Neurophotonics

SignificanceCerebral vascular reactivity is critical parameters of brain homeostasis in health and disease, but the investigational value of brain oxymetry is diminished by anesthesia and mechanical fixation of the mouse scull. AimWe needed to reduce the physical restrictivity of hemodynamic spectroscopy to enable Alzheimers disease (AD) studies in freely-moving mice. ApproachWe combined spectroscopy, spectral analysis software and a magnetic, implantable device to measure vascular reactivity in unanesthetized, freely-moving mice. We measured cerebral blood volume fraction (CBVF) and oxygen saturation (SO2). ResultsWe validated that our system could detect delayed cerebrovascular recovery from hypoxia in an orthotopic xenograft glioma model under anesthetized condition and we also found increased CBVF and impaired vascular reactivity during hypercapnia in a freely-moving mouse model of AD compared to wild-type littermates. ConclusionsOur optomechanical approach to reproducibly getting light into and out of the brain enabled us to successfully measure CBVF and SO2 during hypercapnia in unanesthetized freely-moving mice. We present hardware and software enabling oximetric analysis of metabolic activity, which provides a safe and reliable method for rapid assessment of vascular reactivity in murine disease models as well as CBVF and SO2.

SignificanceHyperspectral imaging sensors have rapidly advanced, aiding in tumor diagnostics for in-vivo brain tumors. Linescan cameras effectively distinguish between pathological and healthy tissue, while snapshot cameras offer a potential alternative to reduce acquisition time. AimOur research compares linescan and snapshot hyperspectral cameras for in-vivo brain tissues and chromophores identification. ApproachWe compared a lines-can pushbroom camera and a snapshot camera using images from 10 patients with various pathologies. Objective comparisons were made using unnormalized and normalized data for healthy and pathological tissues. We utilized Interquartile Range (IQR) for the Spectral Angle Mapping (SAM), the Goodness-of-Fit Coefficient (GFC), and the Root Mean Square Error (RMSE) within the 659.95 to 951.42 nm range. Additionally, we assessed the ability of both cameras to capture tissue chromophores by analyzing absorbance from reflectance information. ResultsThe SAM metric indicates reduced dispersion and high similarity between cameras for pathological samples, with a 9.68% IQR for normalized data compared to 2.38% for unnormalized data. This pattern is consistent across GFC and RMSE metrics, regardless of tissue type. Moreover, both cameras could identify absorption peaks of certain chromophores. For instance, using the absorbance measurements of the linescan camera we obtained SAM values below 0.235 for four peaks, regardless of the tissue and type of data under inspection. These peaks are: one for cytochrome b in its oxidised form at{lambda} = 422 nm, two for HbO2 at{lambda} = 542 nm and{lambda} = 576 nm, and one for water at{lambda} = 976 nm. ConclusionThe spectral signatures of the cameras show more similarity with unnormalized data, likely due to snapshot sensor noise, resulting in noisier signatures post-normalization. Comparisons in this study suggest that snapshot cameras might be viable alternatives to linescan cameras for real-time brain tissues identification.

Two-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations and have the potential to build a framework for network-based therapies, e.g. for rebalancing maladaptive activity patterns in preclinical models of neurological disorders. Here, our goal was to tailor these approaches for this purpose: Firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in layer II/III of mouse visual cortex to tune our detection algorithm towards a 100 % specific identification of AP-related calcium transients. False-positive-free detection was achieved at a sensitivity of approximately 73 %. To further increase specificity, secondly, we minimized photostimulation artifacts as a potential source for false-positives by using extended-wavelength-spectrum laser sources for optogenetic stimulation of the excitatory opsin C1V1. We achieved artifact-free all-optical experiments performing photostimulations at 1100 nm or higher and simultaneous calcium imaging at 920 nm in mouse visual cortex in vivo. Thirdly, we determined the spectral range for maximizing efficacy of optogenetic control by performing 2-P photostimulations of individual neurons with wavelengths up to 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-co-expressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective stimulations of C1V1 in GCaMP-based all-optical interrogations, we increased the translational value of these approaches, e.g. for the use in preclinical applications of network-based therapies. One Sentence SummaryWe maximize translational relevance of 2-P all-optical physiology by increasing specificity, minimizing artifacts and optimizing stimulation efficacy.

Within neuronal circuits, ordered neurotransmission is contingent upon balance between excitatory glutamatergic and inhibitory GABAergic signaling. To study circuit-level processes, the paradigm of 4D cellular physiology has been developed, where, single cells and subcellular structures are studied as individual units in three-dimensional space over a continuous interval rather than as a single moment in time, or as a population-level average. Neurons are excitable cells expressing voltage-gated Ca2+ channels and Ca2+ fluxes subsequent to action potential firing are widely used as markers of neuronal activity. While the imaging of Ca2+ dynamics at the soma is often performed, the imaging of Ca2+ fluxes at presynaptic terminals has often proven to be an experimental challenge: existing imaging modalities suffer from inadequate acquisition speeds, insufficient penetration depths, insufficient spatial resolution to identify axonal structures, or spectral crosstalk issues. To visualize presynaptic Ca2+ dynamics in both excitatory and inhibitory neurons, here we combine advanced lattice light-sheet microscopy with viral delivery of two genetically encoded calcium indicators (GECIs)- jRGECO1a and jGCaMP8f, to perform sequential imaging of Ca2+ dynamics within acute ex vivo slice preparations. Our methodology, Biosensor Lattice light-sheet Imaging of Multidimensional Presynaptic Structure (BLIMPS), includes acute brain slice preparation, mounting on a temperature-controlled flow chamber within a LLSM, and imaging of electrically evoked Ca2+ signals, with high adaptability to a range of genetic and pharmacological disease models. Our technique offers high spectral separation between evoked signals from each of the two GECIs and fast acquisition speeds of 0.1-0.3 KHz. Included within the BLIMPS technique is a robust, open-source data analysis pipeline to track highly responsive neuronal structures such as presynaptic terminals and quantify both the amplitudes and decay rates of evoked fluxes.

BackgroundNear-infrared (NIR) light therapy is used to treat various musculoskeletal disorders. It has been proposed that transcranial NIR light treatment may also be beneficial for Alzheimers disease (AD). However, the ability of NIR light to penetrate the scalp and skull efficiently and induce cytoprotective responses in the brain parenchyma has not been sufficiently examined so far. This study aimed to evaluate whether the amount of NIR light that can penetrate through the human skull can cause a biological effect. MethodsThree commercially available devices (a medical laser emitting light at a wavelength of 905 nm and two LED helmets operating at wavelengths of 810 nm and 1070 nm, respectively) were used to measure the NIR light transmittance through human post-mortem skulls with a thermal power sensor. Furthermore, the biological effects of the fraction of light power that passed through the skull were investigated in a human neuronal cell line and in C. elegans. ResultsThe 905 nm laser achieved transmittances of up to 0.31% (173 {micro}W/cm2) of its input power, and the LED helmets 0.71% (41 {micro}W/cm2; 810 nm) and 0.45% (19 {micro}W/cm2; 1070 nm) of their respective input powers. NIR light exposure at a power density of 134 mW/cm2 was sufficient to activate mitochondrial metabolism in cultured human neurons and C. elegans, as demonstrated by increased cytochrome c oxidase activity and induction of mitochondrial chaperones. However, this stimulatory effect was no longer observed when the applied power density was reduced to 2.5 mW/cm2. ConclusionsMore than 99% of the NIR light emitted by the investigated devices was either absorbed or scattered by the human skull. The residual NIR light that would reach underlying brain structures was too weak to elicit biological effects. In conclusion, NIR light treatment is unlikely to be effective to treat brain diseases such as AD due to the low penetrability of the skull.

SignificanceTwo-photon microscopy is used routinely for in vivo imaging of neural and vascular structure and function in rodents with a high resolution. Image quality, however, often degrades in deeper portions of the cerebral cortex. Strategies to improve deep imaging are therefore needed. We introduce such a strategy using gates of high repetition rate ultrafast pulse trains to increase signal level. AimWe investigate how signal generation, signal-to-noise ratio (SNR), and signal-to-background ratio (SBR) improve with pulse gating while imaging in vivo mouse cerebral vasculature. ApproachAn electro-optic modulator is used with a high-power (6 W) 80 MHz repetition rate ytterbium fiber amplifier to create gates of pulses at a 1 MHz repetition rate. We first measure signal generation from a Texas Red solution in a cuvette to characterize the system with no gating and at a 50%, 25%, and 12.5% duty cycle. We then compare signal generation, SNR, and SBR when imaging Texas Red-labeled vasculature using these conditions. ResultsWe find up to a 6.73-fold increase in fluorescent signal from a cuvette when using a 12.5% duty cycle pulse gating excitation pattern as opposed to a constant 80 MHz pulse train. We verify similar increases for in vivo imaging to that observed in cuvette testing. For deep imaging we find pulse gating to result in a 2.95-fold increase in SNR and a 1.37-fold increase in SBR on average when imaging mouse cortical vasculature at depths ranging from 950 m to 1050 m. ConclusionsWe demonstrate that a pulse gating strategy can either be used to limit heating when imaging superficial brain regions or used to increase signal generation in deep regions. These findings should encourage others to adopt similar pulse gating excitation schemes for imaging neural structure through two-photon microscopy.

IntroductionGamma entrainment using sensory stimuli (GENUS) uses 40Hz-pulsed sensory stimuli to entrain neural activity in the gamma band (30-150Hz). However, the effect of GENUS on low-frequency vascular oscillations has not been fully explored. ObjectivesThe objective of this study is to elucidate the effect of GENUS on vasomotion in healthy mice and potential confounds for future application in disease studies. MethodsHead-fixed, awake C57Bl/6 mice (n=18; 9M 9F) aged between 18 to 60 weeks were subjected to white light of either 40Hz visual flicker (GENUS), or constant stimulus (control). Blood flow was imaged using laser speckle contrast imaging (LSCI) before, during, immediately after 1 hour of stimulus, and 30min after the stimulus termination. ResultsA linear mixed-effects model showed that GENUS enhanced the magnitude of 0.2-0.4Hz blood flow oscillations by 38% during stimulation and by 30% at 30 minutes after stimulation compared to control when controlled for age, sex, and other factors. The effect on vasomotion was distributed across many cortical regions not limited to visual areas and lasted beyond 24 hours post-stimulus. ConclusionThese results support the exploration of GENUS for increasing vasomotion in therapeutic contexts. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/729102v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1deee2eorg.highwire.dtl.DTLVardef@e71833org.highwire.dtl.DTLVardef@1e5c4f0org.highwire.dtl.DTLVardef@1e4832e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Modern optical neuroimaging approaches are expanding our ability to elucidate complex brain function. Diverse imaging contrasts enable direct observation of neural activity with functional sensors along with the induced hemodynamic responses. To date, decoupling the complex interplay of neurovascular coupling and dynamical physiological states has remained challenging when employing single-modality functional neuroimaging tools. We devised a hybrid fluorescence optoacoustic tomography (FLOT) platform combined with a custom data processing pipeline based on statistical parametric mapping, accomplishing the first simultaneous noninvasive observation of both direct and indirect brain-wide activation patterns with optical contrast. Correlated changes in the oxy- and deoxygenated hemoglobin, total hemoglobin, oxygen saturation and rapid GCaMP6f fluorescence signals were observed in response to peripheral sensory stimulation. While the concurrent epifluorescence served to corroborate and complement the functional optoacoustic observations, the latter further aided in decoupling the rapid calcium responses from the slowly varying background in the fluorescence recordings mediated by hemodynamic changes. The hybrid imaging platform expands the capabilities of conventional neuroimaging methods to provide more comprehensive functional readings for studying neurovascular and neurometabolic coupling mechanisms and related diseases.

Wide-field imaging (WFI) is a mesoscopic approach for monitoring cortex-wide activity with high temporal resolution and a broad field of view. Owing to its simple optical configuration and compatibility with chronic preparations, WFI has become an important tool in systems neuroscience and disease-model research. In this chapter, we describe practical protocols for chronic transcranial WFI in mice using two complementary optical signals: genetically encoded calcium indicators (GCaMP) and endogenous flavoprotein autofluorescence. Calcium imaging provides a robust readout of neuronal population activity, whereas flavoprotein imaging reflects mitochondrial redox dynamics and cellular metabolic demand. We detail procedures for animal preparation, skull clearing, headplate implantation, macroscope assembly, synchronized sensory stimulation, triggered image acquisition, and MATLAB-based data analysis. The analysis workflow includes {Delta}F/F normalization, reference-based signal correction, and artifact reduction, followed by trial averaging, atlas registration, and region-of-interest analysis. Because imaging is performed through the intact skull, the protocol enables repeated longitudinal measurements in the same animal over extended periods. This approach is reproducible, cost-effective, and adaptable to studies of cortical physiology and neurological disorders.

Deposition of beta-amyloid (A{beta}) deposits is one major histopathological hallmark of Alzheimers disease (AD). Here, we introduce volumetric multi-spectral optoacoustic tomography (vMSOT), which covers 10x10x10 mm3 field-of-view, capable of 3D whole mouse brain imaging. We show for the first time the optoacoustic properties of oxazine-derivative AOI987 probe, which binds to A{beta}, and the application of vMSOT for the quantification of brain-wide A{beta} deposition. Administration of AOI987 to two common transgenic mouse strains of AD amyloidosis led to a retention of the probe in A{beta}-laden brain regions. Co-registered of vMSOT data to a brain atlas revealed strain-specific pattern of AOI987 uptake. A comparison with ex vivo light-sheet microscopy in cleared mouse brains showed a good correspondence in A{beta} distribution. Lastly, we demonstrate the specificity of the AOI987 probe by immunohistochemistry. vMSOT with AOI987 facilitates preclinical brain region-specific studies of A{beta} spread and accumulation, and the monitoring of putative treatments targeting A{beta}.

Fluorescent imaging enables visualization of the specific molecules of interest with high contrast, and the use of multiple fluorophores in a single tissue sample allows visualization of complex relationships between biological molecules, cell types, and anatomy. The utility of fluorescent imaging in human tissue has been limited by endogenous pigments that can block the light path or emit an autofluorescence, thereby interfering with the specific imaging of target molecules. Although photobleachers have been developed to quench endogenous pigments, the lack of customizability limits their utility for a broad range of applications. Here, we present a high luminous-intensity photobleacher that is based on rigorous simulations of illumination patterns using the laws of radiation, along with the framework to maximize bleaching efficiency. This open-source project is designed to help researchers customize and scale according to the tissue types and the research goals. The photobleacher is applicable to both thin tissue slices and large-volume cleared tissue samples to enable serial three-dimensional imaging of postmortem human brain using multiplexed antibody or oligonucleotide probes. SIGNIFICANCE STATEMENTPhotobleaching is an effective technique for quenching endogenous pigments, enabling multiplexed fluorescent imaging of pigment-rich tissues, such as postmortem human samples. While many photobleaching strategies have been proposed, there is no standard guidance on how to design and use a photobleacher. This study introduces a general strategy for designing an effective, scalable, and customizable photobleacher, and proposes a workflow for properly treating tissues with the photobleacher. The technique enables high-contrast molecular visualization in tissues of various sizes, including large volumetric cleared tissues. Our framework will accelerate the quantitative understanding of human molecular anatomy and is applicable to diverse biological fields, including medical diagnostics.

SignificanceCerebral autoregulation (CA) reflects the dynamic coupling among cerebral blood flow (CBF), intracranial pressure (ICP), and arterial blood pressure (ABP); its failure contributes to secondary brain injury. Existing bedside methods rely on indirect or spatially limited CBF surrogates and cannot resolve microvascular flow dynamics across space, depth, and time. AimTo develop, optimize, and apply a scalable, noncontact time-resolved laser speckle contrast imaging (TR-LSCI) platform for depth-sensitive, high-speed, wide-field CBF imaging during controlled ICP perturbations. ApproachTR-LSCI synchronizes a 20-MHz pulsed laser with a time-gated, single-photon avalanche diode (SPAD) camera (512 x 512 pixels) to detect diffuse photons at varying path lengths, enabling depth-resolved microvascular CBF imaging. Benchtop and mobile TR-LSCI systems were applied in adult rats and a neonatal piglet with synchronized invasive ICP and ABP measurements. ResultsTR-LSCI captured spatially heterogeneous, pulsatile CBF dynamics at up to 52 Hz over large cortical fields of view, with heart rate estimates statistically equivalent to those from ICP and ABP. Multivariable analysis identified reproducible, phase-dependent CA transitions encompassing preserved autoregulation, ABP-driven compensation, and ICP-constrained CBF suppression; notably, CBF alone exhibited distinct phase signatures. ConclusionsTR-LSCI enables dynamic, physiology-informed neurovascular monitoring and supports future bedside CA assessment.

Investigating neural circuit dynamics across distributed brain regions in awake, behaving animals is crucial for understanding complex behavior. Genetically encoded voltage indicators (GEVIs) offer a powerful approach to tracking transmembrane voltage with high temporal and cellular specificity. However, scaling high-sensitivity GEVI recordings across multiple brain regions and multiple animals simultaneously remains a major technical challenge. Furthermore, it is unclear whether soma-targeted GEVIs - typically used for single-cell resolution imaging - can be effectively adapted for fiber photometry. Here, we show that a sCMOS-based widefield imaging system achieves sensitive dual-color multi-site fiber photometry using soma-targeted GEVI indicators with high temporal resolution. We validated this approach in the mouse hippocampal CA1, capturing theta (3-10Hz) and gamma (30-80Hz) rhythms and theta-gamma cross-frequency coupling. Additionally, we recorded high-frequency neural entrainment (>100 Hz) and somatic depolarization induced by electrical stimulation in CA1. Lastly, we tracked synchronized neural activity between the bilateral CA1s as well as multi-site dual-color imaging across CA1 and cortex simultaneously in three freely running mice. This work provides a scalable, accessible platform for high-speed optical electrophysiology in distributed neural circuits. Key points- Implementation of a sCMOS-based widefield imaging setup for sensitive and scalable fiber photometry and cellular imaging. - Demonstration of population multi-site and inter-animal voltage imaging with soma-targeted genetically encoded voltage indicators. - Tracking of high-frequency gamma and high-gamma (>100Hz) neural entrainment

The recent development of optogenetic tools, to manipulate neuronal activity using light, provides opportunities for novel brain-machine interface (BMI) control systems for treating neurological conditions. An issue of critical importance, therefore, is how well light penetrates through brain tissue. We took two different approaches to estimate light penetration through rodent brain tissue. The first employed so-called "nucleated patches" from cells expressing the light-activated membrane channel, channelrhodopsin (ChR2). By recording light-activated currents, we used these nucleated patches as extremely sensitive, microscopic, biological light-meters, to measure light penetration through 300-700{micro}m thick slices of rodent neocortical tissue. The nucleated patch method indicates that the effective illumination drops off with increasing tissue thickness, corresponding to a space constant of 317{micro}m (95% confidence interval between 248-441{micro}m). We compared this with measurements taken from directly visualizing the illumination of brain tissue, orthogonal to the direction of the light. This yielded a contour map of reduced illumination with distance, which along the direction of light delivery, had a space constant,{tau} 453{micro}m. This yields a lower extinction coefficient, {micro}e (the reciprocal of{tau} , [~]3mm-1) than previous estimates, implying better light penetration from LED sources than these earlier studies suggest.

Real-world neuroimaging requires true portability and sensitivity to detect signals from neuronal activity. Conventional methods such as MRI, EEG, or PET are constrained by their size and instrumentation complexity. Near Infra-Red (NIR) based optical methods have the potential for sensitive detection with a smaller footprint. Despite rapid advances in NIR detection there is no known device that is implementable using off-the-shelf integrated microprocessors and possesses the above desired characteristics along with proven sensitivity to detect task evoked responses. Here, we present a Bluetooth Low Energy (BLE)-enabled, high-sensitivity wearable functional near-infrared spectroscopy (fNIRS) device designed for untethered cortical hemodynamic monitoring during naturalistic cognitive tasks. Our fully integrated optical sensing device merges optical data acquisition and wireless transmission into a compact, cable-free platform with a very small footprint. This enables continuous multi-channel recording without placing any constraint on the subjects movement. We validate our device for reliable detection of task-evoked oxy- and deoxy-hemoglobin dynamics in the forearm, primary motor cortex, primary visual cortex, and prefrontal cortex. Subsequently, we capture real-time forebrain activity during a screen-based learning and memory task, revealing robust goal-specific hemodynamic responses. We formulate a method to identify the instances of peak neuronal activity and follow the "task Evoked Instances of Differential Oxygen influx(tEIDO)" as the subject is engaged in a task. These results highlight the potential of our proposed fNIRS device as a mobile neuroimaging solution for next-generation brain-computer interfaces and real-world cognitive monitoring. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/690628v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1f55855org.highwire.dtl.DTLVardef@7a67b8org.highwire.dtl.DTLVardef@2f6146org.highwire.dtl.DTLVardef@9b6e0c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Recently, speckle contrast optical spectroscopy (SCOS) enabled non-invasive, high signal-to-noise-ratio (SNR) human cerebral blood flow (CBF) measurements, relevant for both neuroscience and clinical monitoring of diseases with CBF dysregulation. Single channel SCOS measurements limit the information obtained to only one location on the head. In this work, we develop a multi-channel SCOS system to map spatial heterogeneity in CBF changes during human brain activation. Using a galvanometer, we temporally multiplexed a free-space laser to 7 source fibers positioned at different locations on the head. Diffuse light collected from the tissue is captured by fiber bundles projecting to 17 complementary metal-oxide semiconductor (CMOS) cameras, resulting in 50 source-detector channels measuring optical density (OD) and relative CBF changes covering an area of 7.6 cm by 6.6 cm on the head. We validated the spatial specificity and stability of the system using a liquid flow phantom. We then measured brain activity during a word-color Stroop task in 15 subjects and obtained brain activation maps. The average signal changes in the channel showing the largest activation were 1.7 x 10-2 in {Delta}OD and 6.6% in CBF.

Nerve damage during surgery is a common and serious complication, often leading to chronic pain, functional impairments, and diminished quality of life. However, existing methods for intraoperative nerve identification remain insufficient, especially for detecting small or hidden nerve branches. Here we present a new application of a clinically approved fluorescent agent, sodium fluorescein, to enhance nerve visualization during surgery. Utilizing both clinical and customized imaging systems, fluorescein remarkably improved nerve contrast, revealing structures undetectable with white light, including small branches embedded within tissues. With its established safety profile, low cost, and immediate clinical applicability, sodium fluorescein offers the potential to revolutionize surgical practice by minimizing nerve injuries and improving patient outcomes. Clinical Trial Registration: NCT06054178. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/25321923v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@5d9d90org.highwire.dtl.DTLVardef@1378ecorg.highwire.dtl.DTLVardef@68097dorg.highwire.dtl.DTLVardef@47034b_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionInvestigating cerebral oxygen saturation dynamics in awake animal models remains technically challenging due to motion artifacts and anesthesia-related biases. Here, we introduce a novel high-resolution ultrasound-photoacoustic (PA) imaging approach enabling real-time, non-invasive monitoring of deep cerebrovascular oxygenation dynamics in awake mice with intact skulls. Materials and MethodsSwiss male and female mice (n = 5-6) were head-fixed using a customized holder adapted to the Neurotar Mobile HomeCage floating platform. High-resolution ultrasound combined with PA imaging (VevoLAZR-X, VisualSonics) was used to discriminate oxyhemoglobin, deoxyhemoglobin, and total hemoglobin in multiple brain regions. Cerebrovascular responses were assessed under three paradigms: (i) baseline awake state vs. 2% isoflurane anesthesia, and (ii) right whisker stimulation to probe sensory-driven hemodynamics. ResultsPA imaging successfully resolved deep-brain oxygenation in awake, intact-skull mice. Under isoflurane anesthesia, we observed a rapid and transient increase in cerebrovascular sO{square} (p < 0.01). During whisker stimulation, we detected robust, region-specific increases in total hemoglobin, reflecting localized neurovascular coupling in awake mice. ConclusionsThis study establishes high-resolution PA imaging as a powerful, non-invasive tool to monitor cerebrovascular oxygenation dynamics in awake mice. By integrating baseline, anesthetic, and sensory paradigms, we demonstrate its potential to dissect neurovascular physiology without the confounding effects of anesthesia. These findings provide new opportunities for preclinical neuroscience research and translational applications investigating cerebral oxygen metabolism.

Alzheimers disease pathology typically manifests itself across multiple brain regions yet assessment at this scale in mouse models remains a challenge. This hinders the development of novel therapeutic approaches. Here we introduce a novel fiber photometry approach to monitor amyloid pathology in freely behaving mice. We first demonstrated that flat fiber-based photometry can detect amyloid signals across multiple brain regions under anesthesia after injecting a blood-brain barrier permeable tracer, Methoxy-X04. The depth profile of in vivo fluorescent signals was correlated with postmortem histological plaque signals. After confirming its feasibility ex vivo, we chronically implanted a tapered fiber for depth-resolved fiber photometry in freely behaving mice. After injecting Methoxy-X04, fluorescent signals increased in a depth-specific manner in Alzheimers mice, but not in wild-type littermates. While fiber photometry has been widely adopted to monitor neuronal and non-neuronal activity, our approach expands the capabilities to monitor molecular pathologies such as amyloid plaques, even in a freely behaving condition.

Traditional methods for mapping cerebral blood flow (CBF), such as positron emission tomography and magnetic resonance imaging, offer only isolated snapshots of CBF due to scanner logistics. Speckle contrast optical tomography (SCOT) is a promising optical technique for mapping CBF. However, while SCOT has been established in mice, the method has not yet been demonstrated in humans - partly due to a lack of anatomical reconstruction methods and uncertainty over the optimal design parameters. Herein we develop SCOT reconstruction methods that leverage MRI-based anatomical head models and finite-element modeling of the SCOT forward problem (NIRFASTer). We then simulate SCOT for CBF perturbations to evaluate sensitivity of imaging performance to exposure time and SD-distances. We find image resolution comparable to intensity-based diffuse optical tomography at superficial cortical tissue depth ([~]1.5 cm). Localization errors can be reduced by including longer SD-measurements. With longer exposure times speckle contrast decreases, however, noise decreases faster, resulting in a net increase in SNR. Specifically, extending exposure time from 10s to 10ms increased SCOT SNR by 1000X. Overall, our modeling methods provide anatomically-based image reconstructions that can be used to evaluate a broad range of tissue conditions, measurement parameters, and noise sources and inform SCOT system design.