Abstract

In vivo imaging is often severely compromised by cardiovascular and respiratory motion. Highly successful motion compensation techniques have been developed for clinical imaging (e.g. magnetic resonance imaging) but the use of more advanced techniques for intravital microscopy is largely unexplored. Here, we implement a sequential cardiorespiratory gating scheme (SCG) for averaged microscopy. We show that SCG is very efficient in eliminating motion artifacts, is highly practical, enables high signal-to-noise ratio (SNR) in vivo imaging, and yields large field of views. The technique is particularly useful for high-speed data acquisition or for imaging scenarios where the fluorescence signal is not significantly above noise or background levels.

© 2013 OSA

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2012 (5)

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 Commun3, 1054 (2012).
[CrossRef] [PubMed]

J. Tsao and S. Kozerke, “MRI temporal acceleration techniques,” J. Magn. Reson. Imaging36(3), 543–560 (2012).
[CrossRef] [PubMed]

S. Lee, C. Vinegoni, P. F. Feruglio, and R. Weissleder, “Improved intravital microscopy via synchronization of respiration and holder stabilization,” J. Biomed. Opt.17(9), 096018 (2012).
[CrossRef] [PubMed]

P. Bousso and H. D. Moreau, “Functional immunoimaging: the revolution continues,” Nat. Rev. Immunol.12(12), 858–864 (2012).
[CrossRef] [PubMed]

L. Ritsma, B. Ponsioen, and J. van Rheenen, “Intravital imaging of cell signaling in mice,” Intravital1(1), 2–8 (2012).
[CrossRef]

2011 (3)

M. J. Pittet and R. Weissleder, “Intravital imaging,” Cell147(5), 983–991 (2011).
[CrossRef] [PubMed]

M. R. Looney, E. E. Thornton, D. Sen, W. J. Lamm, R. W. Glenny, and M. F. Krummel, “Stabilized imaging of immune surveillance in the mouse lung,” Nat. Methods8(1), 91–96 (2011).
[CrossRef] [PubMed]

S. Laffray, S. Pagès, H. Dufour, P. De Koninck, Y. De Koninck, and D. Côté, “Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue,” PLoS ONE6(5), e19928 (2011).
[CrossRef] [PubMed]

2010 (2)

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

R. Weigert, M. Sramkova, L. Parente, P. Amornphimoltham, and A. Masedunskas, “Intravital microscopy: a novel tool to study cell biology in living animals,” Histochem. Cell Biol.133(5), 481–491 (2010).
[CrossRef] [PubMed]

2009 (1)

A. D. Scott, J. Keegan, and D. N. Firmin, “Motion in cardiovascular MR imaging,” Radiology250(2), 331–351 (2009).
[CrossRef] [PubMed]

1997 (1)

1995 (1)

T. K. Foo, M. A. Bernstein, A. M. Aisen, R. J. Hernandez, B. D. Collick, and T. Bernstein, “Improved ejection fraction and flow velocity estimates with use of view sharing and uniform repetition time excitation with fast cardiac techniques,” Radiology195(2), 471–478 (1995).
[PubMed]

1993 (1)

J. P. Finn and R. R. Edelman, “Black-blood and segmented k-space magnetic resonance angiography,” Magn. Reson. Imaging Clin. N. Am.1(2), 349–357 (1993).
[PubMed]

1991 (1)

D. J. Atkinson and R. R. Edelman, “Cineangiography of the heart in a single breath hold with a segmented turboFLASH sequence,” Radiology178(2), 357–360 (1991).
[PubMed]

Aisen, A. M.

T. K. Foo, M. A. Bernstein, A. M. Aisen, R. J. Hernandez, B. D. Collick, and T. Bernstein, “Improved ejection fraction and flow velocity estimates with use of view sharing and uniform repetition time excitation with fast cardiac techniques,” Radiology195(2), 471–478 (1995).
[PubMed]

Amornphimoltham, P.

R. Weigert, M. Sramkova, L. Parente, P. Amornphimoltham, and A. Masedunskas, “Intravital microscopy: a novel tool to study cell biology in living animals,” Histochem. Cell Biol.133(5), 481–491 (2010).
[CrossRef] [PubMed]

Atkinson, D. J.

D. J. Atkinson and R. R. Edelman, “Cineangiography of the heart in a single breath hold with a segmented turboFLASH sequence,” Radiology178(2), 357–360 (1991).
[PubMed]

Bernstein, M. A.

T. K. Foo, M. A. Bernstein, A. M. Aisen, R. J. Hernandez, B. D. Collick, and T. Bernstein, “Improved ejection fraction and flow velocity estimates with use of view sharing and uniform repetition time excitation with fast cardiac techniques,” Radiology195(2), 471–478 (1995).
[PubMed]

Bernstein, T.

T. K. Foo, M. A. Bernstein, A. M. Aisen, R. J. Hernandez, B. D. Collick, and T. Bernstein, “Improved ejection fraction and flow velocity estimates with use of view sharing and uniform repetition time excitation with fast cardiac techniques,” Radiology195(2), 471–478 (1995).
[PubMed]

Bousso, P.

P. Bousso and H. D. Moreau, “Functional immunoimaging: the revolution continues,” Nat. Rev. Immunol.12(12), 858–864 (2012).
[CrossRef] [PubMed]

Brunenberg, E. J.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

Collick, B. D.

T. K. Foo, M. A. Bernstein, A. M. Aisen, R. J. Hernandez, B. D. Collick, and T. Bernstein, “Improved ejection fraction and flow velocity estimates with use of view sharing and uniform repetition time excitation with fast cardiac techniques,” Radiology195(2), 471–478 (1995).
[PubMed]

Côté, D.

S. Laffray, S. Pagès, H. Dufour, P. De Koninck, Y. De Koninck, and D. Côté, “Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue,” PLoS ONE6(5), e19928 (2011).
[CrossRef] [PubMed]

De Koninck, P.

S. Laffray, S. Pagès, H. Dufour, P. De Koninck, Y. De Koninck, and D. Côté, “Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue,” PLoS ONE6(5), e19928 (2011).
[CrossRef] [PubMed]

De Koninck, Y.

S. Laffray, S. Pagès, H. Dufour, P. De Koninck, Y. De Koninck, and D. Côté, “Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue,” PLoS ONE6(5), e19928 (2011).
[CrossRef] [PubMed]

Dufour, H.

S. Laffray, S. Pagès, H. Dufour, P. De Koninck, Y. De Koninck, and D. Côté, “Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue,” PLoS ONE6(5), e19928 (2011).
[CrossRef] [PubMed]

Edelman, R. R.

J. P. Finn and R. R. Edelman, “Black-blood and segmented k-space magnetic resonance angiography,” Magn. Reson. Imaging Clin. N. Am.1(2), 349–357 (1993).
[PubMed]

D. J. Atkinson and R. R. Edelman, “Cineangiography of the heart in a single breath hold with a segmented turboFLASH sequence,” Radiology178(2), 357–360 (1991).
[PubMed]

Engels, W.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[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 Commun3, 1054 (2012).
[CrossRef] [PubMed]

S. Lee, C. Vinegoni, P. F. Feruglio, and R. Weissleder, “Improved intravital microscopy via synchronization of respiration and holder stabilization,” J. Biomed. Opt.17(9), 096018 (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 Commun3, 1054 (2012).
[CrossRef] [PubMed]

Finn, J. P.

J. P. Finn and R. R. Edelman, “Black-blood and segmented k-space magnetic resonance angiography,” Magn. Reson. Imaging Clin. N. Am.1(2), 349–357 (1993).
[PubMed]

Firmin, D. N.

A. D. Scott, J. Keegan, and D. N. Firmin, “Motion in cardiovascular MR imaging,” Radiology250(2), 331–351 (2009).
[CrossRef] [PubMed]

Foo, T. K.

T. K. Foo, M. A. Bernstein, A. M. Aisen, R. J. Hernandez, B. D. Collick, and T. Bernstein, “Improved ejection fraction and flow velocity estimates with use of view sharing and uniform repetition time excitation with fast cardiac techniques,” Radiology195(2), 471–478 (1995).
[PubMed]

Glenny, R. W.

M. R. Looney, E. E. Thornton, D. Sen, W. J. Lamm, R. W. Glenny, and M. F. Krummel, “Stabilized imaging of immune surveillance in the mouse lung,” Nat. Methods8(1), 91–96 (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 Commun3, 1054 (2012).
[CrossRef] [PubMed]

Hernandez, R. J.

T. K. Foo, M. A. Bernstein, A. M. Aisen, R. J. Hernandez, B. D. Collick, and T. Bernstein, “Improved ejection fraction and flow velocity estimates with use of view sharing and uniform repetition time excitation with fast cardiac techniques,” Radiology195(2), 471–478 (1995).
[PubMed]

Izatt, J.

Janssen, B. J.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

Keegan, J.

A. D. Scott, J. Keegan, and D. N. Firmin, “Motion in cardiovascular MR imaging,” Radiology250(2), 331–351 (2009).
[CrossRef] [PubMed]

Kozerke, S.

J. Tsao and S. Kozerke, “MRI temporal acceleration techniques,” J. Magn. Reson. Imaging36(3), 543–560 (2012).
[CrossRef] [PubMed]

Krummel, M. F.

M. R. Looney, E. E. Thornton, D. Sen, W. J. Lamm, R. W. Glenny, and M. F. Krummel, “Stabilized imaging of immune surveillance in the mouse lung,” Nat. Methods8(1), 91–96 (2011).
[CrossRef] [PubMed]

Kulkarni, M.

Laffray, S.

S. Laffray, S. Pagès, H. Dufour, P. De Koninck, Y. De Koninck, and D. Côté, “Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue,” PLoS ONE6(5), e19928 (2011).
[CrossRef] [PubMed]

Lamm, W. J.

M. R. Looney, E. E. Thornton, D. Sen, W. J. Lamm, R. W. Glenny, and M. F. Krummel, “Stabilized imaging of immune surveillance in the mouse lung,” Nat. Methods8(1), 91–96 (2011).
[CrossRef] [PubMed]

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 Commun3, 1054 (2012).
[CrossRef] [PubMed]

S. Lee, C. Vinegoni, P. F. Feruglio, and R. Weissleder, “Improved intravital microscopy via synchronization of respiration and holder stabilization,” J. Biomed. Opt.17(9), 096018 (2012).
[CrossRef] [PubMed]

Leenders, P. J.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

Looney, M. R.

M. R. Looney, E. E. Thornton, D. Sen, W. J. Lamm, R. W. Glenny, and M. F. Krummel, “Stabilized imaging of immune surveillance in the mouse lung,” Nat. Methods8(1), 91–96 (2011).
[CrossRef] [PubMed]

Masedunskas, A.

R. Weigert, M. Sramkova, L. Parente, P. Amornphimoltham, and A. Masedunskas, “Intravital microscopy: a novel tool to study cell biology in living animals,” Histochem. Cell Biol.133(5), 481–491 (2010).
[CrossRef] [PubMed]

Megens, R. T. A.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

Moreau, H. D.

P. Bousso and H. D. Moreau, “Functional immunoimaging: the revolution continues,” Nat. Rev. Immunol.12(12), 858–864 (2012).
[CrossRef] [PubMed]

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 Commun3, 1054 (2012).
[CrossRef] [PubMed]

oude Egbrink, M. G.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

Pagès, S.

S. Laffray, S. Pagès, H. Dufour, P. De Koninck, Y. De Koninck, and D. Côté, “Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue,” PLoS ONE6(5), e19928 (2011).
[CrossRef] [PubMed]

Parente, L.

R. Weigert, M. Sramkova, L. Parente, P. Amornphimoltham, and A. Masedunskas, “Intravital microscopy: a novel tool to study cell biology in living animals,” Histochem. Cell Biol.133(5), 481–491 (2010).
[CrossRef] [PubMed]

Pittet, M. J.

M. J. Pittet and R. Weissleder, “Intravital imaging,” Cell147(5), 983–991 (2011).
[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 Commun3, 1054 (2012).
[CrossRef] [PubMed]

Ponsioen, B.

L. Ritsma, B. Ponsioen, and J. van Rheenen, “Intravital imaging of cell signaling in mice,” Intravital1(1), 2–8 (2012).
[CrossRef]

Prinzen, L.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

Reesink, K. D.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

Reitsma, S.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

Ritsma, L.

L. Ritsma, B. Ponsioen, and J. van Rheenen, “Intravital imaging of cell signaling in mice,” Intravital1(1), 2–8 (2012).
[CrossRef]

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 Commun3, 1054 (2012).
[CrossRef] [PubMed]

Scott, A. D.

A. D. Scott, J. Keegan, and D. N. Firmin, “Motion in cardiovascular MR imaging,” Radiology250(2), 331–351 (2009).
[CrossRef] [PubMed]

Sen, D.

M. R. Looney, E. E. Thornton, D. Sen, W. J. Lamm, R. W. Glenny, and M. F. Krummel, “Stabilized imaging of immune surveillance in the mouse lung,” Nat. Methods8(1), 91–96 (2011).
[CrossRef] [PubMed]

Slaaf, D. W.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

Sramkova, M.

R. Weigert, M. Sramkova, L. Parente, P. Amornphimoltham, and A. Masedunskas, “Intravital microscopy: a novel tool to study cell biology in living animals,” Histochem. Cell Biol.133(5), 481–491 (2010).
[CrossRef] [PubMed]

ter Haar Romeny, B. M.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

Thornton, E. E.

M. R. Looney, E. E. Thornton, D. Sen, W. J. Lamm, R. W. Glenny, and M. F. Krummel, “Stabilized imaging of immune surveillance in the mouse lung,” Nat. Methods8(1), 91–96 (2011).
[CrossRef] [PubMed]

Tsao, J.

J. Tsao and S. Kozerke, “MRI temporal acceleration techniques,” J. Magn. Reson. Imaging36(3), 543–560 (2012).
[CrossRef] [PubMed]

van Rheenen, J.

L. Ritsma, B. Ponsioen, and J. van Rheenen, “Intravital imaging of cell signaling in mice,” Intravital1(1), 2–8 (2012).
[CrossRef]

van Zandvoort, M. A.

R. T. A. Megens, S. Reitsma, L. Prinzen, M. G. oude Egbrink, W. Engels, P. J. Leenders, E. J. Brunenberg, K. D. Reesink, B. J. Janssen, B. M. ter Haar Romeny, D. W. Slaaf, and M. A. van Zandvoort, “In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy,” J. Biomed. Opt.15(1), 011108 (2010).
[CrossRef] [PubMed]

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 Commun3, 1054 (2012).
[CrossRef] [PubMed]

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Cell (1)

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Histochem. Cell Biol. (1)

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Figures (8)

Fig. 1
Fig. 1

Scheme of principle for sequential retrospective electrocardiogram (ECG)-gated segmented microscopy. For figure simplicity, we assume here the absence of any respiratory motion. (a) In MRI, a sequence of views is collected in the k-space, by varying the phase encoding gradient. Laser scanning microscopy (LSM) images (IM) are acquired pixel by pixel in the real space, with the excitation scanning laser beam moving along a predefined path (MRI analogue of a group of views). Groups of views (segments S1, S2, etc.) are sequentially collected within a time-gated window TGW, which is coincident with the time window TC, corresponding to the end-diastole. The QRS complex, groups all waves within an ECG generated during ventricular depolarization. R indicates the R phase of the QRS complex. The process is then repeated until the entire real space (LSM) or k-space (MRI) is filled. (b) Image reconstructions are obtained by grouping several consecutive segments and by then transforming them using the function F. In MRI, an image is obtained by applying a transformation (the inverse Fourier transformation IFFT in conjunction with the absolute function ABS (i.e., IFFT + ABS), to each pixel of the image matrix. In LSM, images are constructed directly in the real space and the transformation does not induce any further change after grouping. F can therefore be considered as the identity function (I). (c) Prospective triggered acquisition scheme: data for images are acquired only during the time of a specific triggered window, which is determined by ECG. All acquired data are therefore used for image reconstruction. (d) Retrospective gated acquisition scheme: data for images are continuously acquired together with the ECG recording. Following this non-selective acquisition, only the data that were acquired during the time of a specific gated window, which is determined by ECG, are chosen for image reconstruction. RR indicates the distance between two R phases. IM indicates a generic image.

Fig. 2
Fig. 2

Timing diagram and image reconstruction scheme for non-averaged microscopy in sequential retrospective cardiac-gated segmented microscopy. For the sake of simplicity we assume the absence of any respiratory motion in both time diagrams. We define as TCC the time interval between two cardiac cycles, as TS the acquisition time of a single raw image, as ΔTS the time interval between the end and the beginning of two consecutive acquisitions, and as T’s = Ts + ΔTS + Δ TS the time interval between two consecutive images. Sequentially time-shifted images (Δt = TGW) were acquired, and N individual segments Pi (shown in red) within each image were isolated (corresponding to the time-gated window TGW). Segments were then grouped together ( 1 N i P i ) and a final image was reconstructed. The reconstructed image thus provides a true representation of the heart’s morphology (i.e., a flat virtual section) at the cardiac phase corresponding to the time-gated window TGW. In order to simply illustrate the concept of segmented microscopy we have assumed that the cardiac cycle is constant and that T’s = nTCC – TGW. This implies that the segmented areas from consecutive images will be adjacent to each other.

Fig. 3
Fig. 3

Timing diagram and image reconstruction scheme for averaged microscopy in sequential retrospective cardiac-gated segmented microscopy. Data acquisition was initiated at a specific phase of the cardiac cycle, and M identical segments Pi,j within the time gated window TGW were then extracted from each image. A ‘summation segment’ <P > 1 was obtained by merging all the segments ( <P > 1 = j 1 M P 1,j ). To simply illustrate the concept of segmented averaging microscopy we assume that the cardiac cycle is constant and that T’S = nTCC. This implies that segmented areas from consecutive images will be overlapping on each other in order to obtain an average segment. Data acquisition was subsequently initiated at a different phase of the cardiac cycle, time shifted according to the quantity of TGW from the first acquisition such that T’s = nTCC – TGW, and a second ‘summation segment’ <P > 2 obtained. The process was repeated multiple times (N) until the ‘summation segments’ covered the entire image field to render a final image with high SNR ( 1 N i <P > i ).

Fig. 4
Fig. 4

Experimental setup. LSM, laser scanning microscope; DAQ, data acquisition card; AMP, differential amplifier; VEN mechanical ventilator. Inset: position of stabilizing holder. LSM image acquisition was synchronized with cardiac and respiratory motion, with a DAQ-card registering the signals from the ECG amplifier and a ventilator controling the lung airway pressure.

Fig. 5
Fig. 5

Timing diagram for retrospective gated imaging. Initialization and subsequent ventilation triggers are shown in combination with the measured ECG signal and the recorded lung airway pressure. Inspiration time windows TR, and cardiac time windows TC are located at points of minimum displacement for both motions. The acquisition window TGW corresponds to the intersection between the cardiac time window and the inspiration time windows TC and TR. To note how the length of each time-gate acquisition window TGW is not constant but varies along time. This is independent from the specific (prospective or retrospective) imaging modality but depends only on the physiological parameters. Segments corresponding to these acquisition windows were taken from raw images.

Fig. 6
Fig. 6

In vivo heart imaging using sequential cardiac- and respiratory-gated average segmented microscopy. Images (left to right) illustrate how a sequential average segmented scheme improves the SNR of images. Here, specific raw data segments, corresponding to the gated time window TGW, were collected and added (at its correct temporal position) to the reconstructed image (a summation of all previous data). The accumulation of segments continued until the number of the summation of all views (or segments) in the reconstructed image reached a predefined value. The summation of seven segments is shown. The intensity of the red color and the number of asterisks on the right side of each image represent how many segments have been summed. Fluorescence staining of myocyte nuclei is shown in green (Hoechst 33258); fluorescence lectin staining of the myocardium capillaries is shown in red (Rhodamine labeled Griffoniasimplicifolia lectin, RL-1102, Vector Laboratories). Nuclei in the middle of the image look elongated because the optical imaging plane is sectioning the heart at a different height with respect to the border area due to the natural curved surface of the heart. Cells in the center are representative of myocytes within the tissue, and are surrounded by a rich vasculature network and elongated along the axis of the microcapillaries, as opposite to the nuclei of the cells present in the border area which reside on the surface of the heart.

Fig. 7
Fig. 7

(a) Stabilized image of fluorescently labeled myocyte nuclei (Hoechst 33258). Within the nuclei, subnuclear structures are clearly identifiable, and their locations were used to determine the stabilized imaging resolution (i.e. their position reproducibility over time). (b) The planar position coordinates of the labeled nucleoli shown in (a) (green circle) are plotted for different consecutive segments. The graph shows that the position of the nucleoli in each image was very stable with only a 1.2 and 1.1 micron standard deviation for the x and y coordinates, respectively. This reproducibility, which is the combined result of using the stabilizing holder in conjunction with physiological gating, makes it possible to perform sequential averaged segmented microscopy. STD, standard deviation.

Fig. 8
Fig. 8

Retrospectively gated sequential average segmented microscopy images of fluorescently stained cardiac myocytes (nuclear stain: Hoechst 33258). Left to right: individually triggered segments are summed over time, following the scheme in Fig. 3. For convenience of display, images are not normalized but presented as a sum and displayed on the same scale. The standard deviation of the noise was calculated for each individual averaged image (from left to right: 14.7, 10.0, 8.7, 7.6).

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