Abstract Mapping neuronal activity during the onset and propagation of epileptic seizures can provide a better understanding mechanisms underlying this pathology improve our approaches to development new drugs. Recently, zebrafish has become an important model for studying epilepsy both in basic research drug discovery. Here, we employed transgenic line with pan-neuronal expression genetically-encoded calcium indicator GCaMP6s measure larvae induced by pentylenetretrazole (PTZ). With...

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation neuronal circuit dynamics. However, low efficiency 2P absorption process, speed this technique is typically limited by signal-to-noise-ratio. Here, we...

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a choice method to observe brain function high frame rates at cellular resolution. Inherently LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data...

The development of light-sheet fluorescence microscopy (LSFM) has greatly expanded the experimental capabilities in many biological and biomedical research fields, enabling for example live studies murine zebrafish neural activity or cell growth division. key feature method is selective illumination a sample single plane, providing an intrinsic optical sectioning allowing direct 2D image recording. On other hand, this excitation scheme more affected by absorption scattering artifacts...

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line of complementary metal–oxide–semiconductor camera (CMOS) working rolling shutter mode allows the rejection out-of-focus and scattered light, thus reducing background signal during formation. Most modern CMOS have two shutters, but usually only single illuminating beam is used, halving maximum obtainable frame rate....

Epilepsy accounts for a significant proportion of the world's disease burden. Indeed, many research efforts are produced both to investigate basic mechanism ruling its genesis and find more effective therapies. In this framework, use zebrafish larvae, owing their peculiar features, offers great opportunity. Here, we employ transgenic larvae expressing GCaMP6s in all neurons characterize functional alterations occurring during seizures induced by pentylenetetrazole. Using custom two-photon...

Abstract Two‐photon (2P) excitation is a cornerstone approach widely employed in neuroscience microscopy for deep optical access and sub‐micrometric‐resolution light targeting into the brain. However, besides structural functional imaging, 2P optogenetic stimulations are less routinary, especially 3D. This because of adopted scanning systems, often feebly effective, slow mechanically constricted. Faster illumination can be achieved through acousto‐optic deflectors (AODs) although their...

Light-sheet microscopy (LSM) is a powerful imaging technique that uses planar illumination oriented orthogonally to the detection axis. Two-photon (2P) LSM variant of exploits 2P absorption effect for sample excitation. The light polarization state plays significant, and often overlooked, role in processes. scope this work test whether using different states excitation can affect detected signal levels typical biological samples with spatially unordered dye population. Supported by...

Abstract An inverse procedure is developed and tested to recover functional structural information from global signals of brains activity. The method assumes a leaky-integrate fire model with excitatory inhibitory neurons, coupled via directed network. Neurons are endowed heterogenous current value, which sets their associated dynamical regime. By making use mean-field approximation, the seeks reconstructing activity patterns distribution in-coming degrees, for both as well assigned...

Although it is well known that zebrafish display the behavioural signature of sleep, neuronal correlates this state are not yet completely understood, due to complexity measurements required. For example, when performed with visible excitation light, functional imaging can disrupt day/night cycle induced visual stimulation. To address issue, we developed a custom-made two-photon light-sheet microscope optimized for high-speed volumetric imaging. By employing infra-red light (not larva)...

We present the development of a custom-made two-photon light-sheet microscope optimized for high-speed (5 Hz) volumetric imaging zebrafish larval brain analysis neuronal physiological and pathological activity. High-speed microscopy is challenging to achieve, due constrains on signal-to-noise ratio. To maximize this parameter, we our setup high peak power excitation light, while finely controlling its polarization, implemented remote scanning focal plane record without disturbing sample....

Abstract Objective . Ultrasounds (US) use in neural engineering is so far mainly limited to ablation through high intensity focused ultrasound, but interesting preliminary results show that low frequency ultrasound could be used instead modulate activity. However, the extent of this modulatory ability US still unclear, as vivo studies it hard disentangle contribution responses direct activation neuron by stimulation and indirect due either sensory response mechanical associated US, or...

Bessel beam have attractive properties like their propagation invariance and "self-healing" capabilities. Here we present significant advantages of illumination in structural functional imaging light-sheet microscopy compared to conventional Gaussian illumination.

The ability to simultaneously detect and control neuronal activity grants the capacity unravel causal relationships behind brain processing. Nevertheless, scaling-up this technology whole encephalon of a vertebrate is challenging task, impedes reaching global comprehension how works. To aim, we developed an optical system that enables fast noninvasive functional imaging zebrafish larva and, at same time, allows us optogenetically stimulate arbitrary sets neurons in volume. Our preliminary...

One of the most audacious goals modern neuroscience is unraveling complex web causal relations underlying activity neuronal populations on a whole-brain scale. This endeavor, prohibitive just couple decades ago, has recently become within reach owing to advancements in optical methods and advent genetically encoded indicators/actuators. These techniques, applied translucent larval zebrafish have enabled recording manipulation extensive spanning entire vertebrate brain. Here, we present...

We present a multi-photon system comprising light-sheet microscope for fast whole-brain imaging and an acousto-optic deflector-based light-targeting unit 3D optogenetic stimulation. employed the setup to map habenular functional connectivity in zebrafish larvae.

One of the most audacious goals modern neuroscience is unraveling complex web causal relations underlying activity neuronal populations on a whole-brain scale. This endeavor, which was prohibitive only couple decades ago, has recently become within reach owing to advancements in optical methods and advent genetically encoded indicators/actuators. These techniques, applied translucent larval zebrafish have enabled recording manipulation extensive spanning entire vertebrate brain. Here, we...

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience and is particularly well suited to image the entire brain with high frame rates at single cell resolution. On one hand, LSM employed combination tissue clearing methods like CLARITY which allows for reconstruction of neuronal or vascular anatomy over cm-sized samples. other been paired intrinsically transparent samples real-time recording activity resolution across brain, using calcium indicators GCaMP6. Despite its...

One of the most exciting challenges neurosciences in last few years is real-time recording neuronal activity with single cell resolution across entire brain. Thanks to use optical methods, together animal models which whole encephalon optically accessible, this goal getting within reach. In work, we a transgenic zebrafish line expressing genetically encoded calcium indicator GCaMP6s binding ions leads an increase emitted fluorescence reporter. sensitive reporter family and allows us record...

Most living organisms show highly conserved physiological changes following a 24-hour cycle which goes by the name of circadian rhythm. Among experimental models, effects light-dark have been recently investigated in larval zebrafish. Owing to its small size and transparency, this vertebrate enables optical access entire brain. Indeed, combination organism with light-sheet imaging grants high spatio-temporal resolution volumetric recording neuronal activity. This technique, multiphoton...

Abstract Light-sheet microscopy (LSM) has proven a useful tool in neuroscience to image whole brains with high frame rates at cellular resolution. LSM is employed either combination tissue clearing reconstruct the cyto-architecture over entire mouse brain or intrinsically transparent samples like zebrafish larvae for functional imaging. Inherently LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during imaging, modulate fluorescence...

Light-sheet microscopy excels in fast whole-organ acquisitions either clarified mouse brains or intrinsically transparent zebrafish larva. Here, we present our technical and optical solutions for microscope automation including autofocusing, Bessel beams prospective gating.