Second-harmonic generation imaging of membrane potential with photon counting.
Second-harmonic generation (SHG) can be used for imaging membrane potential in neurons, but poor signal-to-noise (S/N) limits accurate measurements of small voltage transients. We use photon counting to improve the S/N of weak SHG signal detection. Photon counting generates shot-noise limited and integrable signals, eliminates pulse-to-pulse variation, and built-in discriminators reduces the background to practically zero. In single trials, by using photon counting, we obtain a more than a twofold S/N increase over analog voltage detection. Trial-to-trial variability is also reduced by 50%. Finally, we show that, using photon counting, the kinetics of fast events such as action potentials can be recorded more accurately.
Jiang
Jiang
J
HHMI, Department of Biological Sciences, Columbia University, New York, NY 10027, USA. jjiang@ibn.a-star.edu.sg
Yuste
Rafael
R
D000818:Animals; D002478:Cells, Cultured; D004594:Electrophysiology / Q000379:methods*; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D017785:Photons; D014793:Visual Cortex / Q000166:cytology / Q000502:physiology