Overcoming photodamage in second-harmonic generation microscopy: Real-time optical recording of neuronal action potentials

doi:10.1073/pnas.0511338103

Overcoming photodamage in second-harmonic generation microscopy: Real-time optical recording of neuronal action potentials

*School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853; and
^†European Laboratory for Nonlinear Spectroscopy, University of Florence, 50019 Sesto Fiorentino, Italy

Contributed by W. W. Webb, December 31, 2005

Abstract

Second-harmonic generation (SHG) has proven essential for the highest-resolution optical recording of membrane potential (Vm) in intact specimens. Here, we demonstrate single-trial SHG recordings of neuronal somatic action potentials and quantitative recordings of their decay with averaging at multiple sites during propagation along branched neurites at distances up to 350 μm from the soma. We realized these advances by quantifying, analyzing, and thereby minimizing the dynamics of photodamage (PD), a frequent limiting factor in the optical imaging of biological preparations. The optical signal and the PD during SHG imaging of stained cultured Aplysia neurons were examined with intracellular electrode recordings monitoring the resting Vm variations induced by laser-scanning illumination. We found that the PD increased linearly with the dye concentration but grew with the cube of illumination intensity, leading to unanticipated optimization procedures to minimize PD. The addition of appropriate antioxidants in conjunction with an observed Vm recovery after termination of laser scanning further refined the imaging criteria for minimization and control of PD during SHG recording of action potentials. With these advances, the potential of SHG as an effective optical tool for neuroscience investigations is being realized.

Footnotes

^‡To whom correspondence should be addressed. E-mail: www2{at}cornell.edu
Author contributions: L.S. and D.A.D. designed research; L.S. performed research; L.S. analyzed data; and L.S., D.A.D., and W.W.W. wrote the paper.
Conflict of interest statement: No conflicts declared.
↵ § Yuste, R., Nemet, B., Jiang, J., Nuriya, M. & Eisenthal, K., Imaging Neurons and Neural Activity: New Methods Meeting, March 10–13, 2005, Cold Spring Harbor, NY, abstr. 16.
Abbreviations:

Abbreviations:

PD,

photodamage;

SHG,

second-harmonic generation;

Vm,

membrane potential;

AP,

action potential;

TPF,

two-photon fluorescence;

S/N,

signal-to-noise;

dc,

duty cycle.
Freely available online through the PNAS open access option.