Abstract

A new type of computer-controlled optical scanning, high-magnification imaging system with a large field of view is described that overcomes the commonly believed incompatibility of achieving both high magnification and a large field of view. The new system incorporates galvanometer scanners, a CCD camera, and a high-brightness LED source for the fast acquisition of a large number of a high-resolution segmented tile images with a magnification of 800× for each tile. The captured segmented tile images are combined to create an effective enlarged view of a target totaling 1.6  mm×1 .2   mm in area. The speed and sensitivity of the system make it suitable for high-resolution imaging and monitoring of a small segmented area of 320μm×240  μm with 4  μm resolution. Each tile segment of the target can be zoomed up without loss of the high resolution. This new microscope imaging system gives both high magnification and a large field of view. This microscope can be utilized in medicine, biology, semiconductor inspection, device analysis, and quality control.

© 2006 Optical Society of America

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  1. R. W. E. Clarkson, J. L. Heeley, R. Chapman, F. Aillet, R. T. Hay, A. Wyllie, and C. J. Watson, "NF-B inhibits apoptosis in murine mammary epithelia," J. Biol. Chem. 275, 12, 737-12, 742 (2000).
    [CrossRef]
  2. D. Soldati, J. F. Dubremetzb, and M. Lebrunc, "Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii," Int. J. Parasitol. 31, 1293-1302 (2001).
    [CrossRef] [PubMed]
  3. J. Cox and C. J. R. Sheppard, "Scanning optical microscope incorporating a digital framestore and microcomputer," Appl. Opt. 22, 1474-1478 (1983).
    [CrossRef] [PubMed]
  4. D. K. Hamilton and T. Wilson, "Scanning optical microscopy by objective lens scanning," J. Phys. E 19, 52-54 (1986).
    [CrossRef]
  5. P. C. Goodwin and A. Kwiatkowski, "Absolute positioning control: applications in imaging, pharmacological delivery, and functional genomics with live cells," GIT Imag. Microsc. 2, 50-52 (2000).
  6. For each image tile, the main operations and the time that they consume are scanning (2 ms), image acquisition (35 ms), and image saving and imaging compression and saving (55 ms). Stitching and displaying of the full image time take ∼200 ms. The entire imaging and processing time for the full 25-tile image is 25 × 0.092 + 0.2 = 2.5 s.
  7. K. H. Kim, C. Buehler, and P. T. C. So, "High-speed, two-photon scanning microscope," Appl. Opt. 38, 6004-6009 (1999).
    [CrossRef]
  8. Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, "Second-harmonic tomography of tissues," Opt. Lett. 22, 1323-1325 (1997).
    [CrossRef]
  9. Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc. Natl. Acad. Sci. (USA) 96, 10, 854-10, 856 (1999).
    [CrossRef]

2001 (1)

D. Soldati, J. F. Dubremetzb, and M. Lebrunc, "Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii," Int. J. Parasitol. 31, 1293-1302 (2001).
[CrossRef] [PubMed]

2000 (2)

P. C. Goodwin and A. Kwiatkowski, "Absolute positioning control: applications in imaging, pharmacological delivery, and functional genomics with live cells," GIT Imag. Microsc. 2, 50-52 (2000).

R. W. E. Clarkson, J. L. Heeley, R. Chapman, F. Aillet, R. T. Hay, A. Wyllie, and C. J. Watson, "NF-B inhibits apoptosis in murine mammary epithelia," J. Biol. Chem. 275, 12, 737-12, 742 (2000).
[CrossRef]

1999 (2)

K. H. Kim, C. Buehler, and P. T. C. So, "High-speed, two-photon scanning microscope," Appl. Opt. 38, 6004-6009 (1999).
[CrossRef]

Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc. Natl. Acad. Sci. (USA) 96, 10, 854-10, 856 (1999).
[CrossRef]

1997 (1)

1986 (1)

D. K. Hamilton and T. Wilson, "Scanning optical microscopy by objective lens scanning," J. Phys. E 19, 52-54 (1986).
[CrossRef]

1983 (1)

Aillet, F.

R. W. E. Clarkson, J. L. Heeley, R. Chapman, F. Aillet, R. T. Hay, A. Wyllie, and C. J. Watson, "NF-B inhibits apoptosis in murine mammary epithelia," J. Biol. Chem. 275, 12, 737-12, 742 (2000).
[CrossRef]

Alfano, R. R.

Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc. Natl. Acad. Sci. (USA) 96, 10, 854-10, 856 (1999).
[CrossRef]

Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, "Second-harmonic tomography of tissues," Opt. Lett. 22, 1323-1325 (1997).
[CrossRef]

Buehler, C.

Chapman, R.

R. W. E. Clarkson, J. L. Heeley, R. Chapman, F. Aillet, R. T. Hay, A. Wyllie, and C. J. Watson, "NF-B inhibits apoptosis in murine mammary epithelia," J. Biol. Chem. 275, 12, 737-12, 742 (2000).
[CrossRef]

Clarkson, R. W. E.

R. W. E. Clarkson, J. L. Heeley, R. Chapman, F. Aillet, R. T. Hay, A. Wyllie, and C. J. Watson, "NF-B inhibits apoptosis in murine mammary epithelia," J. Biol. Chem. 275, 12, 737-12, 742 (2000).
[CrossRef]

Cox, J.

Dubremetzb, J. F.

D. Soldati, J. F. Dubremetzb, and M. Lebrunc, "Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii," Int. J. Parasitol. 31, 1293-1302 (2001).
[CrossRef] [PubMed]

Goodwin, P. C.

P. C. Goodwin and A. Kwiatkowski, "Absolute positioning control: applications in imaging, pharmacological delivery, and functional genomics with live cells," GIT Imag. Microsc. 2, 50-52 (2000).

Guo, Y.

Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc. Natl. Acad. Sci. (USA) 96, 10, 854-10, 856 (1999).
[CrossRef]

Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, "Second-harmonic tomography of tissues," Opt. Lett. 22, 1323-1325 (1997).
[CrossRef]

Hamilton, D. K.

D. K. Hamilton and T. Wilson, "Scanning optical microscopy by objective lens scanning," J. Phys. E 19, 52-54 (1986).
[CrossRef]

Harris, D.

Hay, R. T.

R. W. E. Clarkson, J. L. Heeley, R. Chapman, F. Aillet, R. T. Hay, A. Wyllie, and C. J. Watson, "NF-B inhibits apoptosis in murine mammary epithelia," J. Biol. Chem. 275, 12, 737-12, 742 (2000).
[CrossRef]

Heeley, J. L.

R. W. E. Clarkson, J. L. Heeley, R. Chapman, F. Aillet, R. T. Hay, A. Wyllie, and C. J. Watson, "NF-B inhibits apoptosis in murine mammary epithelia," J. Biol. Chem. 275, 12, 737-12, 742 (2000).
[CrossRef]

Ho, P. P.

Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc. Natl. Acad. Sci. (USA) 96, 10, 854-10, 856 (1999).
[CrossRef]

Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, "Second-harmonic tomography of tissues," Opt. Lett. 22, 1323-1325 (1997).
[CrossRef]

Kim, K. H.

Kwiatkowski, A.

P. C. Goodwin and A. Kwiatkowski, "Absolute positioning control: applications in imaging, pharmacological delivery, and functional genomics with live cells," GIT Imag. Microsc. 2, 50-52 (2000).

Lebrunc, M.

D. Soldati, J. F. Dubremetzb, and M. Lebrunc, "Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii," Int. J. Parasitol. 31, 1293-1302 (2001).
[CrossRef] [PubMed]

Liu, F.

Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc. Natl. Acad. Sci. (USA) 96, 10, 854-10, 856 (1999).
[CrossRef]

Y. Guo, P. P. Ho, H. Savage, D. Harris, P. Sacks, S. Schantz, F. Liu, N. Zhadin, and R. R. Alfano, "Second-harmonic tomography of tissues," Opt. Lett. 22, 1323-1325 (1997).
[CrossRef]

Sacks, P.

Savage, H.

Savage, H. E.

Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc. Natl. Acad. Sci. (USA) 96, 10, 854-10, 856 (1999).
[CrossRef]

Schantz, S.

Schantz, S. P.

Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc. Natl. Acad. Sci. (USA) 96, 10, 854-10, 856 (1999).
[CrossRef]

Sheppard, C. J. R.

So, P. T. C.

Soldati, D.

D. Soldati, J. F. Dubremetzb, and M. Lebrunc, "Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii," Int. J. Parasitol. 31, 1293-1302 (2001).
[CrossRef] [PubMed]

Watson, C. J.

R. W. E. Clarkson, J. L. Heeley, R. Chapman, F. Aillet, R. T. Hay, A. Wyllie, and C. J. Watson, "NF-B inhibits apoptosis in murine mammary epithelia," J. Biol. Chem. 275, 12, 737-12, 742 (2000).
[CrossRef]

Wilson, T.

D. K. Hamilton and T. Wilson, "Scanning optical microscopy by objective lens scanning," J. Phys. E 19, 52-54 (1986).
[CrossRef]

Wyllie, A.

R. W. E. Clarkson, J. L. Heeley, R. Chapman, F. Aillet, R. T. Hay, A. Wyllie, and C. J. Watson, "NF-B inhibits apoptosis in murine mammary epithelia," J. Biol. Chem. 275, 12, 737-12, 742 (2000).
[CrossRef]

Zhadin, N.

Appl. Opt. (2)

GIT Imag. Microsc. (1)

P. C. Goodwin and A. Kwiatkowski, "Absolute positioning control: applications in imaging, pharmacological delivery, and functional genomics with live cells," GIT Imag. Microsc. 2, 50-52 (2000).

Int. J. Parasitol. (1)

D. Soldati, J. F. Dubremetzb, and M. Lebrunc, "Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii," Int. J. Parasitol. 31, 1293-1302 (2001).
[CrossRef] [PubMed]

J. Biol. Chem. (1)

R. W. E. Clarkson, J. L. Heeley, R. Chapman, F. Aillet, R. T. Hay, A. Wyllie, and C. J. Watson, "NF-B inhibits apoptosis in murine mammary epithelia," J. Biol. Chem. 275, 12, 737-12, 742 (2000).
[CrossRef]

J. Phys. E (1)

D. K. Hamilton and T. Wilson, "Scanning optical microscopy by objective lens scanning," J. Phys. E 19, 52-54 (1986).
[CrossRef]

Opt. Lett. (1)

Proc. Natl. Acad. Sci. (1)

Y. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc. Natl. Acad. Sci. (USA) 96, 10, 854-10, 856 (1999).
[CrossRef]

Other (1)

For each image tile, the main operations and the time that they consume are scanning (2 ms), image acquisition (35 ms), and image saving and imaging compression and saving (55 ms). Stitching and displaying of the full image time take ∼200 ms. The entire imaging and processing time for the full 25-tile image is 25 × 0.092 + 0.2 = 2.5 s.

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Figures (8)

Fig. 1
Fig. 1

Schematic diagram of the optical layout of the scanning microscope: lenses L1 (f = 8 mm, D = 8 mm), L2 (f = 50 mm, D = 25.4 mm), L3 (f = −9 mm, D=9 mm), L4 (f = 30 mm, D = 15 mm), Lf (f = 30 mm, D = 15 mm); LED, light-emitting diode (470 nm).

Fig. 2
Fig. 2

Software flow chart of the computer-controlling system.

Fig. 3
Fig. 3

USAF 1951 target and specification table.

Fig. 4
Fig. 4

Twenty-five segmented tile images of the USAF target.

Fig. 5
Fig. 5

Image of the 12th segmented tile group of the USAF target with an area of 320 μm × 240 μm.

Fig. 6
Fig. 6

Full reassembled segmented image of 25 tiles of the USAF target of an area of 1.6 mm × 1.2 mm.

Fig. 7
Fig. 7

Full reassembled segmented image of 25 tiles of mouse brain tissue.

Fig. 8
Fig. 8

Zoomed image of the area of interest of the mouse brain tissue in the second segmented tile.

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