Dr. Alison G. Tebo Profile

Dr. Alison G. Tebo

at Howard Hughes Medical Institute

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Author

Publications (2)

SPIE Journal Paper | 4 April 2024 Open Access

Blue-shifted genetically encoded Ca²⁺ indicator with enhanced two-photon absorption

Abhi Aggarwal, Smrithi Sunil, Imane Bendifallah, Michael Moon, Mikhail Drobizhev, Landon Zarowny, Jihong Zheng, Sheng-Yi Wu, Alexander Lohman, Alison Tebo, Valentina Emiliani, Kaspar Podgorski, Yi Shen, Robert Campbell

NPh, Vol. 11, Issue 02, 024207, (April 2024) https://doi.org/10.1117/12.10.1117/1.NPh.11.2.024207

KEYWORDS: Calcium, Fluorescence, Proteins, Action potentials, Signal to noise ratio, Photometry, Neurophotonics, Neurons, In vivo imaging, Chromophores

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SignificanceGenetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.AimWe describe the development and applications of T-GECO1—a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1.ApproachWe use protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices.ResultsThe Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M−1 cm−1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold, and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled the detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.ConclusionsT-GECO1 is a high-performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

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Proceedings Article | 13 March 2024 Presentation

Near-infrared co-illumination of fluorescent proteins reduces photobleaching and phototoxicity

Lucie Ludvikova, Emma Simon, Mathieu Deygas, Thomas Panier, Marie-Aude Plamont, Jean Ollion, Alison Tebo, Matthieu Piel, Ludovic Jullien, Lydia Robert, Thomas Le Saux, Agathe Espagne

Proceedings Volume PC12862, PC128620K (2024) https://doi.org/10.1117/12.3008062

KEYWORDS: Photobleaching, Fluorescent proteins, Light sources and illumination, Fluorophores, Visible radiation, Oxygen, In vivo imaging, Fluorescence imaging, Bacteria

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