A method to image brain tissue frozen at autopsy.

Magnetic Resonance Imaging (MRI) can provide the location and signal characteristics of pathological regions within a postmortem tissue block, thereby improving the efficiency of histopathological studies. However, such postmortem-MRI guided histopathological studies have so far only been performed on fixed samples as imaging tissue frozen at the time of extraction, while preserving its integrity, is significantly more challenging. Here we describe the development of cold-postmortem-MRI, which can preserve tissue integrity and help target techniques such as transcriptomics. As a first step, RNA integrity number (RIN) was used to determine the rate of tissue biomolecular degradation in mouse brains placed at various temperatures between -20 °C and +20 °C for up to 24 h. Then, human tissue frozen at the time of autopsy was immersed in 2-methylbutane, sealed in a bio-safe tissue chamber, and cooled in the MRI using a recirculating chiller to determine MRI signal characteristics. The optimal imaging temperature, which did not show significant RIN deterioration for over 12 h, at the same time giving robust MRI signal and contrast between brain tissue types was deemed to be -7 °C. Finally, MRI was performed on human tissue blocks at this optimal imaging temperatures using a magnetization-prepared rapid gradient echo (MPRAGE, isotropic resolution between 0.3-0.4 mm) revealing good gray-white matter contrast and revealing subpial, subcortical, and deep white matter lesions. RINs measured before and after imaging revealed no significant changes (n = 3, p = 0.18, paired t-test). In addition to improving efficiency of downstream processes, imaging tissue at sub-zero temperatures may also improve our understanding of compartment specificity of MRI signal.

Postmortem MRI of Tissue Frozen at Autopsy.

Introduction: Postmortem MRI provides insight into location of pathology within tissue blocks, enabling efficient targeting of histopathological studies. While postmortem imaging of fixed tissue is gaining popularity, imaging tissue frozen at the time of extraction is significantly more challenging. Methods: Tissue integrity was examined using RNA integrity number (RIN), in mouse brains placed between -20 °C and 20 °C for up to 24 hours, to determine the highest temperature that could potentially be used for imaging without tissue degeneration. Human tissue frozen at the time of autopsy was sealed in a tissue chamber filled with 2-methylbutane to prevent contamination of the MRI components. The tissue was cooled to a range of temperatures in a 9.4T MRI using a recirculating aqueous ethylene glycol solution. MRI was performed using a magnetization-prepared rapid gradient echo (MPRAGE) sequence with inversion time of 1400 ms to null the signal from 2-methylbutane bath, isotropic resolution between 0.3-0.4 mm, and scan time of about 4 hours was used to study the anatomical details of the tissue block. Results and Discussion: A temperature of -7 °C was chosen for imaging as it was below the highest temperature that did not show significant RIN deterioration for over 12 hours, at the same time gave robust imaging signal and contrast between brain tissue types. Imaging performed on various human tissue blocks revealed good gray-white matter contrast and revealing subpial, subcortical, and deep white matter lesions typical of multiple sclerosis enabling further spatially targeted studies. Conclusion: Here, we describe a new method to image cold tissue, while maintaining tissue integrity and biosafety during scanning. In addition to improving efficiency of downstream processes, imaging tissue at sub-zero temperatures may also improve our understanding of compartment specificity of MRI signal.

Shaped Magnetogel Microparticles for Multispectral Magnetic Resonance Contrast and Sensing.

Multispectral magnetic resonance imaging (MRI) contrast agents are microfabricated three-dimensional magnetic structures that encode nearby water protons with discrete frequencies. The agents have a unique radiofrequency (RF) resonance that can be tuned by engineering the geometric parameters of these microstructures. Multispectral contrast agents can be used as sensors by incorporating a stimulus-driven shape-changing response into their structure. These geometrically encoded magnetic sensors (GEMS) enable MRI-based sensing via environmentally induced changes to their geometry and their corresponding RF resonance. Previously, GEMS have been made using thin-film lithography techniques in a cleanroom environment. While these approaches offer precise control of the microstructure, they can be a limitation for researchers who do not have cleanroom access or microfabrication expertise. Here, an alternative approach for GEMS fabrication based on soft lithography is introduced. The fabrication scheme uses cheap, accessible materials and simple chemistry to produce shaped magnetic hydrogel microparticles with multispectral MRI contrast properties. The microparticles can be used as sensors by fabricating them out of shape-reconfigurable, "smart" hydrogels. The change in shape causes a corresponding shift in the resonance of the GEMS, producing an MRI-addressable readout of the microenvironment. Proof-of-principle experiments showing a multispectral response to pH change with cylindrical shell-shaped magnetogel GEMS are presented.

Progressive Spread of Beta-amyloid Pathology in an Olfactory-driven Amyloid Precursor Protein Mouse Model.

Years before Alzheimer's disease (AD) is diagnosed, patients experience an impaired sense of smell, and ß-amyloid plaques accumulate within the olfactory mucosa and olfactory bulb (OB). The olfactory vector hypothesis proposes that external agents cause ß-amyloid to aggregate and spread from the OB to connected downstream brain regions. To reproduce the slow accumulation of ß-amyloid that occurs in human AD, we investigated the progressive accumulation of ß-amyloid across the brain using a conditional mouse model that overexpresses a humanized mutant form of the amyloid precursor protein (hAPP) in olfactory sensory neurons. Using design-based stereology, we show the progressive accumulation of ß-amyloid plaques within the OB and cortical olfactory regions with age. We also observe reduced OB volumes in these mice when hAPP expression begins prior-to but not post-weaning which we tracked using manganese-enhanced MRI. We therefore conclude that the reduced OB volume does not represent progressive degeneration but rather disrupted OB development. Overall, our data demonstrate that hAPP expression in the olfactory epithelium can lead to the accumulation and spread of ß-amyloid through the olfactory system into the hippocampus, consistent with an olfactory system role in the early stages of ß-amyloid-related AD progression.

Epidemiology of protozoan and helminthic parasites in wild passerine birds of Britain and Ireland.

Avian endoparasites play important roles in conservation, biodiversity and host evolution. Currently, little is known about the epidemiology of intestinal helminths and protozoans infecting wild birds of Britain and Ireland. This study aimed to determine the rates of parasite prevalence, abundance and infection intensity in wild passerines. Fecal samples (n = 755) from 18 bird families were collected from 13 sites across England, Wales and Ireland from March 2020 to June 2021. A conventional sodium nitrate flotation method allowed morphological identification and abundance estimation of eggs/oocysts. Associations with host family and age were examined alongside spatiotemporal and ecological factors using Bayesian phylogenetically controlled models. Parasites were detected in 20.0% of samples, with corvids and finches having the highest prevalences and intensities, respectively. Syngamus (33%) and Isospora (32%) were the most prevalent genera observed. Parasite prevalence and abundance differed amongst avian families and seasons, while infection intensity varied between families and regions. Prevalence was affected by diet diversity, while abundance differed by host age and habitat diversity. Infection intensity was higher in birds using a wider range of habitats, and doubled in areas with feeders present. The elucidation of these patterns will increase the understanding of parasite fauna in British and Irish birds.

Curves for Curlew: Identifying Curlew breeding status from GPS tracking data.

Identifying the breeding status of cryptic bird species has proved problematic without intense or inherently expensive monitoring. Most, if not all, intensive bird monitoring comes with disturbance risks and many projects now rely on tagging of individuals to provide remote information on movements. Given the importance of breeding status when targeting conservation interventions novel methods are needed. This study aimed to identify breeding status in Eurasian Curlew (Numenius arquata) from GPS tag movement patterns using the "recurse" package in R. This package identifies foci of activity (using K-means clustering) based on revisitations. Using a training data set from an individual of known breeding status, we visually assessed the frequency of revisits to particular locations to identify prebreeding, incubation, chick guarding, and post-breeding stages to an accuracy of a within at most half a day and thus breeding outcomes. Limited validation was provided by additional field observations. Based on our results, we estimate a low daily nest survival rate of 0.935 during incubation, that only a small proportion of individuals successfully raised young, and that there was a high proportion (26%) of non-breeders in the population. The Eurasian Curlew is a species of high conservation concern across Europe, and our results concur with recent studies highlighting that population declines are likely to be driven by low levels of productivity. The acquisition of improved knowledge on the behaviors of individuals at each stage of breeding enables more targeted conservation efforts and reduces the need for additional monitoring visits that may cause increased disturbance and risk of nest failure. We hope that the approach outlined may be developed to provide practitioners who have detailed knowledge of the behavior of their study species with a practical means of assessing the breeding status and outcomes of their study populations.

Multivalent Gd-DOTA Decorated Oligopeptide as Sensitive MRI Molecular Probes for In Vivo Imaging of Brain Connectivity.

One of the most important goals of brain imaging is to define the anatomical connections within the brain. In addition to revealing normal circuitry, studies of neural connections and neuronal transport can show rewiring and degeneration following brain injury and diseases. In this work, a highly sensitive magnetic resonance imaging (MRI)-visible neural tracer that can be used to visualize brain connectivity in vivo is developed. It is based on an oligopeptide with gadolinium chelates appended to the peptide backbone. This peptide construct is a sensitive MRI contrast agent that was conjugated to the classical neurotracer, Cholera-toxin Subunit-B. Injection of this probe enabled it to be used to trace neural connections in vivo. This complements other MRI tracing techniques such as diffusion tensor imaging and manganese-enhanced MRI for neural tracing.

The misunderstood meander: Redesigning MRI meander-line surface coils to reduce noise, increase uniformity, and eliminate image artifacts.

Meander-line, or zig-zag, MRI surface coils theoretically promise spatially uniform fields with optimal field localization close to the coil. In reality, they suffer poorer than expected field localizations and acquired images are often highly inhomogeneous, plagued by repeating stripe-like signal-loss artifacts. We show that both these detrimental effects arise from coil design based on the same invalid approximation in the underlying theory. Here, the conventional approximation is corrected, yielding a modified coil design that validates the new theory by rectifying the above problems. Specifically, an easily implementable coil correction, which amounts to the addition of a single extra turn of wire, is introduced and shown to increase signal uniformity by an order of magnitude, eliminate image artifacts, and reduce unwanted signal interference from deeper within the sample by tightening the coil field localization to close to the coil, as intended for zig-zag designs. With independent optimization of coil size and imaging depth possible, such corrected meander-lines surface coils may be well suited for large area, near-surface imaging and spectroscopy applications.

Sensitive detection of extremely small iron oxide nanoparticles in living mice using MP2RAGE with advanced image co-registration.

Magnetic resonance imaging (MRI) is a widely used non-invasive methodology for both preclinical and clinical studies. However, MRI lacks molecular specificity. Molecular contrast agents for MRI would be highly beneficial for detecting specific pathological lesions and quantitatively evaluating therapeutic efficacy in vivo. In this study, an optimized Magnetization Prepared-RApid Gradient Echo (MP-RAGE) with 2 inversion times called MP2RAGE combined with advanced image co-registration is presented as an effective non-invasive methodology to quantitatively detect T1 MR contrast agents. The optimized MP2RAGE produced high quality in vivo mouse brain T1 (or R1 = 1/T1) map with high spatial resolution, 160 × 160 × 160 µm3 voxel at 9.4 T. Test-retest signal to noise was > 20 for most voxels. Extremely small iron oxide nanoparticles (ESIONPs) having 3 nm core size and 11 nm hydrodynamic radius after polyethylene glycol (PEG) coating were intracranially injected into mouse brain and detected as a proof-of-concept. Two independent MP2RAGE MR scans were performed pre- and post-injection of ESIONPs followed by advanced image co-registration. The comparison of two T1 (or R1) maps after image co-registration provided precise and quantitative assessment of the effects of the injected ESIONPs at each voxel. The proposed MR protocol has potential for future use in the detection of T1 molecular contrast agents.

Multifield and inverse-contrast switching of magnetocaloric high contrast ratio MRI labels.

PURPOSE: Demonstrating multifield and inverse contrast switching of magnetocaloric high contrast ratio MRI labels that either have increasing or decreasing moment versus temperature slopes depending on the material at physiological temperatures and different MRI magnetic field strengths. METHODS: Two iron-rhodium samples of different purity (99% and 99.9%) and a lanthanum-iron-silicon sample were obtained from commercial vendors. Temperature and magnetic field-dependent magnetic moment measurements of the samples were performed on a vibrating sample magnetometer. Temperature-dependent MRI of different iron-rhodium and lanthanum-iron-silicon samples were performed on 3 different MRI scanners at 1 Tesla (T), 4.7T, and 7T. RESULTS: Sharp, first-order magnetic phase transition of each iron-rhodium sample at a physiologically relevant temperature (~37°C) but at different MRI magnetic fields (1T, 4.7T, and 7T, depending on the sample) showed clear image contrast changes in temperature-dependent MRI. Iron-rhodium and lanthanum-iron-silicon samples with sharp, first-order magnetic phase transitions at the same MRI field of 1T and physiological temperature of 37°C, but with positive and negative slope of magnetization versus temperature, respectively, showed clear inverse contrast image changes. Temperature-dependent MRI on individual microparticle samples of lanthanum-iron-silicon also showed sharp image contrast changes. CONCLUSION: Magnetocaloric materials of different purity and composition were demonstrated to act as diverse high contrast ratio switchable MRI contrast agents. Thus, we show that a range of magnetocaloric materials can be optimized for unique image contrast response under MRI-appropriate conditions at physiological temperatures and be controllably switched in situ.

Ex vivo MR microscopy of a human brain with multiple sclerosis: Visualizing individual cells in tissue using intrinsic iron.

PURPOSE: To perform magnetic resonance microscopy (MRM) on human cortex and a cortical lesion as well as the adjacent normal appearing white matter. To shed light on the origins of MRI contrast by comparison with histochemical and immunostaining. METHODS: 3D MRM at a nominal isotropic resolution of 15 and 18 µm was performed on 2 blocks of tissue from the brain of a 77-year-old man who had MS for 47 years. One block contained normal appearing cortical gray matter (CN block) and adjacent normal appearing white matter (NAWM), and the other also included a cortical lesion (CL block). Postmortem ex-vivo MRI was performed at 11.7T using a custom solenoid coil and T2*-weighted 3D GRE sequence. Histochemical and immunostaining were done after paraffin embedding for iron, myelin, oligodendrocytes, neurons, blood vessels, macrophages and microglia, and astrocytes. RESULTS: MRM could identify individual iron-laden oligodendrocytes with high sensitivity (70% decrease in signal compared to surrounding) in CN and CL blocks, as well as some iron-laden activated macrophages and microglia. Iron-deficient oligodendrocytes seemed to cause relative increase in MRI signal within the cortical lesion. High concentration of myelin in the white matter was primarily responsible for its hypointense appearance relative to the cortex, however, signal variations within NAWM could be attributed to changes in density of iron-laden oligodendrocytes. CONCLUSION: Changes in iron accumulation within cells gave rise to imaging contrast seen between cortical lesions and normal cortex, as well as the patchy signal in NAWM. Densely packed myelin and collagen deposition also contributed to MRM signal changes. Even though we studied only one block each from normal appearing and cortical lesions, such studies can help better understand the origins of histopathological and microstructural correlates of MRI signal changes in multiple sclerosis and contextualize the interpretation of lower-resolution in vivo MRI scans.

Sleep Staging Monitoring Based on Sonar Smartphone Technology.

This paper presents the validation results of a new non-contact ultrasonic technology, which employs inaudible Sonar to monitor the movements and respiration of a subject in bed. Sleep monitoring can be achieved by placing a smartphone onto the bedside table and starting a custom app. The app employs sophisticated and novel proprietary algorithms to identify sleep stages: Wake (W), Light Sleep (N1, N2 sleep), Deep Sleep (N3 sleep), Rapid Eye Movement (REM) Sleep or Absence.The sleep staging performance of the app were assessed by testing it against expert manually scored polysomnography (PSG) of 38 subjects gathered in a sleep laboratory. As a secondary assessment, on the same dataset, the performance of the app is compared to that of a reference non-contact device, the S+ by ResMed.Performance across different sleep stage detections was balanced, exceeding the agreement typically reported for actigraphy based devices [1], [2] thanks to a significantly higher sensitivity for all sleep stages. Furthermore, the performance of the app was found to be comparable to the S+ by ResMed product [3], [4].The combination of unobtrusive non-contact sensing and accurate sleep quality assessment, coupled with removal of the requirement to purchase a custom device to enable monitoring of sleep, enables consumers to measure their sleep in the home environment in a zero-cost and accessible manner, while providing sleep staging information not otherwise available with actigraphy based devices.

Magnetocaloric materials as switchable high contrast ratio MRI labels.

PURPOSE: To develop switchable and tunable labels with high contrast ratio for MRI using magnetocaloric materials that have sharp first-order magnetic phase transitions at physiological temperatures and typical MRI magnetic field strengths. METHODS: A prototypical magnetocaloric material iron-rhodium (FeRh) was prepared by melt mixing, high-temperature annealing, and ice-water quenching. Temperature- and magnetic field-dependent magnetization measurements of wire-cut FeRh samples were performed on a vibrating sample magnetometer. Temperature-dependent MRI of FeRh samples was performed on a 4.7T MRI. RESULTS: Temperature-dependent MRI clearly demonstrated image contrast changes due to the sharp magnetic state transition of the FeRh samples in the MRI magnetic field (4.7T) and at a physiologically relevant temperature (~37°C). CONCLUSION: A magnetocaloric material, FeRh, was demonstrated to act as a high contrast ratio switchable MRI contrast agent due to its sharp first-order magnetic phase transition in the DC magnetic field of MRI and at physiologically relevant temperatures. A wide range of magnetocaloric materials are available that can be tuned by materials science techniques to optimize their response under MRI-appropriate conditions and be controllably switched in situ with temperature, magnetic field, or a combination of both.

Neural precursor cells form integrated brain-like tissue when implanted into rat cerebrospinal fluid.

There is tremendous interest in transplanting neural precursor cells for brain tissue regeneration. However, it remains unclear whether a vascularized and integrated complex neural tissue can be generated within the brain through transplantation of cells. Here, we report that early stage neural precursor cells recapitulate their seminal properties and develop into large brain-like tissue when implanted into the rat brain ventricle. Whereas the implanted cells predominantly differentiated into glutamatergic neurons and astrocytes, the host brain supplied the intact vasculature, oligodendrocytes, GABAergic interneurons, and microglia that seamlessly integrated into the new tissue. Furthermore, local and long-range axonal connections formed mature synapses between the host brain and the graft. Implantation of precursor cells into the CSF-filled cavity also led to a formation of brain-like tissue that integrated into the host cortex. These results may constitute the basis of future brain tissue replacement strategies.

Inhibition of the proteasome partially attenuates atrophy in botulinum neurotoxin treated skeletal muscle.

Botulinum neurotoxin type A (BoNT/A) is used as a therapeutic tool to induce chemical denervation of spastically contracted muscles, yet the neurotoxin can also cause skeletal muscle atrophy. The underlying proteolytic mechanisms that induce this atrophy remain unclear. Our previous work has highlighted increased ubiquitin proteasome system (UPS) activity in soleus muscle of male Sprague Dawley rats following hind limb injection of BoNT/A, with the chymotrypsin-like activity of the 20s proteasome the most active. Thus, we chose to inhibit 20s proteasome activity in BoNT/A injected hind limb to determine the effect on soleus muscle atrophy. Epoxomicin is commonly used to inhibit the proteasome in vivo, binding specifically and irreversibly to the 20s proteasome catalytic subunits. Daily subcutaneous injections of epoxomicin abolished BoNT/A-induced elevations in 20s chymotrypsin-like activity both 3 days and 10 days post BoNT/A injection. Furthermore, BoNT/A-induced elevations in polyubiquitination remained elevated in BoNT/A + epoxomicin treated muscle, presumably due to epoxomicin's inhibition of the proteasome causing a back-up of polyubiquitinated proteins. Despite inhibition of the proteasome, epoxomicin was insufficient to significantly attenuate soleus muscle fiber atrophy 3 days following BoNT/A injection however, 10 days of daily epoxomicin injection was sufficient to spare ∼20% of muscle wasting. The mechanism of the remaining 80% of BoNT/A-induced atrophy presumably occurs via mechanisms outside of the 20s proteasome.

Optically controlled on-coil amplifier with RF monitoring feedback.

PURPOSE: To develop a new optically controlled on-coil amplifier that facilitates safe use of multi-channel radiofrequency (RF) transmission in MRI by real-time monitoring of signal phase and amplitude. METHODS: Monitoring was carried out with a 4-channel prototype system by sensing, down sampling, digitizing, and optically transmitting the RF transmit signal to a remote PC to control the amplifiers. Performance was evaluated with benchtop and 7 T MRI experiments. RESULTS: Monitored amplitude and phase were stable across repetitions and had standard deviations of 0.061 µT and 0.0073 rad, respectively. The feedback system allowed inter-channel phase and B1 amplitude to be adjusted within two iterations. MRI experiments demonstrated the feasibility of this approach to perform safe and accurate multi-channel RF transmission and monitoring at high field. CONCLUSION: We demonstrated a 4-channel transceiver system based on optically controlled on-coil amplifiers with RF signal monitoring and feedback control. The approach allows the safe and precise control of RF transmission fields, required to achieve uniform excitation at high field. Magn Reson Med 79:2833-2841, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Magnetic resonance imaging of odorant activity-dependent migration of neural precursor cells and olfactory bulb growth.

Neural progenitors or neuroblasts are produced by precursor cells in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) to the olfactory bulbs (OB) throughout life. In the OB, these adult born neurons either die or replace existing olfactory interneurons, playing a critical role in the stabilization of OB circuitry. Although several aspects of the addition of new neurons into the OB have been studied, it is unclear whether long-distance activity from the OB can regulate the influx of migrating neuroblasts along the RMS. In this study, iron oxide-assisted MRI was used to track the migration of neuroblasts in combination with reversible naris occlusion to manipulate odorant-induced activity. It was found that decreasing olfactory activity led to a decrease in the rate of neuroblast migration along the RMS. Removal of the naris occlusion led to an increase in migratory rate back to control levels, indicating that olfactory activity has regulatory function on neuroblast migration in the RMS. Blocking odorant activity also led to an arrest in OB growth and re-opening the block led to a rapid re-growth returning the bulb size to control levels. Furthermore, pharmacogenetic elimination of the neuroblasts demonstrated that they were required for re-growth of the bulb following sensory deprivation. Together, these results show that sensory activity, neural migration and OB growth are tightly coupled in an interdependent manner.

Breeding density, fine-scale tracking, and large-scale modeling reveal the regional distribution of four seabird species.

Population-level estimates of species' distributions can reveal fundamental ecological processes and facilitate conservation. However, these may be difficult to obtain for mobile species, especially colonial central-place foragers (CCPFs; e.g., bats, corvids, social insects), because it is often impractical to determine the provenance of individuals observed beyond breeding sites. Moreover, some CCPFs, especially in the marine realm (e.g., pinnipeds, turtles, and seabirds) are difficult to observe because they range tens to ten thousands of kilometers from their colonies. It is hypothesized that the distribution of CCPFs depends largely on habitat availability and intraspecific competition. Modeling these effects may therefore allow distributions to be estimated from samples of individual spatial usage. Such data can be obtained for an increasing number of species using tracking technology. However, techniques for estimating population-level distributions using the telemetry data are poorly developed. This is of concern because many marine CCPFs, such as seabirds, are threatened by anthropogenic activities. Here, we aim to estimate the distribution at sea of four seabird species, foraging from approximately 5,500 breeding sites in Britain and Ireland. To do so, we GPS-tracked a sample of 230 European Shags Phalacrocorax aristotelis, 464 Black-legged Kittiwakes Rissa tridactyla, 178 Common Murres Uria aalge, and 281 Razorbills Alca torda from 13, 20, 12, and 14 colonies, respectively. Using Poisson point process habitat use models, we show that distribution at sea is dependent on (1) density-dependent competition among sympatric conspecifics (all species) and parapatric conspecifics (Kittiwakes and Murres); (2) habitat accessibility and coastal geometry, such that birds travel further from colonies with limited access to the sea; and (3) regional habitat availability. Using these models, we predict space use by birds from unobserved colonies and thereby map the distribution at sea of each species at both the colony and regional level. Space use by all four species' British breeding populations is concentrated in the coastal waters of Scotland, highlighting the need for robust conservation measures in this area. The techniques we present are applicable to any CCPF.

Sensory and optogenetically driven single-vessel fMRI.

Magnetic resonance imaging (MRI) sensitivity approaches vessel specificity. We developed a single-vessel functional MRI (fMRI) method to image the contribution of vascular components to blood oxygenation level-dependent (BOLD) and cerebral blood volume (CBV) fMRI signal. We mapped individual vessels penetrating the rat somatosensory cortex with 100-ms temporal resolution by MRI with sensory or optogenetic stimulation. The BOLD signal originated primarily from venules, and the CBV signal from arterioles. The single-vessel fMRI method and its combination with optogenetics provide a platform for mapping the hemodynamic signal through the neurovascular network with specificity at the level of individual arterioles and venules.