Sample Clearing
Sample Clearing - Related Videos
02:08
A Hydrophobic Tissue Clearing Method for a Rat Brain Tissue Sample
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2025
Source: Kirchner, K.N., et al., A Hydrophobic Tissue Clearing Method for Rat Brain Tissue. J. Vis. Exp. (2020).This video demonstrates the method of hydrophobic tissue clearing of a rat brain tissue sample. The sample is sequentially dehydrated in methanol, treated with a dichloromethane and methanol mixture to dissolve lipids and pigments, and transferred to dibenzyl ether for complete tissue clearing, enabling detailed imaging without thin sectioning.
06:52
Extraction and Tissue Clearing Preparation of Mouse Brain-Spinal Cord Samples
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2025
This study presents a protocol for extracting and preparing cleared whole-brain and spinal cord samples with preserved fluorescent signals, enhancing experimental efficiency and data integrity to advance neuroscience research.
10:03
Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP
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Cited by 11 •
2012
Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.
08:25
Analysis of Coronary Vessels in Cleared Embryonic Hearts
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Cited by 11 •
2016
We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.
07:49
Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO
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Cited by 60 •
2014
To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.
FAQs
Refractive-index matching reduces optical mismatch between the remaining tissue and its surrounding clearing medium. That lowers light scattering as light travels through the specimen, so signals from deeper regions can reach the microscope more effectively. The result is improved access to internal structures and more continuous three-dimensional visualization, rather than an image dominated by superficial layers.
Removing or modifying lipids and pigments addresses major sources of optical interference in biological specimens. Lipids can contribute to light scattering, while pigments can interfere with light transmission and visualization. Managing these components helps produce a more transparent or translucent sample, supporting deeper imaging while protocols may also aim to preserve fluorescent labels needed to identify specific structures.
Cleared specimens preserve spatial relationships through a larger volume, whereas thin-section approaches require information from separate slices to be reconstructed. This distinction matters when cells, vessels, or nerves extend across multiple planes. By reducing dependence on physical sectioning, the method can provide a more direct view of how structures are arranged throughout an organ or other biological specimen.
The usefulness of fluorescence after processing depends on whether the clearing protocol preserves the specimen’s fluorescent labels. When labels remain detectable, they can mark selected cells or structures during microscopy and add cellular specificity to the three-dimensional image. Thus, transparency alone is not the only outcome; maintaining interpretable labels can determine which biological relationships the dataset reveals.
At a broad level, preparation includes treating the specimen to remove or modify light-scattering components, placing the processed tissue in a clearing medium that matches its refractive index, and then imaging the result. The workflow is designed to balance optical transparency with label preservation, so the final sample can be examined throughout its depth rather than only at its surface.
Light-sheet and confocal microscopy are identified as useful imaging approaches for cleared samples. Their relevance lies in converting improved optical access into three-dimensional observations of cells, vessels, nerves, and organs. The cleared specimen therefore serves as a preparation step, while microscopy supplies the visualization needed to examine structures and their spatial organization.
It is particularly valuable when the research question concerns organization across depth or the relationship between structures separated in three dimensions. Examples include examining the arrangement of cells, vessels, or nerves within organs. Instead of inferring continuity from surface views or relying mainly on thin slices, researchers can inspect broader spatial patterns in a more continuous specimen.