Introduction to the Optics and the Brain 2023 feature issue.

A feature issue is being presented by a team of guest editors containing papers based on contributed submissions including studies presented at Optics and the Brain, held April 24-27, 2023 as part of Optica Biophotonics Congress: Optics in the Life Sciences, in Vancouver, Canada.

Resting state MRI reveals spontaneous physiological fluctuations in the kidney and tracks diabetic nephropathy in rats.

The kidneys maintain fluid-electrolyte balance and excrete waste in the presence of constant fluctuations in plasma volume and systemic blood pressure. The kidneys perform these functions to control capillary perfusion and glomerular filtration by modulating the mechanisms of autoregulation. An effect of these modulations are spontaneous, natural fluctuations in nephron perfusion. Numerous other mechanisms can lead to fluctuations in perfusion and flow. The ability to monitor these spontaneous physiological fluctuations in vivo could facilitate the early detection of kidney disease. The goal of this work was to investigate the use of resting- state magnetic resonance imaging (rsMRI) to detect spontaneous physiological fluctuations in the kidney. We performed rsMRI of rat kidneys in vivo over 10 minutes, applying motion correction to resolve time series in each voxel. We observed spatially variable, spontaneous fluctuations in rsMRI signal between 0-0.3 Hz, in frequency bands also associated with autoregulatory mechanisms. We further applied rsMRI to investigate changes in these fluctuations in a rat model of diabetic nephropathy. Spectral analysis was performed on time series of rsMRI signal in kidney cortex and medulla. Power from spectra in specific frequency bands from kidney cortex correlated with severity of glomerular pathology caused by diabetic nephropathy. Finally, we investigated the feasibility of using rsMRI of the human kidney in two participants, observing the presence of similar, spatially variable fluctuations. This approach may enable a range of preclinical and clinical investigations of kidney function, and facilitate the development of new therapies to improve outcomes in patients with kidney disease.

Oxytocin-induced birth causes sex-specific behavioral and brain connectivity changes in developing rat offspring.

Despite six decades of the use of exogenous oxytocin for management of labor, little is known about its effects on the developing brain. Motivated by controversial reports suggesting a link between oxytocin use during labor and autism spectrum disorders (ASDs), we employed our recently validated rat model for labor induction with oxytocin to address this important concern. Using a combination of molecular biological, behavioral, and neuroimaging assays, we show that induced birth with oxytocin leads to sex-specific disruption of oxytocinergic signaling in the developing brain, decreased communicative ability of pups, reduced empathy-like behaviors especially in male offspring, and widespread sex-dependent changes in functional cortical connectivity. Contrary to our hypothesis, social behavior, typically impaired in ASDs, was largely preserved. Collectively, our foundational studies provide nuanced insights into the neurodevelopmental impact of birth induction with oxytocin and set the stage for mechanistic investigations in animal models and prospective longitudinal clinical studies.

Multiparametric Radiogenomic Model to Predict Survival in Patients with Glioblastoma.

BACKGROUND: Clinical, histopathological, and imaging variables have been associated with prognosis in patients with glioblastoma (GBM). We aimed to develop a multiparametric radiogenomic model incorporating MRI texture features, demographic data, and histopathological tumor biomarkers to predict prognosis in patients with GBM. METHODS: In this retrospective study, patients were included if they had confirmed diagnosis of GBM with histopathological biomarkers and pre-operative MRI. Tumor segmentation was performed, and texture features were extracted to develop a predictive radiomic model of survival (<18 months vs. ≥18 months) using multivariate analysis and Least Absolute Shrinkage and Selection Operator (LASSO) regularization to reduce the risk of overfitting. This radiomic model in combination with clinical and histopathological data was inserted into a backward stepwise logistic regression model to assess survival. The diagnostic performance of this model was reported for the training and external validation sets. RESULTS: A total of 116 patients were included for model development and 40 patients for external testing validation. The diagnostic performance (AUC/sensitivity/specificity) of the radiomic model generated from seven texture features in determination of ≥18 months survival was 0.71/69.0/70.3. Three variables remained as independent predictors of survival, including radiomics (p = 0.004), age (p = 0.039), and MGMT status (p = 0.025). This model yielded diagnostic performance (AUC/sensitivity/specificity) of 0.77/81.0/66.0 (training) and 0.89/100/78.6 (testing) in determination of survival ≥ 18 months. CONCLUSIONS: Results show that our radiogenomic model generated from radiomic features at baseline MRI, age, and MGMT status can predict survival ≥ 18 months in patients with GBM.

Functional network disorganization and cognitive decline following fractionated whole-brain radiation in mice.

Cognitive dysfunction following radiotherapy (RT) is one of the most common complications associated with RT delivered to the brain, but the precise mechanisms behind this dysfunction are not well understood, and to date, there are no preventative measures or effective treatments. To improve patient outcomes, a better understanding of the effects of radiation on the brain's functional systems is required. Functional magnetic resonance imaging (fMRI) has shown promise in this regard, however, compared to neural activity, hemodynamic measures of brain function are slow and indirect. Understanding how RT acutely and chronically affects functional brain organization requires more direct examination of temporally evolving neural dynamics as they relate to cerebral hemodynamics for bridging with human studies. In order to adequately study the underlying mechanisms of RT-induced cognitive dysfunction, the development of clinically mimetic RT protocols in animal models is needed. To address these challenges, we developed a fractionated whole-brain RT protocol (3Gy/day for 10 days) and applied longitudinal wide field optical imaging (WFOI) of neural and hemodynamic brain activity at 1, 2, and 3 months post RT. At each time point, mice were subject to repeated behavioral testing across a variety of sensorimotor and cognitive domains. Disruptions in cortical neuronal and hemodynamic activity observed 1 month post RT were significantly worsened by 3 months. While broad changes were observed in functional brain organization post RT, brain regions most impacted by RT occurred within those overlapping with the mouse default mode network and other association areas similar to prior reports in human subjects. Further, significant cognitive deficits were observed following tests of novel object investigation and responses to auditory and contextual cues after fear conditioning. Our results fill a much-needed gap in understanding the effects of whole-brain RT on systems level brain organization and how RT affects neuronal versus hemodynamic signaling in the cortex. Having established a clinically-relevant injury model, future studies can examine therapeutic interventions designed to reduce neuroinflammation-based injury following RT. Given the overlap of sequelae that occur following RT with and without chemotherapy, these tools can also be easily incorporated to examine chemotherapy-related cognitive impairment.

Inhibiting metabotropic glutamate receptor 5 after stroke restores brain function and connectivity.

Stroke results in local neural disconnection and brain-wide neuronal network dysfunction leading to neurological deficits. Beyond the hyper-acute phase of ischaemic stroke, there is no clinically-approved pharmacological treatment that alleviates sensorimotor impairments. Functional recovery after stroke involves the formation of new or alternative neuronal circuits including existing neural connections. The type-5 metabotropic glutamate receptor (mGluR5) has been shown to modulate brain plasticity and function and is a therapeutic target in neurological diseases outside of stroke. We investigated whether mGluR5 influences functional recovery and network reorganization rodent models of focal ischaemia. Using multiple behavioural tests, we observed that treatment with negative allosteric modulators (NAMs) of mGluR5 (MTEP, fenobam and AFQ056) for 12 days, starting 2 or 10 days after stroke, restored lost sensorimotor functions, without diminishing infarct size. Recovery was evident within hours after initiation of treatment and progressed over the subsequent 12 days. Recovery was prevented by activation of mGluR5 with the positive allosteric modulator VU0360172 and accelerated in mGluR5 knock-out mice compared with wild-type mice. After stroke, multisensory stimulation by enriched environments enhanced recovery, a result prevented by VU0360172, implying a role of mGluR5 in enriched environment-mediated recovery. Additionally, MTEP treatment in conjunction with enriched environment housing provided an additive recovery enhancement compared to either MTEP or enriched environment alone. Using optical intrinsic signal imaging, we observed brain-wide disruptions in resting-state functional connectivity after stroke that were prevented by mGluR5 inhibition in distinct areas of contralesional sensorimotor and bilateral visual cortices. The levels of mGluR5 protein in mice and in tissue samples of stroke patients were unchanged after stroke. We conclude that neuronal circuitry subserving sensorimotor function after stroke is depressed by a mGluR5-dependent maladaptive plasticity mechanism that can be restored by mGluR5 inhibition. Post-acute stroke treatment with mGluR5 NAMs combined with rehabilitative training may represent a novel post-acute stroke therapy.

The Impact of a Scholarly Concentration Program on Medical Student Research in Pediatrics.

INTRODUCTION: To promote scientific inquiry, medical schools encourage medical students to participate in scholarly concentration programs (SCP). Manuscript publishing, a proxy of productivity, enhances medical student understanding of scientific inquiry. To evaluate an elective medical SCP offered between the first two years of medical school, the pediatrician authors' primary aim was to study the publishing productivity of the program participants in the University of Wisconsin (UW) School of Medicine and Public Health Department of Pediatrics compared to other departments. Secondary aims were to study whether productivity was influenced by the following predictors: (1) self-identified medical student gender, (2) working with a frequent mentor, (3) mentor degree, (4) funding source, and (5) area of research. METHODS: PubMed joint publications from 2002 through 2017 were searched using both medical student and mentor names through 2 years post-graduation. RESULTS: From all UW School of Medicine Public Health departments, 1108 medical students self-selected projects and mentors. One hundred two (9.2%) students chose the Department of Pediatrics. The majority of these students were female (61%) compared to female medical student participation (42%) in other departments (P = 0.0004). The majority of projects were clinical (53%), with basic science (26%) and public/global health (21%) following, though with more public/global health projects chosen in the Department of Pediatrics (P = 0.002) versus other departments. Overall, frequent mentors improved publication rates (P =0.0008), though frequent mentors (P = 0.45) and publication rates (P = 0.60) did not differ between pediatrics and other departments. CONCLUSIONS: Medical students' SCP manuscript productivity benefitted from working with frequent mentors, but productivity in the Department of Pediatrics did not differ from other departments.

Multi-Parametric Radiomic Model to Predict 1p/19q Co-Deletion in Patients with IDH-1 Mutant Glioma: Added Value to the T2-FLAIR Mismatch Sign.

PURPOSE: The T2-FLAIR mismatch sign has shown promise in determining IDH mutant 1p/19q non-co-deleted gliomas with a high specificity and modest sensitivity. To develop a multi-parametric radiomic model using MRI to predict 1p/19q co-deletion status in patients with newly diagnosed IDH1 mutant glioma and to perform a comparative analysis to T2-FLAIR mismatch sign+. METHODS: In this retrospective study, patients with diagnosis of IDH1 mutant gliomas with known 1p/19q status who had preoperative MRI were included. T2-FLAIR mismatch was evaluated independently by two board-certified neuroradiologists. Texture features were extracted from glioma segmentation of FLAIR images. eXtremeGradient Boosting (XGboost) classifiers were used for model development. Leave-one-out-cross-validation (LOOCV) and external validation performances were reported for both the training and external validation sets. RESULTS: A total of 103 patients were included for model development and 18 patients for external testing validation. The diagnostic performance (sensitivity/specificity/accuracy) in the determination of the 1p/19q co-deletion status was 59%/83%/67% (training) and 62.5%/70.0%/66.3% (testing) for the T2-FLAIR mismatch sign. This was significantly improved (p = 0.04) using the radiomics model to 77.9%/82.8%/80.3% (training) and 87.5%/89.9%/88.8% (testing), respectively. The addition of radiomics as a computer-assisted tool resulted in significant (p = 0.02) improvement in the performance of the neuroradiologist with 13 additional corrected cases in comparison to just using the T2-FLAIR mismatch sign. CONCLUSION: The proposed radiomic model provides much needed sensitivity to the highly specific T2-FLAIR mismatch sign in the determination of the 1p/19q non-co-deletion status and improves the overall diagnostic performance of neuroradiologists when used as an assistive tool.

Wide-Field Optical Imaging in Mouse Models of Ischemic Stroke.

Functional neuroimaging is a powerful tool for evaluating how local and global brain circuits evolve after focal ischemia and how these changes relate to functional recovery. For example, acutely after stroke, changes in functional brain organization relate to initial deficit and are predictive of recovery potential. During recovery, the reemergence and restoration of connections lost due to stroke correlate with recovery of function. Thus, information gleaned from functional neuroimaging can be used as a proxy for behavior and inform on the efficacy of interventional strategies designed to affect plasticity mechanisms after injury. And because these findings are consistently observed across species, bridge measurements can be made in animal models to enrich findings in human stroke populations. In mice, genetic engineering techniques have provided several new opportunities for extending optical neuroimaging methods to more direct measures of neuronal activity. These developments are especially useful in the context of stroke where neurovascular coupling can be altered, potentially limiting imaging measures based on hemodynamic activity alone. This chapter is designed to give an overview of functional wide-field optical imaging (WFOI) for applications in rodent models of stroke, primarily in the mouse. The goal is to provide a protocol for laboratories that want to incorporate an affordable functional neuroimaging assay into their current research thrusts, but perhaps lack the background knowledge or equipment for developing a new arm of research in their lab. Within, we offer a comprehensive guide developing and applying WFOI technology with the hope of facilitating accessibility of neuroimaging technology to other researchers in the stroke field.

Arousal as a universal embedding for spatiotemporal brain dynamics.

Neural activity in awake organisms shows widespread and spatiotemporally diverse correlations with behavioral and physiological measurements. We propose that this covariation reflects in part the dynamics of a unified, arousal-related process that regulates brain-wide physiology on the timescale of seconds. Taken together with theoretical foundations in dynamical systems, this interpretation leads us to a surprising prediction: that a single, scalar measurement of arousal (e.g., pupil diameter) should suffice to reconstruct the continuous evolution of multimodal, spatiotemporal measurements of large-scale brain physiology. To test this hypothesis, we perform multimodal, cortex-wide optical imaging and behavioral monitoring in awake mice. We demonstrate that spatiotemporal measurements of neuronal calcium, metabolism, and blood-oxygen can be accurately and parsimoniously modeled from a low-dimensional state-space reconstructed from the time history of pupil diameter. Extending this framework to behavioral and electrophysiological measurements from the Allen Brain Observatory, we demonstrate the ability to integrate diverse experimental data into a unified generative model via mappings from an intrinsic arousal manifold. Our results support the hypothesis that spontaneous, spatially structured fluctuations in brain-wide physiology-widely interpreted to reflect regionally-specific neural communication-are in large part reflections of an arousal-related process. This enriched view of arousal dynamics has broad implications for interpreting observations of brain, body, and behavior as measured across modalities, contexts, and scales.

Homotopic contralesional excitation suppresses spontaneous circuit repair and global network reconnections following ischemic stroke.

Understanding circuit-level manipulations that affect the brain's capacity for plasticity will inform the design of targeted interventions that enhance recovery after stroke. Following stroke, increased contralesional activity (e.g. use of the unaffected limb) can negatively influence recovery, but it is unknown which specific neural connections exert this influence, and to what extent increased contralesional activity affects systems- and molecular-level biomarkers of recovery. Here, we combine optogenetic photostimulation with optical intrinsic signal imaging to examine how contralesional excitatory activity affects cortical remodeling after stroke in mice. Following photothrombosis of left primary somatosensory forepaw (S1FP) cortex, mice either recovered spontaneously or received chronic optogenetic excitation of right S1FP over the course of 4 weeks. Contralesional excitation suppressed perilesional S1FP remapping and was associated with abnormal patterns of stimulus-evoked activity in the unaffected limb. This maneuver also prevented the restoration of resting-state functional connectivity (RSFC) within the S1FP network, RSFC in several networks functionally distinct from somatomotor regions, and resulted in persistent limb-use asymmetry. In stimulated mice, perilesional tissue exhibited transcriptional changes in several genes relevant for recovery. Our results suggest that contralesional excitation impedes local and global circuit reconnection through suppression of cortical activity and several neuroplasticity-related genes after stroke, and highlight the importance of site selection for targeted therapeutic interventions after focal ischemia.

Normal aging in mice is associated with a global reduction in cortical spectral power and network-specific declines in functional connectivity.

Normal aging is associated with a variety of neurologic changes including declines in cognition, memory, and motor activity. These declines correlate with neuronal changes in synaptic structure and function. Degradation of brain network activity and connectivity represents a likely mediator of age-related functional deterioration resulting from these neuronal changes. Human studies have demonstrated both general decreases in spontaneous cortical activity and disruption of cortical networks with aging. Current techniques used to study cerebral network activity are hampered either by limited spatial resolution (e.g. electroencephalography, EEG) or limited temporal resolution (e.g., functional magnetic resonance imaging, fMRI). Here we utilize mesoscale imaging of neuronal activity in Thy1-GCaMP6f mice to characterize neuronal network changes in aging with high spatial resolution across a wide frequency range. We show that while evoked activity is unchanged with aging, spontaneous neuronal activity decreases across a wide frequency range (0.01-4 Hz) involving all regions of the cortex. In contrast to this global reduction in cortical power, we found that aging is associated with functional connectivity (FC) deterioration of select networks including somatomotor, cingulate, and retrosplenial nodes. These changes are corroborated by reductions in homotopic FC and node degree within somatomotor and visual cortices. Finally, we found that whole-cortex delta power and delta band node degree correlate with exploratory activity in young but not aged animals. Together these data suggest that aging is associated with global declines in spontaneous cortical activity and focal deterioration of network connectivity, and that these reductions may be associated with age-related behavioral declines.

STAT3 inhibitor mitigates cerebral amyloid angiopathy and parenchymal amyloid plaques while improving cognitive functions and brain networks.

Previous reports indicate a potential role for signal transducer and activator of transcription 3 (STAT3) in amyloid-ß (Aß) processing and neuritic plaque pathogenesis. In the present study, the impact of STAT3 inhibition on cognition, cerebrovascular function, amyloid pathology, oxidative stress, and neuroinflammation was studied using in vitro and in vivo models of Alzheimer's disease (AD)-related pathology. For in vitro experiments, human brain vascular smooth muscle cells (HBVSMC) and human brain microvascular endothelial cells (HBMEC) were used, and these cultured cells were exposed to Aß peptides followed by measurement of activated forms of STAT3 expression and reactive oxygen species (ROS) generation. Further, 6 months old 5XFAD/APOE4 (5XE4) mice and age-matched negative littermates were used for in vivo experiments. These mice were treated with STAT3 specific inhibitor, LLL-12 for 2 months followed by neurobehavioral and histopathological assessment. In vitro experiments showed exposure of cerebrovascular cells to Aß peptides upregulated activated forms of STAT3 and produced STAT3-mediated vascular oxidative stress. 5XE4 mice treated with the STAT3-specific inhibitor (LLL-12) improved cognitive functions and functional connectivity and augmented cerebral blood flow. These functional improvements were associated with a reduction in neuritic plaques, cerebral amyloid angiopathy (CAA), oxidative stress, and neuroinflammation. Reduction in amyloid precursor protein (APP) processing and attenuation of oxidative modification of lipoprotein receptor related protein-1 (LRP-1) were identified as potential underlying mechanisms. These results demonstrate the broad impact of STAT3 on cognitive functions, parenchymal and vascular amyloid pathology and highlight the therapeutic potential of STAT3 specific inhibition for treatment of AD and CAA.

Multiparametric MRI texture analysis in prediction of glioma biomarker status: added value of MR diffusion.

BACKGROUND: Early identification of glioma molecular phenotypes can lead to understanding of patient prognosis and treatment guidance. We aimed to develop a multiparametric MRI texture analysis model using a combination of conventional and diffusion MRI to predict a wide range of biomarkers in patients with glioma. METHODS: In this retrospective study, patients were included if they (1) had diagnosis of gliomas with known IDH1, EGFR, MGMT, ATRX, TP53, and PTEN status from surgical pathology and (2) had preoperative MRI including FLAIR, T1c+ and diffusion for radiomic texture analysis. Statistical analysis included logistic regression and receiver-operating characteristic (ROC) curve analysis to determine the optimal model for predicting glioma biomarkers. A comparative analysis between ROCs (conventional only vs conventional + diffusion) was performed. RESULTS: From a total of 111 patients included, 91 (82%) were categorized to training and 20 (18%) to test datasets. Constructed cross-validated model using a combination of texture features from conventional and diffusion MRI resulted in overall AUC/accuracy of 1/79% for IDH1, 0.99/80% for ATRX, 0.79/67% for MGMT, and 0.77/66% for EGFR. The addition of diffusion data to conventional MRI features significantly (P < .05) increased predictive performance for IDH1, MGMT, and ATRX. The overall accuracy of the final model in predicting biomarkers in the test group was 80% (IDH1), 70% (ATRX), 70% (MGMT), and 75% (EGFR). CONCLUSION: Addition of MR diffusion to conventional MRI features provides added diagnostic value in preoperative determination of IDH1, MGMT, and ATRX in patients with glioma.

Treatment of positive urine cultures in the neonatal intensive care unit: a guideline to reduce antibiotic utilization.

BACKGROUND: The pediatric definition of bacterial urinary tract infection (UTI) is >50,000 colony forming units (CFU) of a single organism on catheterized culture or 10,000-50,000 CFU with pyuria on urinalysis. LOCAL PROBLEM: The diagnosis of UTI in our NICU is clinician-dependent and not based on the accepted pediatric definition. METHODS: A retrospective review of positive urine cultures between 2015 and 2017 was performed. INTERVENTION: A treatment guideline for positive urine cultures was adopted and PDSA methodology utilized for incremental improvements. RESULTS: For 909 pre-intervention neonates, 26 of 38 positive urine cultures were treated for UTI but only 23% (6/26) met the pediatric definition. For 644 post-guideline neonates, only 7 of 25 positive urine cultures were treated and 86% met guideline criteria with no increase in urosepsis. CONCLUSIONS: A guideline to treat positive urine cultures resulted in a decreased rate of UTI diagnosis and thus prevented unnecessary antibiotic exposure.

Opposed hemodynamic responses following increased excitation and parvalbumin-based inhibition.

Understanding cellular contributions to hemodynamic activity is essential for interpreting blood-based brain mapping signals. Optogenetic studies examining cell-specific influences on local hemodynamics have reported that excitatory activity results in cerebral perfusion and blood volume increase, while inhibitory activity contributes to both vasodilation and vasoconstriction. How specific subpopulations of interneurons regulate the brain's blood supply is less examined. Parvalbumin interneurons are the largest subpopulation of GABAergic neurons in the brain, critical for brain development, plasticity, and long-distance excitatory neurotransmission. Despite their essential role in brain function, the contribution of parvalbumin neurons to neurovascular coupling has been relatively unexamined. Using optical intrinsic signal imaging and laser speckle contrast imaging, we photostimulated awake and anesthetized transgenic mice expressing channelrhodopsin under a parvalbumin promoter. Increased parvalbumin activity reduced local oxygenation, cerebral blood volume, and cerebral blood flow. These "negative" hemodynamic responses were consistent within and across mice and reproducible across a broad range of photostimulus parameters. However, the sign and magnitude of the hemodynamic response resulting from increased parvalbumin activity depended on the type and level of anesthesia used. Opposed hemodynamic responses following increased excitation or parvalbumin-based inhibition suggest unique contributions from different cell populations to neurovascular coupling.

Implementing point of care ultrasound in the neonatal intensive care unit: a safety study.

OBJECTIVE: Point of care ultrasound (POCUS) use is increasing in pediatrics and has been demonstrated to be superior in identifying central catheter tip location in neonatal intensive care units. However, limited data exist regarding cardiorespiratory changes secondary to POCUS in neonates. STUDY DESIGN: A prospective observational equivalence study was performed on 50 POCUS assessments of central catheter tip location in 46 patients ≥23 weeks gestation. Heart rate (HR), respiratory rate (RR), and percent oxygen saturation (SpO2) levels were collected before and after POCUS. Limits of equivalence were set in advance. RESULT: Equivalence was demonstrated in HR, RR, and SpO2 before and after POCUS. HR decreased by 3.24 beats per minute (90% CI: -5.36, -1.14). RR increased by 0.71 breath/min (90% CI: -1.84, +3.27). SpO2 increased by 0.54 percentage points (90% CI: -0.23, +1.31). CONCLUSION: Identifying central catheter tip location using POCUS in neonates appears safe without affecting cardiorespiratory stability.

Problem Solved: Integral Applications of Musculoskeletal Ultrasound.

Musculoskeletal ultrasound has grown substantially in use over the past several years as an indispensable companion to magnetic resonance imaging and other imaging modalities. This article reviews 10 integral applications of musculoskeletal ultrasound as a problem-solving tool with correlative case examples. These applications include the following: (1) accessibility and portability, (2) targeted imaging, (3) dynamic imaging, (4) contralateral comparison, (5) Doppler imaging, (6) increased spatial resolution, (7) solid versus cystic comparison, (8) posttraumatic imaging, (9) postsurgical imaging, and (10) treatment delivery and optimization. The review will help the radiologist recognize the complementary uses of musculoskeletal ultrasound with radiography, computed tomography, and magnetic resonance imaging.