During many decades of serving the life science, materials, medical, forensics, and environmental industries, microscopes have become an icon of laboratory work.
Microscope technology has diverged into three branches. Light microscopes, which view objects illuminated by visible light, have been around for centuries. Related are instruments that image nonvisible infrared or ultraviolet radiation, or ones that view fluorescence or Raman effects. All of these use optics to focus light into a viewing field, detector, or camera lens. Visible light microscopes, by far the most common type, are the subject of this article.
The second major microscope group consists of electron microscopes that use high-energy electrons to visualize objects at very high resolution. The third category, scanning probe microscopes, forms images by scanning a surface with a microscopic probe. Prices of electron and scanning surface microscopes have fallen remarkably over the last twenty years, but these techniques are primarily the domain of basic research groups at universities or high-level corporate R&D.
The technology behind visible light microscopes, arguably the oldest true laboratory instruments, has not changed much in fundamental operation in two hundred years. Microscopes still consist of a light source, optics, and a stage for holding the specimen.
That does not mean that microscopy, and microscopes, are not evolving, says Lorne Davies, group manager at Olympus America (Center Valley, PA). “Some people believe that since microscopy has been around so long, it must be a stagnant industry. It’s not obvious that you can make important changes to an old technology.”
Mr. Davies admits that innovation tends to be incremental rather than earthshaking, but each stage of improvement “enhances what users are looking for, which is crisp, high-quality images.”
How they’ve improved
Advances in optics, mainly of precision lenses, are difficult to come by due to the physical limitations of glass. Improvements do occur, most resulting in improved numerical aperture (the range of angles over which lenses can accept light).
But for medical and diagnostic laboratory workers, a special focus of Olympus, the most critical improvement is ergonomics. Microscopists working in a cytology or pathology laboratory sit at their instruments for many hours at a time. Ergonomically friendly microscopes help users maintain comfortable body positions, minimize repetitive stress, and lessen fatigue on shoulders and eyes.
“Today, all microscopes from reputable manufacturers are very good at producing high-quality images, so ergonomics becomes a differentiator and for some, a pivotal factor in purchase decisions,” Mr. Davies tells Lab Manager. Ease of use is another related feature in high demand.
One exciting development in light microscopy has been the widespread adoption of fluorescence techniques. Fluorescence microscopy requires specialized reagents and equipment, including a camera to capture fleeting events. One fluorescence method, fluorescent in situ hybridization (FISH), enables investigators to identify and locate specific DNA sequences on chromosomes.
Another emerging technique is whole slide or “virtual” microscopy. This involves capturing microscope images and making them available on computer screens for later examination by one or more individuals. Virtual methods are useful for collaborative work, particularly in medical diagnostics.
What should potential buyers look for before purchasing a microscope? Mr. Davies puts a vendor’s reputation, service, and support high on the list. “People have close relationships with their microscopes, and that closeness extends to companies selling them.”
SEM resolution in a light scope?
This past year, Ravikiran Attota, Ph.D., a research engineer at NIST (Gaithersburg, MD), discovered a software technique that he claims provides the resolution of scanning electron microscopy (SEM) or atomic force microscopy (AFM) through a conventional light microscope. The technique, through-focus scanning optical microscopy (TSOM), uses a software trick, and no additional hardware, to reconstruct images rapidly and at low cost.
In fact, Dr. Attota claims that his method improves on SEM and AFM in that it combines their strengths. SEM excels at lateral resolution, while AFM is best for vertical resolution, he says. TSOM achieves both through a paradoxical approach.
Optical microscopes cannot clearly visualize nanometer-scale features because the wavelength of visible light is larger than the object being imaged. Dr. Attota acquires many of these outof- focus, or through-focus images anyway, at different focal points. The computer program he wrote combines the images and reassembles them into a TSOM image with spectacular depth capabilities. Where AFM provides depth resolution of only 1 micron, TSOM easily reaches 100 microns and in tests has produced images with 200-micron depth resolution.
Dr. Attotas group works on nanometrology, the measure of small things, with an emphasis on semiconductors. But TSOM should be of great interest to anyone who does microscopy, he says, particularly those involved in nanomaterials or nanostructures. For life scientists, the technique is particularly applicable to objects that change at different locations, for example cells.
Dr. Attota has been working with Sematech, Intel, and several universities on the new technique.