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.

jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths

Nature Methods volume 17, pages 694–697 (2020)Cite this article

Subjects

Abstract

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Fig. 1: jYCaMP1 is a redshifted variant of jGCaMP7 capable of detecting single action potentials.
Fig. 2: jYCaMP1 enables brighter two-color calcium imaging together with red GECIs, improving correlation analysis of overlapping compartments.

Data availability

Example raw datasets and summary statistics are available through Figshare; https://doi.org/10.25378/janelia.12098361. All other data associated with this study are available from the corresponding author.

Code availability

Custom code is available from the corresponding author, and at https://github.com/KasparP/twoColorUnmixing and github.com/KasparP/SLAP.

References

  1. Grienberger, C. & Konnerth, A. Imaging calcium in neurons. Neuron 73, 862–885 (2012).

    Article  CAS  Google Scholar 

  2. Voigt, F. F. et al. Multiphoton in vivo imaging with a femtosecond semiconductor disk laser. Biomed. Opt. Express 8, 3213 (2017).

    Article  CAS  Google Scholar 

  3. Kazemipour, A. et al. Kilohertz frame-rate two-photon tomography. Nat. Methods 16, 778–786 (2019).

    Article  CAS  Google Scholar 

  4. Wu, J. et al. Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo. Nat. Methods 17, 287–290 (2020).

    Article  CAS  Google Scholar 

  5. Perillo, E. P. et al. Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser. Biomed. Opt. Express 7, 324 (2016).

    Article  Google Scholar 

  6. Kim, D. U. et al. Two-photon microscopy using an Yb(3+)-doped fiber laser with variable pulse widths. Opt. Express 20, 12341 (2012).

    Article  CAS  Google Scholar 

  7. Tilma, B. W. et al. Recent advances in ultrafast semiconductor disk lasers. Light Sci. Appl. 4, e310–e310 (2015).

    Article  CAS  Google Scholar 

  8. Dana, H. et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat. Methods 16, 649–657 (2019).

    Article  CAS  Google Scholar 

  9. Ormö, M. et al. Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence. Science 273, 1392–1395 (1996).

    Article  Google Scholar 

  10. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    Article  CAS  Google Scholar 

  11. Wang, Q., Shui, B., Kotlikoff, M. I. & Sondermann, H. Structural basis for calcium sensing by GCaMP2. Structure 16, 1817–1827 (2008).

    Article  CAS  Google Scholar 

  12. Behnia, R., Clark, D. A., Carter, A. G., Clandinin, T. R. & Desplan, C. Processing properties of ON and OFF pathways for Drosophila motion detection. Nature 5212, 427–430 (2014).

    Article  Google Scholar 

  13. Strother, J. A. et al. The emergence of directional selectivity in the visual motion pathway of Drosophila. Neuron 94, 168–182.e10 (2017).

    Article  CAS  Google Scholar 

  14. Strother, J. A., Nern, A. & Reiser, M. B. Direct observation of ON and OFF pathways in the Drosophila visual system. Curr. Biol. 24, 976–983 (2014).

    Article  CAS  Google Scholar 

  15. Valm, A. M. et al. Applying systems-level spectral imaging and analysis to reveal the organelle interactome. Nature 546, 162–167 (2017).

    Article  CAS  Google Scholar 

  16. Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. Elife 5, e12727 (2016).

    Article  Google Scholar 

  17. Broussard, G. J. et al. In vivo measurement of afferent activity with axon-specific calcium imaging. Nat. Neurosci. 21, 1272–1280 (2018).

    Article  CAS  Google Scholar 

  18. Inoue, M. et al. Rational engineering of XCaMPs, a multicolor GECI Suite for in vivo imaging of complex brain circuit dynamics. Cell 177, 1346–1360.e24 (2019).

    Article  CAS  Google Scholar 

  19. Sun, Y. et al. Neural signatures of dynamic stimulus selection in Drosophila. Nat. Neurosci. 20, 1104–1113 (2017).

    Article  CAS  Google Scholar 

  20. Podgorski, K. & Ranganathan, G. Brain heating induced by near-infrared lasers during multiphoton microscopy. J. Neurophysiol. 116, 1012–1023 (2016).

    Article  CAS  Google Scholar 

  21. Studier, F. W. Stable expression clones and auto-induction for protein production in E. coli. Methods Mol. Biol. 1091, 17–32 (2014).

    Article  CAS  Google Scholar 

  22. Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    Article  CAS  Google Scholar 

  23. Akerboom, J. et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2 (2013).

    Article  CAS  Google Scholar 

  24. Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

    Article  CAS  Google Scholar 

  25. Mütze, J. et al. Excitation spectra and brightness optimization of two-photon excited probes. Biophys. J. 102, 934–944 (2012).

    Article  Google Scholar 

  26. Wardill, T. J. et al. A neuron-based screening platform for optimizing genetically-encoded calcium indicators. PLoS ONE 8, e77728 (2013).

    Article  CAS  Google Scholar 

  27. Chen, J. L., Voigt, F. F., Javadzadeh, M., Krueppel, R. & Helmchen, F. Long-range population dynamics of anatomically defined neocortical networks. Elife 24, e14679 (2016).

    Article  Google Scholar 

Download references

Acknowledgements

We thank H. Davies, C. Morkunas and M. Jeffries for logistical support and S. Di Lisio, K. Ritola, D. Walpita, J. Hasseman and the GENIE Project Team for experimental support. We thank S. Vishwanathan for the gift of Emx1-Cre mice. This work was funded by the Howard Hughes Medical Institute. M.A.M. was supported by the Janelia Graduate Research Fellowship Program. E.J.M., C.-Y.L. and A.A. were supported by the Janelia Undergraduate Scholars Program. Work by J.L.C. and A.M.A. was supported by the National Science Foundation Neuronex Neurotechnology Hub (NEMONIC no. 1707287) and National Institutes of Health BRAIN Initiative Award (no. UF1NS107705).

Author information

Author notes

  1. Manuel Alexander Mohr

    Present address: Department of Biology, Stanford University, Stanford, CA, USA

Authors and Affiliations

  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Manuel Alexander Mohr, Daniel Bushey, Abhi Aggarwal, Jonathan S. Marvin, Jeong Jun Kim, Emiliano Jimenez Marquez, Yajie Liang, Ronak Patel, John J. Macklin, Chi-Yu Lee, Arthur Tsang, Getahun Tsegaye, Douglas S. Kim, Allan M. Wong, Loren L. Looger, Eric R. Schreiter & Kaspar Podgorski

  2. Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland

    Manuel Alexander Mohr

  3. Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

    Abhi Aggarwal

  4. Universidad Nacional Autonoma de Mexico, Mexico, Mexico

    Emiliano Jimenez Marquez

  5. GENIE Project, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Yajie Liang, Arthur Tsang, Getahun Tsegaye, Douglas S. Kim, Allan M. Wong, Loren L. Looger & Eric R. Schreiter

  6. Department of Biology, Boston University, Boston, MA, USA

    Allison M. Ahrens & Jerry L. Chen

Authors
  1. Manuel Alexander Mohr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Bushey
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abhi Aggarwal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jonathan S. Marvin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeong Jun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Emiliano Jimenez Marquez
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yajie Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ronak Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. John J. Macklin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Chi-Yu Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Arthur Tsang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Getahun Tsegaye
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Allison M. Ahrens
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Jerry L. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Douglas S. Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Allan M. Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Loren L. Looger
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Eric R. Schreiter
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Kaspar Podgorski
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.P. conceived and K.P., E.R.S., J.S.M. and M.A.M. refined the idea. J.S.M. and M.A.M. produced Venus-GCaMPs. E.J.M. performed spectral screening with the help of K.P. C.-Y.L. M.A.M. and A.A. characterized proteins in vitro and M.A.M. and D.S.K. in cultured neurons. A.A. performed individual mutation depletion. Y.L. performed mouse virus injections. K.P., J.J.K. and M.A.M. performed mouse in vivo imaging. A.M.W. and M.A.M. generated fly lines and D.B. designed, performed and analyzed fly experiments. A.M.A. and J.L.C. designed and performed multi-area imaging experiments. R.P. and J.J.M. performed multiphoton spectroscopy and analyzed data. M.A.M., K.P., A.A., J.J.K., D.S.K. and E.R.S. analyzed data. M.A.M., K.P. and E.R.S. prepared the manuscript with input from all authors. K.P., E.R.S. and L.L.L. supervised the work.

Corresponding author

Correspondence to Kaspar Podgorski.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Supplementary information

Supplementary Information

Supplementary Figs. 1–9 and Table 1.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mohr, M.A., Bushey, D., Aggarwal, A. et al. jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths. Nat Methods 17, 694–697 (2020). https://doi.org/10.1038/s41592-020-0835-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-020-0835-7

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