Optical Slices

In conventional optical microscopes a thick specimen tends to result in a blurred image, and so researchers have to use thin slices, or sections, of their samples. A new tool called the confocal microscope, however, is able to produce thin sections optically.

It does this by illuminating the sample with laser light through a pinhole, confining the illumination to a single point. An image of the pinhole—a tiny spot—is created on the sample, and the spot reflects back onto the pinhole. If what is being examined is in focus, the light passes through the pinhole and is captured by a detector. A computer collects and digitally combines the series of individual images into a single three-dimensional image.

A big advantage of the confocal microscope is that optical sectioning can be performed in living biological samples without introducing chemicals normally used during tissue preparation. This is a major reason why the confocal microscope has become the preferred optical instrument for examining biological samples. It has, for example, been used to study chemical changes in living cells over time, giving biologists new insights into how plants grow.

Scanning and Tunneling

Some of the most remarkable new microscopes can produce images of the atoms or molecules on the surface of things. The most famous of these is called the scanning tunneling microscope (STM).

The STM takes advantage of a phenomenon that physicists call electron tunneling. The microscope uses a needle, or probe, with an extremely fine tip. It is thought that atoms at the tip's end form a pyramid in which a single atom sits at the top, thus making the point one atom wide. The probe is mounted on a so-called translator, a device that can move the tip with great precision when stimulated by an electric field. The electrons of an atom can be thought of as a sort of cloud around the atom's nucleus. When the probe is brought very close to the sample to be examined, the electron cloud of the tip overlaps with those of the atoms of the sample's surface. If a voltage is applied to the tip, electrons can "tunnel" across the gap between the single atom at the tip and the sample. As the probe scans over the sample's surface, the voltage fluctuates according to variations in the "texture" of the surface (with individual atoms forming hills, surrounded by valleys created by the spaces between them). By measuring this tunneling voltage, atomic-scale images can be produced.

Atomic Force

The STM has a built-in limitation. The surface of the sample has to conduct electricity, and many of the things that scientists would most like to examine, such as biological specimens, are not very conductive. Seeking to get around this obstacle, Binnig, along with Gerber and Stanford University's Calvin Quate, came up in 1985 with a different kind of scanning probe instrument called the atomic force microscope (AFM). In it a very sharp probe is mounted on a soft cantilever spring. The tip rides over the sample's surface almost like a phonograph needle on a record. When the sample is moved back and forth, the entire system acts as a profilometer—an electrical device that measures the roughness of a surface—at atomic and molecular scales. The cantilever's deflection is detected by shining a narrowly focused laser beam on the top of the cantilever. The beam is reflected to a mirror and then to a light detector. A computer converts the light measured by the detector into images of atoms and molecules—images that can be manipulated and enhanced as required.

Дата добавления: 2015-11-26