Abstract

We explore the current limits of the axial resolution of optical sectioning microscopy using a single lens, by combining the resolving power of novel 1.45 numerical aperture oil immersion lenses with superresolving binary aperture filters. We quantify the axial resolution brought about by the increase in semiaperture angle to α max =72.8° and demonstrate an absolute gain in axial resolution through binary pupil filters. Implemented in a confocalized two-photon excitation microscope, the combination of both effectively improves the axial resolution to 330 nm full-width-half-maximum, which is by 34% over that of a standard 1.4 NA system.

© 2002 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]

Bioimaging (1)

T. Wilson and R. Juskaitis, �??The axial response of confocal microscopes with high numerical aperture objective lenses,�?? Bioimaging 3, 35-38 (1995).
[CrossRef]

J. Biomed. Opt. (1)

N. Huse, A. Schönle and S. W. Hell, �??Z-polarized confocal microscopy,�?? J. Biomed. Opt. 6, 480-484 (2001).
[CrossRef]

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

Opt. Commun. (2)

S. Quabis, R. Dorn, M. Eberler, O. Glöckl and G. Leuchs, �??Focusing light to a tighter spot,�?? Opt. Commun. 179, 1-7 (2000).
[CrossRef]

M. Martinez-Corral, P. Andres, C. J. Zapata-Rodriguez and M. Kowalczyk, �??Three-dimensional superresolution by annular binary filters, �?? Opt. Commun. 165, 267-278 (1999).
[CrossRef]

Opt. Express (2)

Opt. Lett. (1)

Phys. Rev. Lett. (1)

L. Novotny, M. R. Beversluis, K. S. Youngworth and T. G. Brown, �??Longitudinal Field Modes Probed by Single Molecules,�?? Phys. Rev. Lett. 86, 5251-5254 (2001).
[CrossRef] [PubMed]

Proc. R. Soc. Lond. A (1)

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

Single Mol. (1)

C. M. Blanca and S. W. Hell, �??Sharp Spherical Focal Spot by Dark Ring 4Pi-Confocal Microscopy, �?? Single Mol. 2, 207-210 (2001).
[CrossRef]

Other (1)

T. Wilson and C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic Press, New York, 1984).

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Figures (3)

Fig. 1.
Fig. 1.

Schematic of the dark ring (DR) filter inserted into the excitation path of a confocal fluorescence microscope operating in transmission mode.

Fig. 2.
Fig. 2.

(a) Theoretical z-responses of a two-photon confocal fluorescence microscope depicting the associated increase in axial resolution as the NA is expanded from 1.40 (bold) to 1.45 (thin). The experimental counterparts in (b) essentially confirm the predicted improvement in axial sectioning. The increase is magnified in the insets plotted with truncated axial coordinates.

Fig. 3.
Fig. 3.

Z-responses of a two-photon DR-confocal microscope. (a) Theoretical responses for NA=1.40 (thin) and NA=1.45 (bold). Panel (b) shows the experimental counterparts to (a). The combination of DR-filter and NA=1.45 lens sharpens the z-response by 34% with respect to that of a standard NA=1.40 confocal microscope, resulting in a full-width-half-maximum of 330 nm.

Equations (1)

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I ( z ) = C [ h exc ( z , r ) ] 2 [ h det ( z , r ) p ( r ) ] rdr

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