Upright Light-Sheet Microscopy with Two-Photon Excitation for Imaging Large Samples

Takashi Saitou; Sota  Takanezawa; Ryosuke Kawakami; Kana Kobayashi-Taguchi; Mai Kamito; Kana Takemoto; Yosuke Niko; Yuji Tsukamoto; Ryotaro Ozaki; Atsushi Tsurumune; Yoshiaki Kamei; Yuzo Umeda; Takeshi Imamura

doi:10.51094/jxiv.1223

##article.authors##

Takashi Saitou Graduate School of Science and Engineering, Ehime University https://orcid.org/0000-0003-0532-8112 https://researchmap.jp/7000015075
Sota Takanezawa Bioscience Sales Division, NIKON SOLUTIONS CO., LTD
Ryosuke Kawakami Graduate School of Medicine, Ehime University
Kana Kobayashi-Taguchi Graduate School of Medicine, Ehime University
Mai Kamito Graduate School of Medicine, Ehime University
Kana Takemoto Graduate School of Medicine, Ehime University
Yosuke Niko Interdisciplinary Science Unit, Kochi University
Yuji Tsukamoto Graduate School of Science and Engineering, Ehime University
Ryotaro Ozaki Graduate School of Science and Engineering, Ehime University
Atsushi Tsurumune Bioscience Sales Division, NIKON SOLUTIONS CO., LTD
Yoshiaki Kamei Graduate School of Medicine, Ehime University
Yuzo Umeda Graduate School of Medicine, Ehime University
Takeshi Imamura Graduate School of Medicine, Ehime University

DOI:

https://doi.org/10.51094/jxiv.1223

キーワード:

Fluorescence imaging、 Light-sheet microscopy、 Two-photon excitation、 in vivo imaging、 Optical clearing

抄録

Light-sheet microscopy has become a valuable tool for 3D imaging of biological samples, offering high resolution, fast imaging speeds, and minimal photodamage. Two-photon excitation further enhances its capabilities by providing deeper tissue penetration and reduced phototoxicity. However, conventional designs with orthogonal illumination and detection geometry often restrict the flexibility needed for imaging large or diverse samples that require varying immersion media. To address limitations in conventional systems for seamless imaging of both living organisms and cleared tissues, we developed an upright two-photon Bessel beam light-sheet microscope. Equipped with a middle-range magnification, multi-immersion objective lens (16x, NA 0.6), the microscope accommodates refractive indices ranging from 1.33 to 1.51. Built upon a previously established two-photon Bessel beam illumination system, this microscope maintains a large field of view while achieving cellular resolution. We demonstrated its versatility by applying it to both live specimen imaging and cleared tissue observation. For imaging optically cleared tissues, we implemented a method in which samples are immersed in a clearing agent and observed using coverslips. This enabled high-speed, large-scale imaging of human breast cancer tissues and mouse brain slices. The microscope’s adaptability to different immersion media and its compatibility with coverslips offers significant flexibility for sample mounting, and this supports the imaging of large samples. By combining a laterally unconstrained configuration with two-photon excitation, this system advances the applicability of light-sheet microscopy across a wide range of biological research fields.

利益相反に関する開示

S.T. and A.T. are employees of NIKON SOLUTIONS CO., LTD. The remaining authors declare that they have no competing interests.

ダウンロード *前日までの集計結果を表示します

ダウンロード実績データは、公開の翌日以降に作成されます。

著者の経歴

Takashi Saitou、Graduate School of Science and Engineering, Ehime University

2009 Ph.D. in Science, Graduate Schoool of Pure and Applied Sciences, University of Tsukuba

2010 Researcher, Graduate School of Engineering Science, Osaka University

2013 Researcher, Graduate School of Medicine, Ehime University

2014 Assistant Professor, Translational Research Center, Ehime University Hospital

2019 Assistant Professor, Graduate School of Medicine, Ehime University

2024 Associate Professor, Graduate School of Science and Engineering, Ehime University

引用文献

Cardone, A., A. Tolino, R. Zarcone and E. Tartaglia (1997). "Prognostic value of desmoplastic reaction and lymphocytic infiltration in the management of breast cancer." Panminerva medica 39(3): 174-177.

Chen, B. C., W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, 3rd, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A. C. Reymann, R. Bohme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart and E. Betzig (2014). "Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution." Science 346(6208): 1257998.

Deguchi, T., K. E. Fujimori, T. Kawasaki, K. Maruyama and S. Yuba (2012). "In vivo visualization of the lymphatic vessels in pFLT4-EGFP transgenic medaka." Genesis 50(8): 625-634.

Dodt, H.-U., U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger and K. Becker (2007). "Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain." Nature methods 4(4): 331-336.

Dunsby, C. (2008). "Optically sectioned imaging by oblique plane microscopy." Optics express 16(25): 20306-20316.

Fahrbach, F. O., P. Simon and A. Rohrbach (2010). "Microscopy with self-reconstructing beams." Nature Photonics 4(11): 780.

Feng, G., R. H. Mellor, M. Bernstein, C. Keller-Peck, Q. T. Nguyen, M. Wallace, J. M. Nerbonne, J. W. Lichtman and J. R. Sanes (2000). "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP." Neuron 28(1): 41-51.

Glaser, A. K., N. P. Reder, Y. Chen, E. F. McCarty, C. Yin, L. Wei, Y. Wang, L. D. True and J. T. Liu (2017). "Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens." Nature biomedical engineering 1(7): 0084.

Glaser, A. K., N. P. Reder, Y. Chen, C. Yin, L. Wei, S. Kang, L. A. Barner, W. Xie, E. F. McCarty and C. Mao (2019). "Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues." Nature communications 10(1): 2781.

Huisken, J. and D. Y. Stainier (2007). "Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM)." Opt Lett 32(17): 2608-2610.

Huisken, J., J. Swoger, F. Del Bene, J. Wittbrodt and E. H. Stelzer (2004). "Optical sectioning deep inside live embryos by selective plane illumination microscopy." Science 305(5686): 1007-1009.

Inoue, K., R. Kawakami, M. Murakami, T. Nakayama, S. Yamamoto, K. Inoue, T. Tsuda, K. Sayama, T. Imamura and D. Kaneno (2022). "Synthesis and photophysical properties of a new push–pull pyrene dye with green-to-far-red emission and its application to human cellular and skin tissue imaging." Journal of Materials Chemistry B 10(10): 1641-1649.

Kalluri, R. and M. Zeisberg (2006). "Fibroblasts in cancer." Nature reviews cancer 6(5): 392-401.

Keller, P. J. (2013). "Imaging morphogenesis: technological advances and biological insights." Science 340(6137): 1234168.

Keller, P. J., A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt and E. H. Stelzer (2010). "Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy." Nat Methods 7(8): 637-642.

Keller, P. J., A. D. Schmidt, J. Wittbrodt and E. H. Stelzer (2008). "Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy." Science 322(5904): 1065-1069.

Kinoshita, M., K. Murata, K. Naruse and M. Tanaka (2009). Medaka: biology, management, and experimental protocols, John Wiley & Sons.

Kobayashi-Taguchi, K., T. Saitou, Y. Kamei, A. Murakami, K. Nishiyama, R. Aoki, E. Kusakabe, H. Noda, M. Yamashita, R. Kitazawa, T. Imamura and Y. Takada (2022). "Computer-Aided Detection of Quantitative Signatures for Breast Fibroepithelial Tumors Using Label-Free Multi-Photon Imaging." Molecules 27(10).

Kumar, A., Y. Wu, R. Christensen, P. Chandris, W. Gandler, E. McCreedy, A. Bokinsky, D. A. Colón-Ramos, Z. Bao and M. McAuliffe (2014). "Dual-view plane illumination microscopy for rapid and spatially isotropic imaging." Nature protocols 9(11): 2555-2573.

McGorty, R., H. Liu, D. Kamiyama, Z. Dong, S. Guo and B. Huang (2015). "Open-top selective plane illumination microscope for conventionally mounted specimens." Optics express 23(12): 16142-16153.

Migliori, B., M. S. Datta, C. Dupre, M. C. Apak, S. Asano, R. Gao, E. S. Boyden, O. Hermanson, R. Yuste and R. Tomer (2018). "Light sheet theta microscopy for rapid high-resolution imaging of large biological samples." BMC biology 16: 1-19.

Mizutani, H., S. Ono, T. Ushiku, Y. Kudo, M. Ikemura, N. Kageyama, N. Yamamichi, M. Fujishiro, T. Someya and M. Fukayama (2018). "Transparency‐enhancing technology allows three‐dimensional assessment of gastrointestinal mucosa: a porcine model." Pathology international 68(2): 102-108.

Orimo, A., P. B. Gupta, D. C. Sgroi, F. Arenzana-Seisdedos, T. Delaunay, R. Naeem, V. J. Carey, A. L. Richardson and R. A. Weinberg (2005). "Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion." Cell 121(3): 335-348.

Planchon, T. A., L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith and E. Betzig (2011). "Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination." Nat Methods 8(5): 417-423.

Power, R. M. and J. Huisken (2017). "A guide to light-sheet fluorescence microscopy for multiscale imaging." Nat Methods 14(4): 360-373.

Prakash, J. and Y. Shaked (2024). "The interplay between extracellular matrix remodeling and cancer therapeutics." Cancer discovery 14(8): 1375-1388.

Richardson, D. S. and J. W. Lichtman (2015). "Clarifying tissue clearing." Cell 162(2): 246-257.

Saitou, T. and T. Imamura (2023). "Extended depth of focus two-photon light-sheet microscopy for in vivo fluorescence imaging of large multicellular organisms at cellular resolution." International Journal of Molecular Sciences 24(12): 10186.

Saitou, T. and T. Imamura (2024). "Enhanced light-sheet illumination by a multi-layered stair-step phase mask." Japanese Journal of Applied Physics 63(10): 108002.

Saitou, T., H. Kiyomatsu and T. Imamura (2018). "Quantitative Morphometry for Osteochondral Tissues Using Second Harmonic Generation Microscopy and Image Texture Information." Sci Rep 8(1): 2826.

Saitou, T., S. Takanezawa, H. Ninomiya, T. Watanabe, S. Yamamoto, Y. Hiasa and T. Imamura (2018). "Tissue Intrinsic Fluorescence Spectra-Based Digital Pathology of Liver Fibrosis by Marker-Controlled Segmentation." Front Med (Lausanne) 5: 350.

Strnad, P., S. Gunther, J. Reichmann, U. Krzic, B. Balazs, G. De Medeiros, N. Norlin, T. Hiiragi, L. Hufnagel and J. Ellenberg (2016). "Inverted light-sheet microscope for imaging mouse pre-implantation development." Nature methods 13(2): 139-142.

Susaki, E. A., C. Shimizu, A. Kuno, K. Tainaka, X. Li, K. Nishi, K. Morishima, H. Ono, K. L. Ode and Y. Saeki (2020). "Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues." Nature communications 11(1): 1982.

Susaki, E. A., K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi and S. Yamaguchi (2014). "Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis." Cell 157(3): 726-739.

Swoger, J., P. Verveer, K. Greger, J. Huisken and E. H. Stelzer (2007). "Multi-view image fusion improves resolution in three-dimensional microscopy." Opt Express 15(13): 8029-8042.

Takanezawa, S., T. Saitou and T. Imamura (2021). "Wide field light-sheet microscopy with lens-axicon controlled two-photon Bessel beam illumination." Nat Commun 12(1): 2979.

Temma, K., R. Oketani, T. Kubo, K. Bando, S. Maeda, K. Sugiura, T. Matsuda, R. Heintzmann, T. Kaminishi and K. Fukuda (2024). "Selective-plane-activation structured illumination microscopy." Nature Methods 21(5): 889-896.

Tomer, R., L. Ye, B. Hsueh and K. Deisseroth (2014). "Advanced CLARITY for rapid and high-resolution imaging of intact tissues." Nature protocols 9(7): 1682-1697.

Truong, T. V., W. Supatto, D. S. Koos, J. M. Choi and S. E. Fraser (2011). "Deep and fast live imaging with two-photon scanned light-sheet microscopy." Nat Methods 8(9): 757-760.

Vettenburg, T., H. I. Dalgarno, J. Nylk, C. Coll-Lladó, D. E. Ferrier, T. Čižmár, F. J. Gunn-Moore and K. Dholakia (2014). "Light-sheet microscopy using an Airy beam." Nat Methods 11(5): 541-544.

Wakamatsu, Y., S. Pristyazhnyuk, M. Kinoshita, M. Tanaka and K. Ozato (2001). "The see-through medaka: a fish model that is transparent throughout life." Proceedings of the National Academy of Sciences 98(18): 10046-10050.

Wu, Y., A. Ghitani, R. Christensen, A. Santella, Z. Du, G. Rondeau, Z. Bao, D. Colón-Ramos and H. Shroff (2011). "Inverted selective plane illumination microscopy (i SPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans." Proceedings of the National Academy of Sciences 108(43): 17708-17713.

Wu, Y., P. Wawrzusin, J. Senseney, R. S. Fischer, R. Christensen, A. Santella, A. G. York, P. W. Winter, C. M. Waterman and Z. Bao (2013). "Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy." Nature biotechnology 31(11): 1032-1038.