Dr Gary Bauchan is the Director of the Electron and Confocal Microscopy Unit at the Agricultural Research Service (ARS), the principal in-house research agency of the United States Department of Agriculture. The Unit is a core facility with the responsibility of providing collaborative assistance to scientists from ARS, Northeast Area and Beltsville Agricultural Research Center (BARC) who have microscopy applications that require high resolution imaging. Dr Bauchan’s team have produced images using electron microscopes of bacteria, fungi, mites, insects, nematodes and parasites along with plant and animal tissues both healthy and diseased. One of his major collaborations is with Dr Ron Ochoa, the world's expert on plant feeding mites.
Biological specimens require special treatment due to the high water content of the samples. Many of the specimens are in liquid cultures or are very soft-bodied and by using classical preparative techniques will either destroy the specimen or distort the specimen producing artefacts. A cryo-prep system is an ultra-fast method to ready the specimens for observation in a SEM especially a high resolution field emission SEM. Thus, specimens are frozen in time to allow for observation of feeding behaviour, mating behaviour, host/parasite interactions, etc. It preserves the natural orientation of ultrafine structures such as setae, antenna, legs, skin texture, sensory organs, waxy coatings and eggs.
Asked about his experience using a Quorum PP2000 Cryo-SEM preparation system on the Hitachi S-4700 field emission scanning electron microscope, Dr Bauchan said “The Quorum system is easy to use, the set-up for imaging is logical, durable, reliable, and maintains ultra-low temperatures for a long period of time. Holders containing pre-frozen samples are transferred into the cryo-prep chamber where they are etched to remove any surface contamination (condensed water vapour) by raising the temperature of the stage from -130 ºC to -90 °C for 10-15 minutes. Following etching, the temperature inside the chamber was lowered below -130 °C, and the specimens were coated with a 10 nm layer of platinum using a magnetron sputter head equipped with a platinum target. The specimens were transferred to a pre-cooled (-130 °C) cryo-stage in the SEM for observation.”
The system has been used in multiple projects by the Unit, many of which have been published with the generation of stunning, colourful images. The use of low temperature SEM has been shown time and again to be the best method for the examination of microscopic biological specimens and their ultrastructure. The work in conjunction with Dr Ochoa has been particularly productive with five papers published this year to date. These have focused on the field of acarology, a branch of zoology dealing with the study of mites and ticks.
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