All-optical neurophysiology FOCUS spatial and temporal precision of optical techniques.
READING NEURONAL ACTIVITY WITH LIGHT The challenge of reading neuronal activity with light was initially addressed through the development of calcium indicators, and more recently, genetically-encoded calcium indicators( GECIs). GECIs exploit the fact that neuronal activity is accompanied by transient increases in intracellular calcium, which changes its fluorescence in response to calcium binding providing an indirect yet robust optical readout of neuronal activity. As genetic tools GECIs provide both cell-type specificity and compatibility with chronic imaging, making them powerful tools for monitoring population dynamics over extended periods.
The use of wide-field single photon illumination allows calcium activity to be imaged across large brain regions and, combined with confocal detection schemes, enables cellular resolution in superficial layers. However, achieving population imaging with single-cell resolution deep within scattering tissue requires the use of two-photon( 2P) microscopy, a technique based on the principle of Ω absorption first predicted by Maria Goeppert Mayer, who was later awarded the Nobel Prize in Physics for her pioneering contributions to quantum mechanics.
While calcium indicators have revolutionized our ability to image neuronal activity, their relatively slow kinetics limit temporal precision, since calcium transients integrate electrical events over hundreds of milliseconds. To overcome this limitation, more recently genetically-encoded voltage indicators( GEVIs) have been developed to directly report membrane potential changes rather than relying on secondary calcium signals. Depending on the molecular design of the GEVI, electrical changes can be read out as variations in fluorescence intensity, emission wavelength, or fluorescence lifetime. When combined with fast optical modalities, like wide field imaging, two-photon random-access scanning, light-sheet, or holographic imaging, GEVIs provide a direct way to monitor the electrical dynamics of neuronal populations at both singlecell and network levels in real time.
Together, calcium and voltage indicators form the foundation of optical neurophysiology, offering a powerful, genetically targetable, and minimally invasive way to observe how neurons communicate and process information across the brain.
CONTROLLING NEURONAL ACTIVITY WITH LIGHT The ability to manipulate neuronal activity is provided by optogenetics, an emerging field that began with the discovery of genes encoding microbial light-sensitive ion transporters like Channelrhodopsin and Halorhodopsin. Expressed in neuronal cells these opsins directly convert absorbed photons into ionic currents, allowing activation or inhibition of neuronal firing with high spatiotemporal precision. Optogenetics has transformed neuroscience, offering an unprecedented, non-invasive way to perturb and control brain function paving the way to dissect neural circuitry. The majority of todays’ optogenetic experiments have used relatively simple illumination methods with visible light used to illuminate large brain regions while genetically restricting expression to certain cell types. Due to that, wide-field illumination can only synchronously activate entire populations of neurons providing rather unphysiological perturbations, while dissecting neural circuitry and contributions of individual neurons requires the development of optical methods capable of illuminating individual or several cells independently.
2P scanning microscopy approaches largely adopted for population calcium imaging cannot
Figure 1. Microbial rhodopsins can be lightactivated ion transporters that render ionic fluxes controllable with light. Genetically targeted to neuronal cells they allow control of neuronal activity with the spatiotemporal precision of the respective illuminaiton.
be directly translated to single-cell optogenetic stimulation for two main reasons. First, the diffraction-limited focal spot employed in conventional scanning microscopy is too small to illuminate the entire soma and efficiently recruit a sufficient number of opsin molecules to reliably trigger an action potential or maintain inhibition. Second, the temporal constraints imposed by sequential scanning hinder a true-parallel control of multiple target cells simultaneously.
SCANLESS HOLOGRAPHIC ILLUMINATION Some limitations of point scanning methods can be overcome through the use of holography, an
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