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

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    [CrossRef]
  2. S. W. Hell, "Double-scanning confocal microscope," European Patent 0491289 (1990).
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]

2006

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

2004

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

2003

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

2002

2001

1999

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

1995

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

1992

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

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

1972

1959

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

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

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

S. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (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.

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

J. Microsc.

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.

J. Opt. Soc. Am. A

Micron

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

Opt. Commun.

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

Proc. R. Soc. London, Ser. A

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

Proc. SPIE

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

Other

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).

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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.

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