Innovative technology EZ Clear: Transparent imaging of whole organs in mice

Seeing is believing
.
Holistic organ clearance imaging allows researchers to explore the three-dimensional spatial structure of organs without affecting tissue structure
.
But complex, laborious procedures, expensive equipment, and the use of dangerous organic solvents have hindered the widespread adoption of
these methods.

The team of Chih-Wei Logan Hsu, PhD, Ph.
D.
, and Joshua D.
Wythe, PhD, of Baylor College of Medicine has developed an innovative technology called EZ Clear, a simple and fast new method that speeds up the process of presenting tissues as optically transparent, enabling 3D imaging
of entire intact tissues and even entire organs in just three simple steps.
Samples are kept room temperature and hydrated throughout the process, preserving endogenous and synthetic fluorescence
without changing sample size.
After imaging, samples treated with EZ Clear can be used for downstream applications such as tissue embedding and cryosectioning, followed by standard histology or immunofluorescence staining without losing fluorescence signals
from endogenous or synthetic reporter labels.
A large number of adult mouse brains treated with EZ Clear can be successfully used for immunolabeling for fluorescence imaging while still retaining signals
from endogenous fluorescent reporter labels.
Overall, EZ Clear's simplicity, speed, and flexibility make it easy to adapt and implement a variety of imaging modalities
in biomedical research.

Their new method, published in the journal eLife, was developed
at Baylor University's Optical Imaging and Life Microscopy Core (OiVM).
Whole-organ imaging has revolutionized biology, making it possible
to explore organs in three-dimensional space without affecting tissue structure.
EZ Clear uses three simple steps: lipid removal, washing, and RI matching, effectively making entire adult mouse organs optically clear
within 48 h.
EZ Clear combines the advantages of solvent-mediated lipid removal and highly water-miscible tetrahydrofuran (THF) to make samples transparent in aqueous solutions, and high RI (n=1.
518) sample mounting and imaging solutions are compatible with
most microscope platforms.
Since the sample was immersed in water throughout the process, there was no significant change
in sample size during tissue processing.

Step 1: Immerse the fixed sample in a degreasing solution consisting of 50% (v/v) THF in sterile milliq water
.
This formula allows tetrahydrofurans to dissolve lipids from tissues while the organs remain hydrated
in an aqueous environment.
2: After lipid removal, the samples are incubated in sterile milliq water for 4 h to wash and remove residual tetrahydrofurans from the tissue; 3: Immerse the tissue in RI-matched imaging aqueous solution (EZ View, RI = 1.
518) at room temperature for 24 h to make it transparent
.
EZ Clear not only makes the whole brain as transparent as 3DISCO and FAST 3D processing, but also has the shortest processing time (48 hours) and the simplest procedure (three steps), which is currently the simplest
tissue processing.
Since the sample remains in an aqueous environment throughout the process, the size of the treated sample remains constant throughout the process and does not shrink or expand
significantly.
In addition, endogenous transgenic fluorescence reporter-labeled activity was retained throughout EZ Clear treatment, and brains using EZ Clear-treated Thy1-EGFP neuron reporter cell lines retained strong signals
in neurons when imaging a total imaging depth of 5 mm by light-sheet microscopy.
EZ Clear is also compatible with organs other than the brain of adult mice, with good transparency
after tissue processing.
Multiple adult mouse organs (eyes, heart, kidneys, testes, and ovaries)
are collected after vascular perfusion of far-red fluorescence-binding tomatolectin (lectin-dylight 649) to label endothelial cells.
Fix the specimen after perfusion and treat
with EZ Clear.
Overall imaging of the sample by LSFM showed that the lectin-649 signal was robust and well
preserved.
These results show that EZ Clear effectively optically transparent adult mouse organs in three simple steps over 48 hours while preserving synthetic and endogenous fluorescent report-labeled signals
.

"Previous methods were complex, laborious, often required expensive equipment, and required the use of harmful organic solvents, all of which hindered widespread adoption
of these methods," Hsu said.
Hsu is co-director of OiVM and assistant professor of
integrative physiology and education, innovation and technology at Baylor University.
"These difficulties prompted us to develop a simpler cleanup process that users could do more easily, saving time and valuable resources and focusing on the actual problems
they wanted to investigate in the system.
" "The beauty of this approach is that you can analyze the sample from a global or macroscopic perspective without disturbing the natural tissues of the tissue or organ," says
Wythe, associate professor of integrative physiology and neurosurgery at Baylor University.
He is also a member of
the Dan L Duncan Comprehensive Cancer Center and the Cardiovascular Institute.

For example, researchers can now see neuronal connections
between the eye and the brain.
If tissue is sliced, this process will disrupt the natural structure of the tissue and be difficult to reconstruct in 3D, limiting our understanding of
the connections between neurons and other surrounding cells over a larger volume or area.
3D imaging bypassed these limitations, and the advent of EZ Clear made 3D imaging accessible
to most modern molecular biology laboratories.
"EZ Clear is also able to preserve endogenous and synthetic labeling methods without changing the sample size
.
The method transparentizes and labels neurons and blood vessels in the brain, as well as blood vessels in the eyes, heart, kidneys, testicles and ovaries, and entire organs
of the lung, liver and pancreas of mice.

"EZ Clear removes previous technical barriers, allowing researchers to examine the organs
of interest they are interested in from macroscopic, whole-organ level to cellular resolution," Wythe said.
"It eliminates previous practical, safe, and economic challenges while providing replicable, high-quality visualization of the whole organ, which is an important perspective because it can provide new insights
into the topic being studied.
"

EZ Clear is faster, cheaper and simpler
than previous processing methods.
Researchers can now learn this easier, replicable organizational process at OiVM
.
They can then be implemented in their own lab, with just three simple steps to get results in 48 hours, compared to other methods that take weeks or even months to complete
.
They can then send the processed organs to OiVM for 3D imaging and analysis
.

Journal Reference:

1. Chih-Wei Hsu, Juan Cerda, Jason M Kirk, Williamson D Turner, Tara L Rasmussen, Carlos P Flores Suarez, Mary E Dickinson, Joshua D Wythe.
   EZ Clear for simple, rapid, and robust mouse whole organ clearing.
   eLife, 2022; 11 DOI: 10.
   7554/eLife.
   77419