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Confocal microscopes.

CONFOCAL MICROSCOPES The CT scan (computerized tomography) revolutionized medical diagnosis with its ability to take two-dimensional x-ray image "slices" of a structure and assemble them into a three-dimensional picture.

Confocal optical microscopy is having a similar impact today on scientific research, as a powerful benchtop tool that can visually probe a translucent specimen in three dimensions.

True optical sections of the sample are taken because the effective focal plane of the objective lens is very thin--as little as 0.5-[mu]m thick for objectives with a high numerical aperture. Any image data from above and below the focal plane are prevented from reaching the detector.

Scientists can scan various levels inside a specimen by simply raising or lowering the focal plane. At each level, new details are revealed. As with the CT scan, software can assemble the confocal image slices into a 3-D structure.

The confocal imaging technique will offer you an impressive list of advantages. For example, a critical examination of a living sample can be done without the need to section or destroy the organism.

In addition, the resolution is superb, down to 0.1 [mu]m; Gerhart Kneissl, sales manager at the Cambridge, MA-based firm, Bio-Rad Laboratories, points out that these instruments offer horizontal resolution as much as 1.4 times that of a conventional optical scope.

"You might say confocal imaging is the bridge between the optical microscope and scanning, electron microscopy," says Frank Lundy, president of Technical Instrument Co. in San Francisco. He explains, "It offers you the world of submicron microscopy at high resolution without the cost of an electron microscope."

Confocals offer useful visual magnification that ranges from 100X to 2,000X--and up to 10,000X with electronic signal detection, which is commonly exploited.

The scanning confocal microscope can give the user information about both surface and subsurface features of a translucent sample. Apparently, the only limitations on this ability are the degree of translucency of the specimen, the working distance of the objective lens used, and the reflectivity of the sample.

The scope can even provide information about a relatively opaque sample if it is very thin (some suppliers claim their scope has been able to look through a thin sheet of copper).

Even if the sample is thick and relatively opaque, as in the case of metals or ceramics, the scope will at least allow a look at the scratches, abrasions, and cracks on the surface. In general, confocal scopes excel at viewing the surface topography of reflective opaque specimens with a high spatial resolution, notes Peter Baurschmidt, manager of microscope systems in the Microscope Div. of Carl Zeiss Inc., Thornwood, NY.

The technique has obvious value in research on tissue samples in cytological and neurological applications. Fluorescence observations in biology and medicine are very hot application areas now for confocal microscopes, with a fluorophore excitable in the extended visible range being used.

Confocals screen out the fogginess normally observed with standard optical scopes when used on living samples (such haze is typically caused by light scattered by the specimen outside of the plane of interest, a problem confocals are designed to avoid).

In the semiconductor field, confocal scopes serve in high-resolution defect analysis and in doing topography profiles. In the material sciences, they are used in research on ceramics, polymers, additives, alloys, and fibers. Metallurgists study friction and wear in metals with them. Confocals are also beginning to see use in dental R&D labs to study tooth wear and decay.

The basic idea in all confocal microscopy, explains Robert Compton, applications specialist of NORAN Instruments in Middleton, WI, is to illuminate a single point at the focal plane of a specimen or sample, and then allow only information from that point to arrive back at the detector, typically a TV camera with a photo-multiplier.

The first aim can be achieved by passing a laser light beam (various lasing materials will work) through the objective lens to the target spot. The reflection (or fluorescence) coming back from the specimen then travels through a pinhole to strike the detector.

The laser beam must be moved along in a raster scan, much like that used on a TV screen. The X-Y plane scanning movement here is typically effected using either a pair of galvanometer-mounted mirrors to direct the beam, or, as in one product, a galvanometer mirror combined with an acousto-optic deflector.

There is another way to illuminate the specimen, however. In this approach, light from a broadband source (halogen, xenon, or mercury lamp) is focused on the target after having been passed through a pinhole lying between the source and the target. The reflected light is then passed through another pinhole lying 180 deg opposite, between the specimen and the detector.

As a motor spins the disk, diametrically opposed clusters of apertures come into action successively. Thus, at any given instant, one pinhole of a matched pair of pinholes is precisely aligned on the illumination side of the spinning disk's axis to act as a point source. At the same time, its opposite number in the reflection path is lined up to act as a point detector.

In either the laser system or the broadband light system, however, only the image of any portion of the specimen lying right in the focal plane of the scope's objective lens will be reflected up to stimulate the detector.

When considering the purchase of one of the dozen or so commercial confocal microscopes now on the market, consider the following points:

* The confocal laser scanning microscope will provide more light intensity (power) in its focused spot than a brordband source-based system. It will not require lenses to correct aberation, either, according to Zeiss's Baurschmidt.

* Also, in fluoroscopy work, the light striking the TV camera detector in a spinning disk system will have to be intensified to be detected--a problem the laser system does not incur.

* On the other hand, the broadband systems are less expensive than the laser systems, and will afford the user true color, whereas the laser image will require pseudocolor assignment to different regions of the image. Laser systems can use fewer fluorchromes.

* The choice of pinhole size is a compromise between the light intensity desired and optical sectioning performance, and therefore it is desirable for this size to be variable or programmable--one advantage of laser systems.

* One can easily obtain vertical sections (i.e., slices in the X-Z and Y-Z planes) with a laser-based system, says Zeiss's Baurschmidt, though he admits the laser scan will be somewhat slower than a broadband source scan.
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Title Annotation:includes related articles
Author:Keeler, Robert
Publication:R & D
Article Type:Cover Story
Date:Apr 1, 1991
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