Abstract

Practical 4Pi microscopy has so far exclusively relied on multiphoton excitation of fluorescence, because the nonlinear suppression of contributions from higher-order sidelobes was mandatory for unambiguous axial superresolution. We show that novel lenses of 74° semiaperture angle enable biological 4Pi microscopy with regular one-photon fluorescence excitation, thus increasing the signal and reducing system complexity and cost. An axial resolution of 95nm, corresponding to a more than fourfold improvement over confocal microscopy, is verified in the imaging of microtubules in mammalian cells.

© 2007 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. S. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
    [CrossRef]
  2. S. W. Hell, "Double-scanning confocal microscope," European Patent 0491289 (1990).
  3. H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
    [CrossRef] [PubMed]
  4. M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, in Proc. SPIE 2412, 147 (1995).
    [CrossRef]
  5. M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
    [CrossRef] [PubMed]
  6. J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
    [CrossRef] [PubMed]
  7. M. Nagorni and S. W. Hell, J. Opt. Soc. Am. A 18, 36 (2001).
    [CrossRef]
  8. S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
    [CrossRef]
  9. S. W. Hell, in Topics in Fluorescence Spectroscopy, J.R.Lakowicz, ed. (Plenum, 1997), p. 361.
  10. M. Martínez-Corral, Micron 34, 319 (2003).
    [CrossRef] [PubMed]
  11. M. Martínez-Corral, A. Pons, and M. T. Caballero, J. Opt. Soc. Am. A 19, 1532 (2002).
    [CrossRef]
  12. B. Richards and E. Wolf, Proc. R. Soc. London, Ser. A 253, 358 (1959).
    [CrossRef]
  13. A. N. Tikhonov and V. Y. Arsenin, Solutions of Ill-Posed Problems (Wiley, 1977).
  14. W. H. Richardson, J. Opt. Soc. Am. 62, 55 (1972).
    [CrossRef]

2006 (1)

J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
[CrossRef] [PubMed]

2004 (1)

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef] [PubMed]

2003 (1)

M. Martínez-Corral, Micron 34, 319 (2003).
[CrossRef] [PubMed]

2002 (1)

2001 (1)

1999 (1)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef] [PubMed]

1995 (1)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, in Proc. SPIE 2412, 147 (1995).
[CrossRef]

1992 (2)

S. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
[CrossRef]

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

1972 (1)

1959 (1)

B. Richards and E. Wolf, Proc. R. Soc. London, Ser. A 253, 358 (1959).
[CrossRef]

Agard, D. A.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef] [PubMed]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, in Proc. SPIE 2412, 147 (1995).
[CrossRef]

Arsenin, V. Y.

A. N. Tikhonov and V. Y. Arsenin, Solutions of Ill-Posed Problems (Wiley, 1977).

Bewersdorf, J.

J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
[CrossRef] [PubMed]

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef] [PubMed]

Caballero, M. T.

Engelhardt, J.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef] [PubMed]

Gugel, H.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef] [PubMed]

Gustafsson, M. G. L.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef] [PubMed]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, in Proc. SPIE 2412, 147 (1995).
[CrossRef]

Hell, S.

Hell, S. W.

J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
[CrossRef] [PubMed]

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef] [PubMed]

M. Nagorni and S. W. Hell, J. Opt. Soc. Am. A 18, 36 (2001).
[CrossRef]

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

S. W. Hell, in Topics in Fluorescence Spectroscopy, J.R.Lakowicz, ed. (Plenum, 1997), p. 361.

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

Jakobs, S.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef] [PubMed]

Martínez-Corral, M.

Nagorni, M.

Pons, A.

Richards, B.

B. Richards and E. Wolf, Proc. R. Soc. London, Ser. A 253, 358 (1959).
[CrossRef]

Richardson, W. H.

Schmidt, R.

J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
[CrossRef] [PubMed]

Sedat, J. W.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef] [PubMed]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, in Proc. SPIE 2412, 147 (1995).
[CrossRef]

Stelzer, E. H. K.

S. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992).
[CrossRef]

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

Storz, R.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef] [PubMed]

Tikhonov, A. N.

A. N. Tikhonov and V. Y. Arsenin, Solutions of Ill-Posed Problems (Wiley, 1977).

Wolf, E.

B. Richards and E. Wolf, Proc. R. Soc. London, Ser. A 253, 358 (1959).
[CrossRef]

Biophys. J. (1)

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, Biophys. J. 87, 4146 (2004).
[CrossRef] [PubMed]

J. Microsc. (2)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, J. Microsc. 195, 10 (1999).
[CrossRef] [PubMed]

J. Bewersdorf, R. Schmidt, and S. W. Hell, J. Microsc. 222, 105 (2006).
[CrossRef] [PubMed]

J. Opt. Soc. Am. (1)

J. Opt. Soc. Am. A (3)

Micron (1)

M. Martínez-Corral, Micron 34, 319 (2003).
[CrossRef] [PubMed]

Opt. Commun. (1)

S. W. Hell and E. H. K. Stelzer, Opt. Commun. 93, 277 (1992).
[CrossRef]

Proc. R. Soc. London, Ser. A (1)

B. Richards and E. Wolf, Proc. R. Soc. London, Ser. A 253, 358 (1959).
[CrossRef]

Proc. SPIE (1)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, in Proc. SPIE 2412, 147 (1995).
[CrossRef]

Other (3)

A. N. Tikhonov and V. Y. Arsenin, Solutions of Ill-Posed Problems (Wiley, 1977).

S. W. Hell, in Topics in Fluorescence Spectroscopy, J.R.Lakowicz, ed. (Plenum, 1997), p. 361.

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

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (4)

Fig. 1
Fig. 1

Axial resolution in 4Pi confocal fluorescence microscopy of type C using one-photon excitation with high-angle immersion lenses: NA 1.20 water, 1.40 oil, and 1.46 oil, as characterized by [(a), left] the axial responses I z ( z ) to an ultrathin plane, and [(a), right] the optical transfer function along the inverse optic axis, OTF ( k z ) . (b) Relative height of the primary axial lobes I lobes of I z ( z ) , and of OTF ( k c ) on the semiaperture angle α. An increase of α by 6 ° (from 68 ° to 74 ° ) leads to a 7% decrease of the first-order maximum in the PSF and by the same token to an increase of the OTF at the critical frequency k c by 7%. Calculations are for excitation at 488 nm and detection at 605 nm ; the pinhole diameter corresponds to half of that of the Airy disk.

Fig. 2
Fig. 2

Measured z response to a fluorescent layer of Qdots (excitation, 488 nm ; emission, 605 nm ). The inset in (b) displays the 4Pi confocal x z image of the layer. The measured FWHM of the z response is 95 nm . The corresponding OTF, shown in (b), is contiguous and well above zero; at the critical frequency we have OTF ( k z ) > 12 % .

Fig. 3
Fig. 3

Side-by-side comparison of focal plane ( x y ) images of 100 nm diameter yellow–green fluorescent beads recorded by (a) one-photon ( λ exc = 488 nm ) and two-photon ( λ exc = 820 nm ) excitation, respectively. The profiles are taken from the same bead. The comparison indicates a slightly improved lateral resolution in the one-photon excitation case.

Fig. 4
Fig. 4

4Pi microscopy at 488 nm one-photon excitation. Axial ( x z ) images show microtubules in a mammalian cell stained with (d)–(f) DY-485XL (emission 560 nm ) and (a)–(c) Alexa 488 (emission 519 nm ). (b), (e) Raw data including the effect of the lobes; (a), (d) standard confocal recording; (c), (f) 4Pi image after lobe removal by three-point deconvolution. (h) The intensity profile through a microtubule exhibits a FWHM of 413 nm in the confocal mode, whereas in the 4Pi case the corresponding value is 95 nm . The Alexa 488 recordings demonstrate (g) that one-photon excitation 4Pi microscopy is possible at low Stokes shifts, thus facilitating dual-color 4Pi imaging with linear excitation.

Metrics