Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning

Proc Natl Acad Sci U S A. 2011 Dec 6;108(49):19504-9. doi: 10.1073/pnas.1109111108. Epub 2011 Nov 9.


Access to three-dimensional structures in the brain is fundamental to probe signal processing at multiple levels, from integration of synaptic inputs to network activity mapping. Here, we present an optical method for independent three-dimensional photoactivation and imaging by combination of digital holography with remote-focusing. We experimentally demonstrate compensation of spherical aberration for out-of-focus imaging in a range of at least 300 μm, as well as scanless imaging along oblique planes. We apply this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging. By bringing extended portions of tilted dendrites simultaneously in-focus, we monitor the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na(+) channels.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Animals
  • CA1 Region, Hippocampal / cytology
  • CA1 Region, Hippocampal / metabolism
  • Calcium / metabolism
  • Dendrites / metabolism
  • Dendrites / physiology*
  • Glutamic Acid / metabolism
  • Hippocampus / anatomy & histology
  • Hippocampus / cytology
  • Hippocampus / metabolism
  • Holography / instrumentation
  • Holography / methods*
  • Imaging, Three-Dimensional / instrumentation
  • Imaging, Three-Dimensional / methods*
  • Ion Channel Gating / drug effects
  • Light*
  • Mice
  • Mice, Inbred Strains
  • Microscopy, Fluorescence
  • Patch-Clamp Techniques
  • Pyramidal Cells / metabolism
  • Pyramidal Cells / physiology
  • Sodium Channel Blockers / pharmacology
  • Sodium Channels / metabolism
  • Tetrodotoxin / pharmacology


  • Sodium Channel Blockers
  • Sodium Channels
  • Glutamic Acid
  • Tetrodotoxin
  • Calcium