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 http://neurospy.org/ under an open source license.
Howard Hughes Medical Institute, Computational Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, United States of America. dejan@salk.edu
Dejan
D
Vucinić
Terrence J
TJ
Sejnowski
D000818:Animals; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000378:metabolism; D007090:Image Interpretation, Computer-Assisted / Q000295:instrumentation / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D007834:Lasers; D036641:Microscopy, Fluorescence, Multiphoton / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D009474:Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D020318:Rats, Long-Evans; D013997:Time Factors; D015018:Ytterbium / Q000737:chemistry
D005456:Fluorescent Dyes; D015018:Ytterbium