Tissue clearing and its applications in neuroscience.
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ABSTRACT:
SUBMITTER: Ueda HR
PROVIDER: S-EPMC8121164 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tissue-clearing techniques have received great attention for volume imaging and for the potential to be applied in optical diagnosis. In principle, tissue clearing is achieved by reducing light scattering through a combination of lipid removal, size change, and matching of the refractive index (RI) between the imaging solution and the tissue. However, the contributions of these major factors in tissue clearing have not been systematically evaluated yet. In this study, we experimentally measured and mathematically calculated the contribution of these factors to the clearing of four organs (brain, liver, kidney, and lung). We found that these factors differentially influence the maximal clearing efficacy of tissues and the diffusivity of materials inside the tissue. We propose that these physical properties of organs can be utilized for the quality control (Q/C) process during tissue clearing, as well as for the monitoring of the pathological changes of tissues.
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Project description:Visualizing biological events and states to resolve biological questions is challenging. Tissue clearing permits three-dimensional multicolor imaging. Here, we describe a pH-adjustable tissue clearing solution, Seebest (SEE Biological Events and States in Tissues), which preserves lipid ultrastructures at an electron microscopy level. Adoption of polyethylenimine was required for a wide pH range adjustment of the tissue clearing solution. The combination of polyethylenimine and urea had a good tissue clearing ability for multiple tissues within several hours. Blood vessels stained with lipophilic carbocyanine dyes were deeply visible using the solution. Adjusting the pH of the solution was important to maximize the fluorescent intensity and suppress dye leakage during tissue clearing. The spatial distribution of doxorubicin and oxidative stress were observable using the solution. Moreover, spatial distribution of liposomes in the liver was visualized. Hence, the Seebest solution provides pH-adjustable, rapid, sufficient tissue clearing, while preserving lipid ultrastructures, which is suitable for drug delivery system evaluations.
| S-EPMC7698078 | biostudies-literature
Project description:Investigation of the internal tissues and organs of a macroscopic organism usually requires destructive processes, such as dissection or sectioning. These processes are inevitably associated with the loss of some spatial information. Recently, aqueous-based tissue clearing techniques, which allow whole-organ or even whole-body clearing of small rodents, have been developed and opened a new method of three-dimensional histology. It is expected that these techniques will be useful tools in the field of zoology, in which organisms with highly diverse morphology are investigated and compared. However, most of these new methods are optimized for soft, non-pigmented organs in small rodents, especially the brain, and their applicability to non-model organisms with hard exoskeletons and stronger pigmentation has not been tested.We explored the possible application of an aqueous-based tissue clearing technique, advanced CUBIC, on small crustaceans. The original CUBIC procedure did not clear the terrestrial isopod, Armadillidium vulgare. Therefore, to apply the whole-mount clearing method to isopods with strong pigmentation and calcified exoskeletons, we introduced several pretreatment steps, including decalcification and bleaching. Thereafter, the clearing capacity of the procedure was dramatically improved, and A. vulgare became transparent. The internal organs, such as the digestive tract and male reproductive organs, were visible through sclerites using an ordinary stereomicroscope. We also found that fluorescent nuclear staining using propidium iodide (PI) helped to visualize the internal organs of cleared specimens. Our procedure was also effective on the marine crab, Philyra sp.In this study, we developed a method to clear whole tissues of crustaceans. To the best of our knowledge, this is the first report of whole-mount clearing applied to crustaceans using an aqueous-based technique. This technique could facilitate morphological studies of crustaceans and other organisms with calcified exoskeletons and pigmentation.
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Project description:Integration of information across heterogeneous sources creates added scientific value. Interoperability of data, tools and models is, however, difficult to accomplish across spatial and temporal scales. Here we introduce the toolbox Parallel Co-Simulation, which enables the interoperation of simulators operating at different scales. We provide a software science co-design pattern and illustrate its functioning along a neuroscience example, in which individual regions of interest are simulated on the cellular level allowing us to study detailed mechanisms, while the remaining network is efficiently simulated on the population level. A workflow is illustrated for the use case of The Virtual Brain and NEST, in which the CA1 region of the cellular-level hippocampus of the mouse is embedded into a full brain network involving micro and macro electrode recordings. This new tool allows integrating knowledge across scales in the same simulation framework and validating them against multiscale experiments, thereby largely widening the explanatory power of computational models.
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Project description:AimsA variety of tissue clearing techniques have been developed to render intact tissue transparent. For thicker samples, additional partial tissue delipidation is required before immersion into the final refractive index (RI)-matching solution, which alone is often inadequate to achieve full tissue transparency. However, it is difficult to determine a sufficient degree of tissue delipidation, excess of which can result in tissue distortion and protein loss. Here, we aim to develop a clearing strategy that allows better monitoring and more precise determination of delipidation progress.MethodsWe combined the detergent sodium dodecyl sulphate (SDS) with OPTIClear, a RI-matching solution, to form a strategy termed Accurate delipidation with Optimal Clearing (Accu-OptiClearing). Accu-OptiClearing allows for a better preview of the final tissue transparency achieved when immersed in OPTIClear alone just before imaging. We assessed for the changes in clearing rate, protein loss, degree of tissue distortion, and preservation of antigens.ResultsPartial delipidation using Accu-OptiClearing accelerated tissue clearing and better preserved tissue structure and antigens than delipidation with SDS alone. Despite achieving similar transparency in the final OPTIClear solution, more lipids were retained in samples cleared with Accu-OptiClearing compared to SDS.ConclusionsCombining the RI-matching solution OPTIClear with detergents, Accu-OptiClearing, can avoid excessive delipidation, leading to accelerated tissue clearing, less tissue damage and better preserved antigens.
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Project description:Collecting comprehensive data sets of the same subject has become a standard in neuroscience research and uncovering multivariate relationships among collected data sets have gained significant attentions in recent years. Canonical correlation analysis (CCA) is one of the powerful multivariate tools to jointly investigate relationships among multiple data sets, which can uncover disease or environmental effects in various modalities simultaneously and characterize changes during development, aging, and disease progressions comprehensively. In the past 10?years, despite an increasing number of studies have utilized CCA in multivariate analysis, simple conventional CCA dominates these applications. Multiple CCA-variant techniques have been proposed to improve the model performance; however, the complicated multivariate formulations and not well-known capabilities have delayed their wide applications. Therefore, in this study, a comprehensive review of CCA and its variant techniques is provided. Detailed technical formulation with analytical and numerical solutions, current applications in neuroscience research, and advantages and limitations of each CCA-related technique are discussed. Finally, a general guideline in how to select the most appropriate CCA-related technique based on the properties of available data sets and particularly targeted neuroscience questions is provided.
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Project description:Tissue optical clearing enables imaging deeper in large volumes with high-resolution. Clear T2 is a relatively rapid clearing method with no use of solvents or detergents, hence poses great advantage on preservation of diverse fluorescent labels. However, this method suffers from insufficient tissue transparency, especially for adult mouse brain blocks. In this work, we develop a rapid and versatile clearing method based on Clear T2 , termed RTF (Rapid clearing method based on Triethanolamine and Formamide), aiming for better clearing capability. The results show that RTF can not only efficiently clear embryos, neonatal brains and adult brain blocks, but also preserve fluorescent signal of both endogenous fluorescent proteins and lipophilic dyes, and be compatible with virus labeling and immunostaining. With the good transparency and versatile compatibility, RTF allows visualization and tracing of fluorescent labeling cells and neuronal axons combined with different imaging techniques, showing potentials in facilitating observation of morphological architecture and visualization of neuronal networks.
| S-EPMC5792593 | biostudies-literature
Project description:Brillouin spectroscopy is a well-established technology in condensed matter physics to characterize the mechanical properties of inert materials, and it has been extended very recently to the study of biological samples. Transparency is beneficial for samples to be properly analyzed by Brillouin spectroscopy. Here, we explored the efficacy of optical tissue clearing techniques to improve the acquisition of Brillouin spectra from biological tissues in order to analyze their biomechanical properties. We describe the first application of Brillouin scattering to optically cleared biological tissues with CUBIC protocol. We conclude that, within the range of error, tissue clearing does not modify the mechanical properties of the studied biological tissues.
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Project description:In situ hybridization (ISH) is a powerful technique that is used to detect the localization of specific nucleic acid sequences for understanding the organization, regulation, and function of genes. However, in most cases, RNA probes are obtained by in vitro transcription from plasmids containing specific promoter elements and mRNA-specific cDNA. Probes originating from plasmid vectors are time-consuming and not suitable for the rapid gene mapping. Here, we introduce a simplified method to prepare digoxigenin (DIG)-labeled non-radioactive RNA probes based on polymerase chain reaction (PCR) amplification and applications in free-floating mouse brain sections. Employing a transgenic reporter line, we investigate the expression of the somatostatin (SST) mRNA in the adult mouse brain. The method can be applied to identify the colocalization of SST mRNA and proteins including corticotrophin-releasing hormone (CRH) and protein kinase C delta type (PKC-?) using double immunofluorescence, which is useful for understanding the organization of complex brain nuclei. Moreover, the method can also be incorporated with retrograde tracing to visualize the functional connection in the neural circuitry. Briefly, the PCR-based method for non-radioactive RNA probes is a useful tool that can be substantially utilized in neuroscience studies.
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Project description:BackgroundSypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use.MethodsHere we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer.ResultsUsing the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate.ConclusionsSypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo.General significanceThe new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.
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