This PDF file contains the front matter associated with SPIE Proceedings Volume 11360, including the Title Page, Copyright information, Table of Contents, author and Conference Committee lists

Recording brain activity at the mesoscopic scale has a strong potential to unveil many new fundamental neuronal operations. Optical imaging offers a unique opportunity to measure brain activity over a large area with high spatio-temporal resolutions (20 μm x 1 ms). However, two major limitations of this imaging technique partially explain the lack of development in this field. The cortex being non-planar, the field's depth limits the region in focus to a small region close to the center of the field of view. This is particularly significant for the highly curved lissencephalic small cortex of non-human primates that are becoming popular in neuroscience experiments. The ideal technique would be a method that compensates for such curvature; it would enable imaging the whole visual system at once, from the primary to the fifth visual cortices, in small non-human primates. Additionally, the signal-to-noise ratio is strongly degraded by the dynamic evolution of the brain curvature due to physiological rhythms (heartbeat, breathing, etc.). This strongly limits the ability to work at a single-trial level and to unravel the real dynamics of neuronal processing, such as spatio-temporal waves. Here in this project, we present an interdisciplinary approach for imaging of the non-human primate cortex, using technologies from astronomical instrumentation to overcome current technological limits. This will be of interest to a wide neuroscientific audience but also will impact the clinical community interested in mapping the nervous activity at the mesoscopic scale. Our current preliminary development involves redesigning the illumination source and the optical design.

Earlier we proposed the method of electrocardiographic signals acousto-optic processing based on application of acousto-optic spectrum analyzers with time integration. This method can be extended also for the electroencephalographic signals processing. However, some features of such signals make some special demands to the acousto-optic device performing the processing. Some elements of electroencephalographic signal which can be the signs of the cerebrum and central nervous system pathologies, can be found and selected only in specific frequency regions which differ from each other by 4…5 orders. Hence, the acousto-optic device which provides the signal processing must operate in very wide frequency range. This range can be attained only using acousto-optic spectrum analyzers with combined space and time integration. Moreover, some specific demands must be made to 2D multielement photodetector used in the mentioned above spectrum analyzers – its accumulation time must bt about 5…20 s because frequency resolution of the device must be of the order of 0,1 Hz. It requires application of the cooler based on Peltier phenomenon. The signal features and the corresponding demands made to the spectrum analyzer, are considered in the paper.

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.

Luminescent nanoparticles are becoming fundamental tools to the field of bioimaging. The optimization of their size, brightness and stability is key for applications ranging from contrast agent assisted surgery to diagnosis and therapeutics. A plethora of formulations have been documented which can be split into inorganic, organic and hybrid categories. While each class has their own advantages and limitations, controlling the interactions occurring between nanoparticles and cellular membranes is of the utmost importance. In particular, a major challenge for various applications, especially molecular imaging of membrane receptors, is to prevent non-specific interactions. Towards this goal, popular strategies based on coating nanoparticles with PEG or zwitterionic moieties have been developed to yield stealth nanoparticles. In this study, we present a series of spontaneously water-soluble and stealth organic nanoparticles. These fluorescent nanoparticles, made from original articulated bis-dipolar dyes, show vanishing interactions with living cells as bare nanoparticles. Moreover, thanks to their brightness and stability, they can be tracked as isolated single emitters in aqueous environments. These stealth nanoparticles thus hold promise for molecular imaging of specific membrane receptors, such as neuronal receptors, after bioconjugation with dedicated targeting agents.

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.

Neuronal networks in living organisms are highly interconnected. Usually, to study their functional roles in healthy conditions, task-evoked neuronal responses are correlated with the behavioral readout in freely moving or head-fixed animals. Recently, optogenetics proved to be a useful tool to manipulate targeted neuronal circuits using light. Optogenetic photostimulation of different cortical motor areas revealed distinct and reproducible motor movements: Rostral Forelimb Area (RFA) is critically involved in controlling grasping-like movements, while Caudal Forelimb Area (CFA) has a role in tap- or locomotion-like movements. In parallel, the development of red-shifted genetically encoded calcium indicators (red-GECIs) like jRCaMP1a allowed to reduce the spectral overlap with the most common optogenetic actuator, channelrhodopsin-2 (ChR2). Therefore, by combining these optical tools it is possible to develop all-optical systems, which are smart approaches for long-term low-invasive studies of neuronal patterns. Here, in order to understand the functional role that cortical ensembles play in motor generation and control, we developed a cross-talk free large-scale all-optical system for unraveling cortical neuronal patterns associated with optogenetically-evoked movements. We demonstrated that the motor cortex exhibits precise inter-regional patterns during movement initiation of grasp- or locomotion-evoked movements. Moreover, the cortical activation covers most of the related light-based optogenetic maps, revealing that a strong local neuronal connectivity is associated with optogenetically-evoked complex movements. To confirm the relevance of local connectivity for the generation of complex movements we used both optogenetic interference and pharmacological inhibition, showing that movement disruption is linked to reduced cortico-cortical coactivation.

We propose a generalized, modular, closed-loop system for objective assessment of human visual parameters. Our system presents periodical visual stimuli to the patient's field of view and analyses the consequent evoked brain potentials elicited in the occipital lobe and recorded through EEG. The analysis of the monitored EEG data is performed in an end-to-end fashion by a convolutional neural network (CNN). We propose a novel CNN architecture for EEG signal analysis that can be trained utilizing the benefits of multi-task learning. The closedloop attribute of our system allows for a real-time adaptation of the subsequent stimuli to further examine a potentially damaged area or increase the granularity of the exploration. Interchangeability is provided in terms of software modules, stimulus type, visual hardware, EEG acquisition device and EEG electrodes. Initially, the system is designed to monitor visual field loss originating from glaucoma or damage to the optic nerve using a virtual reality (VR) headset for the stimuli presentation. The modular architecture of our system paves the way for the assessment and monitoring of other neuro-visual functions.

We still lack a detailed map of the anatomical disposition of neurons in the human brain. A complete map would be an important step for deeply understanding the brain function, providing anatomical information useful to decipher the neuronal pattern in healthy and diseased conditions. Here, we present several important advances towards this goal, obtained by combining a new clearing method, advanced Light Sheet Microscopy and automated machine-learning based image analysis. We perform volumetric imaging of large sequentially stained human brain slices, labelled for two different neuronal markers NeuN and GAD67, discriminating the inhibitory population and reconstructing the brain connectivity.

The brain extracellular space (ECS) is a complex network that constitutes a key microenvironment for cellular communication, homeostasis, and clearance of toxic metabolites¹. Signaling molecules, neuromodulators, and nutrients transit via the ECS, therefore mediating the communication between cells. Despite the relevance of this important part of the brain, its dynamics and structural organization at the nanoscale is still mostly unknown². We have recently demonstrated that single-walled carbon nanotubes (SWCNTs) can be used to image and probe live brain tissue, providing super-resolved maps of the brain ECS and quantitative information on the local diffusion environment^3,4. Here, we propose an important refinement of this approach by implementing a structured illumination technique (named HiLo microscopy⁵) to image fluorescently labelled neuronal structures in parallel to SWCNT NIR imaging. This technique is based on speckle illumination and relies on the acquisition of one structured and one uniform illumination image to obtain images deep into tissues with good optical sectioning. Having access to spatially resolved SWCNT diffusivity around specific neuronal structures will provide more precise insights about the heterogeneity of the brain environment.

Optical coherence tomography (OCT) is a promising method for clarifying the boundaries of the infiltrative brain tumors within surrounding white matter. Since gliomas often tend to grow close to eloquent brain areas, the question of the proximity of the tumor to white matter tracts sharply arise during tumor resection to prevent their damage. Crosspolarization (CP) OCT is a so-called functional extension of OCT that seems to have benefits in visualization of myelin. It looks perspective not just to detect white matter, but also receive information about its condition – the myelination rate and presence of ordered fibers. The aim of this study was to visualize white matter organization of eloquent brain areas with CP OCT using post-processing methods. The ex vivo CP OCT images were collected from autopsy subjects of the human brain. The brain specimens contained white matter of different organization and localization: brainstem, corpus callosum, frontal and parietal tracts, subcortical white matter. Two optical coefficients (attenuation and inter-channel attenuation difference) were calculated for each A-scan and two types of color-coded maps based on them were built. No significant differences based on CP OCT attenuation and inter-channel attenuation difference coefficients were demonstrated between white matter from different brain areas. However, in vivo studies can show conversely results. The detection of white matter microstructure during surgery looks promising therefore additional CP OCT performance build-up can be considered.

Chemically synthesized biomolecules can self-assemble to bioinspired nanostructures of different morphologies such as dots, tubes, spheres, nanofibers and more. They adopt similar basic ordering as their biological counterparts either α-helical or β-sheet peptide/protein conformations. These two fundamental biomolecular architectures exhibit dissimilar physical properties. One of the most interesting physical properties found in biological and bioinspired structures is a new biophotonic phenomenon of visible fluorescence (FL). It has been observed both in neurodegenerative disease-related amyloid fibrils and in synthetic amyloidogenic biorganic di- and tri-aromatic and aliphatic peptide nanostructures. The FL effect has been also found recently in peptide nanodots and hybrid polymer/peptide thin films. All of them have been assembled to β-sheet secondary structure. In this work we report on a new development of FL optical waveguiding in elongated bioinspired fibrillary structures, self-assembled from ultrashort amylodogenic peptides/proteins and hybrid polymer/peptides biomolecules. We show that FL propagation in these two fiber materials of different origin can be described by two completely different mechanisms. One of them is conventional FL propagation in the region of optical transparency of peptide materials in accordance with optical confinement rules. Another model is FL reabsorption mechanism where anomalous long range FL propagation has been found. We show that this intrinsic FL biophotonic waveguiding effects found in different β- sheet biomaterials is considered as a promising tool for precise biomedicine where new biocompatible visible tunable FL optical waveguides can be applied in advanced nanomedical technologies (local bioimaging, light diagnostics, therapy, optogenetitcs and health monitoring).

In this work, we demonstrate the application of two optical imaging systems namely, dual-wavelength laser speckle and integrated spatial frequency domain imaging systems to image the changes in mouse brain tissue properties following acute hyperglycemia. We assume that hyperglycemia alters brain function which in turn can be monitored using these two optical modalities. Hyperglycemia was induced by intraperitoneal injection of an anesthetic ketamine and xylazine combination which is found to increase blood glucose more than threefold relative to normoglycemia. A total of ten mice were used, randomized into two groups of normoglycemia (n = 4) and hyperglycemia (n = 6). Experimental results demonstrated reductions in cerebral blood flow, tissue oxygen saturation, and cerebral metabolic rate of oxygen following acute hyperglycemia. In addition, differences in both cerebral tissue optical properties (absorption and scattering) with increasing glucose level were observed. Overall, experimental result demonstrates the capability of both systems to provide and map various brain tissue metrics following hyperglycemia.

Photon upconversion with the transformation of low-energy photons to high-energy photons is of significant interest for broad applications. Here, we present self-powered, micrometer-scale optoelectronic devices based on III-V materials for high-performance near-infrared to visible upconversion. By taking advantage of its unique photon – “free electron” – photon processes, these thin-film, ultra-miniaturized devices realize fast upconversion that is linearly dependent on incoherent, low-power excitation, with a quantum yield of ~1.5%. By exploiting the advanced manufacturing method, encapsulated, freestanding devices are transferred onto heterogeneous substrates and show desirable biocompatibilities within biological fluids and tissues. These devices as the microscale light sources are implanted in behaving animals, with in vitro and in vivo experiments demonstrating their utility for optogenetic neuromodulation. These results provide routes for high-performance upconversion materials and devices and their unprecedented potential as optical biointerfaces.