Abstract

Using a line scan camera and an acousto-optic deflector (AOD), we constructed a high-speed confocal laser line-scanning microscope that can generate confocal images (512×512 pixels) with up to 191 frames/s without any mechanically moving parts. The line scanner consists of an AOD and a cylindrical lens, which creates a line focus sweeping over the sample. The measured resolutions in z (depth), x (perpendicular to line focus), and y (direction of line focus) directions are 3.3 µm, 0.7 µm and 0.9 µm, respectively, with a 50× objective lens. This confocal microscope may be useful for analyzing fast phenomena during biological and chemical interactions and for fast 3D image reconstruction.

© 2005 Optical Society of America

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References

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2003 (1)

J. Cushion, F. N. Reinholz, and B. A. Patterson, “General purpose control system for scanning laser ophthalmoscopes,” Clin. Exp. Ophthalmol. 31, 241–245 (2003).
[Crossref]

1999 (2)

1998 (1)

C. J. R. Sheppard and X. Q. Mao, “Confocal microscopes with slit apertures,” J. Mod. Opt. 25, 1169 (1998).

1996 (1)

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys. 59, 427–471 (1996).
[Crossref]

1994 (1)

1993 (1)

1988 (1)

G. Q. Xiao, T. R. Corle, and G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[Crossref]

1986 (1)

1980 (1)

Amos, W. B.

W. B. Amos and J. G. White“Direct view confocal imaging systems using a slit aperture,” in Handbook of Biological Confocal Microscopy, J. B. Pawley (Plenum, New York, 1995), pp. 403–415.

Anderson, R. R.

Aziz, D.

Bacskai, B. J.

R. Y. Tsien and B. J. Bacskai“Video-rate confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley (Plenum, New York, 1995), pp. 459–478.

Chou, C.-H.

Corle, T. R.

G. Q. Xiao, T. R. Corle, and G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[Crossref]

T. R. Corle, C.-H. Chou, and G. S. Kino, “Depth response of confocal optical microscope,” Opt. Lett. 11, 770–772 (1986).
[Crossref] [PubMed]

Cushion, J.

J. Cushion, F. N. Reinholz, and B. A. Patterson, “General purpose control system for scanning laser ophthalmoscopes,” Clin. Exp. Ophthalmol. 31, 241–245 (2003).
[Crossref]

Donaldson, L.

Gmitro, A. F.

Hopkins, M. F.

Kino, G. S.

G. Q. Xiao, T. R. Corle, and G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[Crossref]

T. R. Corle, C.-H. Chou, and G. S. Kino, “Depth response of confocal optical microscope,” Opt. Lett. 11, 770–772 (1986).
[Crossref] [PubMed]

Koester, C. J.

Mao, X. Q.

C. J. R. Sheppard and X. Q. Mao, “Confocal microscopes with slit apertures,” J. Mod. Opt. 25, 1169 (1998).

Masters, B. R.

Minsky, M.

M. Minsky, “Microscopy apparatus,” U.S. patent 3,013,467 (December 1961).

Patterson, B. A.

J. Cushion, F. N. Reinholz, and B. A. Patterson, “General purpose control system for scanning laser ophthalmoscopes,” Clin. Exp. Ophthalmol. 31, 241–245 (2003).
[Crossref]

Pawley, J. B.

W. B. Amos and J. G. White“Direct view confocal imaging systems using a slit aperture,” in Handbook of Biological Confocal Microscopy, J. B. Pawley (Plenum, New York, 1995), pp. 403–415.

R. Y. Tsien and B. J. Bacskai“Video-rate confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley (Plenum, New York, 1995), pp. 459–478.

Rahadhyaksha, M.

Reinholz, F. N.

J. Cushion, F. N. Reinholz, and B. A. Patterson, “General purpose control system for scanning laser ophthalmoscopes,” Clin. Exp. Ophthalmol. 31, 241–245 (2003).
[Crossref]

Rouse, A. R.

Sabharwal, Y. S.

Sheppard, C. J. R.

C. J. R. Sheppard and X. Q. Mao, “Confocal microscopes with slit apertures,” J. Mod. Opt. 25, 1169 (1998).

Thaer, A. A.

Tsien, R. Y.

R. Y. Tsien and B. J. Bacskai“Video-rate confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley (Plenum, New York, 1995), pp. 459–478.

Webb, R. H.

White, J. G.

W. B. Amos and J. G. White“Direct view confocal imaging systems using a slit aperture,” in Handbook of Biological Confocal Microscopy, J. B. Pawley (Plenum, New York, 1995), pp. 403–415.

Xiao, G. Q.

G. Q. Xiao, T. R. Corle, and G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[Crossref]

Appl. Opt. (4)

Appl. Phys. Lett. (1)

G. Q. Xiao, T. R. Corle, and G. S. Kino, “Real-time confocal scanning optical microscope,” Appl. Phys. Lett. 53, 716–718 (1988).
[Crossref]

Clin. Exp. Ophthalmol. (1)

J. Cushion, F. N. Reinholz, and B. A. Patterson, “General purpose control system for scanning laser ophthalmoscopes,” Clin. Exp. Ophthalmol. 31, 241–245 (2003).
[Crossref]

J. Mod. Opt. (1)

C. J. R. Sheppard and X. Q. Mao, “Confocal microscopes with slit apertures,” J. Mod. Opt. 25, 1169 (1998).

Opt. Lett. (2)

Rep. Prog. Phys. (1)

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys. 59, 427–471 (1996).
[Crossref]

Other (3)

W. B. Amos and J. G. White“Direct view confocal imaging systems using a slit aperture,” in Handbook of Biological Confocal Microscopy, J. B. Pawley (Plenum, New York, 1995), pp. 403–415.

M. Minsky, “Microscopy apparatus,” U.S. patent 3,013,467 (December 1961).

R. Y. Tsien and B. J. Bacskai“Video-rate confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley (Plenum, New York, 1995), pp. 459–478.

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Figures (6)

Fig. 1.
Fig. 1.

Experimental set-up (a) of the high-speed confocal line-scanning microscope, top view (b) and side view (c) of the line scanner; L’s: convex lens, PBS: polarizing beam splitter, WP: quarter-wave plate, CL’s: cylindrical lens, AOD: acousto-optic deflector, OL: objective lens, S: slit, CCD: line-CCD camera, SP: sagittal plane, TP: transverse plane (Patent pending).

Fig. 2.
Fig. 2.

Image of the air-force target taken by the HSCLM. The image size is 512×512. The separation between the bars in the rectangular box is approximately 4.3 µm.

Fig. 3.
Fig. 3.

Edge response curve and line spread function in x-direction (a) and y-direction (b). The full widths at half maximums in x- and y-directions are 0.7 µm and 0.9 µm, respectively.

Fig. 4.
Fig. 4.

Signal intensity response curve as the microscope stage is moved in z-direction. The full width at half maximum in z-direction is 3.3 µm.

Fig. 5.
Fig. 5.

Images of a micrometer-size structure taken by the scanning electron microscope: (a) by the conventional optical microscope; (b) The stage is moved along the z-axis, and the conventional microscope cannot resolve the depth; (c) These images are taken as the stage is moved by 2 µm along the z-direction. The scale bars correspond to 5 µm.

Fig. 6.
Fig. 6.

Confocal images (a)–(c) taken by the HSCLM and the reconstructed 3D image (d). The images (a), (b), and (c) are obtained as the stage is moved by 1 µm along the z-axis. The scale bars correspond to 5 µm.

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