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Published July 13, 2025 | Early View
Journal Article Open

Refractive Index‐Corrected Light‐Sheet Microscopy for Macro‐View Cardiovascular Imaging

Zhu, Enbo1, 2 ORCID icon
Zhang, Yaran1
Zhao, Peng1
Cho, Jae Min1
Wang, Zhaoqiang1
Li, Yan‐Ruide1
Wang, Jing1
Margolis, Samuel1
Wang, Shaolei1
Yang, Lili1
Chu, Alison1
Zhang, Yuhua3
Gao, Liang1
Hsiai, Tzung K.1, 2, 4 ORCID icon
  • 1. ROR icon University of California, Los Angeles
  • 2. ROR icon VA Greater Los Angeles Healthcare System
  • 3. ROR icon Doheny Eye Institute
  • 4. ROR icon California Institute of Technology

Abstract

Light‐sheet fluorescence microscopy (LSFM) enables rapid data acquisition with minimal phototoxicity, while optical clearing reduces light scattering by matching sample and imaging medium refractive indices (RIs). Emerging clearing methods extend from cells and organoids to entire organs and organisms, prompting macro‐view LSFM microscopes with macro‐objectives for low‐magnification imaging of larger specimens at adequate resolution. In cardiovascular studies, multiple organs often require imaging, yet clearing protocols optimized for different organs alter the sample RIs inconsistently. Standard‐size dipping objectives use correction collars for RI adaptation, but macro‐objectives, owing to their large size and long working distances, are typically dry lenses lacking built‐in RI correction. An RI‐corrected (rc)‐LSFM macro‐view system addresses this limitation by accommodating a broad range of RI values. By integrating axial sweeping, multi‐view imaging, and a closed quartz chamber, the rc‐LSFM improves field of view (up to ≈8.8 mm), isotropy, spatial resolution (≈3 µm), and operational safety. It effectively visualizes microvascular networks in zebrafish embryos and post‐natal mouse retina, traces the stem cell lineage of cardiomyocytes in mouse embryos, and reveals sympathetic nerve innervation in adult mouse aorta. The rc‐LSFM macro‐view system is compatible with various optical clearing protocols for multi‐scale imaging with high isotropy and spatial resolution.

Copyright and License

© 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Funding

  • National Heart, Lung, and Blood Institute. Grant Numbers: R01HL129727, R01HL159970, R01HL165318, T32HL144449
  • National Eye Institute. Grant Numbers: R01EY034218, R01EY024378, R01EY032561

Supplemental Material

Supporting Information: advs70863-sup-0001-SuppMat.pdf

Acknowledgement

The authors thank the University of California, Los Angeles (UCLA) animal facility, zebrafish core facility, and Advanced Light Microscopy core for their support. T.K.H. acknowledges financial support from NIH/NHLBI R01HL129727, NIH/NHLBI R01HL159970, and NIH/NHLBI R01HL165318. Y.Z. (Yuhua Zhang) acknowledges financial support from NIH/NEI R01EY034218 and R01EY024378. A.C. acknowledges financial support from NIH/NEI R01EY032561. E.Z. and J.M.C. acknowledge the T32 UCLA/Caltech Integrated Cardiometabolic Medicine for Bioengineers fellowship (NIH/NHLBI T32HL144449). Y.-R.L. acknowledges the UCLA MIMG M. John Pickett Post-Doctoral Fellow Award and the CIRM-BSCRC Postdoctoral Fellowship.

Ethics

All animal studies were performed in compliance with the IACUC protocol approved by the UCLA Office of Animal Research and in accordance with the guidelines set by the National Institutes of Health. Humane care and use of animals were observed to minimize distress and discomfort. All Zebrafish (Danio rerio) studies were conducted in accordance with animal protocols approved by the UCLA Institutional Animal Care and Use Committee (IACUC) (ARC#: 2015–055).

Conflict of Interest

E.Z. and T.K.H. are inventors on patents relating to this study filed by the University of California, Los Angeles. The remaining authors declare no conflict of interest.

Contributions

E.Z. and T.K.H. conceived the idea for the work. E.Z., Z.W., L.G., and T.K.H. designed the optical system, incorporating suggestions from Y.Z. (Yuhua Zhang). E.Z. constructed the experimental setups and conducted the study, with assistance from Y.Z. (Yaran Zhang), P.Z., J.M.C., Y.-R.L., and guidance from L.Y. and A.C. for mice-related work, as well as support from J.W. and S.G.R. for the zebrafish-related work. E.Z. and T.K.H. wrote the manuscript, with suggestions from S.M., S.W., Y.Z. (Yuhua Zhang) and L.G. All authors had full access to all the data in the study and agreed to submit the manuscript for publication.

Files

Advanced Science - 2025 - Zhu - Refractive Index‐Corrected Light‐Sheet Microscopy for Macro‐View Cardiovascular Imaging.pdf

Additional details

Funding Dates Caltech Custom Metadata
Created:
July 24, 2025
Modified:
July 24, 2025