Translational rapid ultraviolet-excited sectioning tomography for whole-organ multicolor imaging with real-time molecular staining
Abstract
Rapid multicolor three-dimensional (3D) imaging for centimeter-scale specimens with subcellular resolution remains a challenging but captivating scientific pursuit. Here, we present a fast, cost-effective, and robust multicolor whole-organ 3D imaging method assisted with ultraviolet (UV) surface excitation and vibratomy-assisted sectioning, termed translational rapid ultraviolet-excited sectioning tomography (TRUST). With an inexpensive UV light-emitting diode (UV-LED) and a color camera, TRUST achieves widefield exogenous molecular-specific fluorescence and endogenous content-rich autofluorescence imaging simultaneously while preserving low system complexity and system cost. Formalin-fixed specimens are stained layer by layer along with serial mechanical sectioning to achieve automated 3D imaging with high staining uniformity and time efficiency. 3D models of all vital organs in wild-type C57BL/6 mice with the 3D structure of their internal components (e.g., vessel network, glomeruli, and nerve tracts) can be reconstructed after imaging with TRUST to demonstrate its fast, robust, and high-content multicolor 3D imaging capability. Moreover, its potential for developmental biology has also been validated by imaging entire mouse embryos (~2 days for the embryo at the embryonic day of 15). TRUST offers a fast and cost-effective approach for high-resolution whole-organ multicolor 3D imaging while relieving researchers from the heavy sample preparation workload.
Data availability
Data availability. The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information.
Article and author information
Author details
Funding
Research Grants Council, University Grants Committee (16208620)
- Terence TW Wong
Research Grants Council, University Grants Committee (26203619)
- Terence TW Wong
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Kevin M Dean, University of Texas Southwestern Medical Center, United States
Ethics
Animal experimentation: All animal experiments were conducted in conformity with a laboratory animal protocol approved by the Health, Safety and Environment Office of the Hong Kong University of Science and Technology (HKUST) (license number: AEP16212921).
Version history
- Preprint posted: November 13, 2021 (view preprint)
- Received: June 13, 2022
- Accepted: November 4, 2022
- Accepted Manuscript published: November 4, 2022 (version 1)
- Version of Record published: November 17, 2022 (version 2)
Copyright
© 2022, Yu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism:
SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.
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