3D fluorescence imaging is a fundamental tool in the study of functional and developmental biology, but effective imaging is particularly difficult in moving structures such as the beating heart. We have developed a non-invasive real-time optical gating system that is able to exploit the periodic nature of the motion to acquire high resolution 3D images of the normally-beating zebrafish heart without any unnecessary exposure of the sample to harmful excitation light. In order for the image stack to be artefact-free, it is essential to use a synchronization source that is invariant as the sample is scanned in 3D. We therefore describe a scheme whereby fluorescence image slices are scanned through the sample while a brightfield camera sharing the same objective lens is maintained at a fixed focus, with correction of sample drift also included. This enables us to maintain, throughout an extended 3D volume, the same standard of synchronization we have previously demonstrated in and near a single 2D plane. Thus we are able image the complete beating zebrafish heart exactly as if the heart had been artificially stopped, but sidestepping this undesirable interference with the heart and instead allowing the heart to beat as normal.

© 2012 OSA

1. A. S. Forouhar, M. Liebling, A. Hickerson, A. Nasiraei-Moghaddam, H.-J. Tsai, J. R. Hove, S. E. Fraser, M. E. Dickinson, and M. Gharib, “The embryonic vertebrate heart tube is a dynamic suction pump,” Science312, 751–753 (2006). [CrossRef] [PubMed]
2. P. J. Scherz, J. Huisken, P. Sahai-Hernandez, and D. Y. R. Stainier, “High-speed imaging of developing heart valves reveals interplay of morphogenesis and function,” Development (Cambridge, England)135, 1179–1187 (2008). [CrossRef]
3. C.-J. Huang, C.-T. Tu, C.-D. Hsiao, F.-J. Hsieh, and H.-J. Tsai, “Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish,” Dev. Dyn.228, 30–40 (2003). [CrossRef] [PubMed]
4. J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development (Cambridge, England)136, 1963–1975 (2009). [CrossRef]
5. J. Swoger, M. Muzzopappa, H. López-Schier, and J. Sharpe, “4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies,” J. Biophotonics4, 122–134 (2011). [CrossRef]
6. A. C. S. Brau, C. T. Wheeler, L. W. Hedlund, and G. A. Johnson, “Fiber-optic stethoscope: a cardiac monitoring and gating system for magnetic resonance microscopy,” Magn. Reson. Med.47, 314–321 (2002). [CrossRef] [PubMed]
7. B. Hiba, N. Richard, H. Thibault, and M. Janier, “Cardiac and respiratory self-gated cine MRI in the mouse: comparison between radial and rectilinear techniques at 7T,” Magn. Reson. Med.58, 745–753 (2007). [CrossRef] [PubMed]
8. M. Buehrer, J. Curcic, P. Boesiger, and S. Kozerke, “Prospective self-gating for simultaneous compensation of cardiac and respiratory motion,” Magn. Reson. Med.60, 683–690 (2008). [CrossRef] [PubMed]
9. M. Liebling, A. S. Forouhar, M. Gharib, S. E. Fraser, and M. E. Dickinson, “Four-dimensional cardiac imaging in living embryos via postacquisition synchronization of nongated slice sequences,” J. Biomed. Opt.10, 054001 (2005). [CrossRef] [PubMed]
10. J. J. Schoenebeck and D. Yelon, “Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics,” Semin. Cell Dev. Biol.18, 27–35 (2007). [CrossRef] [PubMed]
11. J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J.2, 143–55 (2008). [CrossRef]
12. S. Lee, C. Vinegoni, P. F. Feruglio, L. Fexon, R. Gorbatov, M. Pivoravov, A. Sbarbati, M. Nahrendorf, and R. Weissleder, “Real-time in vivo imaging of the beating mouse heart at microscopic resolution,” Nat. Commun.3, 1054 (2012). [CrossRef] [PubMed]
13. I. V. Larina, K. V. Larin, M. E. Dickinson, and M. Liebling, “Sequential turning acquisition and reconstruction (STAR) method for four-dimensional imaging of cyclically moving structures,” Biomed. Opt. Express3, 650–660 (2012). [CrossRef] [PubMed]
14. J. M. Taylor, C. D. Saunter, G. D. Love, J. M. Girkin, D. J. Henderson, and B. Chaudhry, “Real-time optical gating for three-dimensional beating heart imaging,” J. Biomed. Opt.16, 116021 (2011). [CrossRef] [PubMed]
15. K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78, 023705 (2007). [CrossRef] [PubMed]
16. J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32, 2608–2610 (2007). [CrossRef] [PubMed]

Biomed. Opt. Express

• I. V. Larina, K. V. Larin, M. E. Dickinson, and M. Liebling, “Sequential turning acquisition and reconstruction (STAR) method for four-dimensional imaging of cyclically moving structures,” Biomed. Opt. Express3, 650–660 (2012). [CrossRef] [PubMed]

Dev. Dyn.

• C.-J. Huang, C.-T. Tu, C.-D. Hsiao, F.-J. Hsieh, and H.-J. Tsai, “Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish,” Dev. Dyn.228, 30–40 (2003). [CrossRef] [PubMed]

Development (Cambridge, England)

• J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development (Cambridge, England)136, 1963–1975 (2009). [CrossRef]
• P. J. Scherz, J. Huisken, P. Sahai-Hernandez, and D. Y. R. Stainier, “High-speed imaging of developing heart valves reveals interplay of morphogenesis and function,” Development (Cambridge, England)135, 1179–1187 (2008). [CrossRef]

HFSP J.

• J. Vermot, S. E. Fraser, and M. Liebling, “Fast fluorescence microscopy for imaging the dynamics of embryonic development,” HFSP J.2, 143–55 (2008). [CrossRef]

J. Biomed. Opt.

• J. M. Taylor, C. D. Saunter, G. D. Love, J. M. Girkin, D. J. Henderson, and B. Chaudhry, “Real-time optical gating for three-dimensional beating heart imaging,” J. Biomed. Opt.16, 116021 (2011). [CrossRef] [PubMed]
• M. Liebling, A. S. Forouhar, M. Gharib, S. E. Fraser, and M. E. Dickinson, “Four-dimensional cardiac imaging in living embryos via postacquisition synchronization of nongated slice sequences,” J. Biomed. Opt.10, 054001 (2005). [CrossRef] [PubMed]

J. Biophotonics

• J. Swoger, M. Muzzopappa, H. López-Schier, and J. Sharpe, “4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies,” J. Biophotonics4, 122–134 (2011). [CrossRef]

Magn. Reson. Med.

• A. C. S. Brau, C. T. Wheeler, L. W. Hedlund, and G. A. Johnson, “Fiber-optic stethoscope: a cardiac monitoring and gating system for magnetic resonance microscopy,” Magn. Reson. Med.47, 314–321 (2002). [CrossRef] [PubMed]
• B. Hiba, N. Richard, H. Thibault, and M. Janier, “Cardiac and respiratory self-gated cine MRI in the mouse: comparison between radial and rectilinear techniques at 7T,” Magn. Reson. Med.58, 745–753 (2007). [CrossRef] [PubMed]
• M. Buehrer, J. Curcic, P. Boesiger, and S. Kozerke, “Prospective self-gating for simultaneous compensation of cardiac and respiratory motion,” Magn. Reson. Med.60, 683–690 (2008). [CrossRef] [PubMed]

Nat. Commun.

• S. Lee, C. Vinegoni, P. F. Feruglio, L. Fexon, R. Gorbatov, M. Pivoravov, A. Sbarbati, M. Nahrendorf, and R. Weissleder, “Real-time in vivo imaging of the beating mouse heart at microscopic resolution,” Nat. Commun.3, 1054 (2012). [CrossRef] [PubMed]

Opt. Lett.

• J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32, 2608–2610 (2007). [CrossRef] [PubMed]

Rev. Sci. Instrum.

• K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum.78, 023705 (2007). [CrossRef] [PubMed]

Science

• A. S. Forouhar, M. Liebling, A. Hickerson, A. Nasiraei-Moghaddam, H.-J. Tsai, J. R. Hove, S. E. Fraser, M. E. Dickinson, and M. Gharib, “The embryonic vertebrate heart tube is a dynamic suction pump,” Science312, 751–753 (2006). [CrossRef] [PubMed]

Semin. Cell Dev. Biol.

• J. J. Schoenebeck and D. Yelon, “Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics,” Semin. Cell Dev. Biol.18, 27–35 (2007). [CrossRef] [PubMed]

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