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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Appl. Opt.

Appl. Phys. Lett.

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.

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.

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

Opt. Lett.

Rep. Prog. Phys.

R. H. Webb, �??Confocal optical microscopy,�?? Rep. Prog. Phys. 59, 427�??471 (1996).
[CrossRef]

Other

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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