All-optical neurophysiology FOCUS onto the brain through the GRIN lens or mini-objective.
Following an initial demonstration of a holographic fiberscope in 2014, which used 1P holographic excitation combined with multi-confocal calcium imaging, a new class of endoscopes, called two-photon 2P-FENDO [ 7 ], has been recently developed using 2P excitation. An important property of the fibers used in 2P-FENDO is the inter-core differential delay, designed to reduce inter-core coupling. In the context of 2P excitation, this has the consequence that light injected into multiple cores simultaneously at the fiber entrance emerges at the output with temporal delays on the order of a few picoseconds per meter. As a result, the light produced at the focal plane of the GRIN lens or mini-objective does not form a single, uniform illumination spot. Instead, it appears as a random distribution of small bright points, each corresponding to an individual fiber core. This scattering effectively produces an illumination area equivalent to what would be obtained using a single illuminated core. Because the distribution of these points is sufficiently sparse, the axial resolution remains that of a single core, regardless of the number of cores illuminated providing intrinsic axial confinement without the need for temporal focusing.
Making advantage of this properties 2P-FENDO enable fast calcium imaging and single-cell optogenetic stimulation, enabling all optical experiments in freely moving animals at unprecedented spatiotemporal precision.
CONCLUSION The convergence of advances in genetically encoded indicators, optogenetic actuators, and holographic light-shaping approaches is making it possible to map, manipulate, and decode neuronal circuits across scales, with cellular resolution and millisecond temporal precision, thereby revealing causal links between activity patterns and behavior and giving rise to the field of all-optical neurophysiology. Recent progress in the development of genetically encoded voltage indicators and illumination methods compatible with two-photon voltage imaging has further enriched the field, enabling the readout of neuronal activity with single-spike resolution. Finally, as developments in fiber-based two-photon systems extend these capabilities to freely moving animals, optical neurophysiology is entering a new era in which complex, naturalistic behaviors can be studied with unprecedented resolution and specificity. Ultimately, these approaches are paving the way toward a mechanistic understanding of how distributed neuronal networks give rise to perception, cognition, and action.
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