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A compact multiphoton 3D imaging system for recording fast neuronal activity.

We constructed a simple and compact imaging system designed specifically for the recording of fast neuronal activity in a 3D volume. The system uses an Yb:KYW femtosecond laser we designed for use with acousto-optic deflection. An integrated two-axis acousto-optic deflector, driven by digitally synthesized signals, can target locations in three dimensions. Data acquisition and the control of scanning are performed by a LeCroy digital oscilloscope. The total cost of construction was one order of magnitude lower than that of a typical Ti:sapphire system. The entire imaging apparatus, including the laser, fits comfortably onto a small rig for electrophysiology. Despite the low cost and simplicity, the convergence of several new technologies allowed us to achieve the following capabilities: i) full-frame acquisition at video rates suitable for patch clamping; ii) random access in under ten microseconds with dwelling ability in the nominal focal plane; iii) three-dimensional random access with the ability to perform fast volume sweeps at kilohertz rates; and iv) fluorescence lifetime imaging. We demonstrate the ability to record action potentials with high temporal resolution using intracellularly loaded potentiometric dye di-2-ANEPEQ. Our design proffers easy integration with electrophysiology and promises a more widespread adoption of functional two-photon imaging as a tool for the study of neuronal activity. The software and firmware we developed is available for download at under an open source license.

Pubmed ID: 17684546


  • Vucinić D
  • Sejnowski TJ


PloS one

Publication Data

August 8, 2007

Associated Grants

  • Agency: Howard Hughes Medical Institute, Id:

Mesh Terms

  • Animals
  • Electrophysiology
  • Equipment Design
  • Fluorescent Dyes
  • Image Interpretation, Computer-Assisted
  • Imaging, Three-Dimensional
  • Lasers
  • Microscopy, Fluorescence, Multiphoton
  • Neurons
  • Patch-Clamp Techniques
  • Rats
  • Rats, Long-Evans
  • Time Factors
  • Ytterbium