Abstract

Rapid imaging of multiple focal planes without sample movement may be achieved through remote refocusing, where imaging is carried out in a plane conjugate to the sample plane. The technique is ideally suited to studying the endothelial and smooth muscle cell layers of blood vessels. These are intrinsically linked through rapid communication and must be separately imaged at a sufficiently high frame rate in order to understand this biologically crucial interaction. We have designed and implemented an epifluoresence-based remote refocussing imaging system that can image each layer at up to 20fps using different dyes and excitation light for each layer, without the requirement for optically sectioning microscopy. A novel triggering system is used to activate the appropriate laser and image acquisition at each plane of interest. Using this method, we are able to achieve axial plane separations down to 15 $\mu$m, with a mean lateral stability of $\leq$ 0.32 $\mu$m displacement using a 60x, 1.4NA imaging objective and a 60x, 0.7NA reimaging objective. The system allows us to image and quantify endothelial cell activity and smooth muscle cell activity at a high framerate with excellent lateral and good axial resolution without requiring complex beam scanning confocal microscopes, delivering a cost effective solution for imaging two planes rapidly. We have successfully imaged and analysed Ca$^{2+}$ activity of the endothelial cell layer independently of the smooth muscle layer for several minutes.

Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation, and DOI.

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References

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    [Crossref]
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    [Crossref]
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    [Crossref]
  8. E. J. Botcherby, M. J. Booth, R. Juškaitis, and T. Wilson, “Real-time extended depth of field microscopy,” Opt. Express 16(26), 21843–21848 (2008).
    [Crossref]
  9. B. Endres, A. Staruschenko, M. Schulte, A. Geurts, and O. Palygin, “Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta,” J. Visualized Exp. 10(100), e52734 (2015).
    [Crossref]
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    [Crossref]
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    [Crossref]
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    [Crossref]
  13. P. Salter, M. Baum, I. Alexeev, M. Schmidt, and M. Booth, “Exploring the depth range for three-dimensional laser machining with aberration correction,” Opt. Express 22(15), 17644 (2014).
    [Crossref]
  14. R. Simmonds, P. Salter, A. Jesacher, and M. Booth, “Three dimensional laser microfabrication in diamond using a dual adaptive optics system,” Opt. Express 19(24), 24122–24128 (2011).
    [Crossref]
  15. D. B. Allan, T. Caswell, N. C. Keim, and C. M. van der Wel, “trackpy: Trackpy v0.4.1,” (2018).
  16. J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
    [Crossref]
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    [Crossref]
  18. C. Wilson, C. D. Saunter, J. M. Girkin, and J. G. McCarron, “Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium,” FASEB J. 30(5), 2000–2013 (2016).
    [Crossref]
  19. E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
    [Crossref]

2018 (2)

M. Bawart, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Remote focusing in confocal microscopy by means of a modified alvarez lens,” J. Microsc. 271(3), 337–344 (2018).
[Crossref]

A. Corbett, M. Shaw, A. Yacoot, A. Jefferson, L. Schermelleh, T. Wilson, M. Booth, and P. Salter, “Microscope calibration using laser written fluorescence,” Opt. Express 26(17), 21887–21899 (2018).
[Crossref]

2017 (1)

2016 (2)

C. Wilson, M. D. Lee, and J. G. McCarron, “Acetylcholine released by endothelial cells facilitates flow-mediated dilatation,” The J. Physiol. 594(24), 7267–7307 (2016).
[Crossref]

C. Wilson, C. D. Saunter, J. M. Girkin, and J. G. McCarron, “Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium,” FASEB J. 30(5), 2000–2013 (2016).
[Crossref]

2015 (1)

B. Endres, A. Staruschenko, M. Schulte, A. Geurts, and O. Palygin, “Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta,” J. Visualized Exp. 10(100), e52734 (2015).
[Crossref]

2014 (2)

A. Corbett, R. Burton, G. Bub, P. Salter, S. Tuohy, M. Booth, and T. Wilson, “Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen,” Front. Physiol. 5, 1–9 (2014).
[Crossref]

P. Salter, M. Baum, I. Alexeev, M. Schmidt, and M. Booth, “Exploring the depth range for three-dimensional laser machining with aberration correction,” Opt. Express 22(15), 17644 (2014).
[Crossref]

2012 (2)

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
[Crossref]

2011 (2)

2010 (1)

A. Edelstein, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μmanager,” Curr. Protoc. Mol. Biol. 92, 14–20 (2010).
[Crossref]

2008 (2)

2007 (2)

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, “Aberration-free optical refocussing in high numerical aperture microscopy,” Opt. Lett. 32(14), 2007–2009 (2007).
[Crossref]

W. Göbel, B. Kampa, and F. Helmchen, “Imaging cellular network dynamics in three dimensions using fast 3D laser scanning,” Nat. Methods 4(1), 73–79 (2007).
[Crossref]

2006 (1)

Alexeev, I.

Allan, D. B.

D. B. Allan, T. Caswell, N. C. Keim, and C. M. van der Wel, “trackpy: Trackpy v0.4.1,” (2018).

Amodaj, N.

A. Edelstein, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μmanager,” Curr. Protoc. Mol. Biol. 92, 14–20 (2010).
[Crossref]

Arganda-Carreras, I.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Barnstedt, O.

Baum, M.

Bawart, M.

M. Bawart, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Remote focusing in confocal microscopy by means of a modified alvarez lens,” J. Microsc. 271(3), 337–344 (2018).
[Crossref]

Bernet, S.

M. Bawart, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Remote focusing in confocal microscopy by means of a modified alvarez lens,” J. Microsc. 271(3), 337–344 (2018).
[Crossref]

Booth, M.

Booth, M. J.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
[Crossref]

E. J. Botcherby, M. J. Booth, R. Juškaitis, and T. Wilson, “Real-time extended depth of field microscopy,” Opt. Express 16(26), 21843–21848 (2008).
[Crossref]

Botcherby, E.

Botcherby, E. J.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
[Crossref]

E. J. Botcherby, M. J. Booth, R. Juškaitis, and T. Wilson, “Real-time extended depth of field microscopy,” Opt. Express 16(26), 21843–21848 (2008).
[Crossref]

Bub, G.

A. Corbett, R. Burton, G. Bub, P. Salter, S. Tuohy, M. Booth, and T. Wilson, “Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen,” Front. Physiol. 5, 1–9 (2014).
[Crossref]

Burton, R.

A. Corbett, R. Burton, G. Bub, P. Salter, S. Tuohy, M. Booth, and T. Wilson, “Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen,” Front. Physiol. 5, 1–9 (2014).
[Crossref]

Cardona, A.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Caswell, T.

D. B. Allan, T. Caswell, N. C. Keim, and C. M. van der Wel, “trackpy: Trackpy v0.4.1,” (2018).

Corbett, A.

A. Corbett, M. Shaw, A. Yacoot, A. Jefferson, L. Schermelleh, T. Wilson, M. Booth, and P. Salter, “Microscope calibration using laser written fluorescence,” Opt. Express 26(17), 21887–21899 (2018).
[Crossref]

A. Corbett, R. Burton, G. Bub, P. Salter, S. Tuohy, M. Booth, and T. Wilson, “Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen,” Front. Physiol. 5, 1–9 (2014).
[Crossref]

Débarre, D.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
[Crossref]

Edelstein, A.

A. Edelstein, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μmanager,” Curr. Protoc. Mol. Biol. 92, 14–20 (2010).
[Crossref]

Eliceiri, K.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Endres, B.

B. Endres, A. Staruschenko, M. Schulte, A. Geurts, and O. Palygin, “Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta,” J. Visualized Exp. 10(100), e52734 (2015).
[Crossref]

Frade-Rodriguez, M.

Frise, E.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Geurts, A.

B. Endres, A. Staruschenko, M. Schulte, A. Geurts, and O. Palygin, “Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta,” J. Visualized Exp. 10(100), e52734 (2015).
[Crossref]

Gibson, G. M.

Girkin, J. M.

Göbel, W.

W. Göbel, B. Kampa, and F. Helmchen, “Imaging cellular network dynamics in three dimensions using fast 3D laser scanning,” Nat. Methods 4(1), 73–79 (2007).
[Crossref]

Grewe, B. F.

Helmchen, F.

B. F. Grewe, F. F. Voigt, M. van ’t Hoff, and F. Helmchen, “Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens,” Biomed. Opt. Express 2(7), 2035–2046 (2011).
[Crossref]

W. Göbel, B. Kampa, and F. Helmchen, “Imaging cellular network dynamics in three dimensions using fast 3D laser scanning,” Nat. Methods 4(1), 73–79 (2007).
[Crossref]

Hertenstein, V.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Hoover, K.

A. Edelstein, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μmanager,” Curr. Protoc. Mol. Biol. 92, 14–20 (2010).
[Crossref]

Jefferson, A.

Jesacher, A.

M. Bawart, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Remote focusing in confocal microscopy by means of a modified alvarez lens,” J. Microsc. 271(3), 337–344 (2018).
[Crossref]

R. Simmonds, P. Salter, A. Jesacher, and M. Booth, “Three dimensional laser microfabrication in diamond using a dual adaptive optics system,” Opt. Express 19(24), 24122–24128 (2011).
[Crossref]

Juškaitis, R.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
[Crossref]

E. J. Botcherby, M. J. Booth, R. Juškaitis, and T. Wilson, “Real-time extended depth of field microscopy,” Opt. Express 16(26), 21843–21848 (2008).
[Crossref]

E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, “Aberration-free optical refocussing in high numerical aperture microscopy,” Opt. Lett. 32(14), 2007–2009 (2007).
[Crossref]

Kampa, B.

W. Göbel, B. Kampa, and F. Helmchen, “Imaging cellular network dynamics in three dimensions using fast 3D laser scanning,” Nat. Methods 4(1), 73–79 (2007).
[Crossref]

Kaynig, V.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Keim, N. C.

D. B. Allan, T. Caswell, N. C. Keim, and C. M. van der Wel, “trackpy: Trackpy v0.4.1,” (2018).

Kohl, M. M.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
[Crossref]

Lee, M. D.

C. Wilson, M. D. Lee, and J. G. McCarron, “Acetylcholine released by endothelial cells facilitates flow-mediated dilatation,” The J. Physiol. 594(24), 7267–7307 (2016).
[Crossref]

Longair, M.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

McCarron, J. G.

C. Wilson, M. D. Lee, and J. G. McCarron, “Acetylcholine released by endothelial cells facilitates flow-mediated dilatation,” The J. Physiol. 594(24), 7267–7307 (2016).
[Crossref]

C. Wilson, C. D. Saunter, J. M. Girkin, and J. G. McCarron, “Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium,” FASEB J. 30(5), 2000–2013 (2016).
[Crossref]

Padgett, M. J.

Palygin, O.

B. Endres, A. Staruschenko, M. Schulte, A. Geurts, and O. Palygin, “Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta,” J. Visualized Exp. 10(100), e52734 (2015).
[Crossref]

Patterson, B. A.

Paulsen, O.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
[Crossref]

Pietzsch, T.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Poland, S. P.

Preibisch, S.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Ritsch-Marte, M.

M. Bawart, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Remote focusing in confocal microscopy by means of a modified alvarez lens,” J. Microsc. 271(3), 337–344 (2018).
[Crossref]

Rueden, C.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Saalfeld, S.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Salter, P.

Saunter, C. D.

C. Wilson, C. D. Saunter, J. M. Girkin, and J. G. McCarron, “Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium,” FASEB J. 30(5), 2000–2013 (2016).
[Crossref]

Schermelleh, L.

Schindelin, J.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Schmid, B.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Schmidt, M.

Schulte, M.

B. Endres, A. Staruschenko, M. Schulte, A. Geurts, and O. Palygin, “Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta,” J. Visualized Exp. 10(100), e52734 (2015).
[Crossref]

Shaw, M.

Simmonds, R.

Smith, C. W.

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
[Crossref]

Staruschenko, A.

B. Endres, A. Staruschenko, M. Schulte, A. Geurts, and O. Palygin, “Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta,” J. Visualized Exp. 10(100), e52734 (2015).
[Crossref]

Stuurman, N.

A. Edelstein, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μmanager,” Curr. Protoc. Mol. Biol. 92, 14–20 (2010).
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Tinevez, J.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
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Tomancak, P.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Tuohy, S.

A. Corbett, R. Burton, G. Bub, P. Salter, S. Tuohy, M. Booth, and T. Wilson, “Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen,” Front. Physiol. 5, 1–9 (2014).
[Crossref]

Vale, R.

A. Edelstein, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μmanager,” Curr. Protoc. Mol. Biol. 92, 14–20 (2010).
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van ’t Hoff, M.

van der Wel, C. M.

D. B. Allan, T. Caswell, N. C. Keim, and C. M. van der Wel, “trackpy: Trackpy v0.4.1,” (2018).

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Waddell, S.

White, D.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Wilson, C.

C. Wilson, M. D. Lee, and J. G. McCarron, “Acetylcholine released by endothelial cells facilitates flow-mediated dilatation,” The J. Physiol. 594(24), 7267–7307 (2016).
[Crossref]

C. Wilson, C. D. Saunter, J. M. Girkin, and J. G. McCarron, “Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium,” FASEB J. 30(5), 2000–2013 (2016).
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Wilson, T.

A. Corbett, M. Shaw, A. Yacoot, A. Jefferson, L. Schermelleh, T. Wilson, M. Booth, and P. Salter, “Microscope calibration using laser written fluorescence,” Opt. Express 26(17), 21887–21899 (2018).
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A. Corbett, R. Burton, G. Bub, P. Salter, S. Tuohy, M. Booth, and T. Wilson, “Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen,” Front. Physiol. 5, 1–9 (2014).
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E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
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E. J. Botcherby, M. J. Booth, R. Juškaitis, and T. Wilson, “Real-time extended depth of field microscopy,” Opt. Express 16(26), 21843–21848 (2008).
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E. Botcherby, R. Juškaitis, M. Booth, and T. Wilson, “Aberration-free optical refocussing in high numerical aperture microscopy,” Opt. Lett. 32(14), 2007–2009 (2007).
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Wright, A. J.

Yacoot, A.

Žurauskas, M.

Biomed. Opt. Express (2)

Curr. Protoc. Mol. Biol. (1)

A. Edelstein, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μmanager,” Curr. Protoc. Mol. Biol. 92, 14–20 (2010).
[Crossref]

FASEB J. (1)

C. Wilson, C. D. Saunter, J. M. Girkin, and J. G. McCarron, “Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium,” FASEB J. 30(5), 2000–2013 (2016).
[Crossref]

Front. Physiol. (1)

A. Corbett, R. Burton, G. Bub, P. Salter, S. Tuohy, M. Booth, and T. Wilson, “Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen,” Front. Physiol. 5, 1–9 (2014).
[Crossref]

J. Microsc. (1)

M. Bawart, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Remote focusing in confocal microscopy by means of a modified alvarez lens,” J. Microsc. 271(3), 337–344 (2018).
[Crossref]

J. Visualized Exp. (1)

B. Endres, A. Staruschenko, M. Schulte, A. Geurts, and O. Palygin, “Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta,” J. Visualized Exp. 10(100), e52734 (2015).
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Nat. Methods (2)

W. Göbel, B. Kampa, and F. Helmchen, “Imaging cellular network dynamics in three dimensions using fast 3D laser scanning,” Nat. Methods 4(1), 73–79 (2007).
[Crossref]

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Tinevez, D. White, V. Hertenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref]

Opt. Express (5)

Opt. Lett. (2)

Proc. Natl. Acad. Sci. U. S. A. (1)

E. J. Botcherby, C. W. Smith, M. M. Kohl, D. Débarre, M. J. Booth, R. Juškaitis, O. Paulsen, and T. Wilson, “Aberration-free three-dimensional multiphoton imaging of neuronal activity at khz rates,” Proc. Natl. Acad. Sci. U. S. A. 109(8), 2919–2924 (2012).
[Crossref]

The J. Physiol. (1)

C. Wilson, M. D. Lee, and J. G. McCarron, “Acetylcholine released by endothelial cells facilitates flow-mediated dilatation,” The J. Physiol. 594(24), 7267–7307 (2016).
[Crossref]

Other (1)

D. B. Allan, T. Caswell, N. C. Keim, and C. M. van der Wel, “trackpy: Trackpy v0.4.1,” (2018).

Supplementary Material (2)

NameDescription
» Visualization 1       Rapid imaging of two layers of a fixed carotid artery
» Visualization 2       Rapid imaging of a live artery sample after addition of ACh

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Figures (9)

Fig. 1.
Fig. 1. A schematic of the experimental setup. MO $_{1}$ = 60x 1.4NA Plan Apo oil immersion microscope objective (Nikon), L $_{1}$ = 400mm lens, L $_{2}$ = 250mm lens, BS = 50:50 beamsplitter, MO $_{2}$ = 60x 0.7NA air Plan Fluor microscope objective with a correction collar (Nikon), M $_{2}$ = moving mirror, PM = polychroic mirror reflective at 405,488,561 and 635 nm (Laser 2000), QBF = quad band 405-488-561-640 nm emission filter (Chroma Inc.)
Fig. 2.
Fig. 2. A schematic of the mechanism used to move the refocusing mirror rendered in Autodesk Inventor. The plane adjustment mechanism is highlighted in yellow and red.
Fig. 3.
Fig. 3. (A) Representative TTL trace from optical switches on the oscilloscope. The camera and corresponding laser triggers off the rising edge of each pulse. The red and blue traces indicate different optical switch outputs (B) Relationship between imaging frame rate (Hz) and voltage across the motor (V) at different $z$ separations. Increasing voltages lead to a linear increase in acquisition frame rate. Increasing the voltage any higher than approximately 2V resulted in the camera being unable to acquire the images rapidly enough.
Fig. 4.
Fig. 4. A femtosecond laser-etched [11,12] calibration sample was used to calibrate lateral pixel size and axial plane separation. (A) A diagram of the volume used for x-y-z calibration. The femtosecond-etched features are spaced in a 10  $\mu$ m regularly-spaced grid and start at successive depths of 5  $\mu$ m into the sample. (B) Example images were taken every 5  $\mu$ m over a 15  $\mu$ m axial range in transmission and fluorescence for the features to demonstrate this. (C) Confocal images of the slides taken using a separate apparatus using 488 nm light (D) Images taken with our setup using 488 nm light, compared here to their transmission appearance 15  $\mu$ m apart.
Fig. 5.
Fig. 5. Rat carotid arteries were dissected, opened longitudinally using microscissors and the artery pinned out on a Sylguard block with the endothelium exposed. Arteries were then stained with Cal-520 (5 uM) with 0.02% pluronic F-127 for 30 mins at 37°C. After washing, preparations were placed face down on a 0 grade thickness coverslip fixed to the bottom of a custom bath chamber (3cm x 1.5cm) on 200  $\mu$ m diameter stainless steel pins to stop the preparation touching the coverslip. Preparations were kept in PSS at all times.
Fig. 6.
Fig. 6. (a)0.5  $\mu$ m diameter fluorescent beads were imaged as single planes for the lateral resolution (top panel), and at $\Delta \,z$ $=$ 1  $\mu$ m intervals for the axial resolution (bottom panel). Images were deconvolved with a theoretical PSF, then a line profile was taken across their diameter using Fiji and plotted (light blue) for each bead analysed. The mean was plotted (dark blue) and the FWHM (width of the red area) calculated. Scale bar is 5  $\mu$ m.
Fig. 7.
Fig. 7. The calibration slide in fluorescence mode was imaged every 5  $\mu$ m and the mean apparent spot separation (MASS) calculated for a vertical (red) and horizontal (blue) spot pair (solid line). The mean value was also plotted (dashed line), and example images of the calibration slide shown from 0, 20, 45, 70, 95  $\mu$ m depths.
Fig. 8.
Fig. 8. Representative images of a sample of 6 features from the calibration slide imaged at $\Delta \,z$ $=$ 15  $\mu$ m. A custom-written python script tracked and plotted the trajectories of the dots at $z_{1}$ and $z_{2}$ over time. Images were acquired at 10fps or 20fps framerate, for 50 frames (short term) or 5,000 frames (long term). Bar charts represent the mean displacement of each dot (in pixels) calculated from all trajectories, with error bars showing the standard error on the mean.
Fig. 9.
Fig. 9. Arterial smooth muscle and endothelial cell layers were imaged sequentially using the epifluorescence remote refocussing system. (A) Fixed carotid arteries stained with PI (1.5  $\mu$ M) and SytoGreen (5  $\mu$ M) were imaged on the remote refocussing system at 10fps per plane with an axial separation of 15  $\mu$ m. VSMCs are shown stained with PI in red, and EC stained with SytoGreen in green. (B) A live carotid artery stained with Cal-520 (5  $\mu$ M), imaged at 10 fps with an axial separation of 15  $\mu$ m. (i) The 488 nm and 561 nm laser lines were locked on to the TTL pulse, with the 488 nm laser used to acquire images from ECs ( $z_1$ ) and the 561 nm laser to acquire autofluorescence from the unstained VSMC layer ( $z_2$ ). Brightfield illumination was turned on at the end of the recording session to verify that both imaging planes were inside the arterial tissue. (ii) Ca2+ signals from Cal-520 in ECs were stimulated with ACh (0.5  $\mu$ M) at 5.5 seconds. Individual Ca2+ intensity traces (full lines, colour matched) were plotted as $F/F_0$ , with corresponding VSMC autofluorescence from the same ROIs (dashed lines). (C) A live carotid artery stained with Cal-520 (5  $\mu$ M) and TMRE (120 nM), imaged at 10 fps per plane with an axial separation of 15  $\mu$ m. (i) The 488 nm and 561 nm laser lines were locked on to the TTL pulse, with the 488 nm laser used to acquire images from Cal-520-stained EC Ca2+ signals ( $z_1$ ) and the 561 nm laser to acquire images from the TMRE-stained VSMC mitochondria ( $z_2$ ). (ii) Ca2+ signals from Cal-520 in ECs were stimulated with ACh (0.5  $\mu$ M) at 5.5 seconds. Individual Ca2+ intensity traces (full lines, colour matched) were plotted as $F/F_0$ , with corresponding VSMC mitochondria signal from the same ROIs (dashed lines). Scale bars $=$ 20  $\mu$ m.

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