Abstract

This paper describes a new optically sectioning microscopy technique based on oblique selective plane illumination combined with oblique imaging. This method differs from previous selective plane illumination techniques as the same high numerical aperture lens is used to both illuminate and image the specimen. Initial results obtained using fluorescent pollen grains are presented, together with a measurement of the resolution of the system and an analysis of the potential performance of future systems. Since only the plane of the specimen that is being imaged is illuminated, this technique is particularly suited to time-lapse 3-D imaging of sensitive biological systems where photobleaching and phototoxicity must be kept to a minimum, and it could also be applied to image microfluidic technology for lab-on-a-chip, cytometry and other applications.

© 2008 Optical Society of America

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References

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  1. J. B. Pawley, Handbook of Biological Confocal Microscopy (Plenum Press, New York, 1995).
  2. M. Petran, M. Hadravsky, M. D. Egger, and R. Galambos, "Tandem-Scanning Reflected-Light Microscope," J. Opt. Soc. Am. 58, 661-664 (1968).
    [CrossRef]
  3. A. Egner, V. Andresen, and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
    [CrossRef] [PubMed]
  4. M. A. A. Neil, R. Juskaitis, and T. Wilson, "Method of obtaining optical sectioning by using structured light in a conventional microscope," Opt. Lett. 22, 1905-1907 (1997).
    [CrossRef]
  5. A. Bilenca, A. Ozcan, B. Bouma, and G. Tearney, "Fluorescence coherence tomography," Opt. Express 14, 7134-7143 (2006).
    [CrossRef] [PubMed]
  6. A. Bilenca, T. Lasser, A. Ozcan, R. A. Leitgeb, B. E. Bouma, and G. J. Tearney, "Image formation in fluorescence coherence-gated imaging through scattering media," Opt. Express 15, 2810-2821 (2007).
    [CrossRef] [PubMed]
  7. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, "Optical sectioning deep inside live embryos by selective plane illumination microscopy," Science 305, 1007-1009 (2004).
    [CrossRef] [PubMed]
  8. 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]
  9. E. Fuchs, J. S. Jaffe, R. A. Long, and F. Azam, "Thin laser light sheet microscope for microbial oceanography," Opt. Express 10, 145-154 (2002).
    [PubMed]
  10. P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, "High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy," Nature Methods 4, 311-313 (2007).
    [PubMed]
  11. 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," Nature Methods 4, 331-336 (2007).
    [CrossRef] [PubMed]
  12. T. F. Holekamp, D. Turaga, and T. E. Holy, "Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy," Neuron 57, 661-672 (2008).
    [CrossRef] [PubMed]
  13. M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nature Methods 5, 159-161 (2008).
    [CrossRef] [PubMed]
  14. C. A. Konopka, and S. Y. Bednarek, "Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex," Plant Journal 53, 186-196 (2008).
    [CrossRef]
  15. E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, "Aberration-free optical refocusing in high numerical aperture microscopy," Opt. Lett. 32, 2007-2009 (2007).
    [CrossRef] [PubMed]
  16. E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, "An optical technique for remote focusing in microscopy," Opt. Commun. 281, 880-887 (2008).
    [CrossRef]
  17. C. J. Engelbrecht, and E. H. K. Stelzer, "Resolution enhancement in a light-sheet-based microscope (SPIM)," Opt. Lett. 31, 1477-1479 (2006).
    [CrossRef] [PubMed]

2008 (4)

T. F. Holekamp, D. Turaga, and T. E. Holy, "Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy," Neuron 57, 661-672 (2008).
[CrossRef] [PubMed]

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nature Methods 5, 159-161 (2008).
[CrossRef] [PubMed]

C. A. Konopka, and S. Y. Bednarek, "Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex," Plant Journal 53, 186-196 (2008).
[CrossRef]

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, "An optical technique for remote focusing in microscopy," Opt. Commun. 281, 880-887 (2008).
[CrossRef]

2007 (4)

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, "Aberration-free optical refocusing in high numerical aperture microscopy," Opt. Lett. 32, 2007-2009 (2007).
[CrossRef] [PubMed]

A. Bilenca, T. Lasser, A. Ozcan, R. A. Leitgeb, B. E. Bouma, and G. J. Tearney, "Image formation in fluorescence coherence-gated imaging through scattering media," Opt. Express 15, 2810-2821 (2007).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, "High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy," Nature Methods 4, 311-313 (2007).
[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," Nature Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

2006 (2)

2004 (1)

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

2002 (2)

A. Egner, V. Andresen, and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
[CrossRef] [PubMed]

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

1997 (1)

1993 (1)

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]

1968 (1)

Andresen, V.

A. Egner, V. Andresen, and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
[CrossRef] [PubMed]

Azam, F.

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," Nature Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Bednarek, S. Y.

C. A. Konopka, and S. Y. Bednarek, "Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex," Plant Journal 53, 186-196 (2008).
[CrossRef]

Bilenca, A.

Booth, M. J.

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, "An optical technique for remote focusing in microscopy," Opt. Commun. 281, 880-887 (2008).
[CrossRef]

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, "Aberration-free optical refocusing in high numerical aperture microscopy," Opt. Lett. 32, 2007-2009 (2007).
[CrossRef] [PubMed]

Botcherby, E. J.

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, "An optical technique for remote focusing in microscopy," Opt. Commun. 281, 880-887 (2008).
[CrossRef]

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, "Aberration-free optical refocusing in high numerical aperture microscopy," Opt. Lett. 32, 2007-2009 (2007).
[CrossRef] [PubMed]

Bouma, B.

Bouma, B. E.

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]

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," Nature Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. 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," Nature Methods 4, 331-336 (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," Nature 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," Nature Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Egger, M. D.

Egner, A.

A. Egner, V. Andresen, and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
[CrossRef] [PubMed]

Engelbrecht, C. J.

Fuchs, E.

Galambos, R.

Greger, K.

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

Hadravsky, M.

Hell, S. W.

A. Egner, V. Andresen, and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
[CrossRef] [PubMed]

Holekamp, T. F.

T. F. Holekamp, D. Turaga, and T. E. Holy, "Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy," Neuron 57, 661-672 (2008).
[CrossRef] [PubMed]

Holy, T. E.

T. F. Holekamp, D. Turaga, and T. E. Holy, "Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy," Neuron 57, 661-672 (2008).
[CrossRef] [PubMed]

Huisken, J.

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

Imamoto, N.

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nature Methods 5, 159-161 (2008).
[CrossRef] [PubMed]

Jaffe, J. S.

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," Nature Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Juskaitis, R.

Konopka, C. A.

C. A. Konopka, and S. Y. Bednarek, "Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex," Plant Journal 53, 186-196 (2008).
[CrossRef]

Lasser, T.

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," Nature Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Leitgeb, R. A.

Long, R. A.

Marcello, M.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, "High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy," Nature Methods 4, 311-313 (2007).
[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," Nature Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Neil, M. A. A.

Ozcan, A.

Pampaloni, F.

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

Petran, M.

Sakata-Sogawa, K.

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nature Methods 5, 159-161 (2008).
[CrossRef] [PubMed]

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," Nature Methods 4, 331-336 (2007).
[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]

Stelzer, E. H. K.

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

C. J. Engelbrecht, and E. H. K. 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. K. Stelzer, "Optical sectioning deep inside live embryos by selective plane illumination microscopy," Science 305, 1007-1009 (2004).
[CrossRef] [PubMed]

Swoger, J.

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

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

Tearney, G.

Tearney, G. J.

Tokunaga, M.

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nature Methods 5, 159-161 (2008).
[CrossRef] [PubMed]

Turaga, D.

T. F. Holekamp, D. Turaga, and T. E. Holy, "Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy," Neuron 57, 661-672 (2008).
[CrossRef] [PubMed]

Verveer, P. J.

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

Wilson, T.

Wittbrodt, J.

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

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," Nature Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

J. Microsc. (2)

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]

A. Egner, V. Andresen, and S. W. Hell, "Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment," J. Microsc. 206, 24-32 (2002).
[CrossRef] [PubMed]

J. Opt. Soc. Am. (1)

Nature Methods (3)

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nature Methods 5, 159-161 (2008).
[CrossRef] [PubMed]

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. K. Stelzer, "High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy," Nature Methods 4, 311-313 (2007).
[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," Nature Methods 4, 331-336 (2007).
[CrossRef] [PubMed]

Neuron (1)

T. F. Holekamp, D. Turaga, and T. E. Holy, "Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy," Neuron 57, 661-672 (2008).
[CrossRef] [PubMed]

Opt. Commun. (1)

E. J. Botcherby, R. Juskaitis, M. J. Booth, and T. Wilson, "An optical technique for remote focusing in microscopy," Opt. Commun. 281, 880-887 (2008).
[CrossRef]

Opt. Express (3)

Opt. Lett. (3)

Plant Journal (1)

C. A. Konopka, and S. Y. Bednarek, "Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex," Plant Journal 53, 186-196 (2008).
[CrossRef]

Science (1)

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

Other (1)

J. B. Pawley, Handbook of Biological Confocal Microscopy (Plenum Press, New York, 1995).

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Figures (7)

Fig. 1.
Fig. 1.

(a) experimental geometry for SPIM. (b) simplified experimental geometry for HILO. The green stripe on the fluorescent sample illustrates the region where fluorescence is excited. The final image is stretched axially due to the greater (∝M2) axial magnification of the collection optical system

Fig. 2.
Fig. 2.

Diagram of experimental setup: M, mirror; L, spherical lens; C, cylindrical lens; fx, focal length of lens x; FPx, focal plane of lens x; BFPx, back focal plane of lens x; D, dichroic filter; EM, emission filter; IP, imaging plane for CCD camera. A 10× beam expander was placed in front of the laser source (not shown).

Fig. 3.
Fig. 3.

Schematic showing the geometry of the obliquely imaged plane. A fluorescent cylinder is shown (in blue) with its axis perpendicular to the plane of the coverslip. This cylinder is then illuminated with an oblique sheet of illumination and the region where fluorescence is excited is shown in green. In the bottom left of the figure, the image that would be recorded by a camera placed at IP2 (see Fig. 2) is shown. In the top right of the figure, the image that would be obtained at IP1 is depicted. The axes for the two relevant coordinate systems (xn, yn, zn) for normal microscopic imaging and (xo, yo, zo) for oblique microscopic imaging are also shown.

Fig. 4.
Fig. 4.

Diagram showing the relationship between the illumination and collection angles and numerical apertures: θ, half angle subtended by lens L3; ϕex, half angle subtended by excitation light; ϕem, half angle subtended by collected fluorescence emission. The angle between the centres of the excitation and emission ray bundles is 90°.

Fig. 5.
Fig. 5.

Images of two fluorescent specimens obtained with the OPM system. Images were obtained at both image planes IP1 (a, c) and IP2 (b, d) (see Fig. 2) with the sample in the same position relative to the objective lens L3. (a–b) images of fluorescent pollen grains and (c–d) images of a fluorescently labelled specimen of convallaria. In all images, the oblique illumination is originating from the left hand side of the image. Therefore, for (a, c) the illuminated part of the specimen to the left of centre is in front of the focal plane, with the right hand side being behind the focal plane. Scale bars represent 20 µm.

Fig. 6.
Fig. 6.

3D rendering of an image stack of fluorescent pollen grains obtained using the OPM system. The axes shown are those of a conventional microscope system (Fig. 3) and the rendered image corresponds to a volume in the specimen of 190×80×50 µm3.

Fig. 7.
Fig. 7.

(a) OPM image of the sample of 40 nm fluorescent beads, scale bar 20 µm. Two bead clusters at opposite sides of the sample are indicated by white arrows. The brightness and contrast of this image has been enhanced to enable the fluorescent bead clusters to be seen more clearly. (b) example line profile through a bead in the xo direction and (c) step-response curve obtained from the interface between the coverslip and the bead solution in the sample shown in (a).

Tables (1)

Tables Icon

Table 1: Calculated values for the resolutions achievable using three commercially available microscope objectives with different immersion media. Lens designations are shown in Fig. 2, and angles in Fig. 4. Values of ϕex were chosen to give a useful illumination sheet waist length at the specimen. δxo, δyo are the calculated FWHM of the point spread function in the oblique plane and δzo is the thickness of the illumination sheet. Excitation and emission wavelengths were assumed to be 532 nm and 610 nm respectively, in line with the wavelengths used elsewhere in this paper. NA potential is the potential NA that could be achieved by OPM using the lens specified for L3 and assuming an ideal lens for L7.

Equations (4)

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ϕ em = 2 θ ϕ ex π 2 .
d FWHM λ 2 n sin θ .
w o λ n π θ .
b = 2 z r = 2 π n w o 2 λ ,

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