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Tissue clearing and its applications in neuroscience.

ABSTRACT: State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.

SUBMITTER: Ueda HR

PROVIDER: S-EPMC8121164 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Tissue clearing and its applications in neuroscience.

Ueda Hiroki R HR Ertürk Ali A Chung Kwanghun K Gradinaru Viviana V Chédotal Alain A Tomancak Pavel P Keller Philipp J PJ

Nature reviews. Neuroscience 20200201 2

State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By ...[more]

PMID: 31896771

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Project description:Microelectrode arrays are invaluable tools for investigating the electrophysiological behaviour of neuronal networks with high spatiotemporal precision. In recent years, it has become increasingly common to functionalize such electrodes with highly porous platinum to increase their effective surface area, and hence their signal-to-noise ratio. Although such functionalization significantly improves the electrochemical performance of the electrodes, the impact of various electrode morphologies on biocompatibility and electrophysiological performance in cell cultures remains poorly understood. In this study, we introduce reproducible protocols for depositing highly porous platinum with varying morphologies on microelectrodes designed for neural cell cultures. We also evaluate the impact of morphology and electrode size on the signal-to-noise ratio in recordings from rat cortical neurons cultured on these electrodes. Our results indicate that electrodes with a uniform layer of highly nanoporous platinum offer the best trade-off between biocompatibility, electrochemical, and electrophysiological performance. While more microporous electrodes exhibited lower impedance, nanoporous electrodes detected higher extracellular signal amplitudes from neurons, suggesting reduced distance between perisomatic neuronal areas and the electrodes. Additionally, these nanoporous electrodes showed fewer thickness variations at their edges compared to the more porous electrodes. Such edges can be mechanically broken off during cell culturing and contribute to long-term cytotoxic effects, which is highly undesirable. We hope this work will contribute to better standardization in creating and utilizing nanoporous platinum microelectrodes for neuroscience applications. Improving the accessibility and reproducibility of this technology is crucial for enhancing the quality of electrophysiological data and advancing our understanding of neuronal network function and dysfunction.

Project description:BackgroundInvestigation 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.ResultsWe 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.ConclusionsIn 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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Tissue clearing and its applications in neuroscience.

Publications

Tissue clearing and its applications in neuroscience.

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