Photonics Research, Volume. 10, Issue 6, 06001367(2022)

High-axial-resolution optical stimulation of neurons in vivo via two-photon optogenetics with speckle-free beaded-ring patterns

Cheng Jin1,†, Chi Liu1,†, and Lingjie Kong1,2,*
Author Affiliations
  • 1State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instrument, Tsinghua University, Beijing 100084, China
  • 2IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing 100084, China
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    Two-photon optogenetics has become an indispensable technology in neuroscience, due to its capability in precise and specific manipulation of neural activities. A scanless holographic approach is generally adopted to meet the requirement of stimulating neural ensembles simultaneously. However, the commonly used disk patterns fail in achieving single-neuron resolution, especially in axial dimension, and their inherent speckles decrease stimulation efficiency. Here, we propose a novel speckle-free, beaded-ring pattern for high-axial-resolution optical stimulation of neurons in vivo. Using a dye pool and a fluorescent thin film as samples, we verify that, compared to those with disk patterns, higher axial resolution and better localization ability can be achieved with beaded-ring patterns. Furthermore, we perform two-photon based all-optical physiology with neurons in mouse S1 cortex in vivo, and demonstrate that the axial resolution obtained by beaded-ring patterns can be improved by 24% when stimulating multiple neurons, compared to that of disk patterns.


    In recent decades, optogenetics has played an increasingly important role in probing functional codes of neurons [13]. However, in conventional one-photon stimulation based optogenetics, all neurons in the illumination volume are excited or inhibited, which makes it complex to decipher the neural network. To regulate opsins at single-neuron resolutions specifically, two-photon optogenetics has been demonstrated [4,5].


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    Cheng Jin, Chi Liu, Lingjie Kong. High-axial-resolution optical stimulation of neurons in vivo via two-photon optogenetics with speckle-free beaded-ring patterns[J]. Photonics Research, 2022, 10(6): 06001367

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    Paper Information

    Category: Medical Optics and Biotechnology

    Received: Jan. 10, 2022

    Accepted: Apr. 10, 2022

    Published Online: May. 12, 2022

    The Author Email: Lingjie Kong (



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