Page 13 - C. Biotechnology and molecular imaging

[Part 2] A Novel Tool for Comparative Analyses of Clearing Efficacies
of Tissue Clearance Protocols
1,2
Jiwon Woo 1,2# , Jeong-Yoon Park , and Yong-Eun Cho 1,2*

1 Department of Neurosurgery, The Spine and Spinal cord Institute, Gangnam Severance Hospital, Yonsei University College of Medicine, 211 Eonju-ro, Gangnam-gu, Seoul 06273, Republic of Korea
2 Brain Korea 21 PLUS Project for Medical Science, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea C-9
Abstract
The advent of tissue clearing methods, in conjunction with novel high-resolution imaging techniques, has enabled the visualization of three-dimensional structures with unprecedented depth and detail. Although a variety of clearing
protocols have been developed, little has been done to quantify their efficacies in a systematic, reproducible fashion. Here, we present two novel assays, PACA (Punching-Assisted Clarity Analysis)-Light and PACA-Glow, which use
easily accessible spectroscopy and gel documentation systems to quantify the transparency of multiple cleared tissues simultaneously. We demonstrate the use of PACA-Light and PACA-Glow to compare twenty tissue clearing
protocols on rodent brains. We also show that there are regional differences in tissue transparency in the rodent brain, with cerebellar tissue consistently achieving lower clearance levels compared to the prefrontal or cerebral cortex
across all protocols. This represents the largest comparative study of tissue clearing protocols to date, made possible by the high-throughput nature of our PACA platforms.
Figure 4 Figure 6
Background Comparison of tissue clearance achieved via gelation-based tissue clearing protocols.

● CLARITY
(Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue- Comparison of clearing efficacies of Clear T (A), ScaleA2 (B), CUBIC (C), FOCM
hYdrogel) tissue clearing technologies.
(D), RTF (E), SeeDB (F), 60% TDE (G), and 80% TDE (H) on mouse brain
samples via PACA-Light.
Aim Comparison of clearing efficacies of psPACT (A and B), mPACT (C and D), SWITCH-1 (E and F), SWITCH-4 (G), Figure 7 Comparison of tissue clearance achieved in mouse brains
processed via organic solvent-based clearing protocols.
Understanding the clearance properties of intact organs in a region-specific manner and Tissue-MAP (h) in rodent brain samples (rat: 3-mm thickness, mouse: 1.5-mm thickness) via both PACA-
can allow for further optimization of tissue clearing protocols better suited to a Light and PACA-Glow.
particular region of interest, and in turn, a deeper understanding of the three-
dimensional organization of that region that may not otherwise be uncovered. Figure 5
Results
Figure 1

Comparison of clearing efficacies of
Ethanol BABB (A), 1P BABB (B), tB
BABB (C), 3DISCO (D), iDISCO+ (E),
uDISCO (F), FDISCO (G), Ethanol-ECi
(H), and PEGASOS (I) on mouse brain
samples (1.5-mm thickness) via both
PACA-Light and PACA-Glow.
Comparison of tissue clearance achieved via clearing Figure 8
methods based on hyperhydration or aqueous solutions
with high-refractive indices. Comparison of clearing
efficacies of Clear T (A), Clear T2 (B), ScaleA2 (C), CUBIC
(D), FOCM (E), RTF (F), SeeDB (G), SeeDBp (H), and
80% TDE (I) on rat brain samples (3-mm thickness) via
both PACA-Light and PACA-Glow.
Figure 9
A B E
Schematization of the Punch-based Assistance Clarity-measurement Assay (PACA)
platform.
F
Figure 2
A B Comparison of tissue clearance
achieved in rat brains processed via
(A and B) Absorbance and organic solvent-based clearing
transmittance (%) of 2% protocols. Comparison of clearing
agarose gels of varying efficacies of Ethanol BABB (A), 1-
thickness (1-5mm) at 350-850 propanol BABB (B), tert-butanol BABB
nm. Each color line and bar C D (C), 3DISCO (D), iDISCO+ (E), uDISCO (F), FDISCO (G), Ethanol-ECi (H), and
point to assessment values of PEGASOS (I) on rat brain samples (3-mm thickness) via both PACA-Light and
dH2O (DW, blue) and 2% PACA-Glow.
C agarose gel tablets of differing
thickness. (C) Comparison of G
light emitted by the luminous Methods
disk.
Figure 3 C H
B
A
Investigation of regional differences in tissue transparency achieved in rodent brains via immunofluorescence
studies. Tablets were generated from three distinct regions (B1, B2, B3) of rat and mouse brain samples were
processed via psPACT (A and C) or SWITCH-4 (B) or CUBIC (D). (E and F) The 3D and volumetric imaging of
(A and B) Quantification of light transmitted from the luminous disk compared to a blank psPACT and SWITCH-4 rat brain samples was created from serial single z-images of the blood vessel pattern
control. (C) Comparison of light transmission between an uncleared 4mm-thick mouse using a confocal microscopy in three distinct regions (B1, B2 and B3). (G and H) The 3D and volumetric imaging
brain and a sample cleared via mPACT on a luminous disk in both UV and dark of psPACT and SWITCH-1 mouse brain samples was created from serial single z-images of the blood vessel
conditions. pattern using a confocal microscopy in three distinct regions (B1, B2 and B3).
Reference We developed the absorbance analysis about the optically transparency
Conclusion
of cleared tissues with light attenuation quantification using various
Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, Davidson TJ, et al. Structural clearing methods of current reported. These simple PACA assays require little equipment and minimal hands on
and molecular interrogation of intact biological systems. Nature 2013;497:332-7.
analyze to transmittance of clear tissue and screening the clear-agent in laboratory. In addition, it is provide a
possible that standard transmittance experiment of transparent tissue for three-dimensional deep imaging analysis
at the various optimum transparent conditions. Our data suggest that the PACA platform including the PACA-Light
Contact Information e-mail : jiwonflu@yuhs.ac and the PACA-Glow provides access to optically information that may support to understand anatomical study.