Dataset Information

Expansion-Based Clearing of Golgi-Cox-Stained Tissue for Multi-Scale Imaging.

ABSTRACT: Obtaining fine neuron morphology and connections data is extraordinarily useful in understanding the brain's functionality. Golgi staining is a widely used method for revealing neuronal morphology. However, Golgi-Cox-stained tissue is difficult to image in three dimensions and lacks cell-type specificity, limiting its use in neuronal circuit studies. Here, we describe an expansion-based method for rapidly clearing Golgi-Cox-stained tissue. The results show that 1 mm thick Golgi-Cox-stained tissue can be cleared within 6 hours with a well preserved Golgi-Cox-stained signal. At the same time, we found for the first time that the cleared Golgi-Cox-stained samples were compatible with three-dimensional (3D) immunostaining and multi-round immunostaining. By combining the Golgi-Cox staining with tissue clearing and immunostaining, Golgi-Cox-stained tissue could be used for large-volume 3D imaging, identification of cell types of Golgi-Cox-stained cells, and reconstruction of the neural circuits at dendritic spines level. More importantly, these methods could also be applied to samples from human brains, providing a tool for analyzing the neuronal circuit of the human brain.

SUBMITTER: Shan QH

PROVIDER: S-EPMC8998187 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Expansion-Based Clearing of Golgi-Cox-Stained Tissue for Multi-Scale Imaging.

Shan Qing-Hong QH Qin Xin-Ya XY Zhou Jiang-Ning JN

International journal of molecular sciences 20220325 7

Obtaining fine neuron morphology and connections data is extraordinarily useful in understanding the brain's functionality. Golgi staining is a widely used method for revealing neuronal morphology. However, Golgi-Cox-stained tissue is difficult to image in three dimensions and lacks cell-type specificity, limiting its use in neuronal circuit studies. Here, we describe an expansion-based method for rapidly clearing Golgi-Cox-stained tissue. The results show that 1 mm thick Golgi-Cox-stained tissu ...[more]

PMID: 35408934

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Project description:Metal-based Golgi-Cox (GC) staining is an established method used to visualise neurons with great morphological detail. Although GC stained samples are imaged routinely under transmitted light microscopy, this method is unable to yield information on the three-dimensional structure of dendrites and neurons and thus help reveal the connective properties of the central nervous system. Although a few studies have attempted simultaneous visualisation of GC staining and antigen-specific fluorescent labelling under a confocal reflection technique, the resolution of both confocal reflection and fluorescence modalities used to acquire GC reflection and fluorescently stained antibody signals are still limited by the diffraction limit of light at about 220 nm. Here, we report a confocal reflection super-resolution technique (CRSR) to break this diffraction barrier, which is achieved by minimising the pinhole size from 1 airy unit (AU) to 0.1 AU. This is achieved by minimising or closing the confocal pinhole size and is possible in this reflection modality, unlike fluorescence, because it is not a photon limited technique. Utilising the lowest wavelength of light available in the system (405 nm), the CRSR technique results in ∼30% lateral and axial resolution improvement. We also show that the CRSR technique can be used in conjunction to visualise both GC and immunofluorescence targets to create precise and improved three-dimensional visualisation and analysis. In addition, using these superresolution confocal reflection data sets from GC in CRSR mode significantly reduced the data overestimation, improving the accuracy of statistical analysis of dendritic spine density and average spine dimensions. Combining the 0.1 AU setting with deconvolution routines, the signal-to-noise ratio and resolution could further be improved an additional ∼20-25%, yielding CRSR images with resolutions up to 2-fold over the diffraction limit both laterally and axially. The improved precision of both visualisation and quantification of subdiffraction limited dendritic spines using the CRSR technique may prove to be critical in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease. LAY DESCRIPTION: For over a century, Golgi-Cox (GC) has been a leading staining technique in the field of neuroscience, used to visualise neurons with great morphological detail. GC stained brain or spinal cord samples are conventionally visualised under transmitted light techniques. This limits the view of Golgi-staining to a two-dimensional image. A recent report showed that Golgi staining can be visualised in three-dimensions using the reflection modality of the confocal microscope. This visualisation also allows for the simultaneous acquisition of immunofluorescence signals. However, the reported resolution of Golgi staining confocal reflection is limited by the diffraction limit of light, which is around 220 nm. Here, we report a superresolution confocal reflection technique (CRSR) that achieves superresolution by minimising the pinhole size used in confocal microscopy. The CRSR technique results in ∼30% lateral and axial resolution improvement. Adding a deconvolution step in the final processing could improve the SNR and resolution even further up to 2-fold improvement in resolution over the diffraction limit both laterally and axially. We hope that this improved visualisation will help in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease.

Project description:Recent advances in optical clearing techniques have dramatically improved deep tissue imaging by reducing the obscuring effects of light scattering and absorption. However, these optical clearing methods require specialized equipment or a lengthy undertaking with complex protocols that can lead to sample volume changes and distortion. In addition, the imaging of cleared tissues has limitations, such as fluorescence bleaching, harmful and foul-smelling solutions, and the difficulty of handling samples in high-viscosity refractive index (RI) matching solutions. To address the various limitations of thick tissue imaging, we developed an Aqueous high refractive Index matching and tissue Clearing solution for Imaging (termed AICI) with a one-step tissue clearing protocol that was easily made at a reasonable price in our own laboratory without any equipment. AICI can rapidly clear a 1 mm thick brain slice within 90 min with simultaneous RI matching, low viscosity, and a high refractive index (RI = 1.466), allowing the imaging of the sample without additional processing. We compared AICI with commercially available RI matching solutions, including optical clear agents (OCAs), for tissue clearing. The viscosity of AICI is closer to that of water compared with other RI matching solutions, and there was a less than 2.3% expansion in the tissue linear morphology during 24 h exposure to AICI. Moreover, AICI remained fluid over 30 days of air exposure, and the EGFP fluorescence signal was only reduced to ~65% after 10 days. AICI showed a limited clearing of brain tissue >3 mm thick. However, fine neuronal structures, such as dendritic spines and axonal boutons, could still be imaged in thick brain slices treated with AICI. Therefore, AICI is useful not only for the three-dimensional (3D) high-resolution identification of neuronal structures, but also for the examination of multiple structural imaging by neuronal distribution, projection, and gene expression in deep brain tissue. AICI is applicable beyond the imaging of fluorescent antibodies and dyes, and can clear a variety of tissue types, making it broadly useful to researchers for optical imaging applications.

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.

Dataset Information

Expansion-Based Clearing of Golgi-Cox-Stained Tissue for Multi-Scale Imaging.

Publications

Expansion-Based Clearing of Golgi-Cox-Stained Tissue for Multi-Scale Imaging.

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