Abstract

We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. The method involves acquiring two images, one with nonuniform illumination (in our case, speckle) and another with uniform illumination (in our case, randomized speckle). An evaluation of the local contrast in the speckle-illumination image provides an optically sectioned image with low resolution. This is complemented with high-resolution information obtained from the uniform-illumination image. A fusion of both images leads to a full resolution image that is optically sectioned across all spatial frequencies. This hybrid illumination method is fast, robust, and generalizable to a variety of illumination and imaging configurations.

© 2008 Optical Society of America

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References

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  1. N. Streibl, Optik (Stuttgart) 66, 341 (1984).
  2. J. B. Pawley, Handbook of Biological Confocal Microscopy (Plenum, 2006).
    [CrossRef]
  3. C. Ventalon and J. Mertz, Opt. Lett. 30, 3350 (2005).
    [CrossRef]
  4. C. Ventalon and J. Mertz, Opt. Express 14, 7198 (2006).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  6. J. W. Goodman, Speckle Phenomena: Theory and Applications (Roberts & Company, 2006).
  7. P. A. Stokseth, J. Opt. Soc. Am. 59, 1314 (1969).
    [CrossRef]

2007

2006

2005

1984

N. Streibl, Optik (Stuttgart) 66, 341 (1984).

1969

J. Opt. Soc. Am.

Opt. Express

Opt. Lett.

Optik (Stuttgart)

N. Streibl, Optik (Stuttgart) 66, 341 (1984).

Other

J. B. Pawley, Handbook of Biological Confocal Microscopy (Plenum, 2006).
[CrossRef]

J. W. Goodman, Speckle Phenomena: Theory and Applications (Roberts & Company, 2006).

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Figures (3)

Fig. 1
Fig. 1

Microscope layout. A laser beam is sent through a diffuser plate, which is imaged onto the back aperture of the objective. The resultant sample fluorescence is imaged onto a CCD camera. Two images are acquired: with the diffuser plate stationary (speckle illumination, middle panel), and in rapid motion (uniform illumination, right panel).

Fig. 2
Fig. 2

Hybrid imaging technique applied to a pair of fluorescent pollen grains (Carolina Biological Supply) located at slightly different depths. a, Speckle-illumination image ( I n ) . b, Uniform illumination image ( I u ) . c, Intermediate low-pass image ( I lp ) . d, Intermediate high-pass image ( I hp ) (note: negative values in this panel have been set to zero for ease of presentation). e, Composite full resolution optically sectioned image ( I ) . f, Extended focus image obtained from a maximum intensity projection of 80 slices separated by 0.5 μ m steps. The illumination source was a 488 nm argon laser ( 3 mW at sample). All images in this paper were acquired with an Olympus 60 × 0.9 NA water-immersion objective and an exposure time of 200 ms per image. Panel c was obtained by using sliding resolution areas of size A = 7 × 7   pixels , corresponding to 0.5 μ m 2 in the sample.

Fig. 3
Fig. 3

a, Integrated signal from a thin fluorescent plane as a function of defocus z. b, Uniform-illumination image of 1 μ m beads (Molecular Probes TetraSpeck Slide B). c, Processed hybrid image from speckle and uniform illumination.

Equations (5)

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C N ( ρ ) = σ n ( ρ ) A I n ( ρ ) A ,
I n ( ρ ) [ O A + δ O ( ρ ) ] [ S A + δ S ( ρ ) ] ,
I u ( ρ ) [ O A + δ O ( ρ ) ] S A .
C N 2 C O 2 + C S 2 + C O 2 C S 2 ,
I ( ρ ) = η I lp ( ρ ) + I hp ( ρ ) .

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