Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Spheroids have emerged as in vitro models that reproduce in a great extent the architectural microenvironment found in human tissues. However, the imaging of 3D cell cultures is highly challenging due to its high thickness, which results in a light-scattering phenomenon that limits light penetration. Therefore, several optical clearing methods, widely used in the imaging of animal tissues, have been recently explored to render spheroids with enhanced transparency. These methods are aimed to homogenize the microtissue refractive index (RI) and can be grouped into four different categories, namely (a) simple immersion in an aqueous solution with high RI; (b) delipidation and dehydration followed by RI matching; (c) delipidation and hyperhydration followed by RI matching; and (d) hydrogel embedding followed by delipidation and RI matching. In this review, the main optical clearing methods, their mechanism of action, advantages, and disadvantages are described. Furthermore, the practical examples of the optical clearing methods application for the imaging of 3D spheroids are highlighted.     

Differential prefrontal white matter development in chimpanzees and humans

A comparison of developmental patterns of white matter (WM) within the prefrontal region between humans and nonhuman primates is key to understanding human brain evolution. WM mediates complex cognitive processes and has reciprocal connections with posterior processing regions [1, 2]. Although the developmental pattern of prefrontal WM in macaques differs markedly from that in humans [3], this has not been explored in our closest evolutionary relative, the chimpanzee. The present longitudinal study of magnetic resonance imaging scans demonstrated that the prefrontal WM volume in chimpanzees was immature and had not reached the adult value during prepuberty, as observed in humans but not in macaques. However, the rate of prefrontal WM volume increase during infancy was slower in chimpanzees than in humans. These results suggest that a less mature and more protracted elaboration of neuronal connections in the prefrontal portion of the developing brain existed in the last common ancestor of chimpanzees and humans, and that this served to enhance the impact of postnatal experiences on neuronal connectivity. Furthermore, the rapid development of the human prefrontal WM during infancy may help the development of complex social interactions, as well as the acquisition of experience-dependent knowledge and skills to shape neuronal connectivity.     

Colorization and automated segmentation of human T2 MR brain images for characterization of soft tissues

Characterization of tissues like brain by using magnetic resonance (MR) images and colorization of the gray scale image has been reported in the literature, along with the advantages and drawbacks. Here, we present two independent methods; (i) a novel colorization method to underscore the variability in brain MR images, indicative of the underlying physical density of bio tissue, (ii) a segmentation method (both hard and soft segmentation) to characterize gray brain MR images. The segmented images are then transformed into color using the above-mentioned colorization method, yielding promising results for manual tracing. Our color transformation incorporates the voxel classification by matching the luminance of voxels of the source MR image and provided color image by measuring the distance between them. The segmentation method is based on single-phase clustering for 2D and 3D image segmentation with a new auto centroid selection method, which divides the image into three distinct regions (gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) using prior anatomical knowledge). Results have been successfully validated on human T2-weighted (T2) brain MR images. The proposed method can be potentially applied to gray-scale images from other imaging modalities, in bringing out additional diagnostic tissue information contained in the colorized image processing approach as described.     

光学透明技术在植物多尺度成像中的应用

光学透明技术是一种通过各种化学试剂,将原本不透明的生物样本实现透明化,并在光学显微镜下深度成像的技术。结合多种光学显微成像新技术,光学透明技术可对整个组织进行成像和三维重建,深度剖析生物体内部空间特征与形成机制。近年来,多种植物光学透明技术和多尺度成像技术被陆续研发,并取得了丰硕的研究成果。该文综述了生物体光学透明技术的基本原理和一些新技术,重点介绍基于光学透明技术开发的新型成像方法及其在植物成像与细胞生物学中的应用,为后续植物整体、组织或器官的透明、成像与三维重构及功能研究提供理论依据和技术支持。     

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.     

Q-ball imaging of macaque white matter architecture

Diffusion-weighted magnetic resonance imaging holds substantial promise as a technique for non-invasive imaging of white matter (WM) axonal projections. For diffusion imaging to be capable of providing new insight into the connectional neuroanatomy of the human brain, it will be necessary to histologically validate the technique against established tracer methods such as horseradish peroxidase and biocytin histochemistry. The macaque monkey provides an ideal model for histological validation of the diffusion imaging method due to the phylogenetic proximity between humans and macaques, the gyrencephalic structure of the macaque cortex, the large body of knowledge on the neuroanatomic connectivity of the macaque brain and the ability to use comparable magnetic resonance acquisition protocols in both species. Recently, it has been shown that high angular resolution diffusion imaging (HARDI) can resolve multiple axon orientations within an individual imaging voxel in human WM. This capability promises to boost the accuracy of tract reconstructions from diffusion imaging. If the macaque is to serve as a model for histological validation of the diffusion tractography method, it will be necessary to show that HARDI can also resolve intravoxel architecture in macaque WM. The present study therefore sought to test whether the technique can resolve intravoxel structure in macaque WM. Using a HARDI method called q-ball imaging (QBI) it was possible to resolve composite intravoxel architecture in a number of anatomic regions. QBI resolved intravoxel structure in, for example, the dorsolateral convexity, the pontine decussation, the pulvinar and temporal subcortical WM. The paper concludes by reviewing remaining challenges for the diffusion tractography project.     

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.^1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light³ do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.⁴ Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.     

Mapping connectivity damage in the case of Phineas Gage

White matter (WM) mapping of the human brain using neuroimaging techniques has gained considerable interest in the neuroscience community. Using diffusion weighted (DWI) and magnetic resonance imaging (MRI), WM fiber pathways between brain regions may be systematically assessed to make inferences concerning their role in normal brain function, influence on behavior, as well as concerning the consequences of network-level brain damage. In this paper, we investigate the detailed connectomics in a noted example of severe traumatic brain injury (TBI) which has proved important to and controversial in the history of neuroscience. We model the WM damage in the notable case of Phineas P. Gage, in whom a "tamping iron" was accidentally shot through his skull and brain, resulting in profound behavioral changes. The specific effects of this injury on Mr. Gage's WM connectivity have not previously been considered in detail. Using computed tomography (CT) image data of the Gage skull in conjunction with modern anatomical MRI and diffusion imaging data obtained in contemporary right handed male subjects (aged 25-36), we computationally simulate the passage of the iron through the skull on the basis of reported and observed skull fiducial landmarks and assess the extent of cortical gray matter (GM) and WM damage. Specifically, we find that while considerable damage was, indeed, localized to the left frontal cortex, the impact on measures of network connectedness between directly affected and other brain areas was profound, widespread, and a probable contributor to both the reported acute as well as long-term behavioral changes. Yet, while significantly affecting several likely network hubs, damage to Mr. Gage's WM network may not have been more severe than expected from that of a similarly sized "average" brain lesion. These results provide new insight into the remarkable brain injury experienced by this noteworthy patient.     

Quantification of ethanol methyl 1H magnetic resonance signal intensity following intravenous ethanol administration in primate brain

In vivo ¹H magnetic resonance spectroscopy (MRS) can be used to directly monitor brain ethanol. Previously, studies of human subjects have lead to the suggestion that the ethanol methyl ¹H MRS signal intensity relates to tolerance to ethanol’s intoxicating effects. More recently, the ethanol ¹H MRS signal intensity has been recognized to vary between brain gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) due to differences in T₂ within these environments. The methods presented here extend ethanol MRS techniques to non-human primate subjects. Twelve monkeys were administered ethanol while sedated and positioned within a 3T MRI system. Chemical shift imaging (CSI) measurements were performed following intravenous infusion of 1 g/kg ethanol. Magnetic resonance imaging (MRI) data were also recorded for each monkey to provide volume fractions of GM, WM, and CSF for each CSI spectrum. To estimate co-variance of ethanol MRS intensity with GM, WM, and CSF volume fractions, the relative contribution of each tissue subtype was determined following corrections for radiofrequency pulse profile non-uniformity, chemical shift artifacts, and differences between the point spread function in the CSI data and the imaging data. The ethanol MRS intensity per unit blood ethanol concentration was found to differ between GM, WM, and CSF. Individual differences in MRS intensity were larger in GM than WM. This methodology demonstrates the feasibility of ethanol MRS experiments and analysis in non-human primate subjects, and suggests GM may be a site of significant variation in ethanol MRS intensity between individuals.     

Effect of Simulated Microgravity on Human Brain Gray Matter and White Matter – Evidence from MRI

Background

There is limited and inconclusive evidence that space environment, especially microgravity condition, may affect microstructure of human brain. This experiment hypothesized that there would be modifications in gray matter (GM) and white matter (WM) of the brain due to microgravity.

Method

Eighteen male volunteers were recruited and fourteen volunteers underwent -6° head-down bed rest (HDBR) for 30 days simulated microgravity. High-resolution brain anatomical imaging data and diffusion tensor imaging images were collected on a 3T MR system before and after HDBR. We applied voxel-based morphometry and tract-based spatial statistics analysis to investigate the structural changes in GM and WM of brain.

Results

We observed significant decreases of GM volume in the bilateral frontal lobes, temporal poles, parahippocampal gyrus, insula and right hippocampus, and increases of GM volume in the vermis, bilateral paracentral lobule, right precuneus gyrus, left precentral gyrus and left postcentral gyrus after HDBR. Fractional anisotropy (FA) changes were also observed in multiple WM tracts.

Conclusion

These regions showing GM changes are closely associated with the functional domains of performance, locomotion, learning, memory and coordination. Regional WM alterations may be related to brain function decline and adaption. Our findings provide the neuroanatomical evidence of brain dysfunction or plasticity in microgravity condition and a deeper insight into the cerebral mechanisms in microgravity condition.     

Age-related white matter microstructural differences partly mediate age-related decline in processing speed but not cognition

Aging is associated with declining cognitive performance as well as structural changes in brain gray and white matter (WM). The WM deterioration contributes to a disconnection among distributed brain networks and may thus mediate age-related cognitive decline. The present diffusion tensor imaging (DTI) study investigated age-related differences in WM microstructure and their relation to cognition (episodic memory, visuospatial processing, fluency, and speed) in a large group of healthy subjects (n = 287) covering 6 decades of the human life span. Age related decreases in fractional anisotropy (FA) and increases in mean diffusivity (MD) were observed across the entire WM skeleton as well as in specific WM tracts, supporting the WM degeneration hypothesis. The anterior section of the corpus callosum was more susceptible to aging compared to the posterior section, lending support to the anterior-posterior gradient of WM integrity in the corpus callosum. Finally, and of critical interest, WM integrity differences were found to mediate age-related reductions in processing speed but no significant mediation was found for episodic memory, visuospatial ability, or fluency. These findings suggest that compromised WM integrity is not a major contributing factor to declining cognitive performance in normal aging. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.     

Quantitative assessment of optical clearing methods in various intact mouse organs

Various tissue optical clearing techniques have sprung up for large volume imaging. However, there are few methods showed clearing and imaging data on different organs while most of them were focused on mouse brain, and as a result, it is difficult to select the suitable method for organs in practical applications due to lack of quantitative evaluation and comprehensive comparison. Therefore, it is necessary to evaluate and compare the performances of clearing methods for different organs. In this paper, several typical optical clearing methods were applied, including 3DISCO, uDISCO, SeeDB, FRUIT, CUBIC, ScaleS and PACT to clear intact brain, heart, kidney, liver, spleen, stomach, lung, small intestine, skin and muscle. The clearing efficiency, sample deformation, fluorescence preservation and imaging depth of these methods were quantitatively evaluated. Finally, based on the systemic evaluation of various parameters described above, the appropriate clearing method for specific organ including kidney or intestine was screened out. This paper will provide important references for selection of appropriate clearing methods in related researches.      

High‐resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO)

Optical tissue clearing using dibenzyl ether (DBE) or BABB (1 part benzyl alcohol and 2 parts benzyl benzoate) is easy in application and allows deep‐tissue imaging of a wide range of specimens. However, in both substances, optical clearing and storage times of enhanced green fluorescent protein (EGFP)‐expressing specimens are limited due to the continuous formation of peroxides and aldehydes, which severely quench fluorescence. Stabilisation of purified DBE or BABB by addition of the antioxidant propyl gallate efficiently preserves fluorescence signals in EGFP‐expressing samples for more than a year. This enables longer clearing times and improved tissue transparency with higher fluorescence signal intensity. The here introduced clearing protocol termed stabilised DISCO allows to image spines in a whole mouse brain and to detect faint changes in the activity‐dependent expression pattern of tdTomato.      

Empirical validation of gradient field models for an accurate ADC measured on clinical 3T MR systems in body oncologic applications

PurposeTo empirically corroborate vendor-provided gradient nonlinearity (GNL) characteristics and demonstrate efficient GNL bias correction for human brain apparent diffusion coefficient (ADC) across 3T MR systems and spatial locations.MethodsSpatial distortion vector fields (DVF) were mapped in 3D using a surface fiducial array phantom for individual gradient channels on three 3T MR platforms from different vendors. Measured DVF were converted into empirical 3D GNL tensors and compared with their theoretical counterparts derived from vendor-provided spherical harmonic (SPH) coefficients. To illustrate spatial impact of GNL on ADC, diffusion weighted imaging using three orthogonal gradient directions was performed on a volunteer brain positioned at isocenter (as a reference) and offset superiorly by 10–17 cm (>10% predicted GNL bias). The SPH tensor-based GNL correction was applied to individual DWI gradient directions, and derived ADC was compared with low-bias reference for human brain white matter (WM) ROIs.ResultsEmpiric and predicted GNL errors were comparable for all three studied 3T MR systems, with <1.0% differences in the median and width of spatial histograms for individual GNL tensor elements. Median (±width) of ADC (10⁻³mm²/s) histograms measured at isocenter in WM reference ROIs from three MR systems were: 0.73 ± 0.11, 0.71 ± 0.14, 0.74 ± 0.17, and at off-isocenters (before versus after GNL correction) were respectively 0.63 ± 0.14 versus 0.72 ± 0.11, 0.53 ± 0.16 versus 0.74 ± 0.18, and 0.65 ± 0.16 versus 0.76 ± 0.18.ConclusionThe phantom-based spatial distortion measurements validated vendor-provided gradient fields, and accurate WM ADC was recovered regardless of spatial locations and clinical MR platforms using system-specific tensor-based GNL correction for routine DWI.     

Brain‐derived neurotrophic factor genotype is associated with brain gray and white matter tissue volumes recovery in abstinent alcohol‐dependent individuals

Neuroimaging studies have linked the methionine (Met) allele of the brain‐derived neurotrophic factor (BDNF) gene to abnormal regional brain volumes in several psychiatric and neurodegenerative diseases. However, no neuroimaging studies assessed the effects of this allele on brain morphology in alcohol use disorders and its demonstrated change during abstinence from alcohol. Here we assessed the effects of the BDNF Val66Met (rs6265) polymorphism on regional brain tissue volumes and their recovery during short‐term abstinence in treatment‐seeking alcohol‐dependent individuals. 3D T1 weighted magnetic resonance images from 62 individuals were acquired at 1.5 T at one week of abstinence from alcohol; 41 of the participants were rescanned at 5 weeks of abstinence. The images were segmented into gray matter (GM), white matter (WM) and cerebrospinal fluid and parcellated into regional volumes. The BDNF genotype was determined from blood samples using the TaqMan technique. Alcohol‐dependent Val (Valine)/Met heterozygotes and Val homozygotes had similar regional brain volumes at either time point. However, Val homozygotes had significant GM volume increases, while Val/Met heterozygotes increased predominantly in WM volumes over the scan interval. Longitudinal increases in GM but not WM volumes were related to improvements in neurocognitive measures during abstinence. The findings suggest that functionally significant brain tissue volume recovery during abstinence from alcohol is influenced by BDNF genotype.     

Seeing through Musculoskeletal Tissues: Improving In Situ Imaging of Bone and the Lacunar Canalicular System through Optical Clearing

In situ, cells of the musculoskeletal system reside within complex and often interconnected 3-D environments. Key to better understanding how 3-D tissue and cellular environments regulate musculoskeletal physiology, homeostasis, and health is the use of robust methodologies for directly visualizing cell-cell and cell-matrix architecture in situ. However, the use of standard optical imaging techniques is often of limited utility in deep imaging of intact musculoskeletal tissues due to the highly scattering nature of biological tissues. Drawing inspiration from recent developments in the deep-tissue imaging field, we describe the application of immersion based optical clearing techniques, which utilize the principle of refractive index (RI) matching between the clearing/mounting media and tissue under observation, to improve the deep, in situ imaging of musculoskeletal tissues. To date, few optical clearing techniques have been applied specifically to musculoskeletal tissues, and a systematic comparison of the clearing ability of optical clearing agents in musculoskeletal tissues has yet to be fully demonstrated. In this study we tested the ability of eight different aqueous and non-aqueous clearing agents, with RIs ranging from 1.45 to 1.56, to optically clear murine knee joints and cortical bone. We demonstrated and quantified the ability of these optical clearing agents to clear musculoskeletal tissues and improve both macro- and micro-scale imaging of musculoskeletal tissue across several imaging modalities (stereomicroscopy, spectroscopy, and one-, and two-photon confocal microscopy) and investigational techniques (dynamic bone labeling and en bloc tissue staining). Based upon these findings we believe that optical clearing, in combination with advanced imaging techniques, has the potential to complement classical musculoskeletal analysis techniques; opening the door for improved in situ investigation and quantification of musculoskeletal tissues.     

Compensatory neural reorganization in Tourette syndrome

Children with neurological disorders may follow unique developmental trajectories whereby they undergo compensatory neuroplastic changes in brain structure and function that help them gain control over their symptoms. We used behavioral and brain imaging techniques to investigate this conjecture in children with Tourette syndrome (TS). Using a behavioral task that induces high levels of intermanual conflict, we show that individuals with TS exhibit enhanced control of motor output. Then, using structural (diffusion-weighted imaging) brain imaging techniques, we demonstrate widespread differences in the white matter (WM) microstructure of the TS brain that include alterations in the corpus callosum and forceps minor (FM) WM that significantly predict tic severity in TS. Most importantly, we show that task performance for the TS group (but not for controls) is strongly predicted by the WM microstructure of the FM pathways that lead to the prefrontal cortex and by the functional magnetic resonance imaging blood oxygen level-dependent response in prefrontal areas connected by these tracts. These results provide evidence for compensatory brain reorganization that may underlie the increased self-regulation mechanisms that have been hypothesized to bring about the control of tics during adolescence.     

Optimizing the Magnetization-Prepared Rapid Gradient-Echo (MP-RAGE) Sequence

The three-dimension (3D) magnetization-prepared rapid gradient-echo (MP-RAGE) sequence is one of the most popular sequences for structural brain imaging in clinical and research settings. The sequence captures high tissue contrast and provides high spatial resolution with whole brain coverage in a short scan time. In this paper, we first computed the optimal k-space sampling by optimizing the contrast of simulated images acquired with the MP-RAGE sequence at 3.0 Tesla using computer simulations. Because the software of our scanner has only limited settings for k-space sampling, we then determined the optimal k-space sampling for settings that can be realized on our scanner. Subsequently we optimized several major imaging parameters to maximize normal brain tissue contrasts under the optimal k-space sampling. The optimal parameters are flip angle of 12°, effective inversion time within 900 to 1100 ms, and delay time of 0 ms. In vivo experiments showed that the quality of images acquired with our optimal protocol was significantly higher than that of images obtained using recommended protocols in prior publications. The optimization of k-spacing sampling and imaging parameters significantly improved the quality and detection sensitivity of brain images acquired with MP-RAGE.     

Improvement of low-level light imaging performance using optical clearing method

Low-level light-emitting imaging technique often detects the light emerged at the tissue surface that is generated internally from a specific target. However, in most cases, the high scattering nature of biological tissue limits the sensitivity and spatial resolution of this imaging modality. In this paper, we report that a significant improvement of chemiluminescence (CL) imaging performance in terms of both sensitivity and spatial resolution can be achieved by use of the topical application of glycerol solution onto tissue sample, i.e. optical clearing approach. Monte Carlo (MC) simulation of internally-launched point source shows that the decrease of scattering coefficient of turbid medium, which can be achieved by optical tissue clearing approach, causes stronger peak intensity with a narrower full-width at half-maximum (FWHM). The improvement becomes more significant with the source depth increasing from 1 to 5 mm. The experimental results shows that tissue clearing with 50% glycerol solution could largely improve the brightness and the spatial resolution of CL imaging when the target is covered by biological tissue with a thickness of either 1 or 3mm. This method could have potential applications for the in vivo low-level light imaging techniques.     

A Rapid Optical Clearing Protocol Using 2,2′-Thiodiethanol for Microscopic Observation of Fixed Mouse Brain

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2′-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.