Functional ultrasound imaging of human brain activity through an acoustically transparent cranial window.

Visualization of human brain activity is crucial for understanding normal and aberrant brain function. Currently available neural activity recording methods are highly invasive, have low sensitivity, and cannot be conducted outside of an operating room. Functional ultrasound imaging (fUSI) is an emerging technique that offers sensitive, large-scale, high-resolution neural imaging; however, fUSI cannot be performed through the adult human skull. Here, we used a polymeric skull replacement material to create an acoustic window compatible with fUSI to monitor adult human brain activity in a single individual. Using an in vitro cerebrovascular phantom to mimic brain vasculature and an in vivo rodent cranial defect model, first, we evaluated the fUSI signal intensity and signal-to-noise ratio through polymethyl methacrylate (PMMA) cranial implants of different thicknesses or a titanium mesh implant. We found that rat brain neural activity could be recorded with high sensitivity through a PMMA implant using a dedicated fUSI pulse sequence. We then designed a custom ultrasound-transparent cranial window implant for an adult patient undergoing reconstructive skull surgery after traumatic brain injury. We showed that fUSI could record brain activity in an awake human outside of the operating room. In a video game "connect the dots" task, we demonstrated mapping and decoding of task-modulated cortical activity in this individual. In a guitar-strumming task, we mapped additional task-specific cortical responses. Our proof-of-principle study shows that fUSI can be used as a high-resolution (200 µm) functional imaging modality for measuring adult human brain activity through an acoustically transparent cranial window.

Ultrasonic reporters of calcium for deep tissue imaging of cellular signals.

Calcium imaging has enabled major biological discoveries. However, the scattering of light by tissue limits the use of standard fluorescent calcium indicators in living animals. To address this limitation, we introduce the first genetically encoded ultrasonic reporter of calcium (URoC). Based on a unique class of air-filled protein nanostructures called gas vesicles, we engineered URoC to produce elevated nonlinear ultrasound signal upon binding to calcium ions. With URoC expressed in mammalian cells, we demonstrate noninvasive ultrasound imaging of calcium signaling in vivo during drug-induced receptor activation. URoC brings the depth and resolution advantages of ultrasound to the in vivo imaging of dynamic cellular function and paves the way for acoustic biosensing of a broader variety of biological signals.

A window to the brain: ultrasound imaging of human neural activity through a permanent acoustic window.

Recording human brain activity is crucial for understanding normal and aberrant brain function. However, available recording methods are either highly invasive or have relatively low sensitivity. Functional ultrasound imaging (fUSI) is an emerging technique that offers sensitive, large-scale, high-resolution neural imaging. However, fUSI cannot be performed through adult human skull. Here, we use a polymeric skull replacement material to create an acoustic window allowing ultrasound to monitor brain activity in fully intact adult humans. We design the window through experiments in phantoms and rodents, then implement it in a participant undergoing reconstructive skull surgery. Subsequently, we demonstrate fully non-invasive mapping and decoding of cortical responses to finger movement, marking the first instance of high-resolution (200 µm) and large-scale (50 mmx38 mm) brain imaging through a permanent acoustic window.

Microscale acoustic metamaterials as conformal sonotransparent skull prostheses.

Functional ultrasound imaging enables sensitive, high-resolution imaging of neural activity in freely behaving animals and human patients. However, the skull acts as an aberrating and absorbing layer for sound waves, leading to most functional ultrasound experiments being conducted after skull removal. In pre-clinical settings, craniotomies are often covered with a polymethylpentene film, which offers limited longitudinal imaging, due to the film's poor conformability, and limited mechanical protection, due to the film's low stiffness. Here, we introduce a skull replacement consisting of a microstructured, conformal acoustic window based on mechanical metamaterials, designed to offer high stiffness-to-density ratio and sonotransparency. We test the acoustic window in vivo, via terminal and survival experiments on small animals. Long-term biocompatibility and lasting signal sensitivity are demonstrated over a long period of time (> 4 months) by conducting ultrasound imaging in mouse models implanted with the metamaterial skull prosthesis.

A gut-derived metabolite alters brain activity and anxiety behaviour in mice.

Integration of sensory and molecular inputs from the environment shapes animal behaviour. A major site of exposure to environmental molecules is the gastrointestinal tract, in which dietary components are chemically transformed by the microbiota1 and gut-derived metabolites are disseminated to all organs, including the brain2. In mice, the gut microbiota impacts behaviour3, modulates neurotransmitter production in the gut and brain4,5, and influences brain development and myelination patterns6,7. The mechanisms that mediate the gut-brain interactions remain poorly defined, although they broadly involve humoral or neuronal connections. We previously reported that the levels of the microbial metabolite 4-ethylphenyl sulfate (4EPS) were increased in a mouse model of atypical neurodevelopment8. Here we identified biosynthetic genes from the gut microbiome that mediate the conversion of dietary tyrosine to 4-ethylphenol (4EP), and bioengineered gut bacteria to selectively produce 4EPS in mice. 4EPS entered the brain and was associated with changes in region-specific activity and functional connectivity. Gene expression signatures revealed altered oligodendrocyte function in the brain, and 4EPS impaired oligodendrocyte maturation in mice and decreased oligodendrocyte-neuron interactions in ex vivo brain cultures. Mice colonized with 4EP-producing bacteria exhibited reduced myelination of neuronal axons. Altered myelination dynamics in the brain have been associated with behavioural outcomes7,9-14. Accordingly, we observed that mice exposed to 4EPS displayed anxiety-like behaviours, and pharmacological treatments that promote oligodendrocyte differentiation prevented the behavioural effects of 4EPS. These findings reveal that a gut-derived molecule influences complex behaviours in mice through effects on oligodendrocyte function and myelin patterning in the brain.

Ultrafast amplitude modulation for molecular and hemodynamic ultrasound imaging.

Ultrasound is playing an emerging role in molecular and cellular imaging thanks to new micro- and nanoscale contrast agents and reporter genes. Acoustic methods for the selective in vivo detection of these imaging agents are needed to maximize their impact in biology and medicine. Existing ultrasound pulse sequences use the nonlinearity in contrast agents' response to acoustic pressure to distinguish them from mostly linear tissue scattering. However, such pulse sequences typically scan the sample using focused transmissions, resulting in a limited frame rate and restricted field of view. Meanwhile, existing wide-field scanning techniques based on plane wave transmissions suffer from limited sensitivity or nonlinear artifacts. To overcome these limitations, we introduce an ultrafast nonlinear imaging modality combining amplitude-modulated pulses, multiplane wave transmissions, and selective coherent compounding. This technique achieves contrast imaging sensitivity comparable to much slower gold-standard amplitude modulation sequences and enables the acquisition of larger and deeper fields of view, while providing a much faster imaging framerate of 3.2 kHz. Additionally, it enables simultaneous nonlinear and linear image formation and allows concurrent monitoring of phenomena accessible only at ultrafast framerates, such as blood volume variations. We demonstrate the performance of this ultrafast amplitude modulation technique by imaging gas vesicles, an emerging class of genetically encodable biomolecular contrast agents, in several in vitro and in vivo contexts. These demonstrations include the rapid discrimination of moving contrast agents and the real-time monitoring of phagolysosomal function in the mouse liver.

Ultrasound Technologies for Imaging and Modulating Neural Activity.

Visualizing and perturbing neural activity on a brain-wide scale in model animals and humans is a major goal of neuroscience technology development. Established electrical and optical techniques typically break down at this scale due to inherent physical limitations. In contrast, ultrasound readily permeates the brain, and in some cases the skull, and interacts with tissue with a fundamental resolution on the order of 100 µm and 1 ms. This basic ability has motivated major efforts to harness ultrasound as a modality for large-scale brain imaging and modulation. These efforts have resulted in already-useful neuroscience tools, including high-resolution hemodynamic functional imaging, focused ultrasound neuromodulation, and local drug delivery. Furthermore, recent breakthroughs promise to connect ultrasound to neurons at the genetic level for biomolecular imaging and sonogenetic control. In this article, we review the state of the art and ongoing developments in ultrasonic neurotechnology, building from fundamental principles to current utility, open questions, and future potential.

Pharmaco-fUS: Quantification of pharmacologically-induced dynamic changes in brain perfusion and connectivity by functional ultrasound imaging in awake mice.

There is a critical need for reliable quantitative biomarkers to assess functional brain alterations in mouse models of neuropsychiatric diseases, but current imaging methods measuring drug effects through the neurovascular coupling, face issues including poor sensitivity, drug-induced changes in global brain perfusion and the effects of anesthesia. Here we demonstrate the proof-of-concept of a minimally-invasive fUS imaging approach to detect the acute cholinergic modulatory effects of Scopolamine (ScoP) on functional brain connectivity in awake and behaving mice, through the intact skull. A machine-learning algorithm constructed an ad-hoc pharmacological score from the ScoP-induced changes in connectivity patterns of five mice. The discrimination model shows important ScoP-induced increase of the hippocampo-cortical connectivity. The pharmacological score led to robust discrimination of ScoP treatment from baseline in an independent dataset and showed, in another independent group, dose-dependent specific effects of central cholinergic modulation of functional connectivity, independent from global brain perfusion changes. In conclusion, we introduce pharmaco-fUS as a simple, robust, specific and sensitive modality to monitor drug effects on perfusion and functional connectivity in the awake mouse brain.

4D Functional Imaging of the Rat Brain Using a Large Aperture Row-Column Array.

Functional ultrasound imaging (fUS) recently emerged as a promising neuroimaging modality to image and monitor brain activity based on cerebral blood volume response (CBV) and neurovascular coupling. fUS offers very good spatial and temporal resolutions compared to fMRI gold standard as well as simplicity and portability. It was recently extended to 4D fUS imaging in preclinical settings although this approach remains limited and complex. Indeed 4D fUS requires a 2D matrix probe and specific hardware able to drive the N2 elements of the probe with thousands of electronic channels. Several under-sampling approaches are currently investigated to limit the channel count and spread ultrasound 4D modalities. Among them, the Row Column Addressing (RCA) approach combined with ultrafast imaging is a compelling alternative using only N + N channels. We present a large field of view RCA probe prototype of 128 + 128 channels and 15 MHz central frequency adapted for preclinical imaging. Based on the Orthogonal Plane Wave compounding scheme, we were able to perform 4D vascular brain acquisitions at high volume rate. Doppler volumes of the whole rat brain were obtained in vivo at high rates (23 dB CNR at 156 Hz and 19 dB CNR at 313 Hz). Visual and whiskers stimulations were performed and the corresponding CBV increases were reconstructed in 3D with successful functional activation detected in the superior colliculus and somato-sensorial cortex respectively. This proof of concept study demonstrates for the first time the use of a low-channel count RCA array for in vivo 4D fUS imaging in the whole rat brain.

4D functional ultrasound imaging of whole-brain activity in rodents.

We extended the capabilities of functional ultrasound to whole-brain four-dimensional (4D) neuroimaging. Our multiplane-wave transmission scheme on matrix arrays at thousands of frames per second provides volumetric recordings of cerebral blood volume changes at high spatiotemporal resolution. We illustrated the approach in rats while providing multiple sensory stimuli, for 4D functional connectivity and during instantaneous tracking of epileptiform events.

Suppression of exciton dephasing in sidewall-functionalized carbon nanotubes embedded into metallo-dielectric antennas.

Covalent functionalization of single-walled carbon nanotubes (SWCNTs) is a promising route to enhance the quantum yield of exciton emission and can lead to single-photon emission at room temperature. However, the spectral linewidth of the defect-related E11* emission remains rather broad. Here, we systematically investigate the low-temperature exciton emission of individual SWCNTs that have been dispersed with sodium-deoxycholate (DOC) and polyfluorene (PFO-BPy), are grown by laser vaporization (LV) or by CoMoCat techniques and are functionalized with oxygen as well as 3,5-dichlorobenzene groups. The E11 excitons in oxygen-functionalized SWCNTs remain rather broad with up to 10 meV linewidth while exciton emission from 3,5-dichlorobenzene functionalized SWCNTs is found to be about one order of magnitude narrower. In all cases, wrapping with PFO-BPy provides significantly better protection against pump induced dephasing compared to DOC. To further study the influence of exciton localization on pump-induced dephasing, we have embedded the functionalized SWCNTs into metallo-dielectric antenna cavities to maximize light collection. We show that 0D excitons attributed to the E11* emission of 3,5-dichlorobenzene quantum defects of LV-grown SWCNTs can display near resolution-limited linewidths down to 35 µeV. Interestingly, these 0D excitons give rise to a 3-fold suppressed pump-induced exciton dephasing compared to the E11 excitons in the same SWCNT. These findings provide a foundation to build a unified description of the emergence of novel optical behavior from the interplay of covalently introduced defects, dispersants, and exciton confinement in SWCNTs and might further lead to the realization of indistinguishable photons from carbon nanotubes.