Abstract

We demonstrate a quantitative optical scatter imaging (OSI) technique, based on Fourier filtering, for detecting alterations in the size of particles with wavelength-scale dimensions. We generate our scatter image by taking the ratio of images collected at high and low numerical aperture in central dark-field microscopy. Such an image spatially encodes the ratio of wide to narrow angle scatter and hence provides a measure of local particle size. We validated OSI on sphere suspensions and live cells. In live cells, OSI revealed biochemically induced morphological changes that were not apparent in unprocessed differential interference contrast images. Unlike high-resolution imaging methods, OSI can provide size information for particles smaller than the camera’s spatial resolution.

© 2001 Optical Society of America

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  10. A solvent sample consisting of the medium in which the sample was prepared served to provide the background scatter signal owing to the microscope optics.
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    [CrossRef] [PubMed]
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    [CrossRef]

2000

L. J. Johnson, D. F. Hanley, and N. V. Thakor, J. Neurosci. Methods 98, 21 (2000).
[CrossRef] [PubMed]

1999

1998

1997

M. G. Vander-Heiden, N. S. Chandel, E. K. Williamson, P. T. Shumacker, and C. B. Thompson, Cell 91, 627 (1997).
[CrossRef]

A. Dunn, C. Smithpeter, A. J. Welch, and R. Richards-Kortum, J. Biomed. Opt. 2, 262 (1997).
[CrossRef] [PubMed]

1995

B. Beauvoit, S. M. Evans, T. W. Jenkins, E. E. Miller, and B. Chance, Anal. Biochem. 226, 167 (1995).
[CrossRef] [PubMed]

1993

P. J. Wyatt, Anal. Chim. Acta 272, 1 (1993).
[CrossRef]

1982

D. E. Burger, J. H. Jett, and P. F. Mullaney, Cytometry 2, 327 (1982).
[CrossRef] [PubMed]

1978

Achatz, M.

Ashkenazy, Y.

Beauvoit, B.

B. Beauvoit, S. M. Evans, T. W. Jenkins, E. E. Miller, and B. Chance, Anal. Biochem. 226, 167 (1995).
[CrossRef] [PubMed]

Burger, D. E.

D. E. Burger, J. H. Jett, and P. F. Mullaney, Cytometry 2, 327 (1982).
[CrossRef] [PubMed]

Chance, B.

B. Beauvoit, S. M. Evans, T. W. Jenkins, E. E. Miller, and B. Chance, Anal. Biochem. 226, 167 (1995).
[CrossRef] [PubMed]

Chandel, N. S.

M. G. Vander-Heiden, N. S. Chandel, E. K. Williamson, P. T. Shumacker, and C. B. Thompson, Cell 91, 627 (1997).
[CrossRef]

Deutsch, M.

Drezek, R.

Dunn, A.

R. Drezek, A. Dunn, and R. Richards-Kortum, Appl. Opt. 38, 3651 (1999).
[CrossRef]

A. Dunn, C. Smithpeter, A. J. Welch, and R. Richards-Kortum, J. Biomed. Opt. 2, 262 (1997).
[CrossRef] [PubMed]

Eick, A. A.

Evans, S. M.

B. Beauvoit, S. M. Evans, T. W. Jenkins, E. E. Miller, and B. Chance, Anal. Biochem. 226, 167 (1995).
[CrossRef] [PubMed]

Freyer, J. P.

Hanley, D. F.

L. J. Johnson, D. F. Hanley, and N. V. Thakor, J. Neurosci. Methods 98, 21 (2000).
[CrossRef] [PubMed]

Hielscher, A. H.

Jenkins, T. W.

B. Beauvoit, S. M. Evans, T. W. Jenkins, E. E. Miller, and B. Chance, Anal. Biochem. 226, 167 (1995).
[CrossRef] [PubMed]

Jett, J. H.

D. E. Burger, J. H. Jett, and P. F. Mullaney, Cytometry 2, 327 (1982).
[CrossRef] [PubMed]

Johnson, L. J.

L. J. Johnson, D. F. Hanley, and N. V. Thakor, J. Neurosci. Methods 98, 21 (2000).
[CrossRef] [PubMed]

Johnson, T. M.

Kaplan, P. D.

Marx, J.

For example, see L. J. Miller and J. Marx, Science 281, 1301 (1998).
[CrossRef]

Miller, E. E.

B. Beauvoit, S. M. Evans, T. W. Jenkins, E. E. Miller, and B. Chance, Anal. Biochem. 226, 167 (1995).
[CrossRef] [PubMed]

Miller, L. J.

For example, see L. J. Miller and J. Marx, Science 281, 1301 (1998).
[CrossRef]

Mourant, J. R.

Mullaney, P. F.

D. E. Burger, J. H. Jett, and P. F. Mullaney, Cytometry 2, 327 (1982).
[CrossRef] [PubMed]

Richards-Kortum, R.

R. Drezek, A. Dunn, and R. Richards-Kortum, Appl. Opt. 38, 3651 (1999).
[CrossRef]

A. Dunn, C. Smithpeter, A. J. Welch, and R. Richards-Kortum, J. Biomed. Opt. 2, 262 (1997).
[CrossRef] [PubMed]

Schiffer, Z.

Seelen, W.

Seger, G.

Shen, D.

Shumacker, P. T.

M. G. Vander-Heiden, N. S. Chandel, E. K. Williamson, P. T. Shumacker, and C. B. Thompson, Cell 91, 627 (1997).
[CrossRef]

Smithpeter, C.

A. Dunn, C. Smithpeter, A. J. Welch, and R. Richards-Kortum, J. Biomed. Opt. 2, 262 (1997).
[CrossRef] [PubMed]

Thakor, N. V.

L. J. Johnson, D. F. Hanley, and N. V. Thakor, J. Neurosci. Methods 98, 21 (2000).
[CrossRef] [PubMed]

Thompson, C. B.

M. G. Vander-Heiden, N. S. Chandel, E. K. Williamson, P. T. Shumacker, and C. B. Thompson, Cell 91, 627 (1997).
[CrossRef]

Tirosh, R.

Trappe, V.

Turke, B.

van de Hulst, H. C.

H. C. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1981), Chap.  9.

Vander-Heiden, M. G.

M. G. Vander-Heiden, N. S. Chandel, E. K. Williamson, P. T. Shumacker, and C. B. Thompson, Cell 91, 627 (1997).
[CrossRef]

Weitz, D. A.

Welch, A. J.

A. Dunn, C. Smithpeter, A. J. Welch, and R. Richards-Kortum, J. Biomed. Opt. 2, 262 (1997).
[CrossRef] [PubMed]

Williamson, E. K.

M. G. Vander-Heiden, N. S. Chandel, E. K. Williamson, P. T. Shumacker, and C. B. Thompson, Cell 91, 627 (1997).
[CrossRef]

Wyatt, P. J.

P. J. Wyatt, Anal. Chim. Acta 272, 1 (1993).
[CrossRef]

Anal. Biochem.

B. Beauvoit, S. M. Evans, T. W. Jenkins, E. E. Miller, and B. Chance, Anal. Biochem. 226, 167 (1995).
[CrossRef] [PubMed]

Anal. Chim. Acta

P. J. Wyatt, Anal. Chim. Acta 272, 1 (1993).
[CrossRef]

Appl. Opt.

Cell

M. G. Vander-Heiden, N. S. Chandel, E. K. Williamson, P. T. Shumacker, and C. B. Thompson, Cell 91, 627 (1997).
[CrossRef]

Cytometry

D. E. Burger, J. H. Jett, and P. F. Mullaney, Cytometry 2, 327 (1982).
[CrossRef] [PubMed]

J. Biomed. Opt.

A. Dunn, C. Smithpeter, A. J. Welch, and R. Richards-Kortum, J. Biomed. Opt. 2, 262 (1997).
[CrossRef] [PubMed]

J. Neurosci. Methods

L. J. Johnson, D. F. Hanley, and N. V. Thakor, J. Neurosci. Methods 98, 21 (2000).
[CrossRef] [PubMed]

Science

For example, see L. J. Miller and J. Marx, Science 281, 1301 (1998).
[CrossRef]

Other

H. C. van de Hulst, Light Scattering by Small Particles (Dover, New York, 1981), Chap.  9.

A solvent sample consisting of the medium in which the sample was prepared served to provide the background scatter signal owing to the microscope optics.

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Figures (3)

Fig. 1
Fig. 1

OSI setup. F, objective’s back focal plane; F and F are conjugate Fourier planes. The scattered light (gray beam) is used to image the specimen on the CCD, and the transmitted light (black ray traces) is blocked at F. The inset shows the scattered angles passed at the high- and low-NA settings.

Fig. 2
Fig. 2

OSI images and measurement of the OSIR in aqueous suspensions of polystyrene spheres. Experimental data (open circles, refractive-index ratio m=1.2), theoretical predictions (solid curve, m=1.2; dashed curve, m=1.06). The data in the graph are the mean plus or minus the standard deviation of pixel values in the images shown on the left.

Fig. 3
Fig. 3

A, DIC and OSI images of a representative cell undergoing apoptosis. Top panels, normal cell; bottom panels, the cell 37  min after apoptosis induction. C, cytoplasm; N, nucleus. B, Time course of the OSIR in the cytoplasm of cells undergoing apoptosis (filled circles) compared with control cells kept in the normal growth medium (open circles). Apoptosis was induced at t=0 (arrow). The error bars represent the 95% confidence interval of the mean. The OSIR was normalized to values at t=-7min.

Equations (1)

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OSIR=ϕ=0360°θ=2°67°Fθ,ϕsinθdθdϕϕ=0360°θ=2°10°Fθ,ϕsinθdθdϕ,

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