Methods to Study Single Cells, Cell Membranes, Single Vesicles, and Exocytosis

Focusing on the neuronal process of exocytosis, the Ewing group has pioneered small-volume chemical measurements at single cells, electrochemical detection for capillary electrophoresis, novel approaches for electrochemical imaging of single cells, and new electrochemical strategies to separate individual nanometer vesicles from cells and quantify their contents. The group also pioneered, in collaboration with the Winograd group, many methods for the development and application of mass spectrometry imaging for subcellular and neurochemical analysis.

In the last 5 years, the team has developed new approaches for electrochemical imaging of single cells with complementary chemical imaging with nanometer spatial resolution and we have developed and applied mass spectrometry imaging at the submicrometer level to build on our earlier work (Science 2004 and PNAS 2010)1,2 to understand domains in cell membranes. In the last 2 years we have had incredible success (best of my career) and: (i) developed a new method to measure the contents of vesicles (JACS, 2015; IF 13),3 (ii) developed SIMS imaging of the fly brain to demonstrate that the drug methylphenidate alters the lipid composition (Analyt Chem, 2015; IF 5.8),4 (iii) measured vesicle contents in situ in live cells with a nanoelectrode in a single cell (Angew Chem, 2015; IF 11.7),5 (iv) measured single exocytosis events from varicosities in the fly using optogenetic stimulation (Angew Chem, 2015),6 shown that excited fluorophores in vesicle membranes generate oxidative stress which in turns leads to electroporation and pore opening in electrochemical cytometry (Angew Chem, 2016),7 (v) applied cisplatin to cells and observed the changes in vesicle content and release to propose a mechanism for the cognitive changes in the “chemobrain” observed in cancer patients (Angew Chem, 2016; and back cover: doi/10.1002/anie.201605032/epdf)8 (vi) used the NanoSIMS to measure and image the transmitter dopamine inside a single nanometer vesicle (ACS Nano 2017: IF 13.3),9 and (vii) discovered that zinc changes vesicle content and the fraction released in exocytosis possibly providing a mechanism to explain how zinc affects learning (Angew Chem, 2017 and front cover).10 The intracellular vesicle impact electrochemical cytometry method we have developed allows us to directly compare the contents of vesicles and the material released in the same system and has revolutionized our work in the last year! Our combined work on open and closed exocytosis was recently published in Quarterly Reviews in Biophysics (2016; IF 7.2)11 and the work to understand electrochemical cytometry of vesicles was reviewed in Accounts of Chem Research (2016; IF 22) and Current Opinions in Electrochemistry (2017),12, 13 and a mechanism has been proposed that explains vesicle opening on electrode surfaces.14  We have also published a recent review on analysis and imaging of individual vesicles.15

In addition to the work described above, the group has built a unique mass spectrometry imaging laboratory, second to none in SIMS imaging with an IonTof V instrument, Ionoptika J105 3D Chemical Imager, an AB Sciex Qstar equipped with a C60 ion gun, a Bruker Ultraflextreme MALDI instrument, and now the Cameca NanoSIMS (added recently).

Some Current Research Directions

  • Vesicle impact electrochemical cytometry (VIEC). We are working to examine the mechanism of vesicle opening in VIEC and determine the role of lipids in this mechanism, which could help us understand the effect of lipids on pore opening in exocytosis.
  • New methods for vesicle impact electrochemical cytometry (VIEC). Here we propose new nano fabricated approaches for VIEC, one for high throughput vesicle analysis, and the other to quantify substance in the protein dense core of a single vesicle.
  • Intracellular vesicle impact electrochemical cytometry. We plan to develop new approaches with intracellular vesicle impact electrochemical cytometry (IVIEC) combined with amperometric measurements of exocytosis to measure the fraction of vesicular messenger released and, importantly, we will use these platforms to examine and quantify how lipids (and zinc, which is correlated with learning in mammals) affect the fraction of messenger released during exocytosis, an important step in development of methods to explore the effect of lipids on plasticity.
  • Lipids in signaling plasticity, nanoimaging. We plan to develop methodological paradigms to examine first cell models and then later simple animal models to examine the chemistry associated with dynamic changes in exocytosis. We also plan to examine these cell and animal models using nano mass spectrometry and optical imaging methods.
  • Vesicle structure/function: NanoSIMS combined with IVIEC. We propose to apply our new combined technology with 1) the NanoSIMS, probing to 40 nm spatial resolution and imaging the substructure of vesicles with 2) dynamic amperometric detection of exocytotic release and IVIEC of vesicle content in cells.
  • Drugs, zinc, lipids, and learning/memory. We interested to examine the effects of cognition-enhancing drugs like methylphenidate, modafinil, cocaine (and possibly zinc or lipids in the diet, or glutamate receptor antagonists; e.g. MK-801, CNQX) on the amount and distribution of lipids in the brain, in the active zone of neurons, and in vesicles, as well as the fraction of transmitter released in each exocytosis event, and the level of zinc in vesicles.
  • Model of effectors for short-term memory. Our plan is to assemble the measurements above into a model that will show the simplest chemical steps in the initiation of synaptic plasticity and from there the strengthening to a short-term enhancement otherwise considered short-term memory.


  1. Ostrowski, S. G., et al. Mass Spectrometric Imaging of Highly Curved Membranes During Tetrahymena Mating. Science, 305 (2004) 71-73.
  2. Kurczy, et al. Mass Spectrometry Imaging of Mating Tetrahymena: Changes in Cell Morphology Regulate Lipid Domain Formation. Proc. Natl Acad Sci USA, 107 (2010) 2751-2756.
  3. Dunevall, J. et al. Characterizing the catecholamine content of single mammalian vesicles by collision-adsorption events at an electrode. J Am Chem Soc, 137 (2015) 4344-4346.
  4. Phan, N. T. N., Fletcher, J. S., Ewing, A. G. Lipid Structural Effects of Oral Administration of Methylphenidate in Drosophila Brain by Secondary Ion Mass Spectrometry Imaging
 Anal Chem, 87 (2015) 4063-4071.
  5. Li, X., et al., A. G. Quantitative Measurement of Transmitters in Individual Vesicles in the Cytoplasm of Single Cells with Nanotip Electrodes. Angew Chem Int Ed, 54 (2015) 11978-11982.
  6. Majdi, S. et al. Electrochemical Measurements of Optogenetically Stimulated Quantal Amine Release from Single Nerve Cell Varicosities in Drosophila Larvae. Angew Chem Int Ed, 54 (2015) 13609-13612.
  7. Najafinobar, N. et al. Excited Fluorophores Enhance the Opening of Vesicles at Electrode Surfaces in Vesicle Electrochemical Cytometry. Angew Chem Int Ed, 55 (2016) 15081-15085.
  8. Li, X., Dunevall, J. & Ewing, A. G. Using Single-Cell Amperometry to Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis. Angew Chem Int Ed, 55 (2016) 9041-9044.
  9. Lovric, J. et al. Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles. ACS Nano, 11 (2017) 3446-3455. doi:10.1021/acsnano.6b07233.
  10. Ren, L. et al. Zinc regulates chemical transmitter storage in nanometer vesicles and exocytosis dynamics measured by amperometry. Angew Chem Int Ed 56 (2017) 4970-4975
  11. Ren, L. et al. The Evidence for Open and Closed Exocytosis as the Primary Release Mechanism. Q Rev Biophys, 49 (2016) doi:10.1017/S0033583516000081.
  12. Li, X., Dunevall, J., Ewing, A. G. Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry. Acc Chem Res, 49 (2016) 2347-2354.
  13. J. Dunevall, A. Larsson, S. Majdi, A. G. Ewing, Vesicle impact electrochemical cytometry compared to amperometric exocytosis measurements, Current Opinion in Electrochemistry, (2017) DOI: org/10.1016/j.coelec.2017.07.005.
  14. X. Li, L. Ren, J. Dunevall, D. Ye, H.S. White, M.A. Edwards, A.G. Ewing, On the Nanopore Opening at Flat and Nano-Tip Conical Electrodes during Vesicle Impact Electrochemical Cytometry, ACS Nano, in press (DOI: 10.1021/acsnano.8b00781).
  15. N.T.N. Phan, X. Li and A.G. Ewing, Measuring synaptic vesicles using cellular electrochemistry and nanoscale molecular imaging, Nature Reviews Chemistry, 1 (2017) DOI:10.1038/s41570-017-0048.

Select Recent References

  1. Single-Vesicle Electrochemistry Following Repetitive Stimulation Reveals a Mechanism for Plasticity Changes with Iron Deficiency, Y. Wang, C. Gu and A.G. Ewing, Angewandte Chemie Int. Ed., (2022)
  2. Anionic Species Regulate Chemical Storage in Nanometer Vesicles and Amperometrically Detected Exocytotic Dynamics, X. He and A.G. Ewing, J. Am. Chem. Soc., 144 (2022) 4310–4314,
  3. Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells, T.D.K. Nguyen, L. Mellander, A. Lork, A. Thomen, M. Philipsen, M.E. Kurczy, N.T.N. Phan and A.G. Ewing, ACS Nano 2022, 16, 3, 4831–4842.
  4. Single-Vesicle Electrochemistry Following Repetitive Stimulation Reveals a Mechanism for Plasticity Changes with Iron Deficiency, Y. Wang, C. Gu and A.G. Ewing, Angewandte Chemie Int Ed., (2022)
  5. Simultaneous Counting of Molecules in the Halo and Dense-Core of Nanovesicles by Regulating Dynamics of Vesicle Opening, X. He and A.G. Ewing, Angewandte Chemie Int Ed., (2022).
  6. Concentration of stimulant regulates initial exocytotic molecular plasticity at single cells, X. He and A.G. Ewing, Chemical Science, 13 (2022) 1815-1822,
  7. Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging, S. Rabasco, T.D.K. Nguyen, C. Gu, M.E. Kurczy, N.T.N. Phan, and A.G. Ewing, International Journal of Molecular Sciences, 23 (2022), 1-11,
  8. Quantifying Intracellular Single Vesicular Catecholamine Concentration with Open Carbon Nanopipettes to Unveil the Effect of L-DOPA on Vesicular Structure, K. Hu, K.L.L. Vo, A. Hatamie, and A.G. Ewing, Angewandte Chemie-International Edition, 61 (2022), e202113406,
  9. Anionic Species Regulate Chemical Storage in Nanometer Vesicles and Amperometrically Detected Exocytotic Dynamics, X. He and A.G. Ewing, J. Am. Chem. Soc., 144 (2022) 4310–4314,
  10. Simultaneous Detection of Vesicular Content and Exocytotic Release with Two Electrodes in and at a Single Cell, C. Gu and A.G. Ewing, Chemical Science, 12 (2021) 7393-7400.
  11. Dynamic Visualization and Quantification of Single Vesicle Opening and Content by Coupling Vesicle Impact Electrochemical Cytometry with Confocal Microscopy, Y.N. Zheng, T.D.K. Nguyen, J. Dunevall, N.T.N. Phan, and A.G. Ewing, ACS Measurement Science Au, 1 (2021), 131–138,
  12.  Combined electrochemistry and mass spectrometry imaging to interrogate the mechanism of action of modafinil, a cognition-enhancing drug, at the cellular and sub-cellular level, E. Ranjbari, M.H. Philipsen, Z. Wang, and A.G. Ewing, QRB Discovery, 2 (2021) e6, 1–9.
  13. Electrochemical Measurements Reveal Reactive Oxygen Species in Stress Granules, K. Hu., E. Relton, N. Locker, N.T.N. Phan, and A.G. Ewing, Angewandte Chemie-International Edition, 133 (2021), 15430-15434,
  14. A multimodal electrochemical approach to measure the effect of zinc on vesicular content and exocytosis in a single cell model of ischemia. Y. Wang, C. Gu and A.G. Ewing, QRB Discovery, 2 (2021) e12, 1–8.
  15. Correlating Molecule Count and Release Kinetics with Vesicular Size using Open Carbon Nanopipettes, K. Hu, R. Jia, A. Hatamie, K.L. Le Vo, M.V. Mirkin, A.G. Ewing, J. Am. Chem. Soc., 142 (2020) 16910-16914.
  16. Intracellular Electrochemical Nanomeasurements Reveal that Exocytosis of Molecules at Living Neurons is Subquantal and Complex, A. Larsson, S. Majdi, A. Oleinick, I. Svir,  J. Dunevall, C. Amatore, A.G. Ewing, Angew. Chem. Int. Ed., 59 (2020) 6711-6714.
  17. Plasticity in Exocytosis Revealed Through the Effects of Repetitive Stimuli Affect the Content of Nanometer Vesicles and the Fraction of Transmitter Released, C. Gu, A. Larsson, A.G. Ewing, Proc Natl Acad Sci USA, 116 (2019) 21409-21415.

    Recent Reviews

  1. Measuring Synaptic Vesicles Using Cellular Electrochemistry and Nanoscale Molecular Imaging, N.T.N. Phan, X. Li and A.G. Ewing, Nature Reviews Chemistry, 1 (2017). Peer reviewed.
  2. Vesicle Impact Electrochemical Cytometry Compared to Amperometric Exocytosis Measurements, J. Dunevall, A. Larsson, S. Majdi, A. G. Ewing, Current Opinion in Electrochemistry, 5 (2017) 85-91. Peer reviewed.
  3. Electrochemistry in and of the Fly Brain, S. Majdi, A. Larsson, M. H. Philipsen, A. G. Ewing, Electroanalysis, 30(2018) 999-1010.
  4. Chemical Analysis of Single Cells, P.E. Oomen, M.A. Aref, I. Kaya, N.T.N. Phan, A.G. Ewing, Analytical Chemistry, 91 (2019) 588-621.
  5. Analytical Techniques: Shedding Light upon Nanometer-Sized Secretory Vesicles, E. Ranjbari, S. Majdi, and A.G. Ewing, Trends in Chemistry, 1 (2019) 440-451.
  6. Nano-Electrochemical Analysis Inside a Single Living Cell, X. Zhang, A. Hatamie, A.G. Ewing, Current Opinion in Electrochemistry, 22 (2020) 94-101,
  7. Chemical Analysis of Single Cells and Organelles, K. Hu, T.D.K. Nguyen, S. Rabasco, P.E. Oomen, A.G. Ewing, Analytical Chemistry, 93 (2021), 41-71.
  8. Electrochemistry at and in Single Cells (Chapter 7), A.S. Lima, C. Gu, K. Hu, A.G. Ewing, In Electrochemistry for Bioanalysis, Patel, B., Ed., Elsevier, (2020) pp. 125-160.
  9. Electrochemical Quantification of Neurotransmitters in Single Live Cell Vesicles Shows Exocytosis is Predominantly Partial, Y. Wang, A. Ewing, ChemBioChem, 22 (2021), 807-813.
  10.   Quantitative Chemical Imaging at the Cell Level – SIMS, Fluorescence, and Correlative Techniques, T.D.K. Nguyen, A.A. Lork, A.G. Ewing, N.T.N. Phan, in: Single Cell ‘Omics of Neuronal Cells, edited by J.V. Sweedler, J.H. Eberwine, S.E. Fraser, Neuromethods, Vol 184. Humana, New York, NY.