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Collect 3-D Images Without Sample Prep.

Confocal laser-scanning microscopy complements classical light microscopy and scanning electron microscopy (SEM) by piecing together 3-D images of samples. Although resolution limits prevent the laser-scanning microscope (LSM) from replacing SEM, it has become the microscope of choice for applications that require direct and accurate quantitative analysis of 3-D microstructures without time-consuming sample preparation. With extensive motorization and the possibility to run fully automated routines, the modern confocal LSMs have opened the way for applications ranging from new and exciting research to routine industrial applications.

Microscopists have understood the advantages of a "flying spot microscope" since the early 1950s. In the late 1970s, this technique took advantage of small, reliable lasers and new scanning systems. In 1982, the first commercial LSM was introduced. Today, the basic principle behind confocal LSM still remains the same.

A laser beam is directed through an optical microscope and reaches a sample as a diffraction-limited spot. The microscope objective captures reflected, scattered, or emitted light, which travels through a confocal pinhole to a detector. This pinhole is placed in such a way that only light from the objective lens focal plane can pass through it, blocking out-of-focus contributions. This depth selection creates thin optical sample sections. Moving the laser beam point by point over the sample, together with a precise focusing mechanism, lets researchers acquire 3-D image stacks.

The depth-discrimination capability depends on the numerical aperture, the laser-light wavelength, and the diameter of the confocal pinhole. For a near-zero pinhole diameter, Z resolution (the axial distance for which the intensity reflected from a surface is at least 50% of the maximum value found exactly in the focal plane) is 200 to 300 nm with high-aperture objectives. The capability to detect a surface step on a sample is at least 10 times better than this, since in practice users can distinguish height differences on the order of 10 nm. With a sensitive detector, users can identify small changes in the Z-curve intensity. Users can also examine surfaces that vary in height by several millimeters by using special objectives and long working distances.

With so many laser-light wavelengths and microscope objectives available, users can achieve many different magnifications and field sizes. Large scanned fields provide a quick sample overview. A motorized X-Y stage scans many adjacent fields and allows for 14.25- x 14.25-cm sample area with a 35-[Mu]m pixel size. With UV lasers and high-numerical-aperture objectives, users can achieve 0.1-[micro]m lateral resolution and 10-nm pixel sizes.

Imaging methods, such as reflected light and fluorescence, provide information not fully available with other scanning techniques. With these methods, users can:

* Record non-contact 3-D structural representations without time-consuming sample preparation

* Image the volume inside opaque objects to a depth of a few hundred [Mu]m without destroying them

* Extract multiple profiles from 3-D images or directly record them along a free spline track

* Obtain quantitative data about distances, angles, areas and volumes

* Evaluate statistical values like micro-roughness parameters

* Record surface or volume changes over a few seconds or several hours

Scientific researchers use confocal LSMs to study polymers, metals, and corrosion, as well as for forensics and crack analysis. LSMs are also commonly used in the semiconductor, polymer, printing, ceramics, MEMS, medical/dental, and food industries.

A recent study of binary polymer blends is an example of the use of confocal LSMs in research. By fluorescently labeling one component, the researchers imaged the phase separation in situ. They used these images to follow the time evolution of topographic parameters. With an LSM, the 3-D polymer structure was visible for the first time, and users could confirm the bicontinuity of phase-separated structures that could otherwise only be predicted by theoretical calculations.

For production, confocal LSMs provide accurate and reproducible quality control. For example, a fully automated measuring system analyzes the micro-nozzles that deliver the ink for ink-jet printers. The instrument uses confocal imaging techniques in fluorescence mode to find the top surface, its tilt, the exact entrance and exit opening positions, and the coordinates of the inside walls. From this data analysts reconstruct the nozzle and extract critical parameters.
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Author:Kunath-Fandrei, Gerald; Jena, Carl Zeiss
Publication:R & D
Article Type:Brief Article
Geographic Code:1USA
Date:May 1, 1999
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