Abstract

Wide-field single molecule microscopy is a versatile tool for analyzing dynamics and molecular interactions in biological systems. In extended three-dimensional systems, however, the method suffers from intrinsic out-of-focus fluorescence. We constructed a high-resolution selective plane illumination microscope (SPIM) to efficiently solve this problem. The instrument is an optical sectioning microscope featuring the high speed and high sensitivity of a video microscope. We present theoretical calculations and quantitative measurements of the illumination light sheet thickness yielding 1.7 µm (FWHM) at 543 nm, 2.0 µm at 633 nm, and a FWHM of the axial point spread function of 1.13 µm. A direct comparison of selective plane and epi-illumination of model samples with intrinsic background fluorescence illustrated the clear advantage of SPIM for such samples. Single fluorescent quantum dots in aqueous solution are readily visualized and tracked proving the suitability of our setup for the study of fast and dynamic processes in spatially extended biological specimens.

© 2008 Optical Society of America

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References

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  1. J. P. Siebrasse, D. Grunwald, and U. Kubitscheck, "Single-molecule tracking in eukaryotic cell nuclei," Anal. Bioanal. Chem. (2006).
    [CrossRef] [PubMed]
  2. H. Siedentopf and R. Zsigmondy, "�?ber Sichtbarmachung und Grössenbestimmung ultramikroskopischer Teilchen," Annalen der Physik 4, 1-39 (1903).
  3. A. H. Voie, D. H. Burns, and F. A. Spelman, "Orthogonal-Plane Fluorescence Optical Sectioning - 3-Dimensional Imaging of Macroscopic Biological Specimens," J. Microsc. 170, 229-236 (1993).
    [CrossRef] [PubMed]
  4. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, "Optical sectioning deep inside live embryos by selective plane illumination microscopy," Science 305, 1007-1009 (2004).
    [CrossRef] [PubMed]
  5. J. Huisken and D. Y. Stainier, "Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM)," Opt. Lett. 32, 2608-2610 (2007).
    [CrossRef] [PubMed]
  6. J. A. Buytaert and J. J. Dirckx, "Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution," J Biomed. Opt. 12, 014039 (2007).
    [CrossRef] [PubMed]
  7. H. U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, "Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain," Nat. Methods 4, 331-336 (2007).
    [CrossRef] [PubMed]
  8. W. Alt, "An objective lens for efficient fluorescence detection of single atoms," Optik 113, 142-144 (2002).
    [CrossRef]
  9. B. E. A. Saleh and M. C. Teich, Fundamentals of Photonics (Wiley, 2007).
  10. C. J. Engelbrecht and E. H. Stelzer, "Resolution enhancement in a light-sheet-based microscope (SPIM)," Opt. Lett. 31, 1477-1479 (2006).
    [CrossRef] [PubMed]
  11. J. H. E. Peter W. Millonni, Lasers (Wiley & Sons, 1989).
  12. M. Born and E. Wolf, Principles of Optics (Cambridge University Press, Cambridge, 1997).
  13. D. Wright, P. Greve, J. Fleischer, and L. Austin, "Laser beam width, divergence and beam propagation factor - an international standardization approach," Opt. Quantum Electron. 24, S993-S1000 (1992).
    [CrossRef]
  14. T. Soop, B. Ivarsson, B. Bjorkroth, N. Fomproix, S. Masich, V. C. Cordes, and B. Daneholt, "Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export," Mol. Biol. Cell 16, 5610-5620 (2005).
    [CrossRef] [PubMed]
  15. M. Abramowitz, P. J. Magelhaes, and S. J. Ram, "Image Processing with ImageJ," Biophot. Int. 11, 36-42 (2004).
  16. T. Kues, A. Dickmanns, R. Lührmann, R. Peters, and U. Kubitscheck, "High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking," Proc. Natl. Acad. Sci. U S A 98, 12021-12026 (2001).
    [CrossRef] [PubMed]
  17. D. Grünwald, A. Hoekstra, T. Dange, V. Buschmann, and U. Kubitscheck, "Direct observation of single protein molecules in aqueous solution," ChemPhysChem 7, 812-815 (2006).
    [CrossRef] [PubMed]

2007

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

J. A. Buytaert and J. J. Dirckx, "Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution," J Biomed. Opt. 12, 014039 (2007).
[CrossRef] [PubMed]

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

2006

C. J. Engelbrecht and E. H. Stelzer, "Resolution enhancement in a light-sheet-based microscope (SPIM)," Opt. Lett. 31, 1477-1479 (2006).
[CrossRef] [PubMed]

D. Grünwald, A. Hoekstra, T. Dange, V. Buschmann, and U. Kubitscheck, "Direct observation of single protein molecules in aqueous solution," ChemPhysChem 7, 812-815 (2006).
[CrossRef] [PubMed]

2005

T. Soop, B. Ivarsson, B. Bjorkroth, N. Fomproix, S. Masich, V. C. Cordes, and B. Daneholt, "Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export," Mol. Biol. Cell 16, 5610-5620 (2005).
[CrossRef] [PubMed]

2004

M. Abramowitz, P. J. Magelhaes, and S. J. Ram, "Image Processing with ImageJ," Biophot. Int. 11, 36-42 (2004).

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

2002

W. Alt, "An objective lens for efficient fluorescence detection of single atoms," Optik 113, 142-144 (2002).
[CrossRef]

2001

T. Kues, A. Dickmanns, R. Lührmann, R. Peters, and U. Kubitscheck, "High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking," Proc. Natl. Acad. Sci. U S A 98, 12021-12026 (2001).
[CrossRef] [PubMed]

1993

A. H. Voie, D. H. Burns, and F. A. Spelman, "Orthogonal-Plane Fluorescence Optical Sectioning - 3-Dimensional Imaging of Macroscopic Biological Specimens," J. Microsc. 170, 229-236 (1993).
[CrossRef] [PubMed]

1992

D. Wright, P. Greve, J. Fleischer, and L. Austin, "Laser beam width, divergence and beam propagation factor - an international standardization approach," Opt. Quantum Electron. 24, S993-S1000 (1992).
[CrossRef]

1903

H. Siedentopf and R. Zsigmondy, "�?ber Sichtbarmachung und Grössenbestimmung ultramikroskopischer Teilchen," Annalen der Physik 4, 1-39 (1903).

Abramowitz, M.

M. Abramowitz, P. J. Magelhaes, and S. J. Ram, "Image Processing with ImageJ," Biophot. Int. 11, 36-42 (2004).

Alt, W.

W. Alt, "An objective lens for efficient fluorescence detection of single atoms," Optik 113, 142-144 (2002).
[CrossRef]

Austin, L.

D. Wright, P. Greve, J. Fleischer, and L. Austin, "Laser beam width, divergence and beam propagation factor - an international standardization approach," Opt. Quantum Electron. 24, S993-S1000 (1992).
[CrossRef]

Becker, K.

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

Bjorkroth, B.

T. Soop, B. Ivarsson, B. Bjorkroth, N. Fomproix, S. Masich, V. C. Cordes, and B. Daneholt, "Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export," Mol. Biol. Cell 16, 5610-5620 (2005).
[CrossRef] [PubMed]

Burns, D. H.

A. H. Voie, D. H. Burns, and F. A. Spelman, "Orthogonal-Plane Fluorescence Optical Sectioning - 3-Dimensional Imaging of Macroscopic Biological Specimens," J. Microsc. 170, 229-236 (1993).
[CrossRef] [PubMed]

Buschmann, V.

D. Grünwald, A. Hoekstra, T. Dange, V. Buschmann, and U. Kubitscheck, "Direct observation of single protein molecules in aqueous solution," ChemPhysChem 7, 812-815 (2006).
[CrossRef] [PubMed]

Buytaert, J. A.

J. A. Buytaert and J. J. Dirckx, "Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution," J Biomed. Opt. 12, 014039 (2007).
[CrossRef] [PubMed]

Cordes, V. C.

T. Soop, B. Ivarsson, B. Bjorkroth, N. Fomproix, S. Masich, V. C. Cordes, and B. Daneholt, "Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export," Mol. Biol. Cell 16, 5610-5620 (2005).
[CrossRef] [PubMed]

Daneholt, B.

T. Soop, B. Ivarsson, B. Bjorkroth, N. Fomproix, S. Masich, V. C. Cordes, and B. Daneholt, "Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export," Mol. Biol. Cell 16, 5610-5620 (2005).
[CrossRef] [PubMed]

Dange, T.

D. Grünwald, A. Hoekstra, T. Dange, V. Buschmann, and U. Kubitscheck, "Direct observation of single protein molecules in aqueous solution," ChemPhysChem 7, 812-815 (2006).
[CrossRef] [PubMed]

Deininger, K.

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

Del Bene, F.

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

Deussing, J. M.

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

Dickmanns, A.

T. Kues, A. Dickmanns, R. Lührmann, R. Peters, and U. Kubitscheck, "High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking," Proc. Natl. Acad. Sci. U S A 98, 12021-12026 (2001).
[CrossRef] [PubMed]

Dirckx, J. J.

J. A. Buytaert and J. J. Dirckx, "Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution," J Biomed. Opt. 12, 014039 (2007).
[CrossRef] [PubMed]

Dodt, H. U.

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

Engelbrecht, C. J.

Fleischer, J.

D. Wright, P. Greve, J. Fleischer, and L. Austin, "Laser beam width, divergence and beam propagation factor - an international standardization approach," Opt. Quantum Electron. 24, S993-S1000 (1992).
[CrossRef]

Fomproix, N.

T. Soop, B. Ivarsson, B. Bjorkroth, N. Fomproix, S. Masich, V. C. Cordes, and B. Daneholt, "Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export," Mol. Biol. Cell 16, 5610-5620 (2005).
[CrossRef] [PubMed]

Greve, P.

D. Wright, P. Greve, J. Fleischer, and L. Austin, "Laser beam width, divergence and beam propagation factor - an international standardization approach," Opt. Quantum Electron. 24, S993-S1000 (1992).
[CrossRef]

Grünwald, D.

D. Grünwald, A. Hoekstra, T. Dange, V. Buschmann, and U. Kubitscheck, "Direct observation of single protein molecules in aqueous solution," ChemPhysChem 7, 812-815 (2006).
[CrossRef] [PubMed]

Hoekstra, A.

D. Grünwald, A. Hoekstra, T. Dange, V. Buschmann, and U. Kubitscheck, "Direct observation of single protein molecules in aqueous solution," ChemPhysChem 7, 812-815 (2006).
[CrossRef] [PubMed]

Huisken, J.

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

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

Ivarsson, B.

T. Soop, B. Ivarsson, B. Bjorkroth, N. Fomproix, S. Masich, V. C. Cordes, and B. Daneholt, "Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export," Mol. Biol. Cell 16, 5610-5620 (2005).
[CrossRef] [PubMed]

Jahrling, N.

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

Kubitscheck, U.

D. Grünwald, A. Hoekstra, T. Dange, V. Buschmann, and U. Kubitscheck, "Direct observation of single protein molecules in aqueous solution," ChemPhysChem 7, 812-815 (2006).
[CrossRef] [PubMed]

T. Kues, A. Dickmanns, R. Lührmann, R. Peters, and U. Kubitscheck, "High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking," Proc. Natl. Acad. Sci. U S A 98, 12021-12026 (2001).
[CrossRef] [PubMed]

Kues, T.

T. Kues, A. Dickmanns, R. Lührmann, R. Peters, and U. Kubitscheck, "High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking," Proc. Natl. Acad. Sci. U S A 98, 12021-12026 (2001).
[CrossRef] [PubMed]

Leischner, U.

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

Lührmann, R.

T. Kues, A. Dickmanns, R. Lührmann, R. Peters, and U. Kubitscheck, "High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking," Proc. Natl. Acad. Sci. U S A 98, 12021-12026 (2001).
[CrossRef] [PubMed]

Magelhaes, P. J.

M. Abramowitz, P. J. Magelhaes, and S. J. Ram, "Image Processing with ImageJ," Biophot. Int. 11, 36-42 (2004).

Masich, S.

T. Soop, B. Ivarsson, B. Bjorkroth, N. Fomproix, S. Masich, V. C. Cordes, and B. Daneholt, "Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export," Mol. Biol. Cell 16, 5610-5620 (2005).
[CrossRef] [PubMed]

Mauch, C. P.

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

Peters, R.

T. Kues, A. Dickmanns, R. Lührmann, R. Peters, and U. Kubitscheck, "High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking," Proc. Natl. Acad. Sci. U S A 98, 12021-12026 (2001).
[CrossRef] [PubMed]

Ram, S. J.

M. Abramowitz, P. J. Magelhaes, and S. J. Ram, "Image Processing with ImageJ," Biophot. Int. 11, 36-42 (2004).

Schierloh, A.

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

Siedentopf, H.

H. Siedentopf and R. Zsigmondy, "�?ber Sichtbarmachung und Grössenbestimmung ultramikroskopischer Teilchen," Annalen der Physik 4, 1-39 (1903).

Soop, T.

T. Soop, B. Ivarsson, B. Bjorkroth, N. Fomproix, S. Masich, V. C. Cordes, and B. Daneholt, "Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export," Mol. Biol. Cell 16, 5610-5620 (2005).
[CrossRef] [PubMed]

Spelman, F. A.

A. H. Voie, D. H. Burns, and F. A. Spelman, "Orthogonal-Plane Fluorescence Optical Sectioning - 3-Dimensional Imaging of Macroscopic Biological Specimens," J. Microsc. 170, 229-236 (1993).
[CrossRef] [PubMed]

Stainier, D. Y.

Stelzer, E. H.

C. J. Engelbrecht and E. H. Stelzer, "Resolution enhancement in a light-sheet-based microscope (SPIM)," Opt. Lett. 31, 1477-1479 (2006).
[CrossRef] [PubMed]

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

Swoger, J.

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

Voie, A. H.

A. H. Voie, D. H. Burns, and F. A. Spelman, "Orthogonal-Plane Fluorescence Optical Sectioning - 3-Dimensional Imaging of Macroscopic Biological Specimens," J. Microsc. 170, 229-236 (1993).
[CrossRef] [PubMed]

Wittbrodt, J.

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

Wright, D.

D. Wright, P. Greve, J. Fleischer, and L. Austin, "Laser beam width, divergence and beam propagation factor - an international standardization approach," Opt. Quantum Electron. 24, S993-S1000 (1992).
[CrossRef]

Zieglgansberger, W.

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

Zsigmondy, R.

H. Siedentopf and R. Zsigmondy, "�?ber Sichtbarmachung und Grössenbestimmung ultramikroskopischer Teilchen," Annalen der Physik 4, 1-39 (1903).

Annalen der Physik

H. Siedentopf and R. Zsigmondy, "�?ber Sichtbarmachung und Grössenbestimmung ultramikroskopischer Teilchen," Annalen der Physik 4, 1-39 (1903).

Biophot. Int.

M. Abramowitz, P. J. Magelhaes, and S. J. Ram, "Image Processing with ImageJ," Biophot. Int. 11, 36-42 (2004).

ChemPhysChem

D. Grünwald, A. Hoekstra, T. Dange, V. Buschmann, and U. Kubitscheck, "Direct observation of single protein molecules in aqueous solution," ChemPhysChem 7, 812-815 (2006).
[CrossRef] [PubMed]

J Biomed. Opt.

J. A. Buytaert and J. J. Dirckx, "Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution," J Biomed. Opt. 12, 014039 (2007).
[CrossRef] [PubMed]

J. Microsc.

A. H. Voie, D. H. Burns, and F. A. Spelman, "Orthogonal-Plane Fluorescence Optical Sectioning - 3-Dimensional Imaging of Macroscopic Biological Specimens," J. Microsc. 170, 229-236 (1993).
[CrossRef] [PubMed]

Mol. Biol. Cell

T. Soop, B. Ivarsson, B. Bjorkroth, N. Fomproix, S. Masich, V. C. Cordes, and B. Daneholt, "Nup153 affects entry of messenger and ribosomal ribonucleoproteins into the nuclear basket during export," Mol. Biol. Cell 16, 5610-5620 (2005).
[CrossRef] [PubMed]

Nat. Methods

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

Opt. Lett.

Opt. Quantum Electron.

D. Wright, P. Greve, J. Fleischer, and L. Austin, "Laser beam width, divergence and beam propagation factor - an international standardization approach," Opt. Quantum Electron. 24, S993-S1000 (1992).
[CrossRef]

Optik

W. Alt, "An objective lens for efficient fluorescence detection of single atoms," Optik 113, 142-144 (2002).
[CrossRef]

Proc. Natl. Acad. Sci. U S A

T. Kues, A. Dickmanns, R. Lührmann, R. Peters, and U. Kubitscheck, "High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking," Proc. Natl. Acad. Sci. U S A 98, 12021-12026 (2001).
[CrossRef] [PubMed]

Science

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

Other

J. P. Siebrasse, D. Grunwald, and U. Kubitscheck, "Single-molecule tracking in eukaryotic cell nuclei," Anal. Bioanal. Chem. (2006).
[CrossRef] [PubMed]

B. E. A. Saleh and M. C. Teich, Fundamentals of Photonics (Wiley, 2007).

J. H. E. Peter W. Millonni, Lasers (Wiley & Sons, 1989).

M. Born and E. Wolf, Principles of Optics (Cambridge University Press, Cambridge, 1997).

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Figures (6)

Fig. 1.
Fig. 1.

Comparison of epi-illumination and SPIM. In contrast to (A) epi-illumination microscopy the emission path in (B) SPIM is perpendicular to the excitation beam path. By selective illumination of the focal plane, no out-of-focus fluorescence is generated. This results not only in a strongly reduced background signal and higher resolution, but also in the optical sectioning capability of the instrument.

Fig. 2.
Fig. 2.

Schematic view of the high-resolution SPIM setup. Laser light is guided into a Galilean cylindrical beam expander by mirrors M1 and M2 and a dichroic beam splitter (DB). The beam expander transforms the circular-symmetrical Gaussian beam into an elliptical one. The illumination objective lens focuses the beam into a water chamber (WaCh), and creates a light sheet in the focal plane of the detection objective lens. Positioning of the light sheet can be done with the scanning mirror (SM), which is placed in a conjugated plane of the back focal plane of the illumination objective lens. The sample is moved by a three axis sample scanner, the z-axis is motorized. Fluorescence is collected by a water-dipping objective lens and imaged via a tube lens (TL) onto a CCD-camera. Scattered laser light is blocked by an emission filter (EF). By introducing a dichroic beam splitter into the detection beam path it is possible to use a standard wide-field illumination (dashed box).

Fig. 3.
Fig. 3.

Illumination light sheet for λ0=633nm in water (n=1.33). (A) Dimensions (1/e2 distance) and orientation of the light sheet along the illumination axis (x-direction) over a distance of 300 µm. The axial distance, in which the beam width is not greater than a factor of √2 of its focal value is known as the depth-of-focus. All units in µm. (B) A cut through the elliptical Gaussian intensity distribution of the light sheet at the focus of the detection objective lens. (C) and (D), experimental data and fit (red line) of the Gaussian beam width in the XZ-and XY-plane [9]. All data points were obtained by knife edge measurements (see section 4.1).

Fig. 4.
Fig. 4.

Comparison of calculated and measured PSF. Experimentally determined (A) lateral and (B) axial point spread functions. Fluorescent microspheres were illuminated with a 633 nm He- Ne Laser, and imaged with a water-dipping objective lens (NA 1.0). The data were fitted with a Gaussian function to determine the FWHM (red, dashed lines). The theoretical curves were calculated with Eqs. (7a) and (7c) for the lateral and axial PSFs, respectively.

Fig. 5.
Fig. 5.

Contrast improvement in SPIM versus epi-illumination microscopy. (A) Contrast values of dark red fluorescent 210 nm microspheres were determined using SPIM and epifluorescence microscopy as a function of homogeneous dye (Atto633) concentration in sample. SPIM images showed a strong background reduction, leading to a distinct contrast improvement. The maximum fluorescent intensity was kept at the same level for both cases. (B) Example frames showing the beads upon SPIM and epi-illumination microscopy at a background dye concentration of 460 nM.

Fig. 6.
Fig. 6.

Diffusion of single quantum dots in aqueous solution (A) Single frames illustrating the diffusion of a red fluorescent quantum dot (λem=655nm). The frames were acquired with an integration time of 4 ms. In each frame the position of the dot was determined, and assembled into the trajectory (shown in red). (B) A jump distance histogram was fitted (red) with the probability density function for determination of the diffusion coefficients. The histogram comprised two components, which were due to Qdot monomers (green) and Qdot aggregates (blue).

Tables (1)

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Table 1: Calculated and measured values for the focal height and focal thickness of the light sheet

Equations (10)

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h ( x , y , z ) = g ( x , y , z ) d ( x , y , z )
g ( x , y , z ) = g 0 ( x ) exp ( y 2 2 σ y 2 z 2 2 σ z 2 )
d lat = ( 2 J 1 ( v ) v ) 2
v = 2 π λ 0 n x 2 + y 2 sin α
d ax = ( sin ( u 4 ) u 4 ) 2
u = 8 π λ 0 n · z sin ( α 2 ) 2
h ( x , 0 , 0 ) = ( 2 J 1 ( 2 π λ 0 n · x sin α ) 2 π λ 0 n · x sin α ) 2
h ( 0 , y , 0 ) = exp ( y 2 2 σ y 2 ) · ( 2 J 1 ( 2 π λ 0 n · y sin α ) 2 π λ 0 n · y sin α ) 2
h ( 0 , 0 , z ) = exp ( z 2 2 σ x 2 ) · ( sin ( 2 π λ 0 n · z sin 2 α 2 ) 2 π λ 0 n · z sin 2 α 2 ) 2
p ( r , Δ t ) = i A i 2 D i Δ t r · exp [ r 2 4 D i Δ t ]

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