We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

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Change history

  • Correction 08 March 2018

    In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.


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Download references


We thank G. Paradis and M. Saturno-Condon for help with flow cytometry, F. Chen and L. Kang for help with confocal imaging, N. Ji for assistance with C. elegans imaging, and B. Trout and C. Sudrik for help with spectroscopic analysis of iRFPs. We are grateful to X. Han and K. Hansen (Boston University) for the pCAG-WPRE expression vector, and F. Subach (Moscow Institute of Physics and Technology) for the pWA23h plasmid. We are grateful to E. Costa, D. Estandian, A. Wassie and L. Cai for useful discussions. C.S. acknowledges the Lefler Center for the Study of Neurodegenerative Disorders for support. E.S.B. was supported by the HHMI-Simons Faculty Scholars Program, the IET Harvey Prize, the MIT Media Lab, the New York Stem Cell Foundation-Robertson Award, the Open Philanthropy Project, Human Frontier Science Program RGP0015/2016, and NIH grants 1R43MH109332, 1R24MH106075, 2R01DA029639, 1R01EY023173, 1R01NS087950, 1R01MH103910 and 1R01GM104948, and NIH Director’s Pioneer Award 1DP1NS087724. O.S. was supported by a Simons Fellowship. H.-J.S. was supported by a Samsung Fellowship. D.G. was supported by an NSF Fellowship. Y.-G.Y. was supported by a Samsung Fellowship. L.F. was supported by a Simons Fellowship.

Author information

Author notes

  1. These authors contributed equally: Kiryl D. Piatkevich and Erica E. Jung.


  1. Media Lab, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA

    • Kiryl D. Piatkevich
    • , Erica E. Jung
    • , Changyang Linghu
    • , Demian Park
    • , Ho-Jun Suk
    • , Daniel Goodwin
    • , Nikita Pak
    • , Or Shemesh
    • , Shoh Asano
    • , Young-Gyu Yoon
    • , Limor Freifeld
    •  & Edward S. Boyden
  2. Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    • Christoph Straub
    • , Daniel R. Hochbaum
    • , Jessica L. Saulnier
    •  & Bernardo L. Sabatini
  3. Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA, USA

    • Changyang Linghu
    •  & Young-Gyu Yoon
  4. Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, MA, USA

    • Ho-Jun Suk
  5. Simons Center Data Analysis, Simons Foundation, New York, NY, USA

    • Eftychios Pnevmatikakis
  6. Department of Mechanical Engineering, MIT, Cambridge, MA, USA

    • Nikita Pak
  7. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    • Takashi Kawashima
    • , Chao-Tsung Yang
    •  & Misha B. Ahrens
  8. Picower Institute for Learning & Memory and Department of Brain & Cognitive Sciences, MIT, Cambridge, MA, USA

    • Jeffrey L. Rhoades
    •  & Steven W. Flavell
  9. Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA, USA

    • Clemens Riegler
    •  & Florian Engert
  10. Department of Neurobiology, Faculty of Life Sciences, University of Vienna, Wien, Austria

    • Clemens Riegler
  11. Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, USA

    • Thom Hughes
    •  & Mikhail Drobizhev
  12. Department of Biological Physics, Eotvos University, Budapest, Hungary

    • Balint Szabo
  13. Department of Biological Engineering, MIT, Cambridge, MA, USA

    • Edward S. Boyden
  14. MIT Center for Neurobiological Engineering, MIT, Cambridge, MA, USA

    • Edward S. Boyden
  15. Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA

    • Edward S. Boyden
  16. MIT McGovern Institute for Brain Research, MIT, Cambridge, MA, USA

    • Edward S. Boyden


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K.D.P., E.E.J. and E.S.B. initiated the project, made high-level designs and plans, and interpreted the data. K.D.P., E.E.J., B.S. and O.S. developed the hierarchical multiparameter screening approach. K.D.P. and E.E.J. developed miRFP and together with C.L., M.D., T.H., H.J.S. and S.A. performed its characterization. K.D.P. and E.E.J. developed Archons and, together with D.P., characterized them in cultured cells. C.S., D.R.H., J.L.S. and B.L.S. performed electrophysiology experiments in acute brain slices. K.D.P, E.E.J., D.G., E.P. and C.L. analyzed neuronal culture data. N.P. and Y.G.Y. assisted on imaging setups. E.E.J. and K.D.P. with help from L.F. performed experiments on zebrafish injected by C.T.Y., T.K. and M.B.A. K.D.P., S.W.F. and J.L.R. performed experiments on C. elegans. C.R. and F.E. designed vectors for zebrafish expression. C.L. and E.E.J. performed statistical analysis. K.D.P., E.E.J., C.S., C.L. and E.S.B. wrote the paper with contributions from all of the authors. E.S.B. oversaw all aspects of the project.

Competing interests

B.S. is a founder of the CellSorter startup company. K.D.P., E.E.J. and E.S.B. are inventors on patent applications regarding the molecules described here. B.S., K.D.P., E.E.J. and E.S.B. are inventors on a patent application regarding the screening method developed here.

Corresponding author

Correspondence to Edward S. Boyden.

Electronic supplementary material

  1. Supplementary Tables and Figures

    Supplementary Tables 1–6, Supplementary Figures 1–29

  2. Life Sciences Reporting Summary