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Digital unsharp masking reveals fine detail in images obtained with new spinning-disk confocal microscope.

The confocal microscope scans the specimen point by point with a minute (diffraction-limited) spot of light, and it prevents light originating from all regions other than that minute spot from entering the photodetector; thus it provides a sharp image of the specimen that is not blurred, e.g., by out-of-focus fluorescence (1). The laser point-scanning confocal microscope is especially effective in generating clear, sharp optical sections of fluorescent specimens. However, most point scanning systems require several seconds to capture images that are sufficiently free from photon noise.

In the scanning-disk-type confocal microscopes, multiple pinholes are located on a spinning Nipkow disk. The pinholes simultaneously illuminate many points on the specimen and also filter out the unwanted scattered light originating from regions not illuminated by the focused image of the pinholes. With the scanning-disk type, the image is generated in real time so that the confocal image can be viewed through the eyepiece and the specimen scanned or focused without waiting for the picture to appear on a monitor screen. However, most of these systems were limited by the low throughput of light (ca. 1%) through the pinholes on the Nipkow disk, the presence of residual scan lines, and the imperfect removal of haze originating from fluorescent regions far from focus.

A new system, just introduced, incorporates a second Nipkow spinning disk containing some 20,000 microlenses that are aligned with the same number of pinholes in the main Nipkow disk (Yokogawa Electric Co., CSU-10). The microlenses in this unit improve the throughput of the disk by some 50-fold so that fluorescence images are now bright enough to be seen through the eyepiece under normal room lighting conditions. The new compact unit can be attached to most upright or inverted microscopes and provides stable images with good in-plane as well as z-axis resolution. Its patented pinhole arrangement eliminates all traces of scan lines. The disk in a prototype high-speed confocal unit is also spinning fast enough that, with the unit equipped with an intensified video camera and high-speed video recorder, movies of fluorescent blood cells circulating in exposed vessels have been made by capturing full frame every 4 ms.

We have tested this new confocal system on biological specimens and have added a further improvement to the image by digital unsharp masking. As shown in panels A and B in Figure 1, the new confocal system does provide optical sections of fluorescent objects that are considerably improved over conventional epifluorescence. The sensitivity was high enough to directly visualize and focus 50-nm-diameter fluorescent beads (courtesy of Dr. Stephen Smith). The image still suffers from haze introduced by the out-of-focus fluorescence characteristic of multiple pinhole systems, but the intruding scan lines are totally absent. When we applied a digital unsharp masking algorithm (in MetaMorph, Universal Imaging Corp.), the haze disappeared, and the image became exceptionally crisp as seen in Figure 1C. Unsharp masking (which is simpler and considerably less intensive computationally than digital deconvolution) suppresses the low spatial frequency components in the image and enhances the contrast of the high spatial frequency components, thus giving rise to the type of image improvement demonstrated. Images of the same specimen obtained on a compact point-scanning laser confocal unit (Nikon PCM-2000 with 60/1.40 Plan Apo objective; ca. 10-s scan with 488-nm excitation using similar laser power) displayed less haze but comparable resolution to that with the CSU-10 unit before unsharp masking.

By combining the real-time (and faster-than-video rate) direct-view capability of the microlens-equipped scanning-disk confocal unit with digital unsharp masking, we believe that biologists now have available a powerful new tool for imaging and analyzing dynamic events in cellular and molecular studies.

We thank T. Akiyama of Yokogawa Electric Co., R. Rottenfusser of C. Zeiss Inc., and M. Christianson of Princeton Instruments Co. for loan of equipment, and B. Amos of Cambridge Univ. and M. Terasaki of MBL for providing fluorescent samples. Supported in part by NIH grant R37 GM31617.

Literature Cited

1. Pawley, J, ed. 1995. Handbook of Biological Confocal Microscopy, 2nd edition. Plenum Press, New York.
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Author:Inoue, Shinya; Inoue, Ted
Publication:The Biological Bulletin
Date:Oct 1, 1996
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