Two-photon targeted patching (TPTP) in vivo

Abstract

Two-photon–excited fluorescence laser-scanning microscopy (2PLSM) has provided a wealth of information about the spatiotemporal properties of biological processes at the single cell and population level. Because such nonlinear optical methods allow for imaging deep within biological tissue, 2PLSM can be combined with patch-clamp techniques to obtain electrophysiological recordings from specific fluorescently labeled cells in vivo. Here a protocol referred to as two-photon targeted patching (TPTP) describes a method that may be used to record from cells in the intact animal labeled by virtually any type of fluorophore. We target neurons that have been optically and genetically identified using green fluorescent protein (GFP) expressed under the control of a specific promoter. TPTP when combined with genetic approaches therefore permits electrophysiological recordings from specified neurons and their compartments, including dendrites. This technique may be repeated in the same preparation many times over the course of several hours and is equally applicable to non-neuronal cell types.

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Figure 1: Setup for TPTP.
Figure 2: Monitoring pipette resistance while navigating through upper layers of tissue.
Figure 3: Detecting pipette-cell interactions using visual and electrophysiological cues.
Figure 4: Verification of TPTP.

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Acknowledgements

The authors wish to thank P. Chadderton for his assistance in generating the figures and reading the manuscript and D. Farquarson for providing the design layout of the headplate. T.W.M. is supported by The Welcome Trust and The Human Frontiers Science Program (P.O. and T.W.M.).

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Correspondence to Troy W Margrie.

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Komai, S., Denk, W., Osten, P. et al. Two-photon targeted patching (TPTP) in vivo. Nat Protoc 1, 647–652 (2006). https://doi.org/10.1038/nprot.2006.100

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