Techniques developed for observing soft-bodied organisms with low-temperature scanning electron microscopy are relatively easy to use and have been found to be successful with a wide range of plants, animals, and fungi.
Substrates (plant material, soil particles, arthropod hosts, etc.) containing specimens of interest are transferred onto copper metal sample plates and are affixed with a thin layer of methyl cellulose solution.
The plates are then placed atop a precooled copper bar, and through the process of contact-freeze immobilization, the specimens are rapidly cooled to -196°C and are frozen to the plates.
Next, the plates are transferred to special holding tubes and are stored submerged in liquid nitrogen.
Due to the low surface tension of liquid nitrogen and the extreme hardness of materials cooled to these temperatures, very fragile samples can potentially be shipped by aircraft, in dry shipping dewars from remote study sites. After arrival at the Beltsville Electron Microscopy facility, the copper plates can be stored at -196°C in storage dewars.
Selected samples are transferred to the preparation chamber of an Oxford CT 1500 HF Cryotrans System for sputter coating with platinum. This renders them electrically conductive and they are placed on the precooled (-170°C) stage of a Hitachi S-4100 field emission Scanning Electron Microscope where they are imaged and photographed.
Biologists study photographs of the organisms' morphology and their associations with host plants or animals for identification purposes and with the hope of gaining insights into their behavior and ecology.
SELECTED REFERENCES
Achor, D.S., R. Ochoa, E.F. Erbe, H. Aguilar; W.P. Wergin, C.C.
Childers. 2001. Relative advantages of low temperature versus ambient
temperature scanning electron microscopy in the study of mite
morphology. International Journal of Acarology. 27(1): 3-12.
Becker, H. Mites Get Frozen, Photographed, and Identified. Agricultural Research. October 2000.
Erbe, E. F.; W. P. Wergin; R. Ochoa; J. C. Dickens; B. Moser. 2001.
Low temperature scanning electron microscopy of soft bodied organisms.
Proceedings of the Microscopy Society of America, Microscopy and Microanalysis.
7(2): 1204-1205.
Ochoa, R., E. F. Erbe, J. Pettis and W. P.Wergin. 2000. Examination
of frozen, hydrated mites using low temperature field emission scanning
electron microscopy. Proceedings of the Microscopy Society of America,
Microscopy and Microanalysis. 6(Suppl 2):874-875.
Wergin, W.P., R. Ochoa, E. F. Erbe and D. Joy. 2000. Oscillating
trichobothria in the low temperature SEM: Biological Capacitors or
charging artifacts. Scanning. 22(2):140-141.
Wergin, W P., Ochoa, R., Erbe, E.F., Craemer, C., Raina, A.K. 2000.
Use of low temperature field emission scanning electron microscopy to
examine mites. Scanning. 22(3):145-155.
Wergin, W. P., E. F. Erbe, R. Ochoa. 2001. Versatile and inexpensive
specimen holder for high angle azimuth rotation in a low temperature
scanning electron microscope. Proceedings of the Microscopy Society of
America, Microscopy and Microanalysis. 7(2): 718-719.
