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J Neurophysiol (August 10, 2005). doi:10.1152/jn.00416.2005
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00416.2005v1
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Submitted on April 25, 2005
Accepted on August 4, 2005

Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy

Daniel A. Dombeck, Leonardo Sacconi, Mireille Blanchard-Desce, and Watt W. Webb*

* To whom correspondence should be addressed. E-mail: www2{at}cornell.edu.

Though non-linear microscopy and fast (~1 ms) membrane potential (Vm) recording have proven valuable for neuroscience applications, their potentially powerful combination has not yet been demonstrated for investigations of Vm activity deep in intact tissue. We show that laser illumination of neurons in acute rat brain slices intracellularly filled with FM4-64 dye generates an intense second-harmonic generation (SHG) signal from somatic and dendritic plasma membranes with high contrast >125 µm below the slice surface. The SHG signal provides a linear response to {Delta}Vm of ~7.5%/100 mV. By averaging repeated line-scans (~50), we show the ability to record action potentials (APs) optically with a signal-to-noise ratio (S/N) of ~7-8. We also show recording of fast Vm steps from the dendritic arbor at depths inaccessible with previous methods. The high membrane contrast and linear response of SHG to {Delta}Vm provides the advantage that signal changes are not degraded by background and can be directly quantified in terms of {Delta}Vm. Experimental comparison of SHG and two-photon fluorescence (TPF) Vm recording with the best known probes for each showed that the SHG technique is superior for Vm recording in brain slice applications, with FM4-64 as the best tested SHG Vm probe.




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