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High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device

Abstract

Optogenetic approaches allow the manipulation of neuronal activity patterns in space and time by light, particularly in small animals such as zebrafish. However, most techniques cannot control neuronal activity independently at different locations. Here we describe equipment and provide a protocol for single-photon patterned optical stimulation of neurons using a digital micromirror device (DMD). This method can create arbitrary spatiotemporal light patterns with spatial and temporal resolutions in the micrometer and submillisecond range, respectively. Different options to integrate a DMD into a multiphoton microscope are presented and compared. We also describe an ex vivo preparation of the adult zebrafish head that greatly facilitates optogenetic and other experiments. After assembly, the initial alignment takes about one day and the zebrafish preparation takes <30 min. The method has previously been used to activate channelrhodopsin-2 and manipulate oscillatory synchrony among spatially distributed neurons in the zebrafish olfactory bulb. It can be adapted easily to a wide range of other species, optogenetic probes and scientific applications.

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Figure 1: Fiber-optic stimulation.
Figure 2: Schematic overview of the setup, not drawn to scale.
Figure 3: Beam path configurations.
Figure 4: Beam path between DMD and scan lens.
Figure 5: Ex vivo preparation of the adult zebrafish head.
Figure 6: Visualization of light patterns through the microscope objective.
Figure 7: Light patterns at different focal depths.
Figure 8: Power transmission of the DMD system.
Figure 9: Membrane potential responses of Chr2-expressing neurons evoked by optical stimulation.

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Acknowledgements

This work was supported by the Novartis Research Foundation, the Swiss National Fonds (SNF), the Deutsche Forschungsgemeinschaft (DFG), the Human Frontier Science Program (HFSP), the Whitaker Foundation and Marie Curie Actions. We are grateful to K. Deisseroth for Channelrhodopsin-2 constructs.

Author information

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Authors

Contributions

P.Z. generated transgenic fish, participated in the construction of the DMD device, performed optogenetic experiments in the adult brain, analyzed data and wrote the manuscript. O.F. performed optogenetic experiments in larval zebrafish, composed scripts in Python and wrote the manuscript. J.S. constructed the DMD device, performed optogenetic experiments in adult fish and analyzed data. Y.-P.Z.S. participated in the construction of the DMD device, performed optogenetic experiments in adult fish and analyzed data. R.W.F. conceived the study, designed equipment, participated in the construction of the DMD device and wrote the manuscript.

Corresponding author

Correspondence to Rainer W Friedrich.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Data

Matlab interface for DMD Control. This supplementary data file contains Matlab code, instructions, and test files to control the digital micromirror device via a graphical user interface. The following files are included: (1) Readme_DMDCtrl_Universal.pdf. This file contains important information about the graphical user interface and instructions on how to use it. It includes descriptions of all files in this package. It is recommended to read this file before using the graphical user interface. (2) DMDCtrl_Universal.fig. This Matlab file defines the graphical user interface for control of the digital micromirror device. (3) DMDCtrl_Universal.m. This file contains the matlab code for the graphical user interface. (4) DMDCtrl_Universal.pptx. This file contains screenshots of the graphical user interface to illustrate basic operations. (5) Test_Pattern.mat. This is a Matlab file defining spatio-temporal patterns to test the graphical user interface. (6) Test_Pattern2.mat. This Matlab file contains a second set of spatio-temporal patterns to test the graphical user interface. Both test patterns are described in the file Readme_DMDCtrl_Universal.pdf. (ZIP 752 kb)

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Zhu, P., Fajardo, O., Shum, J. et al. High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device. Nat Protoc 7, 1410–1425 (2012). https://doi.org/10.1038/nprot.2012.072

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