Holographic two-photon activation for synthetic optogenetics

Nature Protocols
Ido CarmiShai Berlin

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 illumin...Continue Reading

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Citations

Mar 12, 2020·Angewandte Chemie·Silvia Castro-FernándezAraceli G Campaña
Jun 18, 2020·Frontiers in Cellular Neuroscience·Wessal Hussein, Shai Berlin
Jul 28, 2021·Methods : a Companion to Methods in Enzymology·Christopher K LaffertyJonathan P Britt

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Methods Mentioned

BETA
genetic modification
chip
transgenic
transfection
fluorescence microscopy
PCR
electrophoresis
fluorescence imaging

Software Mentioned

Genescript
PyMOL
ChemDraw
DH
pClamp
MATLAB

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