Light-based in-vivo brain imaging relies on the transportation of light through highly scattering tissues over long distances. As scattering gradually reduces imaging contrast and resolution, it becomes challenging to visualize structures at greater depths, even when using multiphoton techniques. A recently proposed strategy involves the exploitation of holographic control of light transport through multimode optical fibres, which promises superior imaging performance combined with uniquely atraumatic application. Following the review of the fundamental and technological bases, the talk will introduce a 110μm thin laser-scanning endo-microscope, which enables volumetric imaging of the entire depth of the mouse brain in vivo.

Mapping of human brain function has revolutionized systems neuroscience. Common brain mapping methods such as fMRI offer promising sensitivity yet pose significant methodological challenges in studies of naturally behaving children due to the loud, constraining environment and extreme susceptibility to motion-induced artifacts. Recent developments in high-density diffuse optical tomography (HD-DOT) have demonstrated dramatically improved image quality over traditional fNIRS methods. In this talk, I will discuss challenges associated with mapping brain function in children in natural settings, recent advancements in developing HD-DOT methods, and applications in childhood development, autism spectrum disorder, and critical care environments.

Here, we present a rapid dual-view projection imaging method with two-photon glutamate uncaging capability based on an upright Bessel beam light-sheet microscopy setup. Compared with light-sheet imaging, our projection imaging method can significantly improve the volumetric rate for neural imaging. In addition, an independent laser scanning module is designed for 2P uncaging, allowing simultaneous synaptic resolution of stimulation and 100 Hz volumetric imaging of neural activity in deep tissue. Imaging results from mouse brain slices under 2P glutamate uncaging will be presented.

Advancements in genetically encoded voltage indicators (GEVIs) have made it possible to measure cellular membrane potential changes optically. But performing GEVI imaging in vivo remains highly challenging due to factors such as low GEVI concentrations, modest signal dynamic range, tissue scattering, out-of-focus fluorescence. To address these challenges, we developed a microscopy technique that take advatanges from both widefield targeted illumination and confocal background rejection, enabling high SNR low crosstalk GEVI imaging across millimeter fields-of-view, at supra-kilohertz frame rates, over extended durations, and at high penetration depths. We demonstrate our technique under a variety of imaging conditions across multiple brain regions and with different classes of GEVIs.

We present a two-photon fluorescence microscope designed for high-speed imaging of neural activity in cellular resolution. Our microscope uses line illumination with an adaptive sampling scheme. Instead of building images pixel by pixel via scanning a diffraction-limited spot across the tissue, our scheme only illuminates the regions of interest (i.e., neuronal cell bodies), and samples a large area of them in a single measurement. This significantly increases the imaging speed and reduces the overall laser power on the sample. We characterized the imaging resolution and verified the concept of adaptive sampling through phantom samples. Our approach holds great promise for high-throughput neural activity imaging.

Two-photon microscopy traditionally suffers from slow frame rates due to the necessity of raster scanning the laser excitation spot over the field of view (FOV). We have developed a miniaturized elliptical beam shaper (MEBS) which is compatible with the state-of-the-art open-source two-photon miniscopes for imaging neural activity in freely moving mice. The MEBS enables an elliptical two-photon excitation pattern with a long axis of 5-10 µm, which greatly reduces the number of scanning rows required while maintaining cellular resolution in imaging. As a result, the overall frame rate is increased compared to the diffraction-limited-point scanning method. We demonstrate this high-speed miniaturized two-photon microscope in fluorescence imaging and show a significant frame rate improvement while maintaining cellular resolution across a 400 µm diameter FOV.

We present DeepVIDv2, a resolution-improved self-supervised voltage imaging denoising approach that achieves higher spatial resolution while preserving fast neuronal dynamics. While existing methods enhance signal-to-noise ratio (SNR), they compromise spatial resolution and result in blurry outputs. By disentangling spatial and temporal performance into two parameters, DeepVIDv2 overcomes the tradeoff faced by its predecessor. This advancement enables more effective analysis of high-speed, large-population voltage imaging data.

We present a multimodal approach using functional near-infrared spectroscopy (fNIRS) and optically pumped magnetometers (OPM) for imaging neurovascular coupling (NVC). The study aims to achieve high-density imaging by combining state-of-the-art implementation of the techniques. In-vivo studies during a motor cortex stimulation were carried out inside the 7-layer magnetically shielded room BMSR2 - the single most magnetically-shielded room on Earth. The study demonstrates the feasibility of the proposed approach and paves the way for future mapping and tomography reconstructions using magnetic and optical data.

In this study, we present a physics-informed deep learning model for predicting partial pathlength and absorption changes in human brain using Near-Infrared Spectroscopy (NIRS). Leveraging the multi-layer modified Beer Lamber Law, our model overcomes the limitations of conventional approach that assumes tissue homogeneity. Trained on synthetic data generated from a multi-layer forward model, out model was tested on Monte Carlo simulations of both two and three-layer geometries, demonstrating robust performance despite encountering varying optical properties and anatomical complexities. Future work will focus on refining the model and testing it on multi-layer optical phantoms and human subjects.

Alzheimer’s disease (AD) is a neurodegenerative disorder usually accompanied by buildup of amyloid-beta (Aβ) plaques and neurofibrillary tangles, cerebral vascular morphological and functional changes. Our study presents a combined photoacoustic microscopy and fluorescence microscopy dual-modality imaging system for in-vivo monitoring of biomarkers for AD via cranial window. Photoacoustic microscopy resolves vasculature, blood oxygenation and flow speed at capillary level, while a confocally designed fluorescence microscope, aided by an Aβ targeting dye, maps the distribution of Aβ plaques. Results with age-varied AD mice group showed reduced capillary density, varied flow speed in vein, and increased plaque density compared to wild type mice. This technique provides guidance for longitudinal monitoring of AD progression.

This study presents a novel method using Photoacoustic Computed Tomography (PACT) with a linear ultrasound (US) transducer array to observe visual-evoked hemodynamic responses in the deep mouse brain. By applying advanced data processing techniques, we successfully isolated hemodynamic responses within critical visual regions. The PACT system's high spatial resolution and deep tissue penetration enabled non-invasive examination of visual-evoked brain activity. Our findings demonstrate the potential of PACT for advancing optical neuroimaging and studying synaptic physiology, brain activities, and retinal diseases.

Optoacoustic tomography in the first near infrared window (NIR I) can capture much of the mouse brain with high temporal and mesoscale spatial resolutions, making it an excellent candidate for functional neuroimaging experiments. Using fluorescent contrasts which absorb in the NIR I range, we can obtain concurrent or parallel opotacoustic and fluorescence data. Fluorescence information can then be used to validate or augment the optoacoustic data (hybrid fluorescence and optoacoustic tomography, or FLOT). Using the two modalities, we probe functional neural, vascular, and cerebrospinal fluid flow information throughout the brain.

Alzheimer’s Disease (AD) is a progressive neurodegenerative disease that severely impacts quality of life, so biomarkers that help identify AD are highly sought after. Here, we investigated the retinas of APPSL and 5xFAD mouse models and wild-type littermate controls, provided by QPS Austria GmbH, using a custom optical coherence tomography (OCT) ophthalmoscope. Features of interest including retinal thickness, the visibility of the photoreceptor inner and outer segment junction, and vessel density were quantified from the OCT images, with a number of significant differences between groups. This study identifies potential AD biomarkers and reemphasizes the importance of using proper age-matched controls.

Functional near-infrared spectroscopy (fNIRS) and diffuse correlation spectroscopy (DCS) have shown promise as non-invasive optical methods for cerebral functional imaging. Both approaches currently have limits to sensitivity in adults. Sensitivity can be improved using temporal discrimination, where the laser excitation is of short (~400ps) duration and the detector rejects early photons that have not penetrated into the brain while maintain high sensitivity to those that have. We report here further demonstration of a high-speed Read-Out Integrated Circuit (ROIC) that integrates with a 32x32 Single-Photon Avalanche photo-Detector (SPAD) array that can be either silicon (Si, for visible to infra-red) in indium-phosphide (InP, to allow operation at 1064nm). Data is exfiltrated serially directly to an FPGA where it can be processed in real time. This presentation will include results of recent detector performance tests and phantom demonstrations using this powerful new tool.

We present a novel pipeline for robust optical analysis of fresh human brain tissue. We capture fluorescence signal and characterize optical properties with high sensitivity and high spectral resolution. These in turn allow for quantitative analysis of protoporphyrin IX (PpIX) accumulation in various types of tumor tissue. Our ex vivo protocol for tissue handling was designed to promote high-fidelity replication of in vivo conditions. The on-going consolidation of a fresh ex vivo quantitative dataset from a cohort of 20 patients plus a control cohort lays foundation for the development of imaging devices for intraoperative fluorescence guided resection.

Altered brain vasculature plays a crucial role in neurologic disorders, including Alzheimer's Disease (AD). Recent evidence suggests that vascular changes precede the characteristic cognitive decline in AD, highlighting the potential of cerebrovascular biomarkers as preclinical tools. However, there is a need for improved visualization techniques. Our previous work developed a protocol to visualize brain vasculature in three dimensions using perfusion-based labeling, a modified iDISCO+ clearing protocol, and confocal imaging. Focusing on the 3xTg mouse model, an established AD pathogenesis model, we compare cerebrovascular biomarkers between wild-type and 3xTg mice to understand associated alterations. We use custom MATLAB algorithms and open-source neuron tracing software, neuTube, to segment vessels in fluorescence images and quantify metrics such as diameter, density, and tortuosity. Quantifying these metrics shows promise in uncovering cerebrovascular mechanisms contributing to AD and neurologic conditions, enabling early detection and targeted interventions.

Optical brain imaging has several advantages over other imaging techniques and was used to visualize both the structural and functional aspects of the brain, providing a more complete picture of brain activity. One of the promising techniques is optical coherence tomography (OCT), which uses low-coherence interferometry to obtain three-dimensional depth-resolved imaging of structures. In this research, we proposed a micro-electro-mechanical (MEMS) based miniature OCT probe and conducted brain imaging at the hippocampus area. The designed probe has great potential applied to freely moving mice that can observe the changes in brain structure and vascular, thus aimed at early detection of Alzheimer's disease.

The cerebral vasculature facilitates blood flow to maintain normal function in the brain. Vascular injury can impair the ability of the cerebral vasculature to regulate blood flow and preserve the integrity of the blood-brain barrier. Cerebral microhemorrhages (CMH) are an indicator of structural damage in the brain vasculature. Aging and hypertension are the most common risk factors for CMH. In this study, we analyzed the effect of hypertension on resting-state cerebral blood flow and the development of CMH in a mouse model of aging. A reduction in resting-state cerebral blood flow was observed in hypertension mice. CMH were found to appear nearest to capillary-sized vessels. Together, these findings demonstrate hypertension can impair the function (via reduced resting-state cerebral blood flow) and structure (via formation of CMH) of the brain vasculature.

Diffuse Correlation Spectroscopy (DCS) is an emerging technique capable of measuring cerebral dynamics. To obtain a reliable blood flow index (BFi), DCS often collaborates with near-infrared spectroscopy (NIRS) to provide measurements of static optical properties of tissue. The integration of these two techniques into a single modality has been a subject of ongoing research interest. While dual-comb spectroscopy offers a promising solution, it is often impeded by high costs and complex instrumentation. In this study, we present an innovative approach that employs a single-cavity free-running dual-comb source and balanced detectors to construct two Mach-Zehnder interferometers. Our results indicate that, even in the absence of feedback control loops and single-photon detectors, both static and dynamic properties of bio-phantoms can be simultaneously measured. This approach holds the potential to offer a simplified, cost-effective solution for DCS, NIRS, and optical coherence tomography (OCT).

Diffuse optics often results in ill-posed inverse problems. Here we investigate a time-of-flight (TOF) informed interferometric diffuse optical approach, which applies a series of TOF filters to multiply scattered light collected via the human head. Rather than 1D data sets which are typical of diffuse optics, we obtain a 2D data set at a single source-collector separation, where the dimensions are TOF filter width and correlation time lag. We investigate recovery of optical properties, layer thicknesses, and blood flow indices by fitting a 3-layer model of the human head. We recover reasonable brain-to-scalp blood flow index ratios.

Interferometric diffuse correlation spectroscopy (iDCS) is an emerging technique that enables high quality measurements of cerebral blood flow. By including a reference arm in the optical setup, the SNR of the measured signals are improved relative to traditional DCS. We report here an expansion of our previously demonstrated 1064 nm single channel line scan camera based iDCS system to a multi-source, multiple detection channel approach to enable imaging of the brain perfusion response to functional activation. We confirm the ability to image multiple detectors on a single camera with minimal cross-talk using phantom experiments and show initial functional imaging results.

Diffusing Wave Spectroscopy (DWS) employs non-invasive techniques to study dynamic behaviors within scattering mediums. This involves detecting speckle light patterns from the moving media and gauging speckle decorrelation time for dynamic quantification. A specific form of DWS is Speckle Visibility Spectroscopy (SVS), which employs spatial ensembles by utilizing a high-pixel-count cameras to capture multiple speckles in a single frame. To further increase SNR at low signal level, one combines interferometric techniques with SVS to form interferometric SVS (iSVS). In this talk, I will present the benefits and constraints associated with the utilization of interferometric detection (iSVS) over direct detection (SVS). Surprisingly, we found that iSVS does not always outperform SVS, but it does emerge as the prevailing choice in most cases.

Processing functional near infrared spectroscopy (fNIRS) and diffuse optical tomography (DOT) data poses several challenges including costly software licenses (e.g. MATLAB) and compatibility between a wide variety of data formats (NIRS, SNIRF, BIDS, NIFTI, 4dfp, GIFTI, etc.). NeuroDOT is a toolbox available in both MATLAB and Python that has functions and pipelines for data preprocessing, anatomical light modeling, image reconstruction, analysis, and visualization. NeuroDOT’s standard pipelines provide a self-contained, straightforward method of processing and analysis for fNIRS and DOT data. Additionally, NeuroDOT utilizes cloud-based computing with XNAT to lessen computational burden, promote reproducibility of results, and simplify processing.

In this report, we investigate the effects of array density and modulation frequency on spatial accuracy, resolution, and penetration of frequency domain high-density diffuse optical tomography (FD HD-DOT). The result shows that these metrics are improved by using higher array densities or an optimized modulation frequency, while array density is the stronger parameter. Additionally, the improvement of image quality shows diminishing effects with denser arrays.

Early detection of Alzheimer's disease (AD) is crucial for prompt interventions to preserve brain function. Neuronal hyperexcitability promises a biomarker of early-stage AD. However, in vivo, detection of neuronal hyperexcitability is technically challenging. With a custom-designed optical coherence tomography (OCT), this study demonstrated the feasibility of using retinal intrinsic optical signal (IOS) imaging, also known as optoretinography (ORG), for detecting photoreceptor hyperexcitability associated with early-stage AD. Morphological OCT analysis revealed detectable thinning of retinal thickness in 3xTg-AD mice from 5 months of age. In contrast, functional ORG disclosed increased photoreceptor hyperexcitability of 3xTg-AD mice from 4 months of age.

Measures of vascular dysfunction may serve as valuable clinical markers for the early detection of dementia, and for monitoring its progression. Near-Infrared Spectroscopy (NIRS) can non-invasively measure brain oxygenation by quantifying concentration changes of oxygenated (HbO) and deoxygenated haemoglobin (HbR). NIRS shows much promise for clinical translation: it has few contraindications, it can be performed at the bedside, and it is low-cost. High-Density Diffuse Optical Tomography (HD-DOT) extends this method by combining high-density NIRS with anatomical information to produce volumetric or topographical maps of brain oxygenation. Here we present an observational cohort study for Alzheimer's disease, dementia with Lewy bodies or mild cognitive impairment (n=32), which for the first time evaluates the possibility of using HD-DOT to develop biomarkers for dementia. Our early findings show that visual stimulation elicits a reduction in the magnitude of concentration changes of both HbO and HbR in Alzheimer’s Disease compared to healthy controls.