Abstract

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

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  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]

2012 (2)

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]

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]

2011 (2)

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]

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]

2009 (1)

J. Huisken and D. Y. R. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development (Cambridge, England)136, 1963–1975 (2009).
[CrossRef]

2008 (3)

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]

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]

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]

2007 (4)

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]

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]

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]

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]

2006 (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]

2005 (1)

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]

2003 (1)

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]

2002 (1)

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]

Boesiger, P.

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]

Brau, A. C. S.

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]

Buehrer, M.

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]

Chaudhry, B.

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]

Curcic, J.

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]

Dickinson, M. E.

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]

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]

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]

Feruglio, P. F.

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]

Fexon, L.

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]

Forouhar, A. S.

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]

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]

Fraser, S. E.

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]

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]

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]

Gharib, M.

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]

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]

Girkin, J. M.

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]

Gorbatov, R.

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]

Greger, K.

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]

Hedlund, L. W.

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]

Henderson, D. J.

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]

Hiba, B.

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]

Hickerson, A.

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]

Hove, J. R.

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]

Hsiao, C.-D.

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]

Hsieh, F.-J.

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]

Huang, C.-J.

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]

Huisken, J.

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]

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]

Janier, M.

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]

Johnson, G. A.

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]

Kozerke, S.

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]

Larin, K. V.

Larina, I. V.

Lee, S.

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]

Liebling, M.

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]

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]

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]

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]

López-Schier, H.

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]

Love, G. D.

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]

Muzzopappa, M.

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]

Nahrendorf, M.

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]

Nasiraei-Moghaddam, A.

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]

Pivoravov, M.

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]

Richard, N.

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]

Sahai-Hernandez, P.

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]

Saunter, C. D.

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]

Sbarbati, A.

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]

Scherz, P. J.

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]

Schoenebeck, J. J.

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]

Sharpe, J.

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]

Stainier, D. Y. R.

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]

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]

Stelzer, E. H. K.

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]

Swoger, J.

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]

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]

Taylor, J. M.

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]

Thibault, H.

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]

Tsai, H.-J.

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]

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]

Tu, C.-T.

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]

Vermot, 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]

Vinegoni, C.

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]

Weissleder, R.

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]

Wheeler, C. T.

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]

Yelon, D.

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]

Biomed. Opt. Express (1)

Dev. Dyn. (1)

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) (2)

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. (1)

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. (2)

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 (1)

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. (3)

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. (1)

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. (1)

Rev. Sci. Instrum. (1)

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 (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]

Semin. Cell Dev. Biol. (1)

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]

Supplementary Material (3)

» Media 1: MOV (2943 KB)     
» Media 2: MOV (746 KB)     
» Media 3: MOV (3091 KB)     

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (7)

Fig. 1
Fig. 1

Schematic diagram of synchronization system. Brightfield images are compared against a set of previously-acquired reference frames, and a phase assigned to each received image (“phase recovery”). A fit is then performed on this data in order to predict the time at which the heart will be in the particular desired position (“forward prediction”). This predicted time is transmitted to the timing controller which generates electrical trigger signals for the fluorescence camera and the excitation laser.

Fig. 2
Fig. 2

Optical configuration of the microscope. A free-running camera (CCD1, Prosilica GS650) acquires brightfield images continuously for real-time analysis, and a second fluorescence camera (CCD2, QImaging QIClick) is triggered to acquire gated frames only at the appropriate calculated times. As the sample is moved to change the focal depth for sectioned fluorescence imaging, the f =150 mm tube lens is moved proportionally in order to keep CCD1 focused at a constant depth and provide a fixed reference signal.

Fig. 3
Fig. 3

(a) Ray diagram used in magnification calculation. Two ray paths are shown, for an object at the origin (black rays) and at a shifted location (green rays). (b) Corrective translation (in pixels) determined by our algorithm, showing recovery following manual reset to illustrate robustness of algorithm. Note that the sample was deliberately mounted poorly to induce a faster than normal drift, and the offset was also manually reset at around 5.5 seconds in order to demonstrate the recovery of the software from a large and instantaneous perturbation. (c) Magnification M as a function of tube lens offset (Δz) for our experimental configuration (where the tube lens focal length f2 = 125 mm and the objective focal length f1 = 12.5 mm) showing, for three candidate values of D0, the change in magnification over a substantial range of 200 μm of focal depth.

Fig. 4
Fig. 4

(a) Raw data from brightfield and fluorescence cameras (scale bar 20 μm). The fluorescence camera is only triggered once per heartbeat, taking a single optically sectioned image slice on each heartbeat, scanning down through the ventricle of the heart. (b) Selected fluorescence image slices acquired as part of the image stack (see also Media 1).

Fig. 5
Fig. 5

Still frame from Media 2 illustrating some aspects of the operation of the synchronization algorithm. The movie shows a 100 ms time window around the trigger firing time, slowed down by a factor of 100. The data flow is highlighted in the system diagram as successive brightfield frames are received. Phase recovery and forward prediction is performed for every frame. When the forward prediction indicates that a trigger signal will soon be required (within the next 40 ms), the timing controller is programmed with the required information. This then generates the electrical signal to trigger acquisition of the fluorescence image. At the bottom of the movie two event timelines are shown. The upper timeline shows the brightfield frames being exposed (black) and processed in software (gray). The lower timeline shows the results of the forward prediction analysis (red circle showing predicted time to trigger fluorescence camera). This prediction is improved every time a new brightfield image is received; the marker changes to blue when the algorithm “commits” to a time and programs it into the timing controller, and changes to black when the electrical trigger is actually sent to the fluorescence camera.

Fig. 6
Fig. 6

Left: reconstructed false color image showing a cutaway of a 3D volumetric reconstruction of the ventricle at approximately 5 days after fertilization. The dataset from Fig. 4 was reconstructed in the VolView software package, using intensity-based segmentation to identify the cardiac myocyte tissue labelled with green fluorescent protein (transgenic strain cmlc2:gfp). A cutaway showing the structure of the trabeculae on the inside wall of the ventricle was then rendered in false colour. The ventricle long axis is approximately 140 μm across (see also Media 3). Right: a second reconstruction of a ventricle earlier in the development process, at approximately 4 days after fertilization. Both datasets were taken with the ventricle at end-systole.

Fig. 7
Fig. 7

Image for a single xy plane (left) and two reconstructed slices in the yz plane (center and right) taken from the dataset shown in Figs. 4 and 6 to allow the consistency of the synchronization to be judged visually. The vertical lines indicate the positions where the various perpendicular slices shown intersect each other. Our refocusing scheme maintains a fixed brightfield reference focus, eliminating registration artefacts and systematic bias that might be present in post-processed datasets (see [11, 13]); any residual artefacts in the yz reconstructions are due to slight inaccuracies in the timing of the trigger signals. We note that the spatial resolution of the images degrades gradually with increasing z and x due to scattering and aberrations caused by imaging deeper inside the sample.

Equations (4)

Equations on this page are rendered with MathJax. Learn more.

1 f 1 = n f 1 Δ z / n 1 v z v z = n f 1 2 Δ z + O ( Δ z 2 )
1 f 2 = 1 f 2 + Δ z + 1 v z + D 0 Δ z R = Δ z Δ z = f 2 2 n f 1 2 + O ( Δ z ) .
v y = Δ y v z f 1 v y = Δ y n f 1 Δ z + O ( Δ z 2 )
Δ y v y = f 2 + Δ z v z + D 0 Δ z M = Δ y Δ y = f 2 f 1 ( 1 + f 2 D 0 n f 1 2 Δ z ) + O ( Δ z 2 ) .

Metrics