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Nat Protoc. 2019 Mar;14(3):864-900. doi: 10.1038/s41596-018-0118-2. Epub 2019 Feb 25.

Holographic two-photon activation for synthetic optogenetics.

Author information

1
Department of Neuroscience, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
2
Department of Imaging Physics, Delft University of Technology, Delft, the Netherlands.
3
Department of Chemistry, University of Maine, Orono, ME, USA.
4
Department of Neuroscience, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. shai.berlin@technion.ac.il.

Abstract

Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammalian origin by use of photoswitches, visible light (typically), and genetic modification. Thus, synthetic optogenetics facilitates interrogation of native neuronal signaling mechanisms. However, the poor tissue penetration of visible wavelengths impedes the use of the technique in tissue, as two-photon excitation (2PE) is typically required to access the near-infrared window. Here, we describe an alternative technique that uses 2PE-compatible photoswitches (section 1) for photoactivation of genetically modified glutamate receptors (section 2). Furthermore, for fast, multi-region photoactivation, we describe the use of 2P-digital holography (2P-DH) (section 3). We detail how to combine 2P-DH and synthetic optogenetics with electrophysiology, or with red fluorescence Ca2+ recordings, for all-optical neural interrogation. The time required to complete the methods, aside from obtaining the necessary reagents and illumination equipment, is ~3 weeks.

PMID:
30804570
DOI:
10.1038/s41596-018-0118-2
[Indexed for MEDLINE]

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