Aberration-free holographic microscope for simultaneous imaging and stimulation of neuronal populations

Opt Express. 2023 Sep 25;31(20):33461-33474. doi: 10.1364/OE.498051.


A technical challenge in neuroscience is to record and specifically manipulate the activity of neurons in living animals. This can be achieved in some preparations with two-photon calcium imaging and photostimulation. These methods can be extended to three dimensions by holographic light sculpting with spatial light modulators (SLMs). At the same time, performing simultaneous holographic imaging and photostimulation is still cumbersome, requiring two light paths with separate SLMs. Here we present an integrated optical design using a single SLM for simultaneous imaging and photostimulation. Furthermore, we applied axially dependent adaptive optics to make the system aberration-free, and developed software for calibrations and closed-loop neuroscience experiments. Finally, we demonstrate the performance of the system with simultaneous calcium imaging and optogenetics in mouse primary auditory cortex in vivo. Our integrated holographic system could facilitate the systematic investigation of neural circuit function in awake behaving animals.

MeSH terms

  • Animals
  • Calcium*
  • Holography* / methods
  • Mice
  • Neurons / physiology
  • Photons
  • Software


  • Calcium