Abstract

One of the main challenges in three-dimensional microscopy is to overcome the lack of isotropy of the spatial resolution, which results from the axially-elongated shape of the point spread function. Such anisotropy gives rise to images in which significant axially-oriented structures of the sample are not resolved. In this paper we achieve an important improvement in z resolution in two-photon excitation microscopy through spatial modulation of the incident beam. Specifically, we demonstrate that the design and implementation of a simple shaded ring performs quasi-isotropic three-dimensional imaging and that the corresponding loss in luminosity can be easily compensated by most available femtosecond lasers. The outcome looks particularly relevant to nano-fabrication and optical manipulation.

© 2005 Optical Society of America

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References

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Appl. Opt. (1)

Appl. Phys. Lett. (1)

C. M. Blanca, J. Bewersdorf and S. W. Hell, "Single sharp spot in fluorescence microscopy of two opposing lenses," Appl. Phys. Lett. 79, 2321-2323 (2001).
[CrossRef]

J. Mod. Opt. (2)

S. S. Sherif and P. Török, "Pupil plane masks for super-resolution in high-numerical-aperture focusing," J. Mod. Opt. 51, 2007-2019 (2004).

S. Lindek, J. Swoger and E. H. K. Stelzer, "Single-lens theta microscopy: resolution, efficiency and working distance," J. Mod. Opt. 46, 843-858 (1999).

J. Opt. Soc. Am. A (1)

Microsc. Res. Tech. (1)

C. Ibáñez-López, I. Escobar, G. Saavedra and M. Martínez-Corral, "Optical sectioning improvement in two-color excitation scanning microscopy," Microsc. Res. Tech. 64, 96-102 (2004).
[CrossRef] [PubMed]

Mikrosk. Anat. (1)

Abbe, E. Arch. Mikrosk. Anat. 9, 413�??468 (1873).
[CrossRef]

Nature (2)

S. Kawata, H.-B. Sun, T. Tanaka and K. Takada, "Finer features for functional microdevices," Nature 412, 697-698 (2001).
[CrossRef] [PubMed]

D. G. Grier, "A revolution in optical manipulation," Nature 424, 810-816 (2003).
[CrossRef] [PubMed]

Opt. Commun. (1)

O. Haeberlé, �??Focusing of light through a stratified medium: a practical approach for computing microscope point spread functions. Part I: Conventional microscopy,�?? Opt. Commun. 216, 55-63 (2003).
[CrossRef]

Opt. Express (3)

Opt. Lett. (2)

Phys. Rev. Lett. (4)

M. Dyba and S. W. Hell, "Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef] [PubMed]

T. R. M. Sales, "Smallest focal spot," Phys. Rev. Lett. 81, 3844-3847 (1998).
[CrossRef]

J. Miao, T. Ishikawa, B. Johnson, E. K. Anderson, B. Lai and K. O. Hodgson, "High resolution 3D x-ray diffraction microscopy," Phys. Rev. Lett. 89, 088303 (2002).
[CrossRef] [PubMed]

J. Miao, T. Ohsuna, O. Terasaki, K. O. Hodgson and M. A. O�??Keefe, "Atomic resolution three-dimensional electron diffraction microscopy," Phys. Rev. Lett. 89, 155502 (2002).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sc. (1)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner and S. W. Hell, "Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission," Proc. Natl. Acad. Sc. 97, 8206-8210 (2000).
[CrossRef]

Science (3)

W. Denk, J. H. Strickler and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

B. Bailey, D. L. Farkas, D. Lansing-Taylor and F. Lanni, "Enhancement resolution in fluorescence microscopy by standing-wave excitation," Science 366, 44-48 (1993).

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 (4)

J. B. Pawley, ed., Handbook of biological confocal microscopy. Plenum Press, New York, 1995.

The term superresolution introduced here is understood in the sense of Rayleigh criterion; i. e., as the narrowness of the PSF or, equivalently, the enhancement of the OTF for frequencies under the cut-off frequency.

B. Richards and E. Wolf, Proceedings of the Royal Society (London) A 253, 358 (1959).
[CrossRef]

The eccentricity is defined here as e = �??(1-b^2 / a^2) , where a and b account for the lengths of the semimajor and semiminor axes, respectively.

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Figures (5)

Fig. 1.
Fig. 1.

Amplitude transmittance of the SR filter. The transmittance of the shaded ring is 19%.

Fig. 2.
Fig. 2.

Plot of the numerically evaluated 3-D PSF, in the meridian plan φ = 0 , corresponding to: (a) Standard 1.2 NA objective with clear pupil; and (b) Same objective but with the SR filter inserted as the aperture stop. Axes labels are expressed in microns.

Fig. 3.
Fig. 3.

-Schematic geometry of the TPE scanning microscope.

Fig. 4.
Fig. 4.

- 2D gray-scale sections of the experimental 3-D PSFs as displayed by LabView. (a) PSF obtained with the standard 1.2 NA objective with clear pupil; and (b) PSF obtained with the same objective but with the SR filter inserted as the aperture stop.

Fig. 5.
Fig. 5.

- Normalized axial and lateral PSF produced by the Olympus 1.2 NA objective (x). With empty dots (o) we show the experimental results obtained when the SR filter is inserted in the illumination path.

Equations (5)

Equations on this page are rendered with MathJax. Learn more.

E ( r , z , φ ) = [ I 0 ( r , z ) + I 2 ( r , z ) cos 2 φ ] i + I 2 ( r , z ) sin 2 φ j + i 2 I 1 ( r , z ) cos φ k
I 0 ( r , z ) = 0 α P ( θ ) cos θ ( 1 + cos θ ) J 0 ( k r sin θ ) exp ( ik z cos θ ) sin θ d θ
I 1 ( r , z ) = 0 α P ( θ ) cos θ sin θ J 1 ( k r sin θ ) exp ( ik z cos θ ) sin θ d θ
I 2 ( r , z ) = 0 α P ( θ ) cos θ ( 1 cos θ ) J 2 ( k r sin θ ) exp ( ik z cos θ ) sin θ d θ
PSF ( r , z , φ ) = E ( r , z , φ ) 4 .

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