Skip to main content

Thank you for visiting 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.

  • Brief Communication
  • Published:

Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR

An Author Correction to this article was published on 28 February 2019

A Publisher Correction to this article was published on 02 January 2019

This article has been updated


Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

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

Access options

Buy this article

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

Fig. 1: SF-iGluSnFR.A184S detects orientation-selective glutamate transients in ferret visual cortex.
Fig. 2: SF-iGluSnFR.S72A permits resolution of multiple glutamate-release events in cultured neurons and in single cerebellar granule cell boutons.
Fig. 3: Utility of SF-Venus-iGluSnFR in cultured neurons and in vivo.

Similar content being viewed by others

Data availability

All data from this study are available upon request. All constructs have been deposited at Addgene (#106174–106206; hSynapsin1, FLEX-hSynapsin1, FLEX-CAG, GFAP promoters; some fusions with the red fluorescent protein mRuby3 are available). Sequences have been deposited in GenBank (MH392460, MH392461, MH392462, MH392463, MH392464, and MH392465). AAV is available from Addgene.

Change history

  • 28 February 2019

    The version of this paper originally published cited a preprint version of ref. 12 instead of the published version (Proc. Natl. Acad. Sci. USA 115, 5594–5599; 2018), which was available before this Nature Methods paper went to press. The reference information has been updated in the PDF and HTML versions of the article.

  • 02 January 2019

    In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors’ original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.


  1. Marvin, J. S. et al. Nat. Methods 10, 162–170 (2013).

    Article  CAS  Google Scholar 

  2. Park, S. J. H., Kim, I.-J., Looger, L. L., Demb, J. B. & Borghuis, B. G. J. Neurosci. 34, 3976–3981 (2014).

    Article  Google Scholar 

  3. Brunert, D., Tsuno, Y., Rothermel, M., Shipley, M. T. & Wachowiak, M. J. Neurosci. 36, 6820–6835 (2016).

    Article  CAS  Google Scholar 

  4. O’Herron, P. et al. Nature 534, 378–382 (2016).

    Article  Google Scholar 

  5. Xie, Y. et al. J. Neurosci. 36, 1261–1272 (2016).

    Article  Google Scholar 

  6. Bao, H. et al. Nat. Struct. Mol. Biol. 23, 67–73 (2016).

    Article  CAS  Google Scholar 

  7. Rosa, J. M. et al. eLife 4, 728 (2015).

    Article  Google Scholar 

  8. Enger, R. et al. Cereb. Cortex 25, 4469–4476 (2015).

    Article  Google Scholar 

  9. Jiang, R., Diaz-Castro, B., Looger, L. L. & Khakh, B. S. J. Neurosci. 36, 3453–3470 (2016).

    Article  CAS  Google Scholar 

  10. Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Nat. Biotechnol. 24, 79–88 (2006).

    Article  Google Scholar 

  11. Marvin, J. S. & Hellinga, H. W. Nat. Struct. Biol. 8, 795–798 (2001).

    Article  CAS  Google Scholar 

  12. Helassa, N. et al. Proc. Natl Acad. Sci. USA 115, 5594–5599 (2018).

    Article  CAS  Google Scholar 

  13. Dodge, F. A. Jr. & Rahamimoff, R. J. Physiol. (Lond.) 193, 419–432 (1967).

    Article  CAS  Google Scholar 

  14. Abrahamsson, T., Cathala, L., Matsui, K., Shigemoto, R. & Digregorio, D. A. Neuron 73, 1159–1172 (2012).

    Article  CAS  Google Scholar 

  15. Valera, A. M., Doussau, F., Poulain, B., Barbour, B. & Isope, P. J. Neurosci. 32, 3267–3280 (2012).

    Article  CAS  Google Scholar 

  16. van Beugen, B. J., Gao, Z., Boele, H.-J., Hoebeek, F. & De Zeeuw, C. I. Front. Neural Circuits 7, 95 (2013).

    PubMed  PubMed Central  Google Scholar 

  17. Tang, S., Liu, J., Krasieva, T. B., Chen, Z. & Tromberg, B. J. J. Biomed. Opt. 14, 030508 (2009).

    Article  Google Scholar 

  18. Kazemipour, A. et al. bioRxiv Preprint at (2018).

  19. Borghuis, B. G. et al. J. Neurosci. 31, 2855–2867 (2011).

    Article  CAS  Google Scholar 

  20. Pologruto, T. A., Sabatini, B. L. & Svoboda, K. Biomed. Eng. Online 2, 13 (2003).

    Article  Google Scholar 

  21. Wilson, D. E., Whitney, D. E., Scholl, B. & Fitzpatrick, D. Nat. Neurosci. 19, 1003–1009 (2016).

    Article  CAS  Google Scholar 

  22. Peirce, J. W. J. Neurosci. Methods 162, 8–13 (2007).

    Article  Google Scholar 

  23. Schindelin, J. et al. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

  24. Sage, D., Prodanov, D., Tinevez, J.-Y. & Schindelin, J. MIJ: making interoperability between ImageJ and Matlab possible. Poster presented at the ImageJ User Developer Conference, Luxembourg, 24–26 October 2012.

  25. Woitecki, A. M. H. et al. J. Neurosci. 36, 2561–2570 (2016).

    Article  CAS  Google Scholar 

  26. Silver, R. A., Cull-Candy, S. G. & Takahashi, T. J. Physiol. (Lond.) 494, 231–250 (1996).

    Article  CAS  Google Scholar 

Download references


We thank J. Macklin (Janelia Research Campus) for two-photon spectra, and K. Ritola and Janelia Virus Services for AAV production. E.C. was a Janelia Undergraduate Scholar.

Author information

Authors and Affiliations



J.S.M., L.L.L., A.N.T., and E.C. carried out protein engineering; B.S., D.E.W., and D.F. conducted experiments on ferret visual cortex; J.A.M., S.S., and D.D. carried out neuronal culture analysis; J.P.L. assessed brightness in somatosensory cortex; K.P. and A.K. carried out high-speed imaging of the yellow variant; H.B. and E.R.C. assessed stopped-flow kinetics; N.R., F.J.U.Q., S.S.-H.W., A.W.H., and D.A.D. conducted cerebellum experiments; V.J.D. and B.G.B. conducted retina experiments; and J.S.M. and L.L.L. wrote the manuscript.

Corresponding author

Correspondence to Loren L. Looger.

Ethics declarations

Competing interests

J.S.M. and L.L.L. are named on US patent 9719992, which pertains to original iGluSnFR.

Additional information

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

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14

Reporting Summary

Supplementary Video 1

Example of two-photon imaging of dendritic SFiGluSnFR.A184S activity during visual stimulation. Video is shown at 4× normal speed.

Supplementary Video 2

High-speed two-photon imaging of SF-Venus-iGluSnFR.A184V using 1,030-nm fiber laser excitation. Neurons in culture were imaged by scanned line angular projection microscopy at a 1,016-Hz frame rate. Glutamate was uncaged at two locations, 10 ms apart, starting at t = 400 ms. Saturation denotes ΔF/F0. Blue-tinted regions denote areas where excitation was blocked during high-speed imaging. Recording is a single trial, without averaging. Representative example of 4 trials.

Supplementary Video 3

Example two-photon recording of dendrites in mouse visual cortex labeled with SF-Venus-iGluSnFR.A184S, at a frame rate of 3.41 Hz. Drifting grating visual stimuli of 8 different directions, lasting 2.3 s each, were presented at 5-s intervals. Field-of-view width: 90 μm.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Marvin, J.S., Scholl, B., Wilson, D.E. et al. Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR. Nat Methods 15, 936–939 (2018).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research