Laser microscopy opens a new dimension

Laser-based microscopes already have probed the nanometer realm using near-field methods—and in the process have become even more invaluable as tools for inspection and research. Equally important are recent developments in laser microscopes that generate three-dimensional (3-D) images of microscopic objects. Applications benefiting from these results include research into current flow in semiconductors and into the movement of biological microorganisms.

Confocal microscopes

In contrast to conventional microscopes that image only in a single focal plane, confocal laser-scanning microscopes (CLSMs) allow sharp 3-D imaging of objects.¹ In such devices, light from a laser, which is first focused by a lens, passes through a pinhole aperture before being focused on the specimen. Light from the specimen, either reflected or fluorescence, then passes through the same focusing lens, which acts as both objective and condenser. A beamsplitter diverts this light to another pinhole aperture. The light passing through the second pinhole is then converted to an electrical signal by a photomultiplier tube (PMT).

The confocal system allows only light from the focal plane to pass through because the pinhole apertures block light above or below that plane. This is in contrast to a conventional microscope that allows out-of-focus images from off-focal-plane layers to be seen.

To produce a 3-D image with the confocal system, the device is scanned over the entire specimen at a fixed height to provide a single-layer image; scanning is then repeated after a vertical shift as small as 50 nm. Multiple-layer scans are combined by a computer into a single 3-D image that can be viewed several ways.

Such confocal microscopes have found applications in biological research and medical diagnostics. For example, because human skin is translucent, living skin can be imaged in three dimensions, allowing fast diagnosis of a variety of ailments. These advanced capabilities also are of interest to the cosmetics industry. Recently, researchers at cosmetics firm L'Oreal (Aulany-sous-bois, France), working with Nolan Instruments Inc. (Middletown, WI), developed a confocal laser microscope that provides sharp 3-D images of living skin.²

To prevent blurring due to movements by the patient or blood flow, the confocal scanner uses a rotating polygonal mirror for the scans and takes 30 optical sections per second to rapidly build the 3-D image. The light source is a krypton/argon laser emitting at 488, 568, and 647 nm. Both reflected light and fluorescence data are collected simultaneously, with a dichroic filter separating the two. It takes about 10 s for a complete in-depth scan. Data processing on a workstation consumes another 5 min. The researchers found that the blue 488-nm line gave the sharpest images, with resolutions in the area of 1 µm.

Another major application is materials inspection. With the production of increasingly microscopic parts, 3-D inspection is an essential step in checking dimensions. Similarly, confocal imaging techniques can measure the depth and shape of drilled holes or check the texture of fabric for imperfections.

Theta microscopy

For many biological and medical applications, confocal microscopy has a limitation. Resolution along the optical axis in the vertical direction is often two- or three-times worse than across the axis horizontally. This can seriously affect performance when the fine structure of cells is near the resolution limit.

To address this limitation, a research team at the European Molecular Biology Laboratory (Heidelberg, Germany) is developing a modification of confocal microscopes called a single-lens theta microscope.³ In this device, the illuminating beam is no longer along the optical axis, but instead pointed at right angles to that axis. While an ellipsoidal region along the axis is detected and another ellipsoidal region across the axis is illuminated, only a smaller spherical region where the ellipsoids overlap is both illuminated and detected. This is the data that contributes to the output signal. In this case, the vertical resolution drops to the same level as the horizontal resolution.

The illumination beam passing through the illumination pinhole is deflected at a dichroic mirror and focused onto a focal point above the surface of a plane mirror. The fluorescent beam, which is detected perpendicularly to the illumination axis via an oblique mirror, passes the dichroic deflector and is focused into the detection pinhole with the subsequent detection optics. The scattered or fluorescent light emerging horizontally is reflected by a prism up the optical axis to the detector, so illumination and detection occur at right angles.

Monitoring molecules in 3-D

Confocal and near-field microscopy can be combined to allow the imaging of individual molecules in three dimensions. In near-field microscopy, the resolution can be made significantly smaller than a wavelength of light by locating the specimen in the near field of a tiny aperture. Researchers at the University of Amsterdam (Amsterdam, The Netherlands) and the University of Leiden (Leiden, The Netherlands) use a selective spectroscopic method to marry confocal and near-field techniques.⁴

The key is the excitation of individual molecules one by one in a sample. The scientists tune a single-mode dye laser to the fluorescent excitation frequency of the test pentacene molecules. The actual frequency an individual molecule absorbs is affected by its immediate environment, so no two molecules in the sample region (which is about 3 µm on a side) have identical excitation frequencies. As the frequency of the dye laser is scanned, the molecules emit light one by one.

Scientists determine the location of each molecule within the volume defined by the focal region of the confocal microscope by calculating the centroid of the light detected through the microscope. While the diameter of the light distribution is determined by the wavelength of light used, the centroid that indicates the position of the molecular point source can be determined to a far higher accuracy. In this way, the technique can produce vertical accuracy of 120 nm and horizontal accuracy of 50 nm.

While the team performed its initial test on molecules in a frozen block of solvent, it hopes to soon test the same technique on biological samples to map the distribution of a given species of molecule within a single cell.

Detecting microscopic motion

In biological studies, while the movement of objects under the microscope may be crucial, detecting this motion requires painstaking comparison of images. Researchers at the Institute of Electrical Engineering, National Dong Hwa University, (Hualien, Taiwan), have shown that optical-phase conjugation can provide an automatic way of detecting and highlighting motions.⁵ In this technique, a laser beam passing through an object interacts with a reference beam in a photorefractive crystal to produce a phase-conjugate beam. This beam is identical in phase and amplitude to the image beam, but travels in the opposite direction. When such a beam passes through the object, the phase distortions cancel out, again producing an undisturbed collimated beam.

This process can be used to detect motion because the photorefractive crystal takes from milliseconds to seconds to respond to changes in the image beam (depending on beam intensity). If parts of the object have moved faster than the time it takes for the crystal to react, the phase changes will no longer cancel out. Areas that have moved will be visible in the output beam, while areas that are unchanged will be invisible.

The Taiwanese researchers used a self-pumped phase conjugator. Here, the interference of the image and reference beam creates regions in the photorefractive crystal where the index of refraction is greater or smaller than the average, since the index of refraction for the crystal (typically barium titanate) varies in response to light intensity. The change in index of refraction in turn alters the interference patterns and the index-of-refraction patterns until a stabilized grating pattern emerges. To speed up this process, which can take minutes, the scientists added a stabilizing laser beam that sets up the stable grating, and then modified the grating slightly with the weak laser beam from the object. Only about 1 mW of laser power is required.

Mapping microcurrents

In the inspection of electronic and optoelectronic devices, electrical defects are more important than visible structure. As a result, probes that induce currents in microscopic regions are increasingly being used for such inspection; electron beams often induce the currents. There are limitations, though. These techniques require a high vacuum and will not work with low-conductivity materials, where charge from the e-beam itself builds up.

Recently, scanning-laser microscopy in the form of optical-beam-induced-current microscopy, also called laser-beam-induced-current microscopy, is gaining acceptance as a way to avoid these problems.6 The technique relies on a photoelectric effect in which the laser beam generates electron-hole pairs on the surface of a semiconductor, which in turn generates currents through contacts to the semiconductor. Scanning the laser beam produces a map of induced currents that indicates the location of defects. Depending on the specific application, lasers ranging from the infrared to ultraviolet can be used, with ultraviolet giving greater resolution and infrared greater penetration.

REFERENCES