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All-optical neurophysiology
optical method originally introduced by Dennis Gabor( Nobel Prize in Physics, 1971). Holography works by modulating the wavefront of a laser beam so that the light field in the focal plane reproduces a user-defined spatial pattern. The modern implementation of this technique, known as Computer-Generated Holography( CGH) [ 2 ], relies on Spatial Light Modulators( SLMs) that can dynamically shape the phase of the incident beam. By computing holograms that correspond to specific positions, CGH allows the creation of multiple focal spots or customized illumination patterns within a 3D volume [ 3 ], each capable of stimulating individual neurons or neuronal ensembles.
When combined with 2P excitation, high-power pulsed lasers, and targeting strategies that restrict opsin expression preferentially to the neuronal soma, these approaches enable deep-tissue, parallel, and cell-specific optogenetic control, a powerful combination we refer to as circuit optogenetics [ 4 ].
Using CGH to create larger illumination areas covering the entire soma typically leads to a loss of axial resolution, which scales proportionally with the lateral spot size. To overcome this limitation, CGH can be combined with a technique known as temporal focusing( TF) [ 5 ], where ultrashort laser pulses are spectrally dispersed before being recombined at the focal plane. Because the pulse duration, and therefore the photon density, is restored only at the focal plane, excitation remains both temporally and spatially confined and allows the creation of extended excitation discs that uniformly illuminate the entire cell body while maintaining axial resolution comparable to that of a single diffraction-limited spot.
The combination of CGH and TF thus enables precise, parallel, and volumetric stimulation of multiple neurons in three dimensions, with subcellular spatial accuracy and millisecond temporal precision. After the demonstration of the first system for holographic 2P light patterning combined with TF, multiple variants have been developed to further enhance performance. These include approaches to reduce holographic speckle using generalized phase contrast, to extend the generation of temporally focused shapes in 3D, and to project light patterns at kilohertz rates, enabling sub-millisecond control of relative spike timing and increasing the number of targetable cells.
More recently, holographic light shaping has emerged as a key strategy for fast, multi-target 2P voltage imaging, allowing parallel, highspeed recording of voltage signals to detect neuronal activity with high contrast across multiple neurons [ 6 ]. This recent achievement underlines that holographic 2P illumination allows both 2P optogenetics and 2P voltage imaging rendering it an ideal illumination modality to set up all-optical neurophysiology approaches.
ALL-OPTICAL NEUROPHYSIOLOGY To date, most demonstrations of circuits optogenetics have been performed in head-restrained mice, where both imaging and photostimulation beams are delivered through a high – numerical-aperture objective via a cranial window. In these
Figure 2. Three dimensional 2P holography for for targeting multiple cells with soma-sized spots. Inset showing intensity profiles of TF vs. No-TF spots. preparations, animals can be either anesthetized or awake allowed to run on a spherical treadmill or a rotating wheel, while their neuronal activity is simultaneously monitored and manipulated.
Such experiments have successfully linked the activity of defined neuronal populations to sensory processing, motor control, and decision-making behaviors, providing direct evidence for the causal role of specific cells and circuits in brain function. These studies illustrate that precise spatiotemporal control of neuronal activity, enabled by techniques such as 2P holographic optogenetics, can reproduce physiologically relevant patterns of network activity, allowing researchers not only to observe but also to manipulate the flow of information through brain circuits.
However, many brain circuits, especially those involved in spatial navigation, social interaction, or goal-directed behavior, operate under naturalistic conditions that require freely moving animals. In recent years, 2P miniscopes have emerged as an extremely powerful approach for imaging calcium activity in freely moving mice or rats. In these devices, the scanning head used in conventional 2P microscopy is miniaturized to a size compatible with the animal’ s head, typically weighing less than 3 grams.
An alternative to miniaturized microscopes is to leave the complexity of the optical system on the optical table and use only a fiber and a focusing element, such as a GRIN lens or a mini-objective, as a relay between the output of the optical system and the animal’ s head via an adapter weighing less than 1 gram. To transmit holographic excitation patterns, the fibers used must belong to the class of fiber bundles composed of 15,000 to 30,000 individual multimode fibers allowing the holographic pattern to be faithfully transmitted to the output, which is then focused
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