Live Hydrated Specimens
noise even at high magnifi cations. But high magnifi cations were not needed for most live, uncoated samples; indeed, most images were obtained at magnifi cations of only a few hundred times. T is was partly because low magnifi cations captured large fractions of the organism and partly to maintain a large depth of fi eld. Using the older fi lm recording technique, the fi nal outcome of an image was not usually known until the fi lm was developed and a contact sheet made, taking several hours. Modern digital image acquisition makes SEM imaging more assured because it is possible to extensively vary scanning parameters and see the resulting image immediately, even during the scan. If there is a problem, we can correct it and quickly acquire another image. It is also useful to do a low-resolution, fast scan to check the situation, and if things look good, proceed with a slower, high-resolution fi nal scan.
It is interesting to note that over many years of observations no serious SEM damage was ever observed from the outgassing of hydrated specimens. In fact, in a few cases insects and spiders were still alive aſt er examination and were returned to the garden.
Conclusion
Images of live, uncoated biological samples provide additional scientifi c data due to the variance of surface chemical and elemental composition, making for a more interesting and informative photographic quality than images of fi xed, dried specimens with a uniform metal coating.
The technique described here still has usefulness today, especially for those who may not have access to an ESEM, VP-SEM, or a Cryo-SEM. And unlike using a VP-SEM or
ESEM, where short working distances are required, images may be recorded at any working distance for excellent depth of fi eld.
Copyright Statement
All images in this article are copyright © by David Scharf and may not be used without permission. To see more images by David Scharf visit
www.scharfphoto.com .
References [1] D Stokes , Principles and Practices of Variable Pressure/ Environmental Scanning Electron Microscopy , Wiley , Hoboken, NJ , 2008 , p. 13 .
[2] D Scharf , Journal of Psychedelic Drugs 6 (1974) cover image. [3] “Small Wonders of a Magic Eye – Images of D. Scharf,” Newsweek , March 3, 1975, p. 52–53.
[4] McGraw-Hill , Encyclopedia of Science and Technology , 1975 Yearbook, New York, cover and opening page.
[5] D Scharf , T e National Academy of Sciences, “A Closer Look,” one-man exhibition November 17, 1975–January 31, 1976.
[6] “Electronic Voyage T rough an Invisible World,” National Geographic , Feb. 1977.
[7] D Scharf , Magnifi cations: Photography with the Scanning Electron Microscope, Schocken Books, New York, 1977.
[8] D Scharf , National Geographic , European editions (U.S. edition contains an alternate D. Scharf image of the same subject), cover image, July 2007.
[9] D Scharf , Wideband Multi-detector Color Synthesized Scanning Electron Microscope. U.S. Patent 5,212,383, fi led July 29, 1991, published May 18, 1993.
For the best imaging conditions, I always use Evactroncleaning.
2017 January •
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