Tracking the neurodevelopmental trajectory of beta band oscillations with optically pumped magnetometer-based magnetoencephalography.

Neural oscillations mediate the coordination of activity within and between brain networks, supporting cognition and behaviour. How these processes develop throughout childhood is not only an important neuroscientific question but could also shed light on the mechanisms underlying neurological and psychiatric disorders. However, measuring the neurodevelopmental trajectory of oscillations has been hampered by confounds from instrumentation. In this paper, we investigate the suitability of a disruptive new imaging platform - optically pumped magnetometer-based magnetoencephalography (OPM-MEG) - to study oscillations during brain development. We show how a unique 192-channel OPM-MEG device, which is adaptable to head size and robust to participant movement, can be used to collect high-fidelity electrophysiological data in individuals aged between 2 and 34 years. Data were collected during a somatosensory task, and we measured both stimulus-induced modulation of beta oscillations in sensory cortex, and whole-brain connectivity, showing that both modulate significantly with age. Moreover, we show that pan-spectral bursts of electrophysiological activity drive task-induced beta modulation, and that their probability of occurrence and spectral content change with age. Our results offer new insights into the developmental trajectory of beta oscillations and provide clear evidence that OPM-MEG is an ideal platform for studying electrophysiology in neurodevelopment.

Histological Basis of Laminar MRI Patterns in High Resolution Images of Fixed Human Auditory Cortex.

Functional magnetic resonance imaging (fMRI) studies of the auditory region of the temporal lobe would benefit from the availability of image contrast that allowed direct identification of the primary auditory cortex, as this region cannot be accurately located using gyral landmarks alone. Previous work has suggested that the primary area can be identified in magnetic resonance (MR) images because of its relatively high myelin content. However, MR images are also affected by the iron content of the tissue and in this study we sought to confirm that different MR image contrasts did correlate with the myelin content in the gray matter and were not primarily affected by iron content as is the case in the primary visual and somatosensory areas. By imaging blocks of fixed post-mortem cortex in a 7 T scanner and then sectioning them for histological staining we sought to assess the relative contribution of myelin and iron to the gray matter contrast in the auditory region. Evaluating the image contrast in [Formula: see text]-weighted images and quantitative [Formula: see text] maps showed a reasonably high correlation between the myelin density of the gray matter and the intensity of the MR images. The correlation with T1-weighted phase sensitive inversion recovery (PSIR) images was better than with the previous two image types, and there were clearly differentiated borders between adjacent cortical areas in these images. A significant amount of iron was present in the auditory region, but did not seem to contribute to the laminar pattern of the cortical gray matter in MR images. Similar levels of iron were present in the gray and white matter and although iron was present in fibers within the gray matter, these fibers were fairly uniformly distributed across the cortex. Thus, we conclude that T1- and [Formula: see text]-weighted imaging sequences do demonstrate the relatively high myelin levels that are characteristic of the deep layers in primary auditory cortex and allow it and some of the surrounding areas to be reliably distinguished.

Mapping quantal touch using 7 Tesla functional magnetic resonance imaging and single-unit intraneural microstimulation.

Using ultra-high field 7 Tesla (7T) functional magnetic resonance imaging (fMRI), we map the cortical and perceptual responses elicited by intraneural microstimulation (INMS) of single mechanoreceptive afferent units in the median nerve, in humans. Activations are compared to those produced by applying vibrotactile stimulation to the unit's receptive field, and unit-type perceptual reports are analyzed. We show that INMS and vibrotactile stimulation engage overlapping areas within the topographically appropriate digit representation in the primary somatosensory cortex. Additional brain regions in bilateral secondary somatosensory cortex, premotor cortex, primary motor cortex, insula and posterior parietal cortex, as well as in contralateral prefrontal cortex are also shown to be activated in response to INMS. The combination of INMS and 7T fMRI opens up an unprecedented opportunity to bridge the gap between first-order mechanoreceptive afferent input codes and their spatial, dynamic and perceptual representations in human cortex.

Increase in the iron content of the substantia nigra and red nucleus in multiple sclerosis and clinically isolated syndrome: a 7 Tesla MRI study.

PURPOSE: To study iron deposition in the substantia nigra (SN) and red nuclei (RN), in patients with clinically isolated syndrome (CIS) and relapsing remitting MS (RRMS) and healthy controls (HC). MATERIALS AND METHODS: Iron deposition was assessed using susceptibility maps and T2*-w images acquired at high resolution MRI at 7 Tesla (T). Mean intensities were calculated within circular regions of interest in the SN (d/v, dorsal/ventral) and RN on high resolution T2*-w, quantitative susceptibility maps and their product for: RRMS, CIS and HC (N = 14, 21, 27, respectively). RESULTS: Magnetic susceptibility was significantly greater in SNd and RN in RRMS compared with HC (P = 0.04 [0.001, 0.48] and P = 0.01 [0.005, 0.05]), with intermediate values for the CIS group. 1/T2*-w did not show significant inter-group differences (for SNv, SNd, RN, respectively: P = 0.5 [-0.352, 0976], P = 0.35 [-0.208, 0.778], P = 0.16 [-0.114, 0.885] for RRMS versus HC) and the T2*-susceptibility product maps showed the difference only for RN (P = 0.01, [0.009, 0.062]). Changes were independent of EDSS and disease duration. CONCLUSION: MR changes consistent with iron accumulation occurring in the SN and RN of CIS patients can be identified using susceptibility mapping; this may provide an additional method of monitoring early MS development.

Neuroimaging paradigms for tonotopic mapping (I): the influence of sound stimulus type.

Although a consensus is emerging in the literature regarding the tonotopic organisation of auditory cortex in humans, previous studies employed a vast array of different neuroimaging protocols. In the present functional magnetic resonance imaging (fMRI) study, we made a systematic comparison between stimulus protocols involving jittered tone sequences with either a narrowband, broadband, or sweep character in order to evaluate their suitability for the purpose of tonotopic mapping. Data-driven analysis techniques were used to identify cortical maps related to sound-evoked activation and tonotopic frequency tuning. Principal component analysis (PCA) was used to extract the dominant response patterns in each of the three protocols separately, and generalised canonical correlation analysis (CCA) to assess the commonalities between protocols. Generally speaking, all three types of stimuli evoked similarly distributed response patterns and resulted in qualitatively similar tonotopic maps. However, quantitatively, we found that broadband stimuli are most efficient at evoking responses in auditory cortex, whereas narrowband and sweep stimuli offer the best sensitivity to differences in frequency tuning. Based on these results, we make several recommendations regarding optimal stimulus protocols, and conclude that an experimental design based on narrowband stimuli provides the best sensitivity to frequency-dependent responses to determine tonotopic maps. We forward that the resulting protocol is suitable to act as a localiser of tonotopic cortical fields in individuals, or to make quantitative comparisons between maps in dedicated tonotopic mapping studies.

Visualization of nigrosome 1 and its loss in PD: pathoanatomical correlation and in vivo 7 T MRI.

OBJECTIVE: This study assessed whether high-resolution 7 T MRI allowed direct in vivo visualization of nigrosomes, substructures of the substantia nigra pars compacta (SNpc) undergoing the greatest and earliest dopaminergic cell loss in Parkinson disease (PD), and whether any disease-specific changes could be detected in patients with PD. METHODS: Postmortem (PM) midbrains, 2 from healthy controls (HCs) and 1 from a patient with PD, were scanned with high-resolution T2*-weighted MRI scans, sectioned, and stained for iron and neuromelanin (Perl), TH, and calbindin. To confirm the identification of nigrosomes in vivo on 7 T T2*-weighted scans, we assessed colocalization with neuromelanin-sensitive T1-weighted scans. We then assessed the ability to depict PD pathology on in vivo T2*-weighted scans by comparing data from 10 patients with PD and 8 age- and sex-matched HCs. RESULTS: A hyperintense, ovoid area within the dorsolateral border of the otherwise hypointense SNpc was identified in the HC brains on in vivo and PM T2*-weighted MRI. Location, size, shape, and staining characteristics conform to nigrosome 1. Blinded assessment by 2 neuroradiologists showed consistent bilateral absence of this nigrosome feature in all 10 patients with PD, and bilateral presence in 7/8 HC. CONCLUSIONS: In vivo and PM MRI with histologic correlation demonstrates that high-resolution 7 T MRI can directly visualize nigrosome 1. The absence of nigrosome 1 in the SNpc on MRI scans might prove useful in developing a neuroimaging diagnostic test for PD.

Increased iron accumulation occurs in the earliest stages of demyelinating disease: an ultra-high field susceptibility mapping study in Clinically Isolated Syndrome.

OBJECTIVE: To determine, using ultra-high field magnetic resonance imaging (MRI), whether changes in iron content occur in the earliest phases of demyelinating disease, by quantifying the magnetic susceptibility of deep grey matter structures in patients with Clinically Isolated Syndrome (CIS) that is suggestive of multiple sclerosis (MS), as compared with age-matched healthy subjects. METHODS: We compared 19 CIS patients to 20 age-matched, healthy controls. Scanning of the study subjects was performed on a 7T Philips Achieva system, using a 3-dimensional, T2*-weighted gradient echo acquisition. Phase data were first high-pass filtered, using a dipole fitting method, and then inverted to produce magnetic susceptibility maps. Region of interest (ROI) analysis was used to estimate magnetic susceptibility values for deep grey matter structures (caudate nucleus, putamen, globus pallidus, the thalamus and its pulvinar). RESULTS: Significantly increased relative susceptibilities were found in the CIS group, compared with controls, for the caudate nucleus (p = < 0.01), putamen (p < 0.01), globus pallidus (p < 0.01) and pulvinar (p < 0.05). We found no significant nor consistent trends in the relationship between susceptibility and age for either the study controls or CIS patients, in any ROI (r(2) < 0.5; p > 0.05). In CIS patients, the time elapsed since the clinical event and the Expanded Disability Status Scale (EDSS) scores were not correlated with iron levels in any ROI (r(2) < 0.5; p > 0.05); however, a moderate correlation (r(2) = 0.3; p < 0.01) was found between the T1 lesion load and the mean susceptibility of the caudate nucleus. CONCLUSION: CIS patients showed an increased iron accumulation, as measured using susceptibility mapping of the deep grey matter, suggesting that iron changes did occur at the earlier stages of CIS disease.

Correspondence of human visual areas identified using functional and anatomical MRI in vivo at 7 T.

PURPOSE: To study the correspondence of anatomically and functionally defined visual areas (primary visual cortex, V1, and motion selective area V5/human MT+) by using structural magnetic resonance imaging (MRI) and functional MRI (fMRI) in vivo at 7 T. MATERIALS AND METHODS: Four subjects participated in this study. High-resolution (≈0.4 mm isotropic) anatomical MRI was used to identify cortical regions based on their distinct cortical lamination. The optimal contrast for identifying heavily myelinated layers within gray matter was quantitatively assessed by comparing T(1)-weighted magnetization-prepared rapid gradient echo (MPRAGE) and T(2)*-weighted, 3D fast-low angle shot (FLASH) imaging. Retinotopic mapping was performed using GE-based fMRI at 1.5 mm isotropic resolution to identify functional areas. RESULTS: T(2)*-weighted FLASH imaging was found to provide a significantly higher contrast-to-noise ratio, allowing visualization of the stria of Gennari in every slice of a volume covering the occipital cortex in each of the four subjects in this study. The independently derived boundary of V1, identified in the same subjects using retinotopic mapping by fMRI, closely matched the border of anatomically defined striate cortex in the human brain. Evidence of banding was also found within the functionally defined V5 area; however, we did not find a good correlation of this area, or the functionally identified subregion (MT), with the banded area. CONCLUSION: High-resolution T(2)*-weighted images acquired at 7 T can be used to identify myelinated bands within cortical gray matter in reasonable measurement times. Regions where a myelinated band was identified show a high degree of overlap with the functionally defined V1 area.

High resolution magnetic susceptibility mapping of the substantia nigra in Parkinson's disease.

PURPOSE: To determine if tissue magnetic susceptibility is a more direct marker of tissue iron content than other MR markers of iron. This study presents the first quantitative, in vivo measurements of the susceptibility of the substantia nigra in patients with Parkinson's disease. MATERIALS AND METHODS: Nine patients and 11 controls were studied at 7 Tesla. Susceptibility maps were created by inverting the filtered phase maps associated with T2* weighted images. RESULTS: On average, patients showed an increase in susceptibility of the pars compacta compared with controls, which correlates with the predicted increase in brain iron in Parkinson's disease. A rostral-caudal gradient in susceptibility was also observed in controls and patients. CONCLUSION: Susceptibility mapping may provide a new tool for studying the development of Parkinson's disease.

PEG coating reduces NMR relaxivity of Mn(0.5)Zn(0.5)Gd(0.02)Fe(1.98)O4 hyperthermia nanoparticles.

PURPOSE: To investigate both T1 and T2 MR relaxation enhancement of Gd substituted Zn-Mn ferrite magnetic nanoparticles. Both uncoated and polyethylene glycol (PEG) coated particles were used. MATERIALS AND METHODS: Chemical co-precipitation was used to synthesize particles in the form Mn(0.5)Zn(0.5)Gd(0.2)Fe(1.98)O(4) suitable for hyperthermia applications. Physical characterization of the magnetic nanoparticles included SEM, TEM, ICP, and SQUID. T1 and T2 measurements were performed at 1.5 Tesla (T). RESULTS: The saturation magnetization was 12.86 emu/g while the particle's magnetic moment was 1.86 × 10(-19) J/T. The particle size increased due to coating, while 1/T1 and 1/T2 relaxivities (26°C) decreased from 2.5 to 0.7 and from 201.3 to 76.6 s(-1) mM(-1), respectively, at a magnetic field 1.5T. CONCLUSION: The reduction in both 1/T1 and 1/T2 is attributed to increased distance of closest approach between the protons and the magnetic core caused by the shielding provided by the high molecular weight PEG. 1/T2 data are compared with existing theoretical models using a modified radius that takes into account both possible agglomeration of the particles and increased inter-particle separation induced by PEG coating.

Structural properties of the corticospinal tract in the human brain: a magnetic resonance imaging study at 7 Tesla.

Several fibre tracts can be accurately located using conventional Magnetic Resonance Images (MRI) of the human brain, including the corticospinal tract (CST), which appears as a T (1)-weighted hypointense/T (2)-weighted hyperintense patch in the posterior part of the posterior-limb of the internal capsule (PLIC). Here we use high-field MRI (7T) to assess the quantitative MRI properties of the CST at the PLIC level in 22 healthy young male participants. We used three different imaging modalities: the T (1) and T (2) relaxation times (T (1) and T (2)) and the Magnetization Transfer Ratio (MTR). These measurements obtained in the CST were compared with those in the anterior two-thirds of the PLIC. We observed longer T (1) and T (2) and lower MTR in the CST region compared with the adjacent (control) PLIC region. This effect is consistent with the presence of sparsely distributed, large-diameter fibres described in previous histological studies and, as such, might reflect lower myelin density and/or different morphology of fibres in the CST.

Quantification of blood velocity and flow rates in the uterine vessels using echo planar imaging at 0.5 Tesla.

PURPOSE: To measure uterine artery and vein blood velocity and flow rate profiles using MRI during normal pregnancy. MATERIALS AND METHODS: A two-shot velocity magnitude-encoded echo planar imaging (EPI) sequence is used at a magnetic field 0.5T. Data analysis procedures, necessary to overcome problems associated with low signal to noise ratio (SNR), and a spatial resolution comparable to the vessel size were used. RESULTS: The measured blood flow values averaged over nine volunteers for the mean velocity are 5.33 and 3.97 cm/s and for the unilateral flow rate are 203 and 274 mL/min (for the arteries and veins respectively). Values for the flow rate are consistent with ultrasound Doppler studies. Arterial velocity measurements are more pulsatile than venous ones and validation calculations performed on average velocity values would suggest that the nature of blood flow in the uterine vessels is laminar. CONCLUSION: This study presents the first report of noninvasive quantitative measurements of uterine artery and vein blood velocity and flow rate profiles using MRI during normal pregnancy. Consistent and reproducible measurements have been obtained by subject specific sequence optimization and data analysis procedures.

A functional-magnetic-resonance-imaging investigation of cortical activation from moving vibrotactile stimuli on the fingertip.

Using a 100-element tactile stimulator on the fingertip during functional-magnetic-resonance imaging, brain areas were identified that were selectively activated by a moving vibrotactile stimulus (the sensation of a moving line being dragged over the fingertip). Activation patterns elicited by tactile motion, contrasted to an equivalent stationary stimulus, were compared in six human subjects with those generated by a moving visual stimulus, contrasted to an equivalent stationary stimulus. Results provide further evidence for a neuroanatomical convergence of tactile-motion processing and visual-motion processing in humans. The sites of this convergence are found to lie in the middle temporal complex (hMT+V5), an area with known specialization for visual-motion processing, and in the intraparietal area of the posterior parietal cortex. In an advance on previous studies, the present study includes separate delineation of activations for moving tactile stimuli and activations for moving visual stimuli. Results suggest that the two sets of activations are not entirely collocated. Compared to the visual-motion activations, the tactile-motion activations are found to lie nearer the midline of the brain and further superior.

Improved artifact correction for combined electroencephalography/functional MRI by means of synchronization and use of vectorcardiogram recordings.

PURPOSE: To demonstrate that two methodological developments (synchronization of the MR scanner and electroencephalography [EEG] clocks and use of the scanner's vectorcardiogram [VCG]) improve the quality of EEG data recorded in combined EEG/functional MRI experiments in vivo. MATERIALS AND METHODS: EEG data were recorded using a 32-channel system, during simultaneous multislice EPI acquisition carried out on a 3 Tesla scanner. Recordings were made on three subjects in the resting state and on five subjects using a block paradigm involving visual stimulation with a 10-Hz flashing checkerboard. RESULTS: Gradient artifacts were significantly reduced in the EEG data recorded in vivo when synchronization and a TR equal to a multiple of the EEG clock period were used. This was evident from the greater attenuation of the signal at multiples of the slice acquisition frequency. Pulse artifact correction based on R-peak markers derived from the VCG was shown to offer a robust alternative to the conventionally used ECG-based method. Driven EEG responses at frequencies of up to 60 Hz due to the visual stimulus could be more readily detected in data recorded with EEG and MR scanner clock synchronization. CONCLUSION: Synchronization of the scanner and EEG clocks, along with VCG-based R-peak detection is advantageous in removing gradient and pulse artifacts in combined EEG/fMRI recordings. This approach is shown to allow the robust detection of high frequency driven activity in the EEG data.

High resolution SE-fMRI in humans at 3 and 7 T using a motor task.

OBJECT: The sensitivity of spin echo (SE) experiments to blood oxygenation level dependent (BOLD) contrast was explored in a study of the same six subjects carried out at 3 and 7 T. MATERIALS AND METHODS: Multi-slice, single shot, spin echo, echo planar images with a voxel size of 1 x 1 x 3 mm3 were acquired at three different echo times, during execution of a simple motor task. RESULTS: Significant activation was observed at all echo times at both field strengths. Analysis of the fractional signal change as a function of echo time indicated that the change in relaxation rate, Delta R2, at 7 T was -0.51 +/- 0.14 s (-1), which was 1.3 times larger than the value found at 3 T. Measurements of the percentage signal change on activation and temporal signal to noise ratio showed that there was an increase in the BOLD contrast to noise ratio (CNR) at 7 versus 3 T by a factor of 1.9. There was no overlap of areas of significant activation in the SE data acquired at either field strength with the site of large veins. CONCLUSION: SE-BOLD CNR in motor cortex was found to increase significantly at 7 T compared with 3 T.

Properties of the ballistocardiogram artefact as revealed by EEG recordings at 1.5, 3 and 7 T static magnetic field strength.

Electroencephalogram (EEG) data recorded simultaneously with functional magnetic resonance imaging (fMRI) suffer from severe artefacts. The ballistocardiogram (BCG) artefact in particular is as yet poorly understood and different BCG removal strategies have been proposed. In the present study, EEG data were recorded from four participants in three different MRI scanners with field strengths of 1.5, 3 and 7 T, with the aim of investigating the impact of the static magnetic field strength on the BCG artefact and independent component analysis (ICA). The results confirm that the amplitude of the BCG artefact is a function of the static magnetic field strength. Moreover, the spatial variability of the BCG artefact substantially increased at higher magnetic field strengths. A comparison of ICA before and after channel-wise BCG correction revealed that typical independent components could be more easily identified when ICA was applied after channel-wise BCG correction. Further analysis of EEG and electrocardiogram recordings points towards the contribution of at least two different processes to the origin of the BCG, which are blood movement or axial head rotation on the one hand and electrode movement at lateral sites of the head on the other. This is summarized in a preliminary BCG model that may help to explain recent inconsistencies regarding the usefulness of ICA for BCG removal. It may also guide the future development of more advanced BCG removal procedures.

Thresholds for perceiving metallic taste at high magnetic field.

PURPOSE: To perform an initial characterization of the metallic taste effect observed by some workers when moving around an MRI scanner. MATERIALS AND METHODS: A total of 21 subjects performed controlled movements in the stray field of a 7-T scanner. Rates of change of magnetic flux were recorded during the study using a custom-built three-axis coil unit connected to a data logger. RESULTS: Relatively normal movements could generate switched fields of 2 T/second. Of the 21 subjects, 12 detected a metallic taste, but the threshold at which it was perceived varied greatly between subjects, with the minimum dB/dt value at which such a taste was detected being 1.3 T/second. The threshold also depended on the direction of movement. CONCLUSION: This study indicates that 50% of subjects will perceive a metallic taste for head shaking with a period of 1.5 seconds (magnetic field in an anterior/posterior direction) causing a dB/dt of 2.3 +/- 0.3 T/second. The presence of dental fillings is not a requirement for the sensation of metallic taste.

Pharmaceutical applications of magnetic resonance imaging (MRI).

Magnetic resonance imaging (MRI) is a powerful imaging modality that provides internal images of materials and living organisms on a microscopic and macroscopic scale. It is non-invasive and non-destructive, and one of very few techniques that can observe internal events inside undisturbed specimens in situ. It is versatile, as a wide range of NMR modalities can be accessed, and 2D and 3D imaging can be undertaken. Despite widespread use and major advances in clinical MRI, it has seen limited application in the pharmaceutical sciences. In vitro studies have focussed on drug release mechanisms in polymeric delivery systems, but isolated studies of bioadhesion, tablet properties, and extrusion and mixing processes illustrate the wider potential. Perhaps the greatest potential however, lies in investigations of pharmaceuticals in vivo, where pilot human and animal studies have demonstrated we can obtain unique insights into the behaviour of gastrointestinal, topical, colloidal, and targeted drug delivery systems.

Correction of spatial distortion in EPI due to inhomogeneous static magnetic fields using the reversed gradient method.

PURPOSE: To derive and implement a method for correcting spatial distortion caused by in vivo inhomogeneous static magnetic fields in echo-planar imaging (EPI). MATERIALS AND METHODS: The reversed gradient method, which was initially devised to correct distortion in images generated by spin-warp MRI, was adapted to correct distortion in EP images. This method provides point-by-point correction of distortion throughout the image. EP images, acquired with a 3 T MRI system, of a phantom and a volunteer's head were used to test the correction method. RESULTS: Good correction was observed in all cases. Spatial distortion in the uncorrected images ranged up to 4 pixels (12 mm) and was corrected successfully. CONCLUSION: The correction was improved by the application of a nonlinear interpolation scheme. The correction requires that two EP images be acquired at each slice position. This increases the acquisition time, but an improved signal-to-noise ratio (SNR) is seen in the corrected image. The local SNR gain decreases with increasing distortion. In many EPI acquisition schemes, multiple images are averaged at each slice position to increase the SNR; in such cases the reversed gradient correction method can be applied with no increase in acquisition duration.

Localised mapping of water movement and hydration inside a developing bioadhesive bond.

Few studies have investigated the internal processes involved in bioadhesive bond formation, particularly where mucus and hydrated polymer contribute jointly to bond structure. This paper reports the first study to spatially map the internal environment within a developing bioadhesive bond, utilising nuclear magnetic resonance (NMR) microscopy to measure localised water self-diffusion coefficients (SDC) and confocal laser scanning microscopy (CLSM) to estimate mucin concentration. In a model bioadhesive bond formed between an alginate matrix and mucin gel, characteristic profiles were observed in which fluorescence measurements showed a region of increasing mucin concentration in the mucus layer region adjacent to the matrix, corresponding closely with a zone of restricted water SDC in the diffusion profiles. These regions extended 144 microm (a normal human gastric layer thickness [Clin. Sci. 95 (1998) 97]) into the mucin layer after just 30 s, increasing to 800 microm after 30 min. The formation of a hydrated polymer layer at the matrix surface, confirmed visually, was also reflected in corresponding gradient changes. The results suggest a progressive dehydration of the mucus gel during bond formation, and the study demonstrates how together, these microscopies can provide non-invasive, quantitative, spatial and time-resolved evidence of internal hydration behaviour during bioadhesive bond formation.