Imaging neuromodulators with high spatiotemporal resolution using genetically encoded indicators


Multiple aspects of neural activity, from neuronal firing to neuromodulator release and signaling, underlie brain function and ultimately shape animal behavior. The recently developed and constantly growing toolbox of genetically encoded sensors for neural activity, including calcium, voltage, neurotransmitter and neuromodulator sensors, allows precise measurement of these signaling events with high spatial and temporal resolution. Here, we describe the engineering, characterization and application of our recently developed dLight1, a suite of genetically encoded dopamine (DA) sensors based on human inert DA receptors. dLight1 offers high molecular specificity, requisite affinity and kinetics and great sensitivity for measuring DA release in vivo. The detailed workflow described in this protocol can be used to systematically characterize and validate dLight1 in increasingly intact biological systems, from cultured cells to acute brain slices to behaving mice. For tool developers, we focus on characterizing five distinct properties of dLight1: dynamic range, affinity, molecular specificity, kinetics and interaction with endogenous signaling; for end users, we provide comprehensive step-by-step instructions for how to leverage fiber photometry and two-photon imaging to measure dLight1 transients in vivo. The instructions provided in this protocol are designed to help laboratory personnel with a broad range of experience (at the graduate or post-graduate level) to develop and utilize novel neuromodulator sensors in vivo, by using dLight1 as a benchmark.

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Fig. 1: General strategy for GPCR-based sensor development.
Fig. 2: Outline of the workflow for sensor characterization and validation.
Fig. 3: Experimental setup for in vivo dopamine imaging with fiber photometry.
Fig. 4: Representative traces of dopamine dynamics during reward-based learning.
Fig. 5: In vivo validation of dLight1 with optogenetic control of dopamine release.
Fig. 6: In vivo two-photon imaging of dopamine dynamics in behaving mice.

Data availability

All DNA plasmids and viruses mentioned in this protocol can be obtained from either the Tian laboratory at UC Davis or Addgene under a materials transfer agreement. All data present in this article are available from the authors upon request. Implemented and curated computer codes have been deposited in github (


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This work was supported by NIH BRAIN Initiative grants U01NS090604, U01NS013522, DP2MH107056 and U01NS103571 (L.T.); grants DP2NS083038, R01NS085938 and P30CA014195 (A.N.); BRAIN Initiative grants U01NS013522 (J.T.W. and M.v.Z.), and NIH grant DP2NS087949 and NIH/NIA grant R01AG047664 (V.G.). K.M. is a DFG research fellow and recipient of a Catharina Foundation postdoctoral scholar award. V.G. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute.

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T.P. and L.T. wrote the manuscript with contributions from J.R.C. and V.G. (fiber photometry and optogenetics), G.J.B. (rAAV preparation and cloning), R.L. (structural modeling), A.M. and M.v.Z. (TIRF microscopy, FACS and cAMP measurements), K.M. and A.N. (in vivo two-photon imaging in behaving mice), and J.W. (ex vivo two-photon imaging).

Corresponding authors

Correspondence to John Williams or Axel Nimmerjahn or Mark von Zastrow or Viviana Gradinaru or Lin Tian.

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Competing interests

T.P. and L.T. are co-inventors on a patent application (WO/2018/098262A1) for the technology described in this paper. L.T. is the co-founder of Seven Biosciences.

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Peer review information Nature Protocols thanks Thomas Knopfel and other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

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Patriarchi, T. et al. Science 360, eaat4422 (2018)

Integrated supplementary information

Supplementary Fig. 1 Design of GPCR-based sensors using the universal cpGFP module.

Shown is a sequence alignment of the contiguous regions of transmembrane helixes 5 and 6 (TM5, TM6) used to develop fluorescent sensors from 10 different GPCRs. The third intracellular loop of the receptors is deleted in the sensors (region shown in orange shading) and replaced with the universal cpGFP module consisting of the indicated residues surrounding cpGFP (indicated in the green box). The complete sequence for the cpGFP module is available from Addgene or in our original publication16). Adapted with permission from16, American Association for the Advancement of Science.

Supplementary Fig. 2 Comparison of locomotion-related dopamine transients using dLight1.1 or dLight1.2 in behaving mice with two-photon microscopy.

a, Schematics of two-photon imaging of head-fixed mouse during treadmill locomotion. b, Mean ΔF/F for all significant positive-going transients in mice expressing either dLight1.1 (n = 2 mice, 131 transients) or dLight1.2 (n = 2 mice, 31 transients). c-d, Mean transient ΔF/F during rest and run for all fields in dLight1.1 (n = 2 mice, 5 fields) and dLight1.2 (n = 2 mice, 8 fields). Adapted with permission from16, American Association for the Advancement of Science.

Supplementary Fig. 3 Comparison between dLight1.3b and GRAB-DA1m in neurons.

a, Representative images of primary cultured neurons (DIV15) expressing either dLight1.3b or GRAB-DA1m. Cells were transduced by directly applying ~1010 viral particles either AAV9.Synapsin.dLight1.3b or AAV9.Synapsin.GRAB-DA1m onto the medium 14 d prior to imaging (AAVs produced by the UC Davis Viral Vector Core Facility). Cells were imaged under identical conditions (laser intensity, pinhole size, etc.) with a 40x oil-based objective on a Zeiss 710 confocal microscope. Intensity profile plots representative of the lines drawn across the neuronal dendrites are show as insets. Scale bars, 20 µm. b, Basal fluorescence and signal to noise calculations were performed on Fiji (Image J) from regions of interest manually drawn on the neuronal membrane. At least n=3 neurons from 2 separate experiments were included in the analysis. Data are shown as individual ROI values ± SEM. ****p<0.0001, n.s.= not significant, unpaired student’s t-test. c, Time-lapse images were acquired at ~1 second intervals. Drugs (dopamine, DA; haloperidol, Halo; SCH23390, SCH) were directly applied on the cells at the time points and concentrations indicated in the graphs. n≥2 neurons for each sensor. Data are shown as mean ± SEM.

Supplementary Fig. 4 Equipment setup for photometry recordings.

a, Photograph of optical components in the fiber photometry setup described in 51 and 18. b, Photograph of data processor with inputs from photodetector and external TTL and with outputs for LED modulation. Optical components in (a) are located inside the rackbox below. c, Photograph of a mouse expressing dLight1.1 during freely moving behavior inside an operant box. This water-deprived mouse is obtaining sucrose water reward from lickometer. All procedures were performed in accordance with the guidelines of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) and by the Office of Laboratory Animal Resources at the California Institute of Technology.

Supplementary Fig. 5 Example raw photometry traces.

a, Raw photometry signals demodulated from 490-nm (blue) and 405-nm (violet) channel. Notice photobleaching in both channels. b, Photometry signals after 405-nm signal is fitted to 490-nm signal by applying a least-squares fit. c, ΔF/F trace is calculated for each recording session as. (490-nm signal – fitted 405-nm signal)/fitted 405-nm signal. Notice this can effectively remove the contribution of photobleaching and potential contamination from motion artifacts. All procedures were performed in accordance with the guidelines of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) and by the Office of Laboratory Animal Resources at the California Institute of Technology.

Supplementary Fig. 6 Equipment setup of perfusion system for titrations on cells.

a, The perfusion system setup for performing neuromodulator titrations during one-photon imaging of sensor fluorescence is based on an inverted confocal microscope (7). A display (1) connected with a digital controller (2) for the eight-valve perfusion system (3), an eight-channel perfusion inlet (4) and a single channel outlet (5) connected to a peristaltic pump (6) for removal of excess buffer from the dish. b, Close up view of the perfusion inlet and outlet setup on the stage adaptor containing the imaged dish.

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Patriarchi, T., Cho, J.R., Merten, K. et al. Imaging neuromodulators with high spatiotemporal resolution using genetically encoded indicators. Nat Protoc 14, 3471–3505 (2019).

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