Biology subjects

Dunn, A. K.

Publications and source records attributed to Dunn, A. K..

15 recordsLinked to original sources

bioRxiv2026

Label-Free, Physics-Constrained Learning for Multi-Exposure Speckle Imaging Parameter Estimation

Multi-exposure speckle imaging (MESI) estimates flow-related parameters by fitting a physics-based speckle contrast model to measurements acquired over multiple exposure times. In standard pipelines, parameters are recovered via nonlinear least-squares fitting at each pixel, which is computationally expensive and can yield spatially inconsistent maps when uncertainty in the estimated speckle contrast variance [Formula] (from camera noise and finite spatial/temporal sampling used to compute speckle contrast) is amplified by independent pixel wise inversion. This work reframes MESI parameter estimation as identification of a globally shared inverse operator of the analytical forward model, exploiting the fact that a single physical mapping governs all pixels while noise drives large variance in independent pixel wise inversion. Rather than solving millions of iterative optimizations, a single parameterized inverse mapping is learned directly from a single acquired MESI dataset. Physics consistency is enforced by embedding the fixed MESI forward model as an analysis-by-synthesis layer that re-synthesizes speckle contrast curves from the predicted parameters. Training is self-supervised: the inverse mapping is optimized by minimizing a reconstruction loss between measured and re-synthesized speckle contrast curves, which constrains estimates to the set of physically admissible MESI curves without requiring ground truth parameter labels. Experiments on a numerical MESI phantom with known ground truth and on in vivo mouse cortex data show that the proposed method produces more stable inverse correlation time (ICT) maps (1/{tau}c) and improved spatial coherence relative to conventional per-pixel fitting, while substantially reducing inference time by replacing iterative optimization with a single feed-forward evaluation.

neuroscience↗

bioRxiv2025

Improving retention time of Indocyanine Green for in vivo two-photon microscopy using liposomal encapsulation

We propose a technique for improving the retention time of Indocyanine Green (ICG), an FDA-approved, widely available, near infrared (NIR) probe for in vivo imaging of the cerebrovascular structure in rodents. We present a synthesis process for liposomal nanoparticles that, when used to encapsulate ICG, significantly increase the circulation time of the vascular label. Further, we conduct in vivo imaging experiments with unencapsuated (free) ICG and liposomal ICG (L-ICG) and compare the retention of ICG in the vascular network. Our work tackles one of the most fundamental limitations in using ICG i.e. rapid clearance, for the purpose of two-photon microscopy.

neuroscience↗

bioRxiv2024

Non-Degenerate Two-Photon Imaging of Deep Rodent Cortex using Indocyanine Green in the water absorption window

We present a novel approach for deep vascular imaging in rodent cortex at excitation wavelengths susceptible to water absorption using two-photon microscopy with photons of dissimilar wavelengths. We demonstrate that non-degenerate two-photon excitation (ND-2PE) enables imaging in the water absorption window from 1400-1550 nm using two synchronized excitation sources at 1300 nm and 1600 nm that straddle the absorption window. We explore the brightness spectra of indocyanine green (ICG) and assess its suitability for imaging in the water absorption window. Further, we demonstrate in vivo imaging of the rodent cortex vascular structure up to 1.2 mm using ND-2PE. Lastly, a comparative analysis of ND-2PE at 1435 nm and single-wavelength, two-photon imaging at 1300 nm and 1435 nm is presented. Our work extends the excitation range for fluorescent dyes to include water absorption regimes and underscores the feasibility of deep two-photon imaging at these wavelengths.

neuroscience↗

bioRxiv2023

Microvascular plasticity in stroke recovery: Longitudinal snapshots, network statistical analysis, and dynamics

This research article quantitatively investigates neuro-microvascular network remodeling dynamics following stroke using a novel in vivo two-photon angiography (cubic millimeter volume, weekly snapshots) and high throughput (thousands of connected capillaries) vascular vectorization method. The results suggest distinct temporal patterns of cere-brovascular plasticity, with acute remodeling peaking at one week post-stroke. The network architecture then gradually stabilizes, returning to a new steady state after four weeks. These findings align with previous literature on neuronal plasticity, highlighting the correlation between neuronal and neurovascular remodeling. Quantitative analysis of neurovascular networks using length- and strand-based statistical measures reveals intri-cate changes in network anatomy and topology. The distance and strand-length statistics show significant alterations, with a peak of plasticity observed at one week post-stroke, followed by a gradual return to baseline. The orientation statistic plasticity peaks at two weeks, gradually approaching the (conserved across subjects) stroke signature. The underlying mechanism of the vascular response (angiogenesis vs. tissue deformation), however, is yet unelucidated, requiring network registration advancements. Overall, the combination of two-photon angiography, vectorization, reconstruction/visualization, and statistical analysis enables both qualitative and quantitative assessments of neu-rovascular remodeling dynamics, demonstrating an impactful method for investigating neuro-microvascular network disorders and the therapeutic modes of action thereof. Understanding the timing and nature of neurovascular remodeling allows for optimized interventions, including personalized medicine for stroke rehabilitation. Additionally, the evaluation of pharmaceutical interventions using these tools may facilitate targeted drug development. Furthermore, neurovascular coupling dynamics have implications for neurodegenerative diseases, brain aging, and the field of brain-computer interfaces.

neuroscience↗

bioRxiv2023

Pulse train gating to improve signal generation for in vivo two-photon fluorescence microscopy

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.

neuroscience↗

bioRxiv2022

A deep learning approach for improving two-photon vascular imaging speeds

A potential method for tracking neurovascular disease progression over time in preclinical models is multiphoton fluorescence microscopy (MPM), which can image cerebral vasculature with capillary-level resolution. However, obtaining high-quality, three-dimensional images with a traditional point scanning MPM is time-consuming and limits sample sizes for chronic studies. Here, we present a convolutional neural network-based algorithm for fast upscaling of low-resolution or sparsely sampled images and combine it with a segmentation-less vectorization process for 3D reconstruction and statistical analysis of vascular network structure. In doing so, we also demonstrate that the use of semi-synthetic training data can replace the expensive and arduous process of acquiring low- and high-resolution training pairs without compromising vectorization outcomes, and thus open the possibility of utilizing such approaches for other MPM tasks where collecting training data is challenging. We applied our approach to large field of view images and show that our method generalizes across imaging depths, disease states and other differences in neurovasculature. Our pre-trained models and lightweight architecture can be used to reduce MPM imaging time by up to fourfold without any changes in underlying hardware, thereby enabling deployability across a range of settings.

bioengineering↗

bioRxiv2022

Using pressure-driven flow systems to evaluate laser speckle contrast imaging

SignificanceMicrofluidic flow phantom studies are commonly used for characterizing the performance of laser speckle contrast imaging (LSCI) instruments. The selection of the flow control system is critical for the reliable generation of flow during testing. The majority of recent LSCI studies using microfluidics used syringe pumps for flow control. AimWe quantified the uncertainty in flow generation for a syringe pump and a pressure-regulated flow system. We then assessed the performance of both LSCI and multi-exposure speckle imaging (MESI) using the pressure-regulated flow system across a range of flow speeds. ApproachThe syringe pump and pressure-regulated flow systems were evaluated during stepped flow profile experiments in a microfluidic device using an inline flow sensor. The uncertainty associated with each flow system was calculated and used to determine the reliability for instrument testing. The pressure-regulated flow system was then used to characterize the relative performance of LSCI and MESI during stepped flow profile experiments while using the inline flow sensor as reference. ResultsThe pressure-regulated flow system produced much more stable and reproducible flow outputs compared to the syringe pump. The expanded uncertainty for the syringe pump was 8-20x higher than that of the pressure-regulated flow system across the tested flow speeds. Using the pressure-regulated flow system, MESI outperformed single-exposure LSCI at all flow speeds and closely mirrored the flow sensor measurements, with average errors of 4.6 {+/-} 2.6% and 15.7 {+/-} 4.6%, respectively. ConclusionsPressure-regulated flow systems should be used instead of syringe pumps when assessing the performance of flow measurement techniques with microfluidic studies. MESI offers more accurate relative flow measurements than traditional LSCI across a wide range of flow speeds.

bioengineering↗

bioRxiv2022

Subventricular zone cytogenesis provides trophic support for neural repair

Stroke enhances proliferation of neural precursor cells within the subventricular zone (SVZ) and induces ectopic migration of newborn cells towards the site of injury. Here we characterize the identity of cells arising from the SVZ after stroke and provide insight into their function by uncovering a mechanism through which they facilitate neural repair and functional recovery. Using genetic lineage tracing, we show that SVZ-derived cells that migrate towards stroke- induced cortical lesions in mice are predominantly undifferentiated precursors, suggesting that the main function of post-injury cytogenesis is not cell replacement. We find that SVZ-derived cells are a unique cellular source of trophic factors that instruct neural repair. Chemogenetic ablation of neural precursor cells or conditional knockout of VEGF in the adult neural stem cell lineage impairs neuronal and vascular reparative responses and worsens functional recovery after stroke. In addition, normal aging markedly diminishes the cytogenic response to stroke, resulting in worse functional recovery. Therapeutic replacement of VEGF in peri-infarct cortex is sufficient to induce neural repair and functional recovery in mice with arrested cytogenesis. These findings indicate that the SVZ cytogenic response following brain injury is a source of trophic support that drives neural repair and recovery.

neuroscience↗

bioRxiv2021

An evaluation of resonant scanning as a high-speed imaging technique for two-photon imaging of cortical vasculature

We demonstrate a simple, low-cost two-photon microscope design with both galvo-galvo and resonant-galvo scanning capabilities. We quantify and compare the signal-to-noise ratios and imaging speeds of the galvo-galvo and resonant-galvo scanning modes when used for murine neurovascular imaging. The two scanning modes perform as expected under shot-noise limited detection and are found to achieve comparable signal-to-noise ratios. Resonant-galvo scanning is capable of reaching desired signal-to-noise ratios using less acquisition time when higher excitation power can be used. Given equal excitation power and total pixel dwell time between the two methods, galvo-galvo scanning outperforms resonant-galvo scanning in image quality when detection deviates from being shot-noise limited.

neuroscience↗

bioRxiv2021

Diamond Raman Laser and Yb Fiber Amplifier for In Vivo Multiphoton Fluorescence Microscopy

Here we introduce a fiber amplifier and a diamond Raman laser that output high powers (6.5 W, 1.3 W) at valuable wavelengths (1060 nm, 1250 nm) for multiphoton excitation of red-shifted fluorophores. These custom excitation sources are both simple to construct and cost-efficient in comparison to similar custom and commercial alternatives. Furthermore, they operate at a repetition rate (80 MHz) that allows fast image acquisition using resonant scanners. We demonstrate our systems compatibility with fast resonant scanning, the ability to acquire neuronal images, and the capability to image vasculature at deep locations (>1 mm) within the mouse cerebral cortex.

neuroscience↗

bioRxiv2021

A large lipoprotein mediates target specificity for T6SS-dependent killing

Interbacterial competition is prevalent in host-associated microbiota, where it can shape community structure and function, impacting host health in both positive and negative ways. However, the factors that permit bacteria to discriminate among their various neighbors for targeted elimination of competitors remain elusive. We identified a specificity factor in Vibrio species that is used to target specific competitors for elimination. Here, we describe this specificity factor, which is associated with the broadly-distributed type VI secretion system (T6SS), by studying symbiotic Vibrio fischeri, which use the T6SS to compete for colonization sites in their squid host. We demonstrate that a large lipoprotein (TasL) allows V. fischeri cells to restrict T6SS-dependent killing to certain genotypes by selectively integrating competitor cells into aggregates while excluding other cell types. TasL is also required for T6SS-dependent competition within juvenile squid, indicating the adhesion factor is active in the host. Because TasL homologs are found in other host-associated bacterial species, this newly-described specificity factor has the potential to impact microbiome structure within diverse hosts.

microbiology↗

bioRxiv2020

Visualizing anesthesia-induced vasodilation of cerebral vasculature using multi-exposure speckle imaging

BackgroundAnesthetized animal models are used extensively during neurophysiological and behavioral studies despite systemic effects from anesthesia that undermine both accurate interpretation and translation to awake human physiology. The majority of work examining the impact of anesthesia on cerebral blood flow (CBF) has been restricted to before and after measurements with limited spatial resolution. New MethodWe used multi-exposure speckle imaging (MESI), an advanced form of laser speckle contrast imaging (LSCI), to characterize the dynamics of isoflurane anesthesia induction on cerebral vasculature and blood flow in the mouse brain. ResultsThe large anatomical changes caused by isoflurane are depicted with wide-field imagery and video highlighting the induction of general anesthesia. Within minutes of exposure, both vessel diameter and blood flow increased drastically compared to the awake state and remained elevated for the duration of imaging. An examination of the dynamics of anesthesia induction reveals that blood flow increased faster in arteries than in veins or parenchyma regions. Comparison with Existing MethodsMESI offers robust hemodynamic measurements across large fields-of-view and high temporal resolutions sufficient for continuous visualization of cerebrovascular events featuring major changes in blood flow. ConclusionThe large alterations caused by isoflurane anesthesia to the cortical vasculature and CBF are readily characterized using MESI. These changes are unrepresentative of normal physiology and provide further evidence that neuroscience experiments would benefit from transitioning to un-anesthetized awake animal models.

neuroscience↗

bioRxiv2020

Establishing the Effect of Vascular Structure on Laser Speckle Contrast Imagining

Laser Speckle Contrast Imaging (LSCI) is a powerful tool for non-invasive, real-time imaging of blood flow in tissue. However, the effect of tissue geometry on the form of the electric field autocorrelation function and speckle contrast values is yet to be investigated. In this paper, we present an ultrafast forward model for simulating a speckle contrast image with the ability to rapidly update the image for a desired illumination pattern and flow perturbation. We demonstrate the first simulated speckle contrast image and compare it against experimental results. We simulate three mouse-specific cerebral cortex decorrelation time images and implement three different schemes for analyzing the effects of homogenization of vascular structure on correlation decay times. Our results indicate that dissolving structure and assuming homogeneous geometry creates up to ∼ 10x shift in the correlation function decay times and alters its form compared with the case for which the exact geometry is simulated. These effects are more pronounced for point illumination and detection imaging schemes. Further analysis indicates that correlated multiple scattering events, on average, account for 50% of all dynamic scattering events for a detector over a vessel region and 31% that of a detector over parenchyma region, highlighting the significance of accurate modeling of the three-dimensional vascular geometry for accurate blood flow estimates.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗

bioRxiv2020

Multimodal mapping of neural activity and cerebral blood flow reveals long-lasting neurovascular dissociations after small-scale strokes

Neurovascular coupling, the close spatial and temporal relationship between neural activity and hemodynamics, is disrupted in pathological brain states. To understand the altered neurovascular relationship in brain disorders, longitudinal, simultaneous mapping of neural activity and hemodynamics is critical yet challenging to achieve. Here, we employ a multimodal neural platform in a mouse model of stroke and realize long-term, spatially-resolved tracking of intracortical neural activity and cerebral blood flow in the same brain regions. We observe a pronounced neurovascular dissociation that occurs immediately after small-scale strokes, becomes the most severe a few days after, lasts into chronic periods, and varies with the level of ischemia. Neuronal deficits extend spatiotemporally whereas restoration of cerebral blood flow occurs sooner and reaches a higher relative value. Our findings reveal the neurovascular impact of mini-strokes and inform the limitation of neuroimaging techniques that infer neural activity from hemodynamic responses.

neuroscience↗

bioRxiv2017

In vivo multiphoton fluorescence imaging with polymer dots

Deep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep multiphoton imaging has never before been demonstrated. Here we characterize the multiphoton properties of three pdot variants (CNPPV, PFBT, and PFPV) and demonstrate deep imaging of cortical microvasculature in C57 mice. Specifically, we measure the two-versus three-photon power dependence of these pdots and observe a clear three-photon excitation signature at wavelengths longer than 1300 nm, and a transition from two-photon to three-photon excitation within a 1060 - 1300 nm excitation range. Furthermore, we show that pdots enable in vivo two-photon imaging of cerebrovascular architecture in mice up to 850 m beneath the pial surface using 800 nm excitation. In contrast with traditional multiphoton probes, we also demonstrate that the broad multiphoton absorption spectrum of pdots permits imaging at longer wavelengths ({lambda}ex = 1,060 and 1225 nm). These wavelengths approach an ideal biological imaging wavelength near 1,300 nm and confer compatibility with a high-power ytterbium-fiber laser and a high pulse energy optical parametric amplifier, resulting in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths of 900 m and 1300 m. Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, which will undoubtedly unlock the ability to interrogate deep structures in vivo.

bioengineering↗