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Characterizing specimen induced aberrations for high NA adaptive optical microscopy

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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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Supplementary Material (1)

Media 1: AVI (2414 KB)     

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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)

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

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Table 2. Correction benefit for different degrees of correction.

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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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