Abstract
Functional imaging microscopy based on voltage-sensitive dyes (VSDs) has proven effective for revealing spatiotemporal patterns of activity in vivo and in vitro. Microscopy based on two-photon excitation (TPE) of fluorescent VSDs offers the possibility of three-dimensional recording of membrane potential changes on subcellular length scales hundreds of microns below the brain’s surface. Here we describe progress in monitoring membrane voltage using TPE of VSD fluorescence, and detail an application of this emerging technology in which action potentials (APs) were recorded in single trials from individual mammalian nerve terminals in situ. Prospects for, and limitations of this method are reviewed.
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Notes
- 1.
For a more detailed theoretical description of nonlinear optical phenomena including two-photon absorption, we refer the reader to Boyd (1992). Additionally, Masters and So (2008) provide a thorough theoretical and historical background for multiphoton excitation microscopy, and, further, provide an English translation of Maria Goeppert-Mayer’s original theoretical treatment (Goeppert-Mayer 1931).
- 2.
In the case where the transition to the nearest real excited state represents the absorption of visible-wavelength photons, virtual states lying roughly half-way can exist for a duration on the order of femtoseconds. Not surprisingly, then, mode-locked lasers that deliver intense pulses of photons confined to femtosecond duration bundles can efficiently populate these virtual states.
- 3.
- 4.
Here we are assuming that near-infrared wavelengths, used for TPE, are far from the wavelength at which the compound’s one-photon absorption is maximal.
- 5.
That is at a point where the absolute value of the derivative of the absorption spectrum, with respect to wavelength, is high.
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Supported by USPHS grants NS40966 and NS16824 (B.M.S.) and by a Bristol-Myers Squibb Postdoctoral Fellowship in Basic Neuro-sciences at The Rockefeller University (J.A.N.F.)
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Fisher, J.A.N., Salzberg, B.M. (2010). Monitoring Membrane Voltage Using Two-Photon Excitation of Fluorescent Voltage-Sensitive Dyes. In: Canepari, M., Zecevic, D. (eds) Membrane Potential Imaging in the Nervous System. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-6558-5_11
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