Overcoming photodamage in second-harmonic generation microscopy: Real-time optical recording of neuronal action potentials
Open Access
- 17 February 2006
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 103 (9), 3124-3129
- https://doi.org/10.1073/pnas.0511338103
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.Keywords
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