Abstract

Aberrations are known to severely compromise image quality in optical microscopy, especially when high numerical aperture (NA) lenses are used in confocal fluorescence microscopy (CFM) and two-photon microscopy (TPM). The method of adaptive optics may correct aberrations and restore diffraction limited operation. So far the problem of aberrations that occur in the imaging of biological specimens has not been quantified. However, this information is essential for the design of adaptive optics systems. We have therefore built an interferometer incorporating high NA objective lenses to measure the aberrations introduced by biological specimens. The measured wavefronts were decomposed into their Zernike mode content in order both to classify and quantify the aberrations. We calculated the potential benefit of correcting different numbers of Zernike modes using different NAs in an adaptive CFM by comparing the signal levels before and after correction. The results indicate that adaptive correction of low order Zernike modes can provide significant benefit for many specimens. The results also show that quantitative fluorescence microscopy may be strongly affected by specimen induced aberrations in non-adaptive systems.

© 2004 Optical Society of America

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References

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Appl. Opt. (3)

J. Cell Biol. (1)

E.M.M. Manders, H. Kimura and P.R. Cook �??Direct labelling of DNA in living cells reveals the dynamics of chromosome formation.�?? J. Cell Biol., 144:813-823, 1999.
[CrossRef] [PubMed]

J. Micromech. and Microeng. (1)

G. Vdovin, P.M. Sarro, and S. Middelhoek. �??Technology and applications of micromachined adaptive mirrors.�?? J. Micromech. and Microeng. 9, R8, 1999.
[CrossRef]

J. Microscopy (7)

M. A. A. Neil, R. Ju¡skaitis, M. J. Booth, T. Wilson, T. Tanaka, and S. Kawata. �??Adaptive aberration correction in a two-photon microscope.�?? J. Microscopy 200, 105�??108, 2000.
[CrossRef]

L. Sherman, J. Y. Ye, O. Albert, and T. B. Norris. �??Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror.�?? J. Microscopy 206, 65�??71, 2002.
[CrossRef]

M. Schwertner, M.J. Booth, and T. Wilson. �??Simulation of specimen induced aberrations for objects with spherical and cylindrical symmetry.�?? J. Microscopy 215, 271�??280, 2004.
[CrossRef]

S. Hell, G. Reiner, C. Cremer, and E.H.K. Stelzer. �??Aberrations in confocal fluorescence microscopy induced by mismatches�?? in refractive index. J. Microscopy 169, 391�??405, 1993.

T. Wilson and A.R. Carlini. �??The effects of aberrations on the axial response of confocal imaging�?? systems. J. Microscopy 154, 243�??256, 1989.
[CrossRef]

M.J. Booth, M.A.A. Neil, and T. Wilson. �??Aberration correction for confocal imaging in refractive-indexmismatched media.�?? J. Microscopy 192, 90�??98, 1998.
[CrossRef]

M.J. Booth and T. Wilson. �??Strategies for the compensation of specimen induced aberration in confocal microscopy of skin.�?? J. Microscopy 200, 68�??74, 2000.
[CrossRef]

J. Opt. Soc. Am. (2)

M. A. A. Neil, M. J. Booth, and T. Wilson. �??New modal wavefront sensor: a theoretical analysis.�?? J. Opt. Soc. Am. 17, 1098�??1107, 2000.
[CrossRef]

R.J. Noll. �??Zernike polynomials and atmospheric turbulence.�?? J. Opt. Soc. Am. 66, 207�??277, 1976.
[CrossRef]

J. Opt. Soc. Am. A (2)

J. Refractive Surgery (1)

B.C. Platt and R. Shack. �??History and priciples of Shack-Hartmann wavefront sensing.�?? J. Refractive Surgery 17, 573�??577, 2001.

J.Microscopy (1)

M. Schwertner, M. J. Booth, M.A.A. Neil, and T. Wilson. �??Measurement of specimen-induced aberrations of biological samples using phase stepping interferometry.�?? J.Microscopy 213, 11�??19, 2004.
[CrossRef]

Journal of Biomedical Optics (1)

M. J. Booth and T. Wilson. �??Refractive-index-mismatch induced aberrations in single-photon and two-photon microscopy and the use of aberration correction.�?? Journal of Biomedical Optics, 6(3):266�??272, 2001.
[CrossRef] [PubMed]

National Academy of Science (1)

M.J. Booth, M. A. A. Neil, R. Ju¡škaitis, and T.Wilson. �??Adaptive aberration correction in a confocal microscope.�?? Proceedings of the National Academy of Science, 99:5788�??5792, 2002.
[CrossRef]

Opt. Express (2)

Opt. Lett. (2)

Optics Express (1)

C. Paterson, I. Munro and J. C. Dainty �??A low cost adaptive optics system using a membrane mirror�?? Optics Express, 6(9):175-185, 2000.
[CrossRef] [PubMed]

Proc. SPIE (1)

M.J. Booth. �??Direct measurement of Zernike aberration modes with a modal wave front sensor.�?? Proc. SPIE, Advanced Wavefront Control: Methods, Devices, and Applications, 5162:79�??90, 2003.

Science (1)

W. J. Denk, J. P. Strickler, andW.W.Webb. �??Two-photon laser scanning fluorescence microscopy.�?? Science 248, 73�??76, 1990.
[CrossRef] [PubMed]

Other (7)

J. W. Hardy. �??Adaptive Optics for Astronomical Telescopes�??. Oxford University Press, 1998.

A. Dunn. �??Light scattering properties of cells�??. Dissertation, University of Texas, Austin, <a href= "http://www.nmr.mgh.harvard.edu/~adunn/papers/dissertation/node7.html">http://www.nmr.mgh.harvard.edu/~adunn/papers/dissertation/node7.html</a>, 1998.

J.B. Pawley. �??Handbook of Biological Confocal Microscopy�??. Plenum, New York, 1995.

T. Wilson and C.J.R. Sheppard. �??Theory and practice of Scanning Optical Microscopy�??. Academic Press, London, 1984.

D. C. Ghiglia and M. D. Pritt. �??Two Dimensional Phase Unwrapping. Theory, Algorithms and Software.�?? Wiley, 1998.

M. Born and E. Wolf. �??Principles of Optics�??. Pergamon Press, 6th edition, 1983.

J. E. Greivenkamp and J. H. Bruning. �??Phase shifting interferometry.�?? Optical shop testing, ed. by D. Malacara, Wiley New York, 1992.

Supplementary Material (1)

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Figures (6)

Fig. 1.
Fig. 1.

Phase stepping interferometer for aberration measurement (see text). (PBS - polarising beam splitter, BS - beam splitter.) The specimen is mounted between two opposing high NA lenses and scanned laterally by means of a computer controlled stage. Some intermediate lenses have been omitted for clarity.

Fig. 2.
Fig. 2.

Left: transmitted light image of the specimen number 5 (C. elegans). The red box indicates the scanned region of 50×50 µm. Right: video of the disturbance of the wavefront in the pupil plane of the lens as the focal spot scans across the specimen. Here the complex wavefront consisting of the amplitude A(r,θ) and the wrapped phase function ϕ(r,θ) is displayed. The color encodes the phase whereas the brightness corresponds to the amplitude of the wavefront. The green dot within the red frame in the lower left corner of the video indicates the relative position within the scanned area. (AVI-video file, size 2.4 MB.)

Fig. 3.
Fig. 3.

Specific interferogram examples from a particular position within the 16×16 grid that was recorded for each specimen. The color encodes the phase, the brightness corresponds to the amplitude. The numbers (1) to (6) are the specimen numbers listed in Table 1; The upper part shows the measured initial wavefront, the lower part a simulated correction of the Zernike modes up to i=22.

Fig. 4.
Fig. 4.

Zernike mode pseudo images of the specimen specimen number 5, C. elegans. The Zernike mode amplitudes Mi of the modes 2 to 12 (in Zernike mode units, see definition in equation 3) are depicted.

Fig. 5.
Fig. 5.

Mean and standard deviation of the Zernike mode amplitudes, in Zernike mode units, for the C. elegans - specimen 5. The modes 2 to 22 are shown.

Fig. 6.
Fig. 6.

Maps of the initial Strehl ratio Sini , the Strehl ratio Scorr after correction up to Zernike mode 22, and the derived signal correction factor Fsig . The distribution of Fsig is shown in a histogram for each of the specimens. The non-uniform histogram intervals are: A:[0, 1.5); B:[1.5, 3); C:[3, 5); D:[5, 10); E:[10, 40); F: [40,∞]. The vertical axis shows percentage of pixels within the range. The maximum of the range for each Fsig plot is shown below the plot and values larger than this maximum are shown in white.

Tables (3)

Tables Icon

Table 1. Specimen list (PBS : phosphate buffered saline, measurements approximate).

Tables Icon

Table 2. Correction benefit for different degrees of correction.

Tables Icon

Table 3. Correction benefit at different numerical apertures.

Equations (9)

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P ( r , θ ) = A ( r , θ ) exp ( j ψ ( r , θ ) )
ϕ ( r , θ ) = ψ ( r , θ ) mod 2 π .
M i = 1 π 0 1 0 2 π ψ ( r , θ ) Z i ( r , θ ) r d θ d r .
S = 0 1 0 2 π A ( r , θ ) exp ( j ψ ( r , θ ) ) r d r d θ 2 ( 0 1 0 2 π A ( r , θ ) r d r d θ ) 2 .
S 1 Var ( ψ ( r , θ ) ) = 1 i = 5 M i 2 .
S h ( 0 , 0 , 0 ) ,
I CFM ( x , y , z ) = h il 2 ( x , y , z ) .
I TPM ( x , y , z ) = h il 2 ( x , y , z ) .
F sig = ( S corr S ini ) 2 .

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