Abstract

Dynamic speckle illumination (DSI) provides a simple and robust technique to obtain fluorescence depth sectioning with a widefield microscope. We report a significant improvement to DSI microscopy based on a statistical image-processing algorithm that incorporates spatial wavelet prefiltering. The resultant gain in sectioning strength leads to a fundamentally improved scaling law for the out-of-focus background rejection.

© 2007 Optical Society of America

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References

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  1. T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984).
  2. G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (2000).
    [CrossRef]
  3. R. Juskaitis, T. Wilson, M. A. A. Neil, and M. Kozubek, Nature 383, 804 (1996).
    [CrossRef] [PubMed]
  4. P. J. Verveer, G. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, J. Microsc. 189, 192 (1998).
    [CrossRef]
  5. M. A. A. Neil and T. Wilson, Opt. Lett. 22, 1905 (1997).
    [CrossRef]
  6. M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, J. Microsc. 197, 1 (2000).
    [CrossRef] [PubMed]
  7. C. Ventalon and J. Mertz, Opt. Lett. 30, 3350 (2005).
    [CrossRef]
  8. J. W. Goodman, Speckle Phenomena: Theory and Applications (Roberts & Company, 2006).
  9. C. Ventalon and J. Mertz, Opt. Express 14, 7198 (2006).
    [CrossRef] [PubMed]

2006 (1)

2005 (1)

2000 (2)

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (2000).
[CrossRef]

M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, J. Microsc. 197, 1 (2000).
[CrossRef] [PubMed]

1998 (1)

P. J. Verveer, G. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, J. Microsc. 189, 192 (1998).
[CrossRef]

1997 (1)

1996 (1)

R. Juskaitis, T. Wilson, M. A. A. Neil, and M. Kozubek, Nature 383, 804 (1996).
[CrossRef] [PubMed]

Bastiaens, P. I. H.

M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, J. Microsc. 197, 1 (2000).
[CrossRef] [PubMed]

Corle, T. R.

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (2000).
[CrossRef]

Goodman, J. W.

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

Hanley, G. S.

P. J. Verveer, G. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, J. Microsc. 189, 192 (1998).
[CrossRef]

Jovin, T. M.

P. J. Verveer, G. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, J. Microsc. 189, 192 (1998).
[CrossRef]

Juskaitis, R.

M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, J. Microsc. 197, 1 (2000).
[CrossRef] [PubMed]

R. Juskaitis, T. Wilson, M. A. A. Neil, and M. Kozubek, Nature 383, 804 (1996).
[CrossRef] [PubMed]

Kino, G. S.

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (2000).
[CrossRef]

Kozubek, M.

R. Juskaitis, T. Wilson, M. A. A. Neil, and M. Kozubek, Nature 383, 804 (1996).
[CrossRef] [PubMed]

Mertz, J.

Neil, M. A. A.

M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, J. Microsc. 197, 1 (2000).
[CrossRef] [PubMed]

M. A. A. Neil and T. Wilson, Opt. Lett. 22, 1905 (1997).
[CrossRef]

R. Juskaitis, T. Wilson, M. A. A. Neil, and M. Kozubek, Nature 383, 804 (1996).
[CrossRef] [PubMed]

Sheppard, C.

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984).

Squire, A.

M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, J. Microsc. 197, 1 (2000).
[CrossRef] [PubMed]

Van Vliet, L. J.

P. J. Verveer, G. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, J. Microsc. 189, 192 (1998).
[CrossRef]

Ventalon, C.

Verbeek, P. W.

P. J. Verveer, G. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, J. Microsc. 189, 192 (1998).
[CrossRef]

Verveer, P. J.

P. J. Verveer, G. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, J. Microsc. 189, 192 (1998).
[CrossRef]

Wilson, T.

M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, J. Microsc. 197, 1 (2000).
[CrossRef] [PubMed]

M. A. A. Neil and T. Wilson, Opt. Lett. 22, 1905 (1997).
[CrossRef]

R. Juskaitis, T. Wilson, M. A. A. Neil, and M. Kozubek, Nature 383, 804 (1996).
[CrossRef] [PubMed]

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984).

Xiao, G. Q.

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (2000).
[CrossRef]

Appl. Phys. Lett. (1)

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (2000).
[CrossRef]

J. Microsc. (2)

P. J. Verveer, G. S. Hanley, P. W. Verbeek, L. J. Van Vliet, and T. M. Jovin, J. Microsc. 189, 192 (1998).
[CrossRef]

M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, J. Microsc. 197, 1 (2000).
[CrossRef] [PubMed]

Nature (1)

R. Juskaitis, T. Wilson, M. A. A. Neil, and M. Kozubek, Nature 383, 804 (1996).
[CrossRef] [PubMed]

Opt. Express (1)

Opt. Lett. (2)

Other (2)

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

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984).

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Figures (3)

Fig. 1
Fig. 1

a, DSI microscope layout. MO, microscope objective; BS, dichroic beamsplitter. b–f, Images of a fluorescent pollen grain obtained with an Olympus 40 × , 1.3 NA oil objective. b, Widefield image obtained with an arbitrary speckle pattern. Spatial prefiltering is performed by convolving with a 2D wavelet filter, c, that removes uniform background and extracts in-focus speckle grains, d. Final DSI images are obtained by computing the rms of an image sequence illuminated with random speckle patterns for each image, without, e, or with, f, wavelet prefiltering. DSI images e and f are obtained from the same data set. Scale bar, 2 μ m . For the gradients in images b–f, green and orange (bottom of scale d) correspond to positive and negative values, respectively; black corresponds to zero. g, Temporal (blue, topmost plots) and spatiotemporal (red) DSI signals measured from a thin fluorescent plane as a function of defocus z, acquired with an Olympus 40 × , 0.65 NA objective. Traces are shown on linear (left) and logarithmic scales (right). In the right-hand panel, the experimental traces are fitted with straight lines of slope 1 and 3 2 for temporal and spatiotemporal DSI (respectively).

Fig. 2
Fig. 2

Images of an excised mouse olfactory bulb labeled with synapto-pHluorin, a pH-sensitive GFP targeted to presynaptic terminals of sensory neurons, acquired with an Olympus, 20 × , 0.95 NA water objective. Axial sections of a glomerulus are acquired at depths 50 μ m , a–c, and 90 μ m , d–f, below the tissue surface, obtained with standard widefield, a,d, and DSI microscopy using temporal, b,e, and spatiotemporal, c,f, postprocessing with 10 images (same data sets for each row). Standard widefield images are obtained from the average (as opposed to the rms) of the DSI raw image sequence. Scale bar, 20 μ m .

Fig. 3
Fig. 3

Axon fibers in a GFP-labeled olfactory bulb (same sample and experimental conditions as in Fig. 2). Images are obtained with standard widefield, a, and DSI microscopy using temporal, b, and spatiotemporal, c,d, postprocessing, using 10, a, b, c, and 30, d, raw images. The exposure time per raw image was 500 ms , imposed by the low maximum output power delivered by our laser ( 3 mW at the sample). Scale bar, 20 μ m .

Equations (5)

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I d ( r d ) = PSF d ( r d r ) C ( r ) I s ( r ) d 3 r ,
I s ( ρ , z ) I s ( ρ , z ) ¯ = I s ¯ 2 [ 1 + PSF i ( ρ ρ , 0 ) ] I s ¯ 2 [ 1 + A s δ ( ρ ρ ) ] ,
I s ( r ) I s ( r ) ¯ I s ¯ 2 [ 1 + A s L s δ ( r r ) ] .
I DSI 2 ( r d ) I s ¯ 2 A s L s PSF d ( r d r ) 2 C ( r ) 2 d 3 r .
PSF w ( ρ , z ) = W ( ρ ) PSF d ( ρ ρ , z ) d 2 ρ ,

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