Abstract

We present a simple modification to a conventional wide-field fluorescence microscope that provides depth discrimination in thick tissues. The technique consists of illuminating a sample with a sequence of independent speckle patterns and displaying the rms of the resultant sequence of fluorescence images. The advantage of speckle illumination is that it provides diffraction-limited illumination granularity that is highly contrasted even in scattering media. We demonstrate quasi-confocal imaging in a mouse olfactory bulb labeled with green fluorescent protein.

© 2005 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. T. Wilson, R. Juskaitis, M. A. A. Neil, and M. Kozubek, Opt. Lett. 21, 1879 (1996).
    [CrossRef] [PubMed]
  3. M. A. A. Neil and T. Wilson, Opt. Lett. 22, 1905 (1997).
    [CrossRef]
  4. M. A. A. Neil, A. Squire, R. Juskaitis, P. I. H. Bastiaens, and T. Wilson, J. Microsc. 197, 1 (2000).
    [CrossRef] [PubMed]
  5. M. G. Somekh, C. W. See, and J. Goh, Opt. Commun. 174, 75 (2000).
    [CrossRef]
  6. M. C. Pitter, C. W. See, and M. G. Somekh, Opt. Lett. 29, 1200 (2004).
    [CrossRef] [PubMed]
  7. J. Walker, Opt. Commun. 189, 221 (2001).
    [CrossRef]
  8. S. Jiang and J. G. Walker, Opt. Commun. 238, 1 (2004).
    [CrossRef]
  9. J. W. Goodman, Statistical Optics (Wiley, 1985).

2004

2001

J. Walker, Opt. Commun. 189, 221 (2001).
[CrossRef]

2000

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

M. G. Somekh, C. W. See, and J. Goh, Opt. Commun. 174, 75 (2000).
[CrossRef]

1997

1996

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]

Goh, J.

M. G. Somekh, C. W. See, and J. Goh, Opt. Commun. 174, 75 (2000).
[CrossRef]

Goodman, J. W.

J. W. Goodman, Statistical Optics (Wiley, 1985).

Jiang, S.

S. Jiang and J. G. Walker, Opt. Commun. 238, 1 (2004).
[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]

T. Wilson, R. Juskaitis, M. A. A. Neil, and M. Kozubek, Opt. Lett. 21, 1879 (1996).
[CrossRef] [PubMed]

Kozubek, M.

Neil, M. A. A.

Pitter, M. C.

See, C. W.

M. C. Pitter, C. W. See, and M. G. Somekh, Opt. Lett. 29, 1200 (2004).
[CrossRef] [PubMed]

M. G. Somekh, C. W. See, and J. Goh, Opt. Commun. 174, 75 (2000).
[CrossRef]

Sheppard, C.

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

Somekh, M. G.

M. C. Pitter, C. W. See, and M. G. Somekh, Opt. Lett. 29, 1200 (2004).
[CrossRef] [PubMed]

M. G. Somekh, C. W. See, and J. Goh, Opt. Commun. 174, 75 (2000).
[CrossRef]

Squire, A.

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

Walker, J.

J. Walker, Opt. Commun. 189, 221 (2001).
[CrossRef]

Walker, J. G.

S. Jiang and J. G. Walker, Opt. Commun. 238, 1 (2004).
[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]

T. Wilson, R. Juskaitis, M. A. A. Neil, and M. Kozubek, Opt. Lett. 21, 1879 (1996).
[CrossRef] [PubMed]

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

J. Microsc.

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

Opt. Commun.

M. G. Somekh, C. W. See, and J. Goh, Opt. Commun. 174, 75 (2000).
[CrossRef]

J. Walker, Opt. Commun. 189, 221 (2001).
[CrossRef]

S. Jiang and J. G. Walker, Opt. Commun. 238, 1 (2004).
[CrossRef]

Opt. Lett.

Other

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

J. W. Goodman, Statistical Optics (Wiley, 1985).

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Figures (4)

Fig. 1
Fig. 1

Experimental setup. Laser light emanating from a rotating diffuser plate is imaged onto the back aperture of an objective and produces speckled illumination in the sample. The resultant fluorescence is imaged by a CCD camera. The inset depicts the laser light in the sample around the objective focal plane ( z = 0 ) . Expansion lenses f 1 and f 2 are chosen such that the laser beam fills the objective back aperture.

Fig. 2
Fig. 2

Theoretical (solid curve) and measured (dots) rms signal from a thin plane of Fluorescein (thickness less than 1.5 μ m ) as a function of the axial position z c .

Fig. 3
Fig. 3

Simultaneous (a) DSI and (b) conventional wide-field images of a fluorescent pollen grain. The field size is 40 μ m × 40 μ m .

Fig. 4
Fig. 4

Simultaneous (left) DSI and (right) conventional wide-field images of GFP targeted to presynaptic terminals of sensory neurons in the glomeruli of an excised (nonfixed) mouse olfactory bulb. The field size is 150 μ m × 150 μ m for each image.

Equations (7)

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I d ( ρ d ) = PSF det ( ρ d ρ , z ) C ( ρ , z ) I s ( ρ , z ) d 2 ρ d z ,
I s ( ρ , z ) I s ( ρ , z ) = I s 2 { 1 + PSF ill ( Δ ρ , 0 ) } ,
R det ( Δ ρ , z c ) = PSF det ( ρ d ρ , z c ) × PSF det ( ρ d ρ + Δ ρ , z c ) d 2 ρ
V ( ρ d ) = I s 2 C 2 R det ( Δ ρ , z c ) PSF ill ( Δ ρ , 0 ) d 2 Δ ρ .
PSF ( ρ , z ) = 1 1 + ζ 2 exp [ 2 ρ 2 w 0 2 ( 1 + ζ 2 ) ] ,
R det ( Δ ρ , z c ) = π w 0 2 4 ( 1 + ζ c 2 ) exp [ Δ ρ 2 w 0 2 ( 1 + ζ c 2 ) ] .
rms = I s C A 3 + 2 ζ c 2 .

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