.L., K.C., A.S. and Y.K. designed the project. K.L., K.C. and A.S. performed experiments. K.L., K.C., A.S. and Y.K. analysed the data. K.L., K.C., A.S. and Y.K. wrote the paper. All authors discussed the results and gave input on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Yamuna Krishnan.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–13, Supplementary Tables 1–2 and Supplementary References

  2. Reporting Summary

About this article

Publication history