We will present a new method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. Our method exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method typically reduces photobleaching effects 4-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins.
We describe a 3D holographic two-photon optogenetics setup based on acousto-optic deflectors combined with a custom-made compact light-sheet microscope setup. The system allows performing 3D-targeted perturbations of neuronal activity while simultaneously imaging the whole brain neuronal response over large field-of-view. This random-access holographic light patterning method offers high-speed sequential activation of dense neuronal assemblies spread over large areas while maintaining constant lateral and axial resolution. Here, we report on a systematic characterization of the stimulated neurons response and perform functional imaging on zebrafish (Danio Rerio) larvae while stimulating single neurons.
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