Excitation wavelength optimization improves photostability of ASAP-family GEVIs

Fang Xu,Pak-Ming Lau,Michael Z Lin,Dong-Qing Shi,Guo-Qiang Bi

doi:10.1186/s13041-018-0374-7

Fang Xu, Pak-Ming Lau + Show 3 more

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https://doi.org/10.1186/s13041-018-0374-7

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Recent interest in high-throughput recording of neuronal activity has motivated rapid improvements in genetically encoded calcium or voltage indicators (GECIs or GEVIs) for all-optical electrophysiology. Among these probes, the ASAPs, a series of voltage indicators based on a variant of circularly permuted green fluorescent protein (cpGFP) and a conjugated voltage sensitive domain (VSD), are capable of detecting both action potentials and subthreshold neuronal activities. Here we show that the ASAPs, when excited by blue light, undergo reversible photobleaching. We find that this fluorescence loss induced by excitation with 470-nm light can be substantially reversed by low-intensity 405-nm light. We demonstrate that 405-nm and 470-nm co-illumination significantly improved brightness and thereby signal-to-noise ratios during voltage imaging compared to 470-nm illumination alone. Illumination with a single wavelength of 440-nm light also produced similar improvements. We hypothesize that reversible photobleaching is related to cis-trans isomerization and protonation of the GFP chromophore of ASAP proteins. Amino acids that influence chromophore isomerization are potential targets of point mutations for future improvements.

Highlights

Understanding neural circuit function requires untangling contextual neural activity, ideally by simultaneous monitoring the activation of all individual neurons in a population
ASAPs retain the fluorescent properties of circularly permuted green fluorescent protein (cpGFP), which emits green fluorescence when excited by blue light
We found that ASAPs exhibited fluorescence loss with biphasic kinetics when exposed to blue excitation light centered at 470-nm (Fig. 1a), with the rapid phase lasting less than one second followed by a near-steady state with very slow photobleaching (Fig. 1b)

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Introduction

Understanding neural circuit function requires untangling contextual neural activity, ideally by simultaneous monitoring the activation of all individual neurons in a population. We found that ASAPs exhibited fluorescence loss with biphasic kinetics when exposed to blue excitation light centered at 470-nm (Fig. 1a), with the rapid phase lasting less than one second followed by a near-steady state with very slow photobleaching (Fig. 1b).

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