Biology subjects

Schaffer, C. B.

Publications and source records attributed to Schaffer, C. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Depth-Dependent Advantages of Three-Photon Microscopy for Imaging Through Intact Murine Cortical Bone and Into the Marrow

Bone adapts to its mechanical and physiological environment through the coordinated actions of osteocytes and marrow-resident cell populations. Because these processes depend on complex interactions within native tissue microenvironments, intravital imaging offers a unique opportunity to reveal cellular behaviors that cannot be fully captured ex vivo. However, the highly scattering nature of mineralized bone limits imaging depth and direct observation of cells within intact tissue. Two-photon (2P) microscopy has enabled important advances in bone biology, while three-photon (3P) microscopy has been proven to extend imaging depth and image quality in skeletal tissues. Here, we directly compared the performance of 2P and 3P microscopy in ex vivo mouse long bones by quantifying laser attenuation, signal-to-noise ratio, signal-to-background ratio, and spatial resolution, as well as assessed the impact of wave-front correction. We found that 2P and 3P microscopy generated comparable image quality through approximately 50 {micro}m of cortical bone. Beyond this depth, however, 3P microscopy provided superior brightness, contrast, and resolution, enabling improved visualization of structures deep within the cortex and at the cortical-marrow interface. To assess compatibility with intravital imaging, we evaluated endogenous markers of cellular stress during 3P imaging. Although prolonged continuous imaging decreased osteocyte spontaneous calcium signaling magnitude and increased autofluorescence, short intermittent imaging bouts produced negligible evidence of cellular damage compared to controls. Leveraging the enhanced penetration depth of 3P microscopy, we further show visualization of immune cell migration within the marrow cavity through intact cortical bone in both the third metatarsal (MT3) and tibia. Together, these findings affirm 3P microscopy as a powerful tool for studying cellular dynamics in living bone. By extending imaging beyond superficial cortical regions and enabling direct visualization of marrow-resident cells through intact bone, 3P microscopy expands opportunities to investigate osteocyte biology, marrow niche function, and skeletal adaptation in vivo.

bioengineering↗

bioRxiv2026

Reprogrammed neutrophils with impaired transit mechanics drive multi-organ capillary stalling after stroke

Beyond the focal brain lesion, stroke causes systemic complications including cardiac failure, pneumonia, renal injury, and sustained immune dysfunction. The source of this multiorgan vulnerability remains unresolved. By imaging over 16,000 vessels of healthy, inflamed and ischemic brains, we identify a circulating neutrophil subpopulation reprogrammed by stroke into a pathological stalling phenotype, occluding capillaries in the brain, heart, kidneys, retina and lungs. Combining transcriptomics, genetic models, integrated microfluidics, cell mechanics assays, and in vivo imaging, we show that this subpopulation exhibits an atypical morphology, increased actin polymerization, and heightened adhesion that impair transit through capillary networks. This phenotype is present in patients with stroke, transmissible by adoptive transfer, and selectively sensitive to inhibition of the Src-family kinase Fgr. Both pharmacological and genetic inactivation of Fgr normalize neutrophil adhesion, reduce capillary stalls, and improve neurological recovery after stroke. These findings identify immune cell transit failure as a systemic driver of post-stroke pathology and a therapeutic target to improve both cerebral and multiorgan outcomes.

neuroscience↗

bioRxiv2026

Scattering-enabled epi-quantitative phase imaging reveals subcellular detail in organoids and deep mouse brains

Imaging subcellular structures deep within thick, turbid biological tissues remains fundamentally limited by light scattering, which distorts optical wavefronts and degrades contrast, resolution, and sensitivity. These limitations hinder quantitative interrogation of complex biological systems where resolving dynamic microenvironments at subcellular resolution is critical. Here, we introduce scattering-enabled epi-quantitative phase imaging (SEEQPI), a label-free method that leverages tissue scattering and provides subcellular spatial resolution, nanometer-scale spatiotemporal phase sensitivity, and millimeter-scale imaging depth in murine brains. SEEQPI is enabled by common-path phase-shifting confocal epi-interferometry with near-infrared illumination and the scattering-enabled phase reconstruction algorithm. SEEQPI requires low illumination power, minimizing tissue damage while enabling high-speed imaging of biological dynamics. We demonstrate simultaneous, colocalized imaging of subcellular structures with SEEQPI, third-harmonic generation, and three-photon fluorescence microscopy in liver cancer spheroids and in vivo mouse brains. SEEQPI enables quantitative, longitudinal studies of dry mass dynamics in intact, living biological systems.

bioengineering↗

bioRxiv2025

Label-free Tracking of Subcortical White Matter Degradation In Vivo Using Third Harmonic Generation Microscopy in a Mouse Model of Multiple Sclerosis

Early detection and characterization of myelin degradation in the white matter (WM) is important for understanding neurodegenerative diseases such as multiple sclerosis (MS) and dementia. Here, we demonstrate a label-free in vivo imaging technique using third harmonic generation (THG) microscopy at 1320-nm excitation, which enables visualization of subcellular myelin structural changes deep in the mouse brain. By applying longitudinal THG imaging of the same axons in the cuprizone mouse model of MS, we captured the formation, evolution, and regression of myelin blisters in the corpus callosum through intact cortex - a depth inaccessible to other optical methods. We also confirmed the utility of conducting THG imaging in parallel with three-photon excited fluorescence imaging, which allowed us to determine that myelin blistering events were not correlated with the location of microglia cell bodies. Further, we used post-mortem immunohistochemistry to establish the ability to identify and measure the intranodal distance at nodes of Ranvier using in vivo THG imaging, and we found increased intranodal distances after cuprizone administration. We also developed a novel quantitative metric based on spatial concentration of the brightest THG signal that occurs before more overt changes like myelin blistering. Overall, THG microscopy offers a powerful method for detailed tracking of subcortical myelin dynamics, providing new opportunities to investigate disease mechanisms and potential therapeutic interventions in MS and other neurodegenerative diseases.

bioengineering↗

bioRxiv2025

Psilocybin Prolongs the Neurovascular Coupling Response in Mouse Visual Cortex

Psilocybin has profound therapeutic potential for various mental health disorders, but its mechanisms of action are unknown. Functional MRI studies have reported the effects of psilocybin on brain activity and connectivity; however, these measurements rely on neurovascular coupling to infer neural activity changes and assume that blood flow responses to neural activity are not altered by psilocybin. Using two-photon excited fluorescence imaging in the visual cortex of awake mice to simultaneously measure neural activity and capillary blood flow dynamics, we found that psilocybin administration prolonged the increase in visual stimulus-evoked capillary blood flow - an effect which was reduced by pretreatment with a 5-HT2AR antagonist - despite not causing changes in the stimulus-evoked neural response. Multi-modal widefield imaging also showed that psilocybin extends the stimulus-evoked vascular responses in surface vessels with no observed effect on the population neural response. Computational simulation with a whole-brain neural mass model showed that prolonged neurovascular coupling responses can lead to spurious increases in BOLD-based measures of functional connectivity. Together, these findings demonstrate that psilocybin broadens neurovascular responses in the brain and highlights the importance of accounting for these effects when interpreting human neuroimaging data of psychedelic drug action.

neuroscience↗

bioRxiv2025

Neutrophil arrest in myocardial capillaries drives hypoxia and impairs diastolic function in a mouse model of heart failure with preserved ejection fraction

Impairments in myocardial blood flow have been recognized for decades in human patients with and animal models of heart failure with preserved ejection fraction (HFpEF), but the underlying mechanisms and roles in pathogenesis remain poorly understood. Using intravital cardiac microcopy in a two-hit mouse model of HFpEF that combines high fat diet and inhibition of nitric oxide synthase, we identified an increase in slow or non-flowing neutrophils in capillaries compared to Control mice. In other mouse models of disease (e.g., in the brain of Alzheimers Disease mice), the presence of such stalled neutrophils leads to organ-wide decreases in perfusion and oxygenation. Administration of antibodies against the neutrophil surface protein Ly6G to deplete neutrophils reduced the number of arrested neutrophils in myocardial capillaries, leading to improvements in myocardial hypoxia, diastolic function, and exercise capacity. This study identifies a previously uncharacterized cellular mechanism that explains myocardial blood flow deficits in mouse models of HFpEF, and demonstrates that improving myocardial blood flow improves cardiac function, without necessitating reversal of pathologic remodeling. Restoring myocardial perfusion by decreasing neutrophil arrest in myocardial capillaries may provide a strategy for improving heart function in HFpEF patients in the future.

physiology↗

bioRxiv2024

Three-photon excited fluorescence microscopy enables imaging of blood flow, neural structure and inflammatory response deep into mouse spinal cord in vivo

Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 m into the mouse spinal cord, in vivo. We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 m of the murine spinal cord, in vivo.

neuroscience↗

bioRxiv2023

Bidirectional Regulation of Motor Circuits Using Magnetogenetic Gene Therapy

Here we report a novel suite of magnetogenetic tools, based on a single anti-ferritin nanobody-TRPV1 receptor fusion protein, which regulated neuronal activity when exposed to magnetic fields. AAV-mediated delivery of a floxed nanobody-TRPV1 into the striatum of adenosine 2a receptor-cre driver mice resulted in motor freezing when placed in an MRI or adjacent to a transcranial magnetic stimulation (TMS) device. Functional imaging and fiber photometry both confirmed activation of the target region in response to the magnetic fields. Expression of the same construct in the striatum of wild-type mice along with a second injection of an AAVretro expressing cre into the globus pallidus led to similar circuit specificity and motor responses. Finally, a mutation was generated to gate chloride and inhibit neuronal activity. Expression of this variant in subthalamic nucleus in PitX2-cre parkinsonian mice resulted in reduced local c-fos expression and motor rotational behavior. These data demonstrate that magnetogenetic constructs can bidirectionally regulate activity of specific neuronal circuits non-invasively in-vivo using clinically available devices. TeaserA novel magnetogenetics toolbox to regulate neural circuits in-vivo.

neuroscience↗

bioRxiv2023

RNA structures and dynamics with A resolution revealed by x-ray free electron lasers

RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free electron laser sources to reveal the formation and ready identification of [A] scale features in structured and unstructured RNAs. New structural signatures of RNA secondary and tertiary structures are identified through wide angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base paired intermediate to assume a triple helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. In addition to understanding how RNA triplexes form and thereby function as dynamic signaling elements, this new method can vastly increase the rate of structure determination for these biologically essential, but mostly uncharacterized macromolecules.

biophysics↗

bioRxiv2023

A quasi-analytic solution for real-time multi-exposure speckle imaging of tissue perfusion

Laser speckle contrast imaging (LSCI) is a widefield imaging technique that enables high spatiotemporal resolution measurement of blood flow. Laser coherence, optical aberrations, and static scattering effects restrict LSCI to relative and qualitative measurements. Multi-exposure speckle imaging (MESI) is a quantitative extension of LSCI that accounts for these factors but has been limited to post-acquisition analysis due to long data processing times. Here we propose and test a real-time quasi-analytic solution to fitting MESI data, using both simulated and real-world data from a mouse model of photothrombotic stroke. This rapid estimation of multi-exposure imaging (REMI) enables processing of full-frame MESI images at up to 8 Hz with negligible errors relative to time-intensive least-squares methods. REMI opens the door to real-time, quantitative measures of perfusion change using simple optical systems.

bioengineering↗

bioRxiv2023

MousePZT: A Simple, Reliable, Low-Cost Device for Vital Sign Monitoring and Respiratory Gating in Mice Under Anesthesia

Small animal studies in biomedical research often require anesthesia to reduce pain or stress experienced by research animals and to minimize motion artifact for imaging or other measurements. Anesthetized animals should be closely monitored to avoid complications and unintended effects of altered physiology during such procedures. Many currently available monitoring devices can be expensive, invasive, or interfere with experimental design. Here, we present a low-cost device, based on a simple piezoelectric sensor, with a custom circuit and computer software that allows for measurements of both respiratory rate and heart rate in a non-invasive, minimal contact manner. We find the accuracy of the MousePZT device in measuring respiratory and heart rate under anesthesia and with pharmacologically induced changes in heart rate match those of commercial or more invasive systems. We also demonstrate that changes in respiratory rate can precede changes in heart rate associated with alterations in anesthetic depth. Additional circuitry on the device outputs a respiration locked trigger signal for respiratory-gating of imaging or other data acquisition that has high sensitivity and specificity for detecting respiratory cycles. We provide detailed construction documents and all necessary microcontroller and computer software, enabling straightforward adoption of this device.

physiology↗

bioRxiv2023

Vascular oxidative stress causes neutrophil arrest in brain capillaries, leading to decreased cerebral blood flow and contributing to memory impairment in a mouse model of Alzheimer's disease

INTRODUCTIONIn this study, we explore the role of oxidative stress produced by NOX2-containing NADPH oxidase as a molecular mechanism causing capillary stalling and cerebral blood flow deficits in the APP/PS1 mouse model of AD. METHODSWe inhibited NOX2 in APP/PS1 mice by administering a 10 mg/kg dose of the peptide inhibitor gp91-ds-tat i.p., for two weeks. We used in vivo two-photon imaging to measure capillary stalling, penetrating arteriole flow, and vascular inflammation. We also characterized short-term memory function and gene expression changes in cerebral microvessels. RESULTSWe found that after NOX2 inhibition capillary stalling, as well as parenchymal and vascular inflammation, were significantly reduced. In addition, we found a significant increase in penetrating arteriole flow, followed by an improvement in short-term memory, and downregulation of inflammatory gene expression pathways. DISCUSSIONOxidative stress is a major mechanism leading to microvascular dysfunction in AD, and represents an important therapeutic target.

neuroscience↗

bioRxiv2021

Circumscribing laser cuts attenuate seizure propagation in a mouse model of focal epilepsy

In partial onset epilepsy, seizures arise focally in the brain and often propagate, causing acute behavior changes, chronic cognitive decline, and increased mortality. Patients frequently become refractory to medical management, leaving neurosurgical resection of the seizure focus as a primary treatment, which can cause neurologic deficits. In the cortex, focal seizures are thought to spread through horizontal connections in layers II/III, suggesting that selectively severing these connections could block seizure propagation while preserving normal columnar circuitry and function. We induced focal neocortical epilepsy in mice and used tightly-focused femtosecond-duration laser pulses to create a sub-surface, opencylinder cut surrounding the seizure focus and severing cortical layers II-IV. We monitored seizure propagation using electrophysiological recordings at the seizure focus and at distant electrodes for 3-8 months. With laser cuts, only 5% of seizures propagated to the distant electrodes, compared to 85% in control animals. Laser cuts also decreased the number of seizures that were initiated, so that the average number of propagated seizures per day decreased from 42 in control mice to 1.5 with laser cuts. Physiologically, these cuts produced a modest decrease in cortical blood flow that recovered within days and, at one month, left a ~20-m wide scar with increased gliosis and localized inflammatory cell infiltration but minimal collateral damage. When placed over motor cortex, cuts did not cause notable deficits in a skilled reaching task. Femtosecond laser produced sub-surface cuts hold promise as a novel neurosurgical approach for intractable focal cortical epilepsy, as might develop following traumatic brain injury. Once sentence summaryIn a mouse model of focal epilepsy, sub-surface laser-produced cuts encircling the seizure focus attenuate propagation without behavioral impairment.

neuroscience↗

bioRxiv2021

Inhibition of peripheral VEGF signaling rapidly reduces leucocyte obstructions in brain capillaries and increases cortical blood flow in an Alzheimers disease mouse model

Increased incidence of stalled capillary blood flow caused by adhesion of leucocytes to the brain microvascular endothelium leads to a 17% reduction of cerebral blood flow (CBF) and exacerbates short-term memory loss in multiple mouse models of Alzheimers disease (AD). Here, we report that Vascular Endothelial Growth Factor (VEGF) signaling at the luminal side of the brain microvasculature plays an integral role in the capillary stalling phenomenon of the APP/PS1 mouse model. Administration of the anti-mouse VEGF-A164 antibody, an isoform that inhibits blood brain barrier (BBB) hyperpermeability, reduced the number of stalled capillaries within an hour of injection, leading to an immediate increase in average capillary blood flow but not capillary diameter. VEGF-A inhibition also reduced the overall eNOS protein concentrations, increased occludin levels, and decreased the penetration of circulating Evans Blue dye across the BBB into the brain parenchyma, suggesting increased BBB integrity. Capillaries prone to neutrophil adhesion after anti-VEGF-A treatment also had lower occludin concentrations than flowing capillaries. Taken together, our findings demonstrate that VEGF-A signaling in APP/PS1 mice contributes to aberrant eNOS/occludin- associated BBB permeability, increases the incidence of capillary stalls, and leads to reductions in CBF. Reducing leucocyte adhesion by inhibiting luminal VEGF signaling may provide a novel and well-tolerated strategy for improving brain microvascular blood flow in AD patients.

neuroscience↗

bioRxiv2020

Voluntary running does not increase capillary blood flow but promotes neurogenesis and short-term memory in the APP/PS1 mouse model of Alzheimers disease

Exercise exerts a beneficial effect on the major pathological and clinical symptoms associated with Alzheimer s disease in humans and mouse models of the disease. While numerous mechanisms for such benefits from exercise have been proposed, a clear understanding of the causal links remains elusive. Recent studies also suggest that cerebral blood flow in the brain of both Alzheimer s patients and mouse models of the disease is decreased and that the cognitive symptoms can be improved when blood flow is restored. We therefore hypothesized that the mitigating effect of exercise on the development and progression of Alzheimer s disease may be mediated through an increase in the otherwise reduced brain blood flow. To test this idea, we examined the impact of three months of voluntary wheel running in [~]1-year-old APP/PS1 mice on short-term memory function, brain inflammation, amyloid deposition, and cerebral blood flow. Our findings that exercise led to improved memory function, a trend toward reduced brain inflammation, markedly increased neurogenesis in the dentate gyrus, and no changes in amyloid-beta deposits are consistent with other reports on the impact of exercise on the progression of Alzheimer s related symptoms in mouse models. Notably, we did not observe any impact of wheel running on overall cortical blood flow nor on the incidence of non-flowing capillaries, a mechanism we recently identified as one contributing factor to cerebral blood flow deficits in mouse models of Alzheimer s disease. Overall, our results replicate previous findings that exercise is able to ameliorate certain aspects of Alzheimer s disease pathology, but show that this benefit does not appear to act through increases in cerebral blood flow.

neuroscience↗

bioRxiv2019

High fat diet worsens pathology and impairment in an Alzheimers mouse model, but not by synergistically decreasing cerebral blood flow

Obesity is linked to increased risk for and severity of Alzheimers disease (AD). Cerebral blood flow (CBF) reductions are an early feature of AD and are also linked to obesity. We showed that non-flowing capillaries, caused by adhered neutrophils, underlie the CBF reduction in mouse models of AD. Because obesity could exacerbate the vascular inflammation likely underlying this neutrophil adhesion, we tested links between obesity and AD by feeding APP/PS1 mice a high fat diet (Hfd) and evaluating behavioral, physiological, and pathological changes. We found trends toward poorer memory performance in APP/PS1 mice fed a Hfd, impaired social interactions with either APP/PS1 genotype or a Hfd, and synergistic impairment of sensory-motor function in APP/PS1 mice fed a Hfd. The Hfd led to increases in amyloid-beta monomers and plaques in APP/PS1 mice, as well as increased brain inflammation. These results agree with previous reports showing obesity exacerbates AD-related pathology and symptoms in mice. We used a crowd-sourced, citizen science approach to analyze imaging data to determine the impact of the APP/PS1 genotype and a Hfd capillary stalling and CBF. Surprisingly, we did not see an increase in the number of non-flowing capillaries or a worsening of the CBF deficit in APP/PS1 mice fed a Hfd as compared to controls, suggesting capillary stalling is not a mechanistic link between a Hfd and increased severity of AD in mice. Reducing capillary stalling by blocking neutrophil adhesion improved CBF and short-term memory function in APP/PS1 mice, even when fed a Hfd. Significance statementObesity, especially in mid-life, has been linked to increased risk for and severity of Alzheimers disease. Here, we show that blocking adhesion of white blood cells leads to increases in brain blood flow that improve cognitive function, regardless of whether mice are obese or not.

neuroscience↗

bioRxiv2019

Non-invasive multiphoton imaging of neural structure and activity in Drosophila

We developed a multiphoton imaging method to capture neural structure and activity in behaving flies through the intact cuticles. Our measurements show that the fly head cuticle has surprisingly high transmission at wavelengths > 900 nm, and the difficulty of through-cuticle imaging is due to the air sacs and/or fat tissue underneath the head cuticle. By compressing the air sacs, we performed deep multiphoton imaging of fly brains through the intact cuticle. Our anatomical and functional imaging results show that 2- and 3-photon imaging are comparable in superficial regions such as the mushroom body, but 3-photon imaging is superior in deeper regions such as the central complex and beyond. We further demonstrated 2-photon through-cuticle functional imaging of odor-evoked calcium responses from the mushroom body {gamma}-lobes in behaving flies short-term and long-term (12 consecutive hours). The through-cuticle imaging method developed here extends the time limits of in vivo imaging in flies, and opens up new ways to capture neural structure and activity from the intact fly brain.

neuroscience↗

bioRxiv2019

Neutrophil-mediated dynamic capillary stalls in ischemic penumbra: persistent traffic jams after reperfusion contribute to injury

Ever since the introduction of thrombolysis and the subsequent expansion of endovascular treatments for acute ischemic stroke, it remains to be identified why the actual outcomes are less favorable despite recanalization. Here, by high spatio-temporal resolution imaging of capillary circulation in mice, we introduce the pathological phenomenon of dynamic flow stalls in cerebral capillaries, occurring persistently in the salvageable penumbra after recanalization. These stalls, which are distinct from permanent cellular plugs that can lead to no-flow, were temporarily and repetitively occurring in the capillary network, impairing the overall circulation like small focal traffic jams. In vivo microscopy in the ischemic penumbra revealed leukocytes traveling through capillary lumen or getting stuck, while red blood cell flow was being disturbed in the neighboring segments, within 3 hours after stroke onset. Stall dynamics could be modulated, by injection of an anti-Ly6G antibody specifically targeting neutrophils. By decreasing the number and duration of stalls, we were able to improve the blood flow in the penumbra within 2-24 hours after reperfusion, increase capillary oxygenation, decrease cellular damage and improve functional outcome. Thereby the dynamic microcirculatory stall phenomenon contributes to the ongoing penumbral injury and is a potential hyperacute stage mechanism adding on previous observations of detrimental effects of activated neutrophils in ischemic stroke.\n\nSignificanceThis work provides in vivo evidence that, even in perfused capillaries, abnormal capillary flow patterns in the form of dynamic stalls can contribute to ongoing tissue injury in the salvageable penumbra in very early hours of cerebral ischemia. These events resembling micro traffic jams in a complex road network, are mediated by passage of neutrophils through the microcirculation and persist despite recanalization of the occluded artery.

neuroscience↗