Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution

J Neurosci Methods. 2008 Jul 30;172(2):178-84. doi: 10.1016/j.jneumeth.2008.04.024. Epub 2008 May 3.


We describe a simple two-photon fluorescence imaging strategy, called targeted path scanning (TPS), to monitor the dynamics of spatially extended neuronal networks with high spatiotemporal resolution. Our strategy combines the advantages of mirror-based scanning, minimized dead time, ease of implementation, and compatibility with high-resolution low-magnification objectives. To demonstrate the performance of TPS, we monitor the calcium dynamics distributed across an entire juvenile rat hippocampus (>1.5mm), at scan rates of 100 Hz, with single cell resolution and single action potential sensitivity. Our strategy for fast, efficient two-photon microscopy over spatially extended regions provides a particularly attractive solution for monitoring neuronal population activity in thick tissue, without sacrificing the signal-to-noise ratio or high spatial resolution associated with standard two-photon microscopy. Finally, we provide the code to make our technique generally available.

Publication types

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

MeSH terms

  • Animals
  • Calcium / analysis
  • Calcium / metabolism
  • Calcium Signaling / physiology
  • Fluorescent Dyes
  • Hippocampus / cytology*
  • Hippocampus / physiology
  • Image Cytometry / instrumentation
  • Image Cytometry / methods*
  • Imaging, Three-Dimensional / instrumentation
  • Imaging, Three-Dimensional / methods
  • Membrane Potentials / physiology
  • Microscopy, Confocal / instrumentation
  • Microscopy, Confocal / methods
  • Microscopy, Fluorescence, Multiphoton / instrumentation
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Nerve Net / cytology*
  • Nerve Net / physiology
  • Neural Pathways / cytology
  • Neural Pathways / physiology
  • Neurons / cytology*
  • Neurons / physiology
  • Optics and Photonics / instrumentation
  • Organ Culture Techniques
  • Photons
  • Rats
  • Rats, Long-Evans
  • Software
  • Time Factors


  • Fluorescent Dyes
  • Calcium