A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals

Neuron. 2001 Sep 27;31(6):903-12. doi: 10.1016/s0896-6273(01)00421-4.


Two-photon microscopy has enabled anatomical and functional fluorescence imaging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a miniaturized head-mounted microscope. Excitation light is conducted to the microscope in a single-mode optical fiber, and images are scanned using vibrations of the fiber tip. Microscope performance was first characterized in the neocortex of anesthetized rats. We readily obtained images of vasculature filled with fluorescently labeled blood and of layer 2/3 pyramidal neurons filled with a calcium indicator. Capillary blood flow and dendritic calcium transients were measured with high time resolution using line scans. In awake, freely moving rats, stable imaging was possible except during sudden head movements.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anesthesia
  • Animals
  • Artifacts
  • Brain / blood supply
  • Brain / ultrastructure*
  • Calcium Signaling
  • Cerebrovascular Circulation
  • Dendrites / ultrastructure
  • Dextrans / pharmacokinetics
  • Equipment Design
  • Fiber Optic Technology / instrumentation
  • Fluorescein-5-isothiocyanate / analogs & derivatives*
  • Fluorescein-5-isothiocyanate / pharmacokinetics
  • Fluorescent Dyes / pharmacokinetics
  • Head Movements
  • Image Processing, Computer-Assisted
  • Lasers
  • Microcirculation
  • Microscopy, Fluorescence / instrumentation*
  • Microscopy, Fluorescence / methods
  • Miniaturization
  • Movement
  • Optical Fibers
  • Organic Chemicals
  • Pyramidal Cells / chemistry
  • Pyramidal Cells / ultrastructure
  • Rats
  • Rats, Sprague-Dawley
  • Wakefulness


  • Dextrans
  • Fluorescent Dyes
  • Organic Chemicals
  • fluorescein isothiocyanate dextran
  • calcium green
  • Fluorescein-5-isothiocyanate