Two-Color Volumetric Imaging of Neuronal Activity of Cortical Columns

Cell Rep. 2019 May 14;27(7):2229-2240.e4. doi: 10.1016/j.celrep.2019.04.075.


To capture the emergent properties of neural circuits, high-speed volumetric imaging of neural activity at cellular resolution is needed. Here, we introduce wavelength multiplexing to perform fast volumetric two-photon imaging of cortical columns (>2,000 neurons in 10 planes at 10 vol/s), using two different calcium indicators, an electrically tunable lens and a spatial light modulator. We image the activity of neuronal populations from layers 2/3 to 5 of primary visual cortex from awake mice, finding a lack of columnar structures in orientation responses and revealing correlations between layers which differ from trial to trial. We also simultaneously image functional correlations between presynaptic layer 1 axons and postsynaptic layer 2/3 neurons. Wavelength multiplexing enhances high-speed volumetric microscopy and can be combined with other optical multiplexing methods to easily boost imaging throughput.

Keywords: beam multiplexing; ensembles; spatial light modulator; two-photon calcium imaging; volumetric imaging; wavefront shaping; wavelength multiplexing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Axons / physiology
  • Calcium / metabolism
  • Female
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Neural Conduction / physiology*
  • Neurons / cytology
  • Neurons / physiology*
  • Synapses / physiology
  • Synaptic Transmission / physiology*
  • Visual Cortex / cytology
  • Visual Cortex / physiology*


  • Calcium