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You're trying to decide which talk to go to. It's 7:00 P.M. of the last day of the conference you traveled many miles to attend. Let's see, there's a gene cloning talk featuring slides with continuous strings of ACTG, a diverse collection of grayscale gels, and some text slides to summarize the key points. In the room next door, there's a cell biology talk featuring bright, multicolored images and dynamic video clips of moving cells. Which one do you really want to attend?
Photomicrography has long been a centerpiece of biological research. But extraordinary advances in the development of electronic devices for capturing, processing, and displaying images have recently transformed the light microscope into an even more powerful resource. The production of beautiful, informative, and quantifiable images has allowed the detailed and accurate exploration of the distribution and behavior of subcellular structures, including individual macromolecules within living cells. It is no exaggeration to say that electronic technology has brought about a renaissance of the light microscope, making it more far-reaching and versatile than ever before.
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| The MSA Journal, Microscopy and Microanalysis |
Microscopy on the Web
The Web is richly informative on many aspects of microscopy and photomicroscopy. An excellent site for general and up-to-date information is that of the Microscopy Society of America (MSA). The MSA is a nonprofit organization that promotes the study and practice of microscopical imaging and analysis. It offers many reference sources, including a library of software for microscopic analysis and imaging, a videotape library featuring instructional videos (mostly for electron microscopy), a list of books displayed at the society's meetings, an email-based discussion forum, and a link to Microscopy and Microanalysis, the journal of the Society, where one can view abstracts of the articles for free. Of particular educational interest are suggestions for precollegiate educational materials and an interactive site for students to query and observe microscopists at work. An extensive listing of meetings, conferences, and short courses on microscopy can also be found at the MSA site. Of outstanding interest are the courses on "Optical Microscopy and Imaging in the Biomedical Sciences" and "Analytical and Quantitative Light Microscopy" offered at the Marine Biological Laboratory in Woods Hole. Finally, the MSA site links to many other microscopy-related sites, such as MicroWorld Resources and News and WWW Virtual Library: Microscopy, two other extensive resources for general microscopy.
Wide-Field Microscopy
Light microscopes can be classified as either wide-field or point-scanning. In wide-field microscopes the whole field of view is illuminated simultaneously, whereas in point-scanning microscopes a small region of the field is illuminated by a point light source that scans the specimen in a regular raster. Two of the most powerful types of wide-field microscopy are video enhanced contrast (VECM) and digital fluorescence imaging microscopy (DFIM). VECM allows the visualization of objects that generate too little contrast to be visible by eye. Studies of cell motility, organelle movement, and cytoskeletal filament dynamics have particularly benefited from VECM. A movie produced by R.S. Bedlack in Leslie
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| Free Software: IMR's 4D Viewer analyzing an embryo of the nematode c. elegans |
Many components within cells cannot be visualized with VECM because they are not easily distinguished from their surrounding material. A much broader range of components, including molecules present in very small amounts, can be studied by fluorescently labeling the component and using DFIM. In conventional wide-field microscopy, images recorded at a given focal plane are contaminated with light emitted and scattered by out-of-focus regions. A clever method to eliminate this contamination relies on obtaining a mathematical description of the contaminating light and then computationally subtracting it from the image. The development of these algorithms has turned DFIM into one of the most powerful forms of modern microscopy. Two examples of this technology are the exhaustive photon reassignment algorithm developed by Fred Fay at the Biomedical Imaging Center in Worcester, Massachusetts, and commercialized by Scanalytics/Signal Analytics Corporation; and the deconvolution algorithm developed by John Sedat and David Agard at the University of California, San Francisco and commercialized as DeltaVision by Applied Precision. The result is a series of very high resolution, single section images that can then be assembled into 3-D structures.
Point-Scanning Microscopy
Confocal and near-field scanning optical microscopes constitute the two main types of point-scanning microscopes. In conventional confocal microscopy, small apertures are positioned in the excitation and emission pathways, such that most of the out-of-focus light emitted and scattered by the specimen is blocked, allowing the detection of only the light coming from a point confocal to the focal point of the specimen. The main advantage of confocal microscopes is their ability to yield direct images of single optical sections. Although its last update is a year old, the 3-D Laser Scanning Confocal Microscopy Web site produced by Lance Ladic is an excellent resource for confocal information. It includes a clear description of the principles underlying confocal microscopy, an impressive collection of free graphics software for PCs, Macintoshes, and Unix/SGI systems, and a long list of other Web sites featuring confocal images and animations.
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| Nomarski (upper) and two-photon (lower) micrographs of living neurons growing on silicon wells from the Pine Laboratory site |
An even more recent development in point-scanning microscopy is the scanning near-field optical microscope (SNOM). The key feature of this microscope is that it overcomes the optical diffraction limit, which has restricted all other forms of light microscopy, by using an extremely small aperture to funnel the excitation beam and an imaging probe that scans very close to the surface of the specimen. Thus, SNOM provides phenomenal spatial resolution which can exceed 30 nm. The Scanning Probe Microscopy Program at Arizona State University contains a good description of the SNOM. In addition, the Topometrix Web site offers information on their latest SNOM products, including the option of adding SNOM to a conventional inverted light microscope.
Technical Tips
Superb technical information on many aspects of light microscopy can be found in Solutions!, an Internet magazine edited by Ted Inoue, an expert on digital microscopy technology. This site features excellent application notes, technical tips, product reviews, and discussions. The Trading Post - a section for buying and selling used equipment - and links to the Web pages of prominent digital microscopy researchers are also available. At this site, one can also order the long-awaited second edition of Video Microscopy by Shinya Inoue, a landmark text in digital microscopy.
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| Fluorescence image composite of the mitochondria (red), actin filaments (green), and nuclei (blue) in a pair of mouse fibroblasts from Molecular Probes |
Examples of other companies that maintain helpful Web sites are the Omega Optical Web site, providing an excellent source for filter information, the Universal Imaging Corporation, developers of one of the first software packages for image analysis, and Photometrics, leaders in digital cameras and developers of the first commercially available high performance CCD. For microscopes and microscope accessories, major Web sites include Olympus, Zeiss, Nikon, and Leica.
The OpticsNet Web site maintained by the Optical Society of America is highly recommended for those interested in the nitty-gritty physical science of light microscopy. For biological applications, explore the Bio-Optics link within this site. Journals covering the science and use of optical technologies can be found at the Photonics site. Of particular interest is Biophotonics magazine, to which one can subscribe for free.
Artistic and Amateur Photomicrography
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| Felice Frankel's microscopic image (Nomarski) of a microrotor's blades |
A mixture of science, art and educational materials can be found at several entertaining Web sites. Molecular Expressions, run by Michael W. Davidson at Florida State University, is perhaps the largest Web site of this type. It contains an enormous collection of photomicrographs mostly obtained with optically polarized transmitted light. The creative highlight of this site is the photo gallery featuring surrealistic landscapes created using multiple-exposure photomicrography. Images are available for commercial use, and a service for custom photomicrography is advertised. But visitors are encouraged to produce their own images: an extensive Microscopy Primer section contains detailed descriptions of the principles underlying polarized microscopy, how to set it up, how to prepare samples, and how to obtain photomicrographs. Also featured at this site is a virtual microscope, under development by the National High Magnetic Field Laboratory, designed for teaching microscopy principles.
Educators will also enjoy the CELLS Alive! Web site, containing illustrated descriptions on a variety of cell biological topics. And for the amateur photomicroscopist, the Microscopy-UK Web site - from the producers of the Micscape Online magazine for amateur microscopists - and the Amateur Microscopy Web site are musts.
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| A Nikon Small World Competition winner: confocal image of a Drosophila wing imaginal disk shot at 500x by Steve Paddock |
Looking into the Future
As technology progresses, the light microscope continues to take on new forms. The Center for Light Microscope Imaging and Biotechnology, directed by D. Lansing Taylor at Carnegie Mellon University, is a leader in the advancement of light microscopy technology for the biomedical sciences. Novel fluorescent reagents including the development of "super bright" and photostable fluorescent labels, fluorophores for labeling antibodies and DNA, energy transfer dyes for multicolor analysis, and optical biosensors for detecting enzymatic activities are being developed at the center. In addition, new imaging technologies such as a high-throughput microscope, a standing-wave fluorescence microscope, and an automated interactive microscope, which attempts to combine the latest techniques in fluorescence microscopy with advanced image processing and pattern recognition software, promise to enhance the light microscope's power even further. Other centers involved in the development of new technologies include the Center for Biomedical Imaging Technology at the University of Connecticut Health Center, the National Center for Microscopy and Imaging Research, and the Integrated Microscopy Resource Center at the University of Wisconsin. The latter center is developing novel techniques for integrating light and electron microscopy. Improved optical sectioning methods for in vivo imaging at the light microscope level are being combined with preparation techniques that allow subsequent high-fidelity visualization by electron microscopy.
This wealth of technological advances and the consequent explosion in the generation of imaging data is reflected in the large and growing number of photomicroscopy Web sites. How does one extract the most benefit from this overwhelming amount of information? An ambitious group of researchers, led by seven European microscopists, recently established the Bioimage Database project, a Web site that attempts to consolidate the huge and rapidly growing set of multidimensional image data generated by all forms of microscopical techniques. The project is still in its initial stages, but a prototype database is to be released this summer. Categorizing and analyzing the huge amounts of sequence data generated by the genome projects are proving to be enormous and very challenging tasks. A comprehensive analysis of the imaging data will be, no doubt, even more challenging. The rewards it promises, however, would seem to be well worth the effort.
Marina Chicurel is a postdoctoral fellow at Harvard Medical School/Children's Hospital and at the Albert Einstein College of Medicine in New York.
The above photo of a transverse zebrafish retinal section was contributed by Karen D. Larison, Molecular Probes, Inc.
Endlinks
History of the Light Microscope - a history of light microscopy from the eighth century B.C. through the mid-19th century.
Tips and Tricks of Microscopy - collection of useful tips from independent contributions and selections from the Microscopy and Confocal List Servers.
Construction of Optical Tweezers - a chapter from Cells: A Laboratory Manual that describes the construction of optical tweezers, an optical trap that can be used to manipulate microscopic objects. Prepared by Steven M. Block.
Singer Lab - example of the power of DFIM. Femino and colleagues in the Singer lab are able to detect single mRNA molecules and obtain quantitative data on the induction of transcription in a single cell.
David Piston Lab - quantitative experts in two-photon excitation microscopy. Research focuses on glucose metabolism and insulin secretion in beta cells.
John Cooper Lab - movies of actin dynamics in yeast using GFP-tagged actin capping protein. A useful primer on movie-making is also included.
Dennis Kunkel's Microscopy - a broad and rich library of images. Explored in detail in the HMS Beagle Site Review.
Web sites mentioned in this column:
Overviews
Wide-Field Microscopy
Point-Scanning Microscopy
Technical Tips
Artistic and Amateur Photomicrography
Looking into the Future
Discussion Groups
on the Web
The Forsburg Lab
Grant-Writing Tips and
Resources
Check Out the Alternatives
Chemical and Biological Warfare Online
Biosupply Resources on the Web