Abstract

We demonstrate that oil immersion lenses with a semi-aperture angle ≥ 74° enable 4Pi confocal fluorescence microscopy of type A with 1-photon excitation. The axial sidelobes amount to < 50 % of the main diffraction maximum, implying that lobe induced artifacts can be removed from the image data. The advancement reported herein enables a relative inexpensive implementation of 4Pi microscopy, providing axially superresolved 3D-imaging in transparent samples. As an example, we show dual-color 4Pi images of double stained Golgi stacks in a mammalian cell with 110 nm axial resolution. The resolution can be further enhanced to values slightly below 100 nm by image deconvolution.

© 2007 Optical Society of America

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  1. S. Hell and E. H. K. Stelzer, "Properties of a 4Pi-confocal fluorescence microscope", J. Opt. Soc. Am. A 9, 2159-2166 (1992).
    [CrossRef]
  2. S. W. Hell, "Double-scanning confocal microscope," European Patent 0491289 (1990).
  3. M. G. L. Gustafsson et al., "Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses," Proc. SPIE 2412, 147-156 (1995).
    [CrossRef]
  4. M. G. L. Gustafsson et al., "I5M: 3D widefield light microscopy with better than 100 nm axial resolution", J. Microsc. 195, 10-16 (1999).
    [CrossRef] [PubMed]
  5. S. W. Hell and E. H. K. Stelzer, "Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation," Opt. Commun. 93, 277-282 (1992).
    [CrossRef]
  6. S. W. Hell, "Toward fluorescence nanoscopy," Nat. Biotechnol. 21, 1347-1355 (2003).
    [CrossRef] [PubMed]
  7. R. Medda et al., "4Pi Microscopy of Quantum Dot-Labeled Cellular Structures," J. Struct. Biol. 156, 517-523 (2006).
    [CrossRef] [PubMed]
  8. T. Staudt et al., "2, 2'-Thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy," Microsc. Res. Tech. 70, 1-9 (2007).
    [CrossRef]
  9. A. Egner et al., "Fast 100-nm resolution 3D-microscope reveals structural plasticity of mitochondria in live yeast," Proc. Natl. Acad. Sci. USA 99, 3370-3375 (2002).
    [CrossRef] [PubMed]
  10. B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system," Proc. R. Soc. Lond. A 253, 358-379 (1959).
    [CrossRef]
  11. G. Hildenbrand et al., "Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy," Biophys. J. 88, 4312-4318 (2005).
    [CrossRef] [PubMed]
  12. H. Gugel et al., "Cooperative 4Pi excitation and detection yields 7-fold sharper optical sections in live cell microscopy," Biophys. J.,  87, 4146-4152 (2004).
    [CrossRef] [PubMed]
  13. M. Nagorni and S. W. Hell, "Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. I. Comparative study of concepts," J. Opt. Soc. Am. A 18, 36-48 (2001).
    [CrossRef]
  14. W. H. Richardson, "Bayesian-based iterative method of image restoration," J. Opt. Soc. Am. 62, 55-59 (1972).
    [CrossRef]
  15. M. Nagorni and S. W. Hell, "Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. II. Power and limitation of nonlinear image restoration," J. Opt. Soc. Am. A 18, 49-54 (2001).
    [CrossRef]

2007

T. Staudt et al., "2, 2'-Thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy," Microsc. Res. Tech. 70, 1-9 (2007).
[CrossRef]

2006

R. Medda et al., "4Pi Microscopy of Quantum Dot-Labeled Cellular Structures," J. Struct. Biol. 156, 517-523 (2006).
[CrossRef] [PubMed]

2005

G. Hildenbrand et al., "Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy," Biophys. J. 88, 4312-4318 (2005).
[CrossRef] [PubMed]

2004

H. Gugel et al., "Cooperative 4Pi excitation and detection yields 7-fold sharper optical sections in live cell microscopy," Biophys. J.,  87, 4146-4152 (2004).
[CrossRef] [PubMed]

2003

S. W. Hell, "Toward fluorescence nanoscopy," Nat. Biotechnol. 21, 1347-1355 (2003).
[CrossRef] [PubMed]

2002

A. Egner et al., "Fast 100-nm resolution 3D-microscope reveals structural plasticity of mitochondria in live yeast," Proc. Natl. Acad. Sci. USA 99, 3370-3375 (2002).
[CrossRef] [PubMed]

2001

1999

M. G. L. Gustafsson et al., "I5M: 3D widefield light microscopy with better than 100 nm axial resolution", J. Microsc. 195, 10-16 (1999).
[CrossRef] [PubMed]

1995

M. G. L. Gustafsson et al., "Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses," Proc. SPIE 2412, 147-156 (1995).
[CrossRef]

1992

S. W. Hell and E. H. K. Stelzer, "Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation," Opt. Commun. 93, 277-282 (1992).
[CrossRef]

S. Hell and E. H. K. Stelzer, "Properties of a 4Pi-confocal fluorescence microscope", J. Opt. Soc. Am. A 9, 2159-2166 (1992).
[CrossRef]

1972

1959

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system," Proc. R. Soc. Lond. A 253, 358-379 (1959).
[CrossRef]

Egner, A.

A. Egner et al., "Fast 100-nm resolution 3D-microscope reveals structural plasticity of mitochondria in live yeast," Proc. Natl. Acad. Sci. USA 99, 3370-3375 (2002).
[CrossRef] [PubMed]

Gugel, H.

H. Gugel et al., "Cooperative 4Pi excitation and detection yields 7-fold sharper optical sections in live cell microscopy," Biophys. J.,  87, 4146-4152 (2004).
[CrossRef] [PubMed]

Gustafsson, M. G. L.

M. G. L. Gustafsson et al., "I5M: 3D widefield light microscopy with better than 100 nm axial resolution", J. Microsc. 195, 10-16 (1999).
[CrossRef] [PubMed]

M. G. L. Gustafsson et al., "Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses," Proc. SPIE 2412, 147-156 (1995).
[CrossRef]

Hell, S.

Hell, S. W.

Hildenbrand, G.

G. Hildenbrand et al., "Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy," Biophys. J. 88, 4312-4318 (2005).
[CrossRef] [PubMed]

Medda, R.

R. Medda et al., "4Pi Microscopy of Quantum Dot-Labeled Cellular Structures," J. Struct. Biol. 156, 517-523 (2006).
[CrossRef] [PubMed]

Nagorni, M.

Richards, B.

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system," Proc. R. Soc. Lond. A 253, 358-379 (1959).
[CrossRef]

Richardson, W. H.

Staudt, T.

T. Staudt et al., "2, 2'-Thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy," Microsc. Res. Tech. 70, 1-9 (2007).
[CrossRef]

Stelzer, E. H. K.

S. W. Hell and E. H. K. Stelzer, "Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation," Opt. Commun. 93, 277-282 (1992).
[CrossRef]

S. Hell and E. H. K. Stelzer, "Properties of a 4Pi-confocal fluorescence microscope", J. Opt. Soc. Am. A 9, 2159-2166 (1992).
[CrossRef]

Wolf, E.

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system," Proc. R. Soc. Lond. A 253, 358-379 (1959).
[CrossRef]

Biophys. J.

G. Hildenbrand et al., "Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy," Biophys. J. 88, 4312-4318 (2005).
[CrossRef] [PubMed]

H. Gugel et al., "Cooperative 4Pi excitation and detection yields 7-fold sharper optical sections in live cell microscopy," Biophys. J.,  87, 4146-4152 (2004).
[CrossRef] [PubMed]

J. Microsc.

M. G. L. Gustafsson et al., "I5M: 3D widefield light microscopy with better than 100 nm axial resolution", J. Microsc. 195, 10-16 (1999).
[CrossRef] [PubMed]

J. Opt. Soc. Am.

J. Opt. Soc. Am. A

J. Struct. Biol.

R. Medda et al., "4Pi Microscopy of Quantum Dot-Labeled Cellular Structures," J. Struct. Biol. 156, 517-523 (2006).
[CrossRef] [PubMed]

Microsc. Res. Tech.

T. Staudt et al., "2, 2'-Thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy," Microsc. Res. Tech. 70, 1-9 (2007).
[CrossRef]

Nat. Biotechnol.

S. W. Hell, "Toward fluorescence nanoscopy," Nat. Biotechnol. 21, 1347-1355 (2003).
[CrossRef] [PubMed]

Opt. Commun.

S. W. Hell and E. H. K. Stelzer, "Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation," Opt. Commun. 93, 277-282 (1992).
[CrossRef]

Proc. Natl. Acad. Sci. USA

A. Egner et al., "Fast 100-nm resolution 3D-microscope reveals structural plasticity of mitochondria in live yeast," Proc. Natl. Acad. Sci. USA 99, 3370-3375 (2002).
[CrossRef] [PubMed]

Proc. R. Soc. Lond. A

B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system," Proc. R. Soc. Lond. A 253, 358-379 (1959).
[CrossRef]

Proc. SPIE

M. G. L. Gustafsson et al., "Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses," Proc. SPIE 2412, 147-156 (1995).
[CrossRef]

Other

S. W. Hell, "Double-scanning confocal microscope," European Patent 0491289 (1990).

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Figures (7)

Fig. 1.
Fig. 1.

(a). Calculated sidelobe height (in % of the main maximum) in the z-response and in the line profile h eff(z) as a function of the semi-aperture angle α. The use of α = 74° instead of 68° decreases the sidelobes in the z-response by an absolute value of 7 %. The lateral and axial FWHM of the main maximum of the PSF for λexc=488 nm and a refractive index of 1.518 is shown in (b). Although the axial FWHM slightly increases for very high angles this is compensated by the increased lateral resolution.

Fig. 2.
Fig. 2.

Convolution of a 1-photon 4Pi PSF of type A with a 100 nm diameter sphere (b), a 100 nm diameter rod (in y-direction) (c) and a 100 nm thick layer (d). PSF conditions: α=74°, λexc=488 nm, and λem=515 nm. The right hand panels show the data in the xz-plane, whereas the left hand panels display line profile data from the optic axis (x,y=0). The sidelobes along the optic axis (z) rise from 27 % for a point-like object to 42 % for a layer in the xy-plane, indicating that laterally extended objects are more difficult to discern axially. The FWHM of the line profile is 108 nm for the point-like object and 127 nm for the 100 nm diameter sphere.

Fig. 3.
Fig. 3.

4Pi-confocal fluorescence microscopy of type A with 1-photon excitation at 568 nm: xz-images of 100 nm diameter fluorescent beads with central emission λem=605 nm. The line profile through one of the beads displays lobes of 35 %, in good agreement with theory. Both the calculated and the measured FWHM are 140 nm. Further calculation shows that the FWHM of the theoretical PSF of a point-like object is 126 nm for these conditions.

Fig. 4.
Fig. 4.

Comparison of 1-photon (λexc=568 nm) confocal and 4Pi confocal axial images (xz) of the distribution of the Cy3-immunolabelled cis-Golgi protein GM 130 in a Vero cell reveals the improved axial resolution of the latter. The 4Pi raw data (bottom) features sidelobes of ∼50 % height. Nevertheless, sidelobe removal by 3-point deconvolution renders virtually artifact-free images of the Golgi stacks (center). Note the substantial resolution improvement of the 4Pi over that of the confocal microscope. (Scale bar 1 μm).

Fig. 5.
Fig. 5.

3D-rendered representation of the distribution of a Rhodamine 6G stained Golgi protein in a Vero cell: confocal versus type A 4Pi confocal microscopy. The 1-photon excitation wavelength was 568 nm in both cases. The xy-image (left) shows the complete data set and the xy-section of the 4Pi Golgi recording presented in the 3D view.

Fig. 6.
Fig. 6.

Dual-color (yellow-red) 1-photon excitation 4Pi confocal microscopy of type A of a double-stained Golgi apparatus in a Vero cell. A trans-Golgi protein was labeled with Alexa546 (green), whereas a protein of the cis-Golgi was stained with an Alexa647-labeled antibody emitting red fluorescence. The panels show the raw data of a 4Pi xz-image, along with the pertinent confocal, and the final 4Pi image after sidelobe removal. The superimposed images (g, h) demonstrate the superior axial resolution of the 4Pi microscope of type A. The results after a Richardson-Lucy deconvolution for confocal and 4Pi imaging are shown in i,j. (Scale bar 1 μm).

Fig. 7.
Fig. 7.

Double-labeled (green-yellow) 4Pi xz-imaging of the Golgi apparatus in a Vero cell. The trans-Golgi was stained with Bodipy FL (green emission) and the cis-Golgi marker protein GM130 was labeled via antibodies with the yellow emitting fluorophore Alexa546, shown as the color red channel. The 4Pi raw data for Bodipy FL show higher sidelobes than the corresponding Alexa546 data. The 4Pi data after 3-point deconvolution and a subtraction of a 10 % offset demonstrate that the deconvolution works sufficiently well. (Scale bar 1 μm).

Equations (1)

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h eff ( r ) = h exc 4 pi h det 4 pi = E exc ( r ) + M ̂ E exc ( M ̂ r ) 2 E det ( r ) 2 p ( r )

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