Nature Methods: Next-Generation Stealth HYBRiD Technology Visualizes Whole Body Tissues of Animals, Helping Scientific Research

Click on the blue word to focus on the opacity of our tissue samples mainly caused by the scattering and absorption of light
.

Scattering occurs in media with different refractive indices
.

The refractive index of the fixed sample tissue is 1.
5, which is higher than that of water
.

Therefore, the current clearing technology balances the refractive index of the tissue by replacing the moisture and lipids in the tissue with an optical clearing agent of a high refractive index medium to achieve a transparent effect
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At present, there are two types of brain tissue clearing technologies.
One is mainly based on organic solvents (tetrahydrofuran, tert-butanol, dichloromethane, etc.
), such as iDISCO, FDISCO, etc.
, which can achieve rapid and stable transparency.
Fluorescence quenching, tissue shrinkage, transparent tissue cannot be stored for a long time, and reagents are toxic
.

Another transparent brain technology based on hydrogel (acrylamide) and water, such as CLARITY technology, has good transparent effect, high signal-to-noise ratio of endogenous fluorescent signal, does not cause tissue shrinkage, but requires electrophoresis equipment, and the clearing time is relatively short.
long
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Although the above techniques can easily achieve clear rodent brains, it is more difficult to observe animal body parts (bones, muscles, blood vessels and connective tissue and other intact tissues)
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In 2016, the team of Ali Ertürk of the University of Munich in Germany developed uDISCO, a technology that makes the whole body of animals transparent.
It achieves transparent mouse organs by reducing the volume of mice by 65%, and the fluorescence intensity can be maintained for several months
.

On March 29, 2022, Li Ye's research team from the Department of Neuroscience of Scripps Research Institute developed HYBRiD, a whole-body tissue transparency technology that is more powerful than uDISCO
.

Figure 1: Clearing of hindlimbs by different clearing techniques The researchers used CLARITY, iDISCO, and FDISCO clearing techniques to clear the hindlimbs of mice.
)
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Further experiments found that dibenzyl ether (DBE) is the main cause of the loss of the fluorescence signal.
The fluorescence signal can be enhanced by replacing DBE with an aqueous high refractive index solution, but the degree of transparency is not high
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However, this suggests that the use of mild reagents to reduce fluorescence quenching is required for clearing large tissues
.

Figure 2: Comparison of HYBRiD technology and uDISCO technical parameters In order to solve the above problems, they first dehydrated the tissue through the organic solvent FDISCO, and then used the water-based CLARITY clearing technology for degreasing, and combined the two to develop the HYBRiD technology
.

The effect of this technology on transparent mouse forelimbs is significantly better than that of uDISCO technology, and its fluorescence intensity is about 100 times more than that of uDISCO technology (Figure 2), indicating little loss of fluorescence signal
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Cre-dependent genetically modified mice are now commonly used in the field of neuroscience, but their potential off-targets also bring a lot of uncertainty to research
.

aP2-Cre mouse is a tool mouse to specifically label adipocytes for the study of diabetic diseases, but its cre enzyme can be leaked and expressed in macrophages and lymphatic system, which leads to misunderstanding of the pathogenesis of diabetes
.

Figure 3: Whole body clearing treatment of mice at different developmental stages Researchers used HYBRiD technology to detect parvalbumin (PV-Cre) and somatostatin (SST) at different developmental stages (new birth, 2nd and 3rd weeks after birth).
-Cre) mice underwent whole-body clearing treatment, and found that PV positive signals were distributed in the inner cochlea and vestibular system, epithelial cells of kidney, pulmonary vasculature, colon and bladder, and dorsal root ganglion neurons
.

The SST positive signals were distributed in the inner ear, dorsal root ganglia, kidney, colon and bladder with different spatial expression patterns, which indicated that HYBRiD technology could be well used to detect the spatial expression of cre enzyme in the whole body
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Figure 4: HYBRiD technology in the entire thoracic cavity of transparent mice HYBRiD technology can also be applied to systemic disease research
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K18-hACE2 mice can specifically express human angiotensin-converting enzyme 2 (hACE2), which is mainly used for the study of the pathogenesis of SARS coronavirus and the development of antiviral therapeutic drugs
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After clearing the K18-hACE2 mice after SARS-CoV-2 infection, the researchers were able to clearly observe the infection distribution characteristics of SARS-CoV-2 throughout the thoracic cavity, revealing the macroscopic and microscopic characteristics of virus infection in the same sample
.

In general, this paper developed a HYBRiD technology that can achieve whole-body tissue transparency by comparing the previous transparency technologies, which can be applied to system tissues (respiratory system, digestive system, circulatory system, nervous system, etc.
) and single organs ( Brain, hindlimb, heart, etc.
) and other large tissues can be imaged, and can be combined with fluorescence microscopic optical section tomography to achieve macroscopic and microscopic structural analysis
.

[References] 1.
https://doi.
org/10.
1038/s41592-022-01427-0 The pictures in the text are from the references