Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for cleared samples.

In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope.

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy.

It is a matter of perspective: Attentional focus rather than dietary restraint drives brain responses to food stimuli.

Brain responses to food are thought to reflect food's rewarding value and to fluctuate with dietary restraint. We propose that brain responses to food are dynamic and depend on attentional focus. Food pictures (high-caloric/low-caloric, palatable/unpalatable) were presented during fMRI-scanning, while attentional focus (hedonic/health/neutral) was induced in 52 female participants varying in dietary restraint. The level of brain activity was hardly different between palatable versus unpalatable foods or high-caloric versus low-caloric foods. Activity in several brain regions was higher in hedonic than in health or neutral attentional focus (p < .05, FWE-corrected). Palatability and calorie content could be decoded from multi-voxel activity patterns (p < .05, FDR-corrected). Dietary restraint did not significantly influence brain responses to food. So, level of brain activity in response to food stimuli depends on attentional focus, and may reflect salience, not reward value. Palatability and calorie content are reflected in patterns of brain activity.

Magnetic resonance imaging at 9.4 T: the Maastricht journey.

The 9.4 T scanner in Maastricht is a whole-body magnet with head gradients and parallel RF transmit capability. At the time of the design, it was conceptualized to be one of the best fMRI scanners in the world, but it has also been used for anatomical and diffusion imaging. 9.4 T offers increases in sensitivity and contrast, but the technical ultra-high field (UHF) challenges, such as field inhomogeneities and constraints set by RF power deposition, are exacerbated compared to 7 T. This article reviews some of the 9.4 T work done in Maastricht. Functional imaging experiments included blood oxygenation level-dependent (BOLD) and blood-volume weighted (VASO) fMRI using different readouts. BOLD benefits from shorter T2* at 9.4 T while VASO from longer T1. We show examples of both ex vivo and in vivo anatomical imaging. For many applications, pTx and optimized coils are essential to harness the full potential of 9.4 T. Our experience shows that, while considerable effort was required compared to our 7 T scanner, we could obtain high-quality anatomical and functional data, which illustrates the potential of MR acquisitions at even higher field strengths. The practical challenges of working with a relatively unique system are also discussed.

Efficient 3D light-sheet imaging of very large-scale optically cleared human brain and prostate tissue samples.

The ability to image human tissue samples in 3D, with both cellular resolution and a large field of view (FOV), can improve fundamental and clinical investigations. Here, we demonstrate the feasibility of light-sheet imaging of ~5 cm3 sized formalin fixed human brain and up to ~7 cm3 sized formalin fixed paraffin embedded (FFPE) prostate cancer samples, processed with the FFPE-MASH protocol. We present a light-sheet microscopy prototype, the cleared-tissue dual view Selective Plane Illumination Microscope (ct-dSPIM), capable of fast 3D high-resolution acquisitions of cm3 scale cleared tissue. We used mosaic scans for fast 3D overviews of entire tissue samples or higher resolution overviews of large ROIs with various speeds: (a) Mosaic 16 (16.4 µm isotropic resolution, ~1.7 h/cm3), (b) Mosaic 4 (4.1 µm isotropic resolution, ~ 5 h/cm3) and (c) Mosaic 0.5 (0.5 µm near isotropic resolution, ~15.8 h/cm3). We could visualise cortical layers and neurons around the border of human brain areas V1&V2, and could demonstrate suitable imaging quality for Gleason score grading in thick prostate cancer samples. We show that ct-dSPIM imaging is an excellent technique to quantitatively assess entire MASH prepared large-scale human tissue samples in 3D, with considerable future clinical potential.

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples.

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

More complex than you might think: Neural representations of food reward value in obesity.

Obesity reached pandemic proportions and weight-loss treatments are mostly ineffective. The level of brain activity in the reward circuitry is proposed to be proportionate to the reward value of food stimuli, and stronger in people with obesity. However, empirical evidence is inconsistent. This may be due to the double-sided nature of high caloric palatable foods: at once highly palatable and high in calories (unhealthy). This study hypothesizes that, viewing high caloric palatable foods, a hedonic attentional focus compared to a health and a neutral attentional focus elicits more activity in reward-related brain regions, mostly in people with obesity. Moreover, caloric content and food palatability can be decoded from multivoxel patterns of activity most accurately in people with obesity and in the corresponding attentional focus. During one fMRI-session, attentional focus (hedonic, health, neutral) was manipulated using a one-back task with individually tailored food stimuli in 32 healthy-weight people and 29 people with obesity. Univariate analyses (p < 0.05, FWE-corrected) showed that brain activity was not different for palatable vs. unpalatable foods, nor for high vs. low caloric foods. Instead, this was higher in the hedonic compared to the health and neutral attentional focus. Multivariate analyses (MVPA) (p < 0.05, FDR-corrected) showed that palatability and caloric content could be decoded above chance level, independently of either BMI or attentional focus. Thus, brain activity to visual food stimuli is neither proportionate to the reward value (palatability and/or caloric content), nor significantly moderated by BMI. Instead, it depends on people's attentional focus, and may reflect motivational salience. Furthermore, food palatability and caloric content are represented as patterns of brain activity, independently of BMI and attentional focus. So, food reward value is reflected in patterns, not levels, of brain activity.

Inconsistencies in atlas-based volumetric measures of the human nucleus basalis of Meynert: A need for high-resolution alternatives.

The nucleus basalis of Meynert (nbM) is the major source of cortical acetylcholine (ACh) and has been related to cognitive processes and to neurological disorders. However, spatially delineating the human nbM in MRI studies remains challenging. Due to the absence of a functional localiser for the human nbM, studies to date have localised it using nearby neuroanatomical landmarks or using probabilistic atlases. To understand the feasibility of MRI of the nbM we set our four goals; our first goal was to review current human nbM region-of-interest (ROI) selection protocols used in MRI studies, which we found have reported highly variable nbM volume estimates. Our next goal was to quantify and discuss the limitations of existing atlas-based volumetry of nbM. We found that the identified ROI volume depends heavily on the atlas used and on the probabilistic threshold set. In addition, we found large disparities even for data/studies using the same atlas and threshold. To test whether spatial resolution contributes to volume variability, as our third goal, we developed a novel nbM mask based on the normalized BigBrain dataset. We found that as long as the spatial resolution of the target data was 1.3 mm isotropic or above, our novel nbM mask offered realistic and stable volume estimates. Finally, as our last goal we tried to discern nbM using publicly available and novel high resolution structural MRI ex vivo MRI datasets. We find that, using an optimised 9.4T quantitative T2⁎ ex vivo dataset, the nbM can be visualised using MRI. We conclude caution is needed when applying the current methods of mapping nbM, especially for high resolution MRI data. Direct imaging of the nbM appears feasible and would eliminate the problems we identify, although further development is required to allow such imaging using standard (f)MRI scanning.

BUDA-MESMERISE: Rapid acquisition and unsupervised parameter estimation for T1 , T2 , M0 , B0 , and B1 maps.

PURPOSE: Rapid acquisition scheme and parameter estimation method are proposed to acquire distortion-free spin- and stimulated-echo signals and combine the signals with a physics-driven unsupervised network to estimate T1 , T2 , and proton density (M0 ) parameter maps, along with B0 and B1 information from the acquired signals. THEORY AND METHODS: An imaging sequence with three 90° RF pulses is utilized to acquire spin- and stimulated-echo signals. We utilize blip-up/-down acquisition to eliminate geometric distortion incurred by the effects of B0 inhomogeneity on rapid EPI acquisitions. For multislice imaging, echo-shifting is applied to utilize dead time between the second and third RF pulses to encode information from additional slice positions. To estimate parameter maps from the spin- and stimulated-echo signals with high fidelity, 2 estimation methods, analytic fitting and a novel unsupervised deep neural network method, are developed. RESULTS: The proposed acquisition provided distortion-free T1 , T2 , relative proton density (M0), B0 , and B1 maps with high fidelity both in phantom and in vivo brain experiments. From the rapidly acquired spin- and stimulated-echo signals, analytic fitting and the network-based method were able to estimate T1 , T2 , M0 , B0 , and B1 maps with high accuracy. Network estimates demonstrated noise robustness owing to the fact that the convolutional layers take information into account from spatially adjacent voxels. CONCLUSION: The proposed acquisition/reconstruction technique enabled whole-brain acquisition of coregistered, distortion-free, T1 , T2 , M0 , B0 , and B1 maps at 1 × 1 × 5 mm3 resolution in 50 s. The proposed unsupervised neural network provided noise-robust parameter estimates from this rapid acquisition.

Effects of mindset on hormonal responding, neural representations, subjective experience and intake.

A person can alternate between food-related mindsets, which in turn may depend on one's emotional state or situation. Being in a certain mindset can influence food-related thoughts, but interestingly it might also affect eating-related physiological responses. The current study investigates the influence of an induced 'loss of control' mindset as compared to an 'in control' mindset on hormonal, neural and behavioural responses to chocolate stimuli. Mindsets were induced by having female chocolate lovers view a short movie during two sessions in a within-subjects design. Neural responses to visual chocolate stimuli were measured using an ultra-high field (7T) scanner. Momentary ghrelin and glucagon-like peptide 1 (GLP-1) levels were determined on five moments and were simultaneously assessed with self-reports on perceptions of chocolate craving, hunger and feelings of control. Furthermore, chocolate intake was measured using a bogus chocolate taste test. It was hypothesized that the loss of control mindset would lead to hormonal, neural and behavioural responses that prepare for ongoing food intake, even after eating, while the control mindset would lead to responses reflecting satiety. Results show that neural activity in the mesocorticolimbic system was stronger for chocolate stimuli than for neutral stimuli and that ghrelin and GLP-1 levels responded to food intake, irrespective of mindset. Self-reported craving and actual chocolate intake were affected by mindset, in that cravings and intake were higher with a loss of control mindset than with a control mindset. Interestingly, these findings suggest that physiology on the one hand (hormonal and neural responses) and behavior and subjective experience (food intake and craving) on the other hand are not in sync, are not equally affected by mindset.

Human larynx motor cortices coordinate respiration for vocal-motor control.

Vocal flexibility is a hallmark of the human species, most particularly the capacity to speak and sing. This ability is supported in part by the evolution of a direct neural pathway linking the motor cortex to the brainstem nucleus that controls the larynx the primary sound source for communication. Early brain imaging studies demonstrated that larynx motor cortex at the dorsal end of the orofacial division of motor cortex (dLMC) integrated laryngeal and respiratory control, thereby coordinating two major muscular systems that are necessary for vocalization. Neurosurgical studies have since demonstrated the existence of a second larynx motor area at the ventral extent of the orofacial motor division (vLMC) of motor cortex. The vLMC has been presumed to be less relevant to speech motor control, but its functional role remains unknown. We employed a novel ultra-high field (7T) magnetic resonance imaging paradigm that combined singing and whistling simple melodies to localise the larynx motor cortices and test their involvement in respiratory motor control. Surprisingly, whistling activated both 'larynx areas' more strongly than singing despite the reduced involvement of the larynx during whistling. We provide further evidence for the existence of two larynx motor areas in the human brain, and the first evidence that laryngeal-respiratory integration is a shared property of both larynx motor areas. We outline explicit predictions about the descending motor pathways that give these cortical areas access to both the laryngeal and respiratory systems and discuss the implications for the evolution of speech.

Dedicated container for postmortem human brain ultra-high field magnetic resonance imaging.

BACKGROUND: The emerging field of ultra-high field MRI (UHF-MRI, 7 Tesla and higher) provides the opportunity to image human brains at a higher resolution and with higher signal-to-noise ratios compared to the more widely available 1.5 and 3T scanners. Scanning postmortem tissue additionally allows for greatly increased scan times and fewer movement issues leading to improvements in image quality. However, typical postmortem neuroimaging routines involve placing the tissue within plastic bags that leave room for susceptibility artifacts from tissue-air interfaces, inadequate submersion, and leakage issues. To address these challenges in postmortem imaging, a custom-built nonferromagnetic container was developed that allows whole brain hemispheres to be scanned at sub-millimeter resolution within typical head-coils. METHOD: The custom-built polymethylmethacrylaat container consists of a cylinder with a hemispheric side and a lid with valves on the adjacent side. This shape fits within common MR head-coils and allows whole hemispheres to be submerged and vacuum sealed within it reducing imaging artifacts that would otherwise arise at air-tissue boundaries. Two hemisphere samples were scanned on a Siemens 9.4T Magnetom MRI scanner. High resolution T2* weighted data was obtained with a custom 3D gradient echo (GRE) sequence and diffusion-weighted imaging (DWI) scans were obtained with a 3D kT-dSTEAM sequence along 48 directions. RESULTS: The custom-built container proved to submerge and contain tissue samples effectively and showed no interferences with MR scanning acquisition. The 3D GRE sequence provided high resolution isotropic T2* weighted data at 250 µm which showed a clear visualization of gray and white matter structures. DWI scans allowed for dense reconstruction of structural white matter connections via tractography. CONCLUSION: Using this custom-built container worked towards achieving high quality MR images of postmortem brain material. This procedure can have advantages over traditional schemes including utilization of a standardized protocol and the reduced likelihood of leakage. This methodology could be adjusted and used to improve typical postmortem imaging routines.

hFRUIT: An optimized agent for optical clearing of DiI-stained adult human brain tissue.

Here, we describe a new immersion-based clearing method suitable for optical clearing of thick adult human brain samples while preserving its lipids and lipophilic labels such as 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). This clearing procedure is simple, easy to implement, and allowed for clearing of 5 mm thick human brain tissue samples within 12 days. Furthermore, we show for the first time the advantageous effect of the Periodate-Lysine-Paraformaldehyde (PLP) fixation as compared to the more commonly used 4% paraformaldehyde (PFA) on clearing performance.

Gray matter network reorganization in multiple sclerosis from 7-Tesla and 3-Tesla MRI data.

OBJECTIVE: The objective of this study was to determine the ability of 7T-MRI for characterizing brain tissue integrity in early relapsing-remitting MS patients compared to conventional 3T-MRI and to investigate whether 7T-MRI improves the performance for detecting cortical gray matter neurodegeneration and its associated network reorganization dynamics. METHODS: Seven early relapsing-remitting MS patients and seven healthy individuals received MRI at 7T and 3T, whereas 30 and 40 healthy controls underwent separate 3T- and 7T-MRI sessions, respectively. Surface-based cortical thickness (CT) and gray-to-white contrast (GWc) measures were used to model morphometric networks, analyzed with graph theory by means of modularity, clustering coefficient, path length, and small-worldness. RESULTS: 7T-MRI had lower CT and higher GWc compared to 3T-MRI in MS. CT and GWc measures robustly differentiated MS from controls at 3T-MRI. 7T- and 3T-MRI showed high regional correspondence for CT (r = 0.72, P = 2e-78) and GWc (r = 0.83, P = 5.5e-121) in MS patients. MS CT and GWc morphometric networks at 7T-MRI showed higher modularity, clustering coefficient, and small-worldness than 3T, also compared to controls. INTERPRETATION: 7T-MRI allows to more precisely quantify morphometric alterations across the cortical mantle and captures more sensitively MS-related network reorganization. Our findings open new avenues to design more accurate studies quantifying brain tissue loss and test treatment effects on tissue repair.

Power of mind: Attentional focus rather than palatability dominates neural responding to visual food stimuli in females with overweight.

Research investigating neural responses to visual food stimuli has produced inconsistent results. Crucially, high-caloric palatable foods have a double-sided nature - they are often craved but are also considered unhealthy - which may have contributed to the inconsistency in the literature. Taking this double-sided nature into account in the current study, neural responses to individually tailored palatable and unpalatable high caloric food stimuli were measured, while participants' (females with overweight: n = 23) attentional focus was manipulated to be either hedonic or neutral. Notably, results showed that the level of neural activity was not significantly different for palatable than for unpalatable food stimuli. Instead, independent of food palatability, several brain regions (including regions in the mesocorticolimbic system) responded more strongly when attentional focus was hedonic than when neutral (p < 0.05, cluster-based FWE corrected). Multivariate analyses showed that food palatability could be decoded from multi-voxel patterns of neural activity (p < 0.05, FDR corrected), mostly with a hedonic attentional focus. These findings illustrate that the level of neural activity might not be proportionate to the palatability of foods, but that food palatability can be decoded from multi-voxel patterns of neural activity. Moreover, they underline the importance of considering attentional focus when measuring food-related neural responses.

The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue.

Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample ( www.mesospim.org ).

Scalable Labeling for Cytoarchitectonic Characterization of Large Optically Cleared Human Neocortex Samples.

Optical clearing techniques and light sheet microscopy have transformed fluorescent imaging of rodent brains, and have provided a crucial alternative to traditional confocal or bright field techniques for thin sections. However, clearing and labeling human brain tissue through all cortical layers and significant portions of a cortical area, has so far remained extremely challenging, especially for formalin fixed adult cortical tissue. Here, we present MASH (Multiscale Architectonic Staining of Human cortex): a simple, fast and low-cost cytoarchitectonic labeling approach for optically cleared human cortex samples, which can be applied to large (up to 5 mm thick) formalin fixed adult brain samples. A suite of small-molecule fluorescent nuclear and cytoplasmic dye protocols in combination with new refractive index matching solutions allows deep volume imaging. This greatly reduces time and cost of imaging cytoarchitecture in thick samples and enables classification of cytoarchitectonic layers over the full cortical depth. We demonstrate application of MASH to large archival samples of human visual areas, characterizing cortical architecture in 3D from the scale of cortical areas to that of single cells. In combination with scalable light sheet imaging and data analysis, MASH could open the door to investigation of large human cortical systems at cellular resolution and in the context of their complex 3-dimensional geometry.

Characterizing Microstructural Tissue Properties in Multiple Sclerosis with Diffusion MRI at 7 T and 3 T: The Impact of the Experimental Design.

The recent introduction of advanced magnetic resonance (MR) imaging techniques to characterize focal and global degeneration in multiple sclerosis (MS), like the Composite Hindered and Restricted Model of Diffusion, or CHARMED, diffusional kurtosis imaging (DKI) and Neurite Orientation Dispersion and Density Imaging (NODDI) made available new tools to image axonal pathology non-invasively in vivo. These methods already showed greater sensitivity and specificity compared to conventional diffusion tensor-based metrics (e.g., fractional anisotropy), overcoming some of its limitations. While previous studies uncovered global and focal axonal degeneration in MS patients compared to healthy controls, here our aim is to investigate and compare different diffusion MRI acquisition protocols in their ability to highlight microstructural differences between MS and control tissue over several much used models. For comparison, we contrasted the ability of fractional anisotropy measurements to uncover differences between lesion, normal-appearing white matter (WM), gray matter and healthy tissue under the same imaging protocols. We show that: (1) focal and diffuse differences in several microstructural parameters are observed under clinical settings; (2) advanced models (CHARMED, DKI and NODDI) have increased specificity and sensitivity to neurodegeneration when compared to fractional anisotropy measurements; and (3) both high (3 T) and ultra-high fields (7 T) are viable options for imaging tissue change in MS lesions and normal appearing WM, while higher b-values are less beneficial under the tested short-time (10 min acquisition) conditions.

Ex vivo diffusion MRI of the human brain: Technical challenges and recent advances.

This review discusses ex vivo diffusion magnetic resonance imaging (dMRI) as an important research tool for neuroanatomical investigations and the validation of in vivo dMRI techniques, with a focus on the human brain. We review the challenges posed by the properties of post-mortem tissue, and discuss state-of-the-art tissue preparation methods and recent advances in pulse sequences and acquisition techniques to tackle these. We then review recent ex vivo dMRI studies of the human brain, highlighting the validation of white matter orientation estimates and the atlasing and mapping of large subcortical structures. We also give particular emphasis to the delineation of layered gray matter structure with ex vivo dMRI, as this application illustrates the strength of its mesoscale resolution over large fields of view. We end with a discussion and outlook on future and potential directions of the field.