Abstract

The diagnostic utility of a conventional transillumination microscope, the most common imaging modality in clinical use today, is limited by the microscope’s resolution. It is, however, possible to achieve lateral resolution well beyond the classical limit by using laterally structured illumination in a wide-field, nonconfocal microscope. In this method, the spatially modulated illumination (SMI) makes high-resolution information that is normally inaccessible visible in the observed image. Previously presented SMI microscopy systems operated in epifluorescence mode. We describe the design, construction, and testing of a novel transillumination SMI microscope. As transillumination is necessary for most medical applications, such as histopathologic evaluation of biopsy tissue and chromosomal analysis, such a system should have a significant diagnostic effect.

© 2005 Optical Society of America

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References

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  1. S. W. Hell, M. Schrader, and T. M. Van Der Voort, J. Microsc. 187, 1 (1997).
    [CrossRef] [PubMed]
  2. T. A. Klar and S. W. Hell, Opt. Lett. 24, 954 (1999).
    [CrossRef]
  3. J. B. Pawley, ed., Handbook of Biological Confocal Microscopy, 2nd ed. (Plenum, 1995).
    [CrossRef]
  4. M. G. L. Gustafson, J. Microsc.(Oxford)  198, 82 (2000).
    [CrossRef]
  5. R. Heintzmann and T. M. Jovin, J. Opt. Soc. Am. A 19, 1599 (2002).
    [CrossRef]
  6. M. A. A. Neil, R. Juskaitis, and T. Wilson, Opt. Lett. 22, 1905 (1997).
    [CrossRef]
  7. R. Heintzmann and C. Cremer, Proc. SPIE 3568, 185 (1998).
    [CrossRef]

2002 (1)

2000 (1)

M. G. L. Gustafson, J. Microsc.(Oxford)  198, 82 (2000).
[CrossRef]

1999 (1)

1998 (1)

R. Heintzmann and C. Cremer, Proc. SPIE 3568, 185 (1998).
[CrossRef]

1997 (2)

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

S. W. Hell, M. Schrader, and T. M. Van Der Voort, J. Microsc. 187, 1 (1997).
[CrossRef] [PubMed]

Cremer, C.

R. Heintzmann and C. Cremer, Proc. SPIE 3568, 185 (1998).
[CrossRef]

Gustafson, M. G. L.

M. G. L. Gustafson, J. Microsc.(Oxford)  198, 82 (2000).
[CrossRef]

Heintzmann, R.

R. Heintzmann and T. M. Jovin, J. Opt. Soc. Am. A 19, 1599 (2002).
[CrossRef]

R. Heintzmann and C. Cremer, Proc. SPIE 3568, 185 (1998).
[CrossRef]

Hell, S. W.

T. A. Klar and S. W. Hell, Opt. Lett. 24, 954 (1999).
[CrossRef]

S. W. Hell, M. Schrader, and T. M. Van Der Voort, J. Microsc. 187, 1 (1997).
[CrossRef] [PubMed]

Jovin, T. M.

Juskaitis, R.

Klar, T. A.

Neil, M. A. A.

Schrader, M.

S. W. Hell, M. Schrader, and T. M. Van Der Voort, J. Microsc. 187, 1 (1997).
[CrossRef] [PubMed]

Van Der Voort, T. M.

S. W. Hell, M. Schrader, and T. M. Van Der Voort, J. Microsc. 187, 1 (1997).
[CrossRef] [PubMed]

Wilson, T.

J. Microsc. (2)

S. W. Hell, M. Schrader, and T. M. Van Der Voort, J. Microsc. 187, 1 (1997).
[CrossRef] [PubMed]

M. G. L. Gustafson, J. Microsc.(Oxford)  198, 82 (2000).
[CrossRef]

J. Opt. Soc. Am. A (1)

Opt. Lett. (2)

Proc. SPIE (1)

R. Heintzmann and C. Cremer, Proc. SPIE 3568, 185 (1998).
[CrossRef]

Other (1)

J. B. Pawley, ed., Handbook of Biological Confocal Microscopy, 2nd ed. (Plenum, 1995).
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Simplified schematic of the SMI microscope, consisting of a white-light source (S), a bandpass green filter (F), a grating (G), an illumination objective ( L 1 ) , an imaging objective ( L 2 ) , and a CCD camera (C). The sample was placed on plane S.

Fig. 2
Fig. 2

A, Raw image from the SMI microscope with vertical illumination. B, Horizontal cross section along the dashed line in A.

Fig. 3
Fig. 3

Processed images of polystyrene microspheres embedded in UV-curing glue. A, Average of the images taken under all illumination phases, which corresponds to a conventional microscope image. C, Processed image from the SMI microscope. B, D, Associated two-dimensional spectra. The black arrows in D highlight the increase in high-frequency spectral content of the SMI image. The black arrows in A and C point to the image of a microsphere where the resolution enhancement is evident; the white arrows indicate phase mismatch artifacts.

Fig. 4
Fig. 4

Cross sections from processed images of polystyrene microspheres taken with A, a standard microscope and B, a SMI microscope. The arrows point to areas where the resolution enhancement is evident.

Equations (8)

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i ( x , y , θ ) = [ f ( x , y ) ( A cos ( ω o x + θ ) + B ) ] h ( x , y ) ,
I 0 ( u , v ) = I [ i ( x , y , 0 ° ) ] = [ A F ( u ω o , v ) + A F ( u + ω o , v ) + B F ( u , v ) ] H ( u , v ) ,
I 180 ( u , v ) = I [ i ( x , y , 180 ° ) ] = [ A F ( u ω o , v ) A F ( u + ω o , v ) + B F ( u , v ) ] H ( u , v ) ,
I 90 ( u , v ) = I [ i ( x , y , 90 ° ) ] = [ j A F ( u ω o , v ) j A F ( u + ω o , v ) + B F ( u , v ) ] H ( u , v ) ,
I 270 ( u , v ) = I [ i ( x , y , 270 ° ) ] = [ j A F ( u ω o , v ) + j A F ( u + ω o , v ) + B F ( u , v ) ] H ( u , v ) ,
I 0 ( u , v ) I 180 ( u , v ) = [ 2 A F ( u + ω o , v ) + 2 A F ( u + ω o , v ) ] H ( u , v ) .
I 90 ( u , v ) I 270 ( u , v ) = [ 2 j A F ( u + ω o , v ) 2 j A F ( u + ω o , v ) ] H ( u , v ) .
I ( u , v ) = 4 A F ( u + ω o , v ) H ( u , v ) .

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