Abstract

A new optical-fluorescence microscopy technique, called HR-OPFOS, is discussed and situated among similar OPFOS-implementations. OPFOS stands for orthogonal-plane fluorescence optical sectioning and thus is categorized as a laser light sheet based fluorescence microscopy method. HR-OPFOS is used to make tomographic recordings of macroscopic biomedical specimens in high resolution. It delivers cross sections through the object under study with semi-histological detail, which can be used to create three-dimensional computer models for finite-element modeling or anatomical studies. The general innovation of this class of microscopy setup consists of the separation of the illumination and observation axes, but now in our setup combined with focal line scanning to improve sectioning resolution. HR-OPFOS is demonstrated on gerbil hearing organs and on mouse and bird brains. The necessary specimen preparation is discussed.

© 2009 Optical Society of America

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    [CrossRef]
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    [CrossRef] [PubMed]
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  6. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932-940 (2005).
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    [CrossRef] [PubMed]
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  9. W. Valk, H. Wit, J. Segenhout, F. Dijk, J. van der Want, and F. Albers, “Morphology of the endolymphatic sac in the guinea pig after an acute endolymphatic hydrops,” Hear. Res. 202, 180-187 (2005).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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  21. S. Lindek and E. H. Stelzer, “Confocal theta microscopy and 4Pi-confocal theta microscopy,” Proc. SPIE 2184, 188-194(1994).
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  23. J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett. 32, 2608-2610 (2007).
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  24. P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4, 311-313 (2007).
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  25. S. Tinling, R. Giberson, and R. Kullar, “Microwave exposure increases bone demineralization rate independent of temperature,” J. Microsc. 215, 230-235 (2004).
    [CrossRef] [PubMed]

2008

R. Hofman, J. Segenhout, J. A. N. Buytaert, J. J. J. Dirckx, and H. Wit, “Morphology and function of Bast's valve; Additional insight in its functioning using 3D-reconstruction,” Eur. Arch. Oto-Rhino-L. 265, 153-157 (2008).
[CrossRef]

J. A. N. Buytaert and J. J. J. Dirckx, “High-resolution virtual optical-sectioning imaging and tomography for 3-D modeling of biomedical specimens,” in Biomedical Optics Topical Meeting, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BWG5.

C. Davis, “Optical imaging of ocean plankton: a fantastic voyage,” in Digital Holography and Three-Dimensional Imaging, OSA Technical Digest (CD) (Optical Society of America, 2008), paper DMB1.

J. G. Ritter, R. Veith, J.-P. Siebrasse, and U. Kubitscheck, “High-contrast single-particle tracking by selective focal plane illumination microscopy,” Opt. Express 16, 7142-7152 (2008).
[CrossRef] [PubMed]

2007

H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. Mauch, K. Deininger, J. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning 3-D imaging technique for biomedical specimens, featuring 2 ?m slicing resolution,” J Biomed. Opt. 12, 014039 (2007).
[CrossRef] [PubMed]

J. A. N. Buytaert and J. J. J. Dirckx, “High-resolution 3-D imaging of middle ear ossicles and soft tissue structures of intact gerbil temporal bones using orthogonal-plane fluorescence optical sectioning,” in Middle Ear Mechanics in Research and Otology, A. Eiber and A. Huber, eds. (World Scientific, 2007), pp. 282-288.
[CrossRef]

B. Masschaele, V. Cnudde, M. Dierick, P. Jacobs, L. V. Hoorebeke, and J. Vlassenbroeck, “UGCT: new x-ray radiography and tomography facility,” Nucl. Instrum. Meth. A 580, 266-269 (2007).
[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]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4, 311-313 (2007).
[PubMed]

2005

W. Valk, H. Wit, J. Segenhout, F. Dijk, J. van der Want, and F. Albers, “Morphology of the endolymphatic sac in the guinea pig after an acute endolymphatic hydrops,” Hear. Res. 202, 180-187 (2005).
[CrossRef] [PubMed]

J. M. Tyszka, S. E. Fraser, and R. E. Jacobs, “Magnetic resonance microscopy: recent advantages and applications,” Curr. Opin. Biotechnol. 16, 93-99 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

S. L. R. Gea, W. F. Decraemer, and J. J. J. Dirckx, “Region of interest micro-CT of the middle ear: A practical approach,” J. X-Ray Sci. Technol. 13, 137-147 (2005).

2004

J. Swoger, J. Bene, F. D. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305, 1007-1009 (2004).
[CrossRef] [PubMed]

S. Tinling, R. Giberson, and R. Kullar, “Microwave exposure increases bone demineralization rate independent of temperature,” J. Microsc. 215, 230-235 (2004).
[CrossRef] [PubMed]

2003

2002

A. Voie, “Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy,” Hear. Res. 171, 119-128 (2002).
[CrossRef] [PubMed]

E. Fuchs, J. Jaffe, R. Long, and F. Azam, “Thin laser light sheet microscope for microbial oceanography,” Opt. Express 10, 145-154 (2002).
[PubMed]

1998

W. J. Weninger, S. Meng, J. Streicher, and G. B. Müller, “A new episcopic method for rapid 3-D reconstruction: applications in anatomy and embryology,” Anat. Embryol. 197, 341-348 (1998).
[CrossRef] [PubMed]

1994

M. M. Henson, O. W. Henson, S. L. Gewalt, J. L. Wilson, and G. A. Johnson, “Imaging the cochlea by magnetic resonance microscopy,” Hear. Res. 75, 75-80 (1994).
[CrossRef] [PubMed]

S. Lindek and E. H. Stelzer, “Confocal theta microscopy and 4Pi-confocal theta microscopy,” Proc. SPIE 2184, 188-194(1994).
[CrossRef]

1993

A. Voie, D. Burns, and F. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170, 229-236 (1993).
[CrossRef] [PubMed]

1911

W. Spalteholz, Uber das Durchsichtigmachen van menschlichen und tierischen Präparaten (Verlag S. Hirzel, 1911).

1903

H. Siedentopf and R. Zsigmondy, “Uber die Sichtbarmachung und Grössenbestimmung ultramikroskopischer Teilchen,” Ann. Phys. 4, (1903).

Albers, F.

W. Valk, H. Wit, J. Segenhout, F. Dijk, J. van der Want, and F. Albers, “Morphology of the endolymphatic sac in the guinea pig after an acute endolymphatic hydrops,” Hear. Res. 202, 180-187 (2005).
[CrossRef] [PubMed]

Azam, F.

Becker, K.

H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. Mauch, K. Deininger, J. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Bene, J.

J. Swoger, J. Bene, F. D. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305, 1007-1009 (2004).
[CrossRef] [PubMed]

Burns, D.

A. Voie, D. Burns, and F. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170, 229-236 (1993).
[CrossRef] [PubMed]

Buytaert, J. A. N.

R. Hofman, J. Segenhout, J. A. N. Buytaert, J. J. J. Dirckx, and H. Wit, “Morphology and function of Bast's valve; Additional insight in its functioning using 3D-reconstruction,” Eur. Arch. Oto-Rhino-L. 265, 153-157 (2008).
[CrossRef]

J. A. N. Buytaert and J. J. J. Dirckx, “High-resolution virtual optical-sectioning imaging and tomography for 3-D modeling of biomedical specimens,” in Biomedical Optics Topical Meeting, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BWG5.

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning 3-D imaging technique for biomedical specimens, featuring 2 ?m slicing resolution,” J Biomed. Opt. 12, 014039 (2007).
[CrossRef] [PubMed]

J. A. N. Buytaert and J. J. J. Dirckx, “High-resolution 3-D imaging of middle ear ossicles and soft tissue structures of intact gerbil temporal bones using orthogonal-plane fluorescence optical sectioning,” in Middle Ear Mechanics in Research and Otology, A. Eiber and A. Huber, eds. (World Scientific, 2007), pp. 282-288.
[CrossRef]

Cnudde, V.

B. Masschaele, V. Cnudde, M. Dierick, P. Jacobs, L. V. Hoorebeke, and J. Vlassenbroeck, “UGCT: new x-ray radiography and tomography facility,” Nucl. Instrum. Meth. A 580, 266-269 (2007).
[CrossRef]

Davis, C.

C. Davis, “Optical imaging of ocean plankton: a fantastic voyage,” in Digital Holography and Three-Dimensional Imaging, OSA Technical Digest (CD) (Optical Society of America, 2008), paper DMB1.

Decraemer, W. F.

S. L. R. Gea, W. F. Decraemer, and J. J. J. Dirckx, “Region of interest micro-CT of the middle ear: A practical approach,” J. X-Ray Sci. Technol. 13, 137-147 (2005).

Deininger, K.

H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. Mauch, K. Deininger, J. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Denk, W.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

Deussing, J.

H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. Mauch, K. Deininger, J. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Dierick, M.

B. Masschaele, V. Cnudde, M. Dierick, P. Jacobs, L. V. Hoorebeke, and J. Vlassenbroeck, “UGCT: new x-ray radiography and tomography facility,” Nucl. Instrum. Meth. A 580, 266-269 (2007).
[CrossRef]

Dijk, F.

W. Valk, H. Wit, J. Segenhout, F. Dijk, J. van der Want, and F. Albers, “Morphology of the endolymphatic sac in the guinea pig after an acute endolymphatic hydrops,” Hear. Res. 202, 180-187 (2005).
[CrossRef] [PubMed]

Dirckx, J. J. J.

R. Hofman, J. Segenhout, J. A. N. Buytaert, J. J. J. Dirckx, and H. Wit, “Morphology and function of Bast's valve; Additional insight in its functioning using 3D-reconstruction,” Eur. Arch. Oto-Rhino-L. 265, 153-157 (2008).
[CrossRef]

J. A. N. Buytaert and J. J. J. Dirckx, “High-resolution virtual optical-sectioning imaging and tomography for 3-D modeling of biomedical specimens,” in Biomedical Optics Topical Meeting, OSA Technical Digest (CD) (Optical Society of America, 2008), paper BWG5.

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning 3-D imaging technique for biomedical specimens, featuring 2 ?m slicing resolution,” J Biomed. Opt. 12, 014039 (2007).
[CrossRef] [PubMed]

J. A. N. Buytaert and J. J. J. Dirckx, “High-resolution 3-D imaging of middle ear ossicles and soft tissue structures of intact gerbil temporal bones using orthogonal-plane fluorescence optical sectioning,” in Middle Ear Mechanics in Research and Otology, A. Eiber and A. Huber, eds. (World Scientific, 2007), pp. 282-288.
[CrossRef]

S. L. R. Gea, W. F. Decraemer, and J. J. J. Dirckx, “Region of interest micro-CT of the middle ear: A practical approach,” J. X-Ray Sci. Technol. 13, 137-147 (2005).

Dodt, H.

H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. Mauch, K. Deininger, J. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Eder, M.

H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. Mauch, K. Deininger, J. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Fraser, S. E.

J. M. Tyszka, S. E. Fraser, and R. E. Jacobs, “Magnetic resonance microscopy: recent advantages and applications,” Curr. Opin. Biotechnol. 16, 93-99 (2005).
[CrossRef] [PubMed]

Fuchs, E.

Gea, S. L. R.

S. L. R. Gea, W. F. Decraemer, and J. J. J. Dirckx, “Region of interest micro-CT of the middle ear: A practical approach,” J. X-Ray Sci. Technol. 13, 137-147 (2005).

Gewalt, S. L.

M. M. Henson, O. W. Henson, S. L. Gewalt, J. L. Wilson, and G. A. Johnson, “Imaging the cochlea by magnetic resonance microscopy,” Hear. Res. 75, 75-80 (1994).
[CrossRef] [PubMed]

Giberson, R.

S. Tinling, R. Giberson, and R. Kullar, “Microwave exposure increases bone demineralization rate independent of temperature,” J. Microsc. 215, 230-235 (2004).
[CrossRef] [PubMed]

Greger, K.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4, 311-313 (2007).
[PubMed]

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

Henson, M. M.

M. M. Henson, O. W. Henson, S. L. Gewalt, J. L. Wilson, and G. A. Johnson, “Imaging the cochlea by magnetic resonance microscopy,” Hear. Res. 75, 75-80 (1994).
[CrossRef] [PubMed]

Henson, O. W.

M. M. Henson, O. W. Henson, S. L. Gewalt, J. L. Wilson, and G. A. Johnson, “Imaging the cochlea by magnetic resonance microscopy,” Hear. Res. 75, 75-80 (1994).
[CrossRef] [PubMed]

Hofman, R.

R. Hofman, J. Segenhout, J. A. N. Buytaert, J. J. J. Dirckx, and H. Wit, “Morphology and function of Bast's valve; Additional insight in its functioning using 3D-reconstruction,” Eur. Arch. Oto-Rhino-L. 265, 153-157 (2008).
[CrossRef]

Hoorebeke, L. V.

B. Masschaele, V. Cnudde, M. Dierick, P. Jacobs, L. V. Hoorebeke, and J. Vlassenbroeck, “UGCT: new x-ray radiography and tomography facility,” Nucl. Instrum. Meth. A 580, 266-269 (2007).
[CrossRef]

Huisken, J.

Jacobs, P.

B. Masschaele, V. Cnudde, M. Dierick, P. Jacobs, L. V. Hoorebeke, and J. Vlassenbroeck, “UGCT: new x-ray radiography and tomography facility,” Nucl. Instrum. Meth. A 580, 266-269 (2007).
[CrossRef]

Jacobs, R. E.

J. M. Tyszka, S. E. Fraser, and R. E. Jacobs, “Magnetic resonance microscopy: recent advantages and applications,” Curr. Opin. Biotechnol. 16, 93-99 (2005).
[CrossRef] [PubMed]

Jaffe, J.

Jährling, N.

H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. Mauch, K. Deininger, J. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Johnson, G. A.

M. M. Henson, O. W. Henson, S. L. Gewalt, J. L. Wilson, and G. A. Johnson, “Imaging the cochlea by magnetic resonance microscopy,” Hear. Res. 75, 75-80 (1994).
[CrossRef] [PubMed]

Kubitscheck, U.

Kullar, R.

S. Tinling, R. Giberson, and R. Kullar, “Microwave exposure increases bone demineralization rate independent of temperature,” J. Microsc. 215, 230-235 (2004).
[CrossRef] [PubMed]

Leischner, U.

H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. Mauch, K. Deininger, J. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Lindek, S.

S. Lindek and E. H. Stelzer, “Confocal theta microscopy and 4Pi-confocal theta microscopy,” Proc. SPIE 2184, 188-194(1994).
[CrossRef]

Long, R.

Marcello, M.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4, 311-313 (2007).
[PubMed]

Masschaele, B.

B. Masschaele, V. Cnudde, M. Dierick, P. Jacobs, L. V. Hoorebeke, and J. Vlassenbroeck, “UGCT: new x-ray radiography and tomography facility,” Nucl. Instrum. Meth. A 580, 266-269 (2007).
[CrossRef]

Mauch, C.

H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. Mauch, K. Deininger, J. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Meng, S.

W. J. Weninger, S. Meng, J. Streicher, and G. B. Müller, “A new episcopic method for rapid 3-D reconstruction: applications in anatomy and embryology,” Anat. Embryol. 197, 341-348 (1998).
[CrossRef] [PubMed]

Müller, G. B.

W. J. Weninger, S. Meng, J. Streicher, and G. B. Müller, “A new episcopic method for rapid 3-D reconstruction: applications in anatomy and embryology,” Anat. Embryol. 197, 341-348 (1998).
[CrossRef] [PubMed]

Pampaloni, F.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4, 311-313 (2007).
[PubMed]

Ritter, J. G.

Schierloh, A.

H. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. Mauch, K. Deininger, J. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Segenhout, J.

R. Hofman, J. Segenhout, J. A. N. Buytaert, J. J. J. Dirckx, and H. Wit, “Morphology and function of Bast's valve; Additional insight in its functioning using 3D-reconstruction,” Eur. Arch. Oto-Rhino-L. 265, 153-157 (2008).
[CrossRef]

W. Valk, H. Wit, J. Segenhout, F. Dijk, J. van der Want, and F. Albers, “Morphology of the endolymphatic sac in the guinea pig after an acute endolymphatic hydrops,” Hear. Res. 202, 180-187 (2005).
[CrossRef] [PubMed]

Siebrasse, J.-P.

Siedentopf, H.

H. Siedentopf and R. Zsigmondy, “Uber die Sichtbarmachung und Grössenbestimmung ultramikroskopischer Teilchen,” Ann. Phys. 4, (1903).

Spalteholz, W.

W. Spalteholz, Uber das Durchsichtigmachen van menschlichen und tierischen Präparaten (Verlag S. Hirzel, 1911).

Spelman, F.

A. Voie, D. Burns, and F. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170, 229-236 (1993).
[CrossRef] [PubMed]

Stainier, D. Y. R.

Stelzer, E. H.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4, 311-313 (2007).
[PubMed]

J. Swoger, J. Bene, F. D. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305, 1007-1009 (2004).
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Eur. Arch. Oto-Rhino-L.

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[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4, 311-313 (2007).
[PubMed]

Nucl. Instrum. Meth. A

B. Masschaele, V. Cnudde, M. Dierick, P. Jacobs, L. V. Hoorebeke, and J. Vlassenbroeck, “UGCT: new x-ray radiography and tomography facility,” Nucl. Instrum. Meth. A 580, 266-269 (2007).
[CrossRef]

Opt. Express

Opt. Lett.

Proc. SPIE

S. Lindek and E. H. Stelzer, “Confocal theta microscopy and 4Pi-confocal theta microscopy,” Proc. SPIE 2184, 188-194(1994).
[CrossRef]

Science

J. Swoger, J. Bene, F. D. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305, 1007-1009 (2004).
[CrossRef] [PubMed]

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[CrossRef]

Supplementary Material (5)

» Media 1: MPG (1598 KB)     
» Media 2: MPG (3900 KB)     
» Media 3: MPG (3629 KB)     
» Media 4: MPG (2718 KB)     
» Media 5: MPG (2980 KB)     

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Figures (7)

Fig. 1
Fig. 1

Schematic drawing of the hyperbolic focus of a cylindrical lens. w 0 is the (minimal) beam waist, w ( z ) is the half beam width at z, and b is the confocal parameter.

Fig. 2
Fig. 2

(a) OPFOS: the hyperbolic focus is approximated to a planar sheet in the confocal parameter range b, with 1 / e 2 thickness d 1 in the center and 2 d 1 near the edges of the sheet. A 2-D image is taken orthogonally in the X Y plane. (b) HR-OPFOS: a larger lens aperture can be used to create a minimal focal line thickness d 2 , as no 2-D sheet is needed. Line scanning of the object along X is performed to obtain a 2-D cross section.

Fig. 3
Fig. 3

Schematic drawing of the (HR-) OPFOS instrument. Using a dichroic mirror (DM), blue laser (BL) or green laser (GL) light is broadened by a beam expander (BE). A double field stop (FS) limits the amount of light along Y and determines the slit aperture of the cylindrical lens (CL). The CL focuses the light hyperbolically through a window in the specimen container into the object (O) under study. At the back of the container a black wall is placed, absorbing all laser light. A second window in the specimen chamber, filled with fluid, allows the CCD with objective lens (OL) to record a cross section. The focus translation stage (FTS) has to be calibrated only once. The object is scanned along X and at different Z depths by a two-axis motorized translation stage (OTS).

Fig. 4
Fig. 4

(a) Cross section of a right gerbil cochlea obtained (every 30 μm in Media 1) with HR-OPFOS of 1.92 mm × 2.55 mm ( 1280 × 1700 pixels). Every image is scanned in 1700 steps of 1.5 μm , with lateral and axial resolution of 2 μm . 1, scala tympani; 2, basilar membrane; 3, scala media; 4, Reissner's membrane; 5, scala vestibuli; 6, modiolus; 7, blood vessels. See for more cross sections. (b) Cross section of a right gerbil stapes obtained with HR-OPFOS of 0.96 mm × 1.88 mm ( 1280 × 2500 pixels) with lateral resolution of 1 μm and axial resolution of 2 μm . 1, duct within incus; 2, incus; 3, lenticular process; 4, articulation; 5, calcified cartilage within stapes head; 6, stapedial artery; 7, stapes footplate; 8, annular ligament footplate.

Fig. 5
Fig. 5

After recording 435 scanned cross sections of 1280 × 1280 pixels with HR-OPFOS in the X Y plane, 2 μm apart, computer software can resection this 3-D data stack to X Z or Y Z with the same image quality. See Media 2 for more cross sections in X Y . In Media 2 an entire stack of 385 HR-OPFOS cross sections ( 1.92 mm × 2.55 mm ) each 2.5 µm apart is shown with 13   sections / s , which amounts to a depth progress of 32.5 µm / s . Readers familiar with the anatomy may see the stapedial muscle attaching to the stapes head, and the stapedial artery running through the crurae.

Fig. 6
Fig. 6

(a) From a 3-D (HR-) OPFOS data stack we can create 3-D reconstructions. The model shown here originates from the data stack in Fig. 5, showing a part of the incus attaching to the stapes, the stapedial artery running through the stapes “legs” and the stapedial muscle connecting at the stapes head (voxels 1.5 μm × 2 μm × 2 μm ). The cube in the image has dimensions 150 μm . See Media 3 for a 3-D impression of the model. (b) This 3-D model shows a reconstruction of a complete ossicle chain, comprising bone structures (malleus, incus, and stapes) and soft tissue structures (annular ligament around the stapes and the stapedial muscle) (voxels 4 μm × 4 μm × 4 μm ).

Fig. 7
Fig. 7

Three OPFOS cross sections ( 4.8 mm × 3.6 mm ) at different depths through a mouse brain are shown. The natural autofluorescence of the specimen clearly allows one to distinguish histological detail. See Media 4 for more cross sections in X Y . The movie shows 24   sections / s , which amounts to a depth progress of 240 µm / s .

Equations (6)

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I ( z ) = I 0 · sinc 2 ( z π D lens λ f ) = I 0 · ( sin ( z π D lens λ f ) z π D lens λ f ) 2 ,
w 0 = 1.4 f λ / D lens ,
d 0 = 2 w 0 ,
d ( x ) = 2 w ( x ) = 2 w 0 1 + ( x x R ) 2 ,
x R = π w 0 2 λ .
b n = 2 n x R = n b .

Metrics