The guiding or focusing of light, crucial in many applications, often relies on bulky optical elements that are difficult to place in delicate mediums like biological tissue. In current systems, the optical components are located outside the sample, limiting possibilities due to geometric constraints. Scattering further complicates the control of light within non-homogeneous media, restricting operating beyond 1 mm of depth. A promising solution involves shaped ultrasound to induce refractive index gradients within the tissue, acting as embedded lenses or waveguides. However, existing methods rely on bulky ultrasonic transducers, introducing invasiveness and fixing the ultrasound geometry. The proposed approach uses photoacoustic generation of pressure waves within the medium, allowing light guiding without geometrical constraints. As it is successfully demonstrated in tissue phantoms with different scattering coefficients, this method offers a promising solution in conditions not feasable with traditional external optical elements.
Real-time imaging of rapid events or fast-evolving processes at the microscale is paramount in applications ranging from neuroscience to industrial optical inspection. However, current microscopy architectures can be limited in speed by the camera frame rate or the need for mechanical scanning. Here, we propose a scanless method that exploits the intrinsic high speed of single-pixel detectors to achieve two-dimensional diffraction-limited imaging at rates as high as 5 kHz. Based on interfering orthogonal light stripes from two acousto-optic deflectors to generate a grid of flickering points - each with its unique modulation frequency -, our approach offers high spatiotemporal resolution compatible with a wide spectral range.
The ability to simultaneously detect and control neuronal activity grants the capacity to unravel the causal relationships behind brain processing. Nevertheless, scaling-up this technology to the whole encephalon of a vertebrate is a challenging task, and this impedes reaching a global comprehension of how the brain works. To this aim, we developed an optical system that enables fast noninvasive functional imaging of the whole brain of the zebrafish larva and, at the same time, allows us to optogenetically stimulate the activity of arbitrary sets of neurons in the volume. Our preliminary results show that with this optical system we can both consistently evoke activation of neurons in the stimulation site and identify distantly-located functionally-connected neurons placed downstream in the activated circuits. The expansibility of this concept paves the way for the brain-wide mapping of functional connectivity in the zebrafish larva.
To understand the brain computation paradigms and the causal interactions in complex neuronal networks, we need methods and technologies to record and perturb neuronal distributions over large fields of view. In this application, two-photon (2P) imaging has become a cornerstone microscopy technique, widely used for deep optical access in biological samples and selective light targeting with submicrometric resolution. In parallel to structural and functional imaging, 2P optogenetics has represented a game-changer, allowing targeted stimulation of specific neural circuits. However, the long commutation times and refresh rates of traditional scanning methods substantially hinder near-simultaneous multi-site 3D stimulation. Acousto-optic deflectors (AODs), owing to their fastest scanning and refresh rates, can fulfil the temporal requirements for concurrent activation of sparsely distributed neurons. Nevertheless, their applicability to 2P optogenetics in large volumes has been limited so far by the massive efficiency drop along the optical axis during their use in axial scanning. To counteract this drawback, a compensation software module is frequently employed to flatten the power distribution throughout the volume. However, the power threshold is reduced to the minimum intensity value addressable, lowering the peak intensity released in the centre of the axial scan.
Here, we propose a unique approach for overcoming this drawback which provided lifted axial power distribution while maintaining a uniform lateral illumination range. We tested this method by the 2P photoactivation of optogenetic actuators in 3D in zebrafish larvae, showing how the probability of evoking an electrophysiological response and the relative neuronal activity amplitude improved by carefully optimizing the light targeting time on different axial planes.
In conclusion, fast and uniform axial light addressing with AODs enables unprecedented 3D 2P optostimulation, formerly not feasible. Furthermore, this approach can be adopted as an upgrade for existing microscopes designed for volumetric imaging, providing 3D multi-site imaging and random-access illumination.
We present the development of a custom-made two-photon light-sheet microscope optimized for high-speed (5 Hz) volumetric imaging of zebrafish larval brain for the analysis of neuronal physiological and pathological activity. High-speed volumetric two-photon light-sheet microscopy is challenging to achieve, due to constrains on the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarization, and we implemented remote scanning of the focal plane to record without disturbing the sample. Two-photon illumination is advantageous for zebrafish larva studies since infra-red excitation does not induce a visual response, that otherwise would affect the neuronal activity. In particular, we were able to record whole-brain neuronal activity of the larva with high temporal- and spatial-resolution during the nocturnal period without affecting the circadian rhythm. Analyzing the spatially resolved power spectra of GCaMP signal, we found significant differences for several frequency bands between the day/night phases in various brain regions. Moreover, we studied the fast dynamics that characterize the acutely induced pathological epileptic activity of the larvae, identifying the brain structures that are more susceptible to the action of the epileptogenic drug. In conclusion, the high speed two-photon light-sheet microscope that we developed is proving to be an important tool to study both the physiological and the pathological activity of the zebrafish larval brain without undesired visual stimulation.
In the past few years, Light Sheet Fluorescence Microscopy (LSFM) has become a cornerstone imaging technique for neuroscience, improving the quality and capabilities of 3D imaging. Selec-tive illumination of a single plane provides intrinsic optical sectioning and fast image recording, while minimizing out-of-focus fluorescence background, sample photo-damage and photobleach-ing. However, images acquired with LSFM are often affected by light absorption or scattering effects, leading to un-even illumination and striping artifacts. Here we present an optical solution for such problem, via fast multi-directional illumination of the sample, based on an Acousto-Optical Deflector (AOD). Using this device, we were able to pivot the beam respect the propagation axis, with a scanning rate faster than the detector acquisition rate, in order to average the shadows attenuation at different angles over time. We also demonstrate that this scanning AOD system is compatible with Digital Scanned laser Light-sheet fluorescence microscopy (DSLM). Furthermore, we provided a theoretical model of the beam pivoting, looking for the optimal beam parameters to optimize the detection efficiency. It has been done because we wanted to adapt such scanning technique to Confocal Light Sheet Microscopy (CLSM), which intrinsically shows an incompatibility between the beam pivoting and the finite size of a digital slit used to spatially filter out spurious signals. We partially solved the problem expanding the beam and using two cylindrical lenses to create an elliptical-Gaussian beam which enables to cover more the digital slit while pivoting the beam. We test its performance by acquiring several mouse brain areas, observing real-time shadows suppression while preserving confocal detection of the signal emitted by specific fluorophores. A comparison between such scanning beam illumination and a traditional static one has been carried out in term of contrast analysis, striping suppression and Point Spread Function response.
Although it is well known that zebrafish display the behavioural signature of sleep, the neuronal correlates of this state are not yet completely understood, due to the complexity of the measurements required. For example, when performed with visible excitation light, functional imaging can disrupt the day/night cycle due to the induced visual stimulation. To address this issue, we developed a custom-made two-photon light-sheet microscope optimized for high-speed volumetric imaging. By employing infra-red light (not visible to the larva) for excitation, we are able to record wholebrain neuronal activity with high temporal- and spatial-resolution without affecting the sleep state. In two-photon light-sheet microscopy the maximum achievable frame rate is limited by the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarisation, and we implemented remote scanning of the focal plane to record without disturbing the sample. Using this setup, as a preliminary result, we characterized the intensity spectra of neuronal calcium traces of 4 days post fertilisation larvae during the day/night phases. We aim to extend these results to multiple brain regions and frequency bands.
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 detection of a complementary metal–oxide–semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acousto-optic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast.
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 the one hand, LSM is employed in combination with tissue clearing methods like CLARITY which allows for the reconstruction of neuronal or vascular anatomy over cm-sized samples. On the other hand, LSM has been paired with intrinsically transparent samples for real-time recording of neuronal activity with single cell resolution across the entire brain, using calcium indicators like GCaMP6.
Despite its intrinsic advantages in terms of high imaging speed and reduced photobleaching, LSM is very sensitive to residual opaque objects present in the sample, which cause dark horizontal stripes in the collected images. In the best case, these artefacts obscure the features of interest in structural imaging; in the worst case, dynamic shadowing introduced by red blood cells significantly alters the fluorescence signal variations related to neuronal activity.
We show how the use of Bessel beams in LSM can dramatically reduce such artefacts even in conventional one-sided illumination schemes, thanks to their “self-healing” properties. On the functional side, Bessel-beam LSM allows recording neuronal activity traces without any disturbing flickering caused by the movement of red blood cells. On the structural side, our proposed method is capable of obtaining anatomical information across the entire volume of whole mouse brains allowing tracing blood vessels and neuronal projections also in poorly cleared specimens.
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