Invertebrates and Insects
Butterfly wing, prepared by cryo-SEM
Images courtesy of Yangzhou University, China
Critical point dried aphid
There are more than 300 species of aphid within the UK alone, feeding on a variety of plants. Identification can be difficult and highly dependent upon subtle taxonomic structural features.
SEM is an ideal technique for the isolation of these features, but the aphids themselves present problems in preparation for the technique. Unlike many insects that can be readily prepared for SEM by air-drying with artefact, aphids are highly hydrated and susceptible to shrinkage and distortion.
Even critical point dried specimens undergo considerable gross structural changes. As shown in these images, the abdomen shrinks and collapses, and most of the thin ribbons of wax, which are present on many aphid species, are entirely dissolved away by acetone dehydration.
Low-temperature SEM (cryo-SEM) is therefore a preferred method of preparing these very delicate organisms - see frozen hydrated aphid for comparison.
Cryo-SEM prepared aphid
In comparison with the critical point dried aphid, this image shows that there is no distortion of the abdomen nor any other parts of the aphid following cryo preparation. The delicate patterns on the surface of the abdomen (probably chitin or wax) are an important taxonomic feature are well preserved.
Cryo-SEM: European house dust mites
Feeding European house dust mite (Dermatophagoides pteronyssinus).
The specimen was rapidly frozen in slushy nitrogen, sublimated at -90°C for 4 minutes, sputter coated with 5 nm of platinium and observed in the SEM at -140°C
Cryo-SEM: millipede ansdmotile hypopus mite
The mite, identified by the Natural History Museum in London as a motile hypopus , was imaged as it was "taking a ride" on a millipede to search for richer feeding grounds.
The image was prepared using a Quorum Technologies PP3000T Cryo-SEM Preparation System attached to a Carl Zeiss Sigma FE-SEM.
Images courtesy of the Image and Analysis Unit at the School of Pharmacy and Biomolecular Sciences, University of Brighton.
Cryo-SEM: mite (Brevipalpus yothersii)
This mite was collected in Florida, USA and has been shown to carry the citrus laprosis virus that can kill citrus trees (oranges, lemons, limes, grapefruit, etc.). Until now (2015) it has not been widely detected in the US, however the host mite is present in some citrus orchards. The close-up image of the skin (known as the integument) shows the wax-like microplates which are distinctive to this species of mites and can be used as a morphological trait to separate and identify mite species.
The microplates are wax-like and so they can only be observed using cryo–SEM. Chemical techniques (e.g. critical point drying) will dissolve the wax-like structures. Variable pressure/ environmental SEM using a tungsten source cannot clearly image these beam-sensitive structures so it is imperative to use cryo preparation with together with field emission SEM.
Images courtesy of USDA Beltsville and Hitachi High Technologies North America.
Cryo-SEM: brown marmorated stink bug (Halyomorpha halys)
The stink bug is agricultural pest introduced into the US from China, Taiwan and Japan and has become a season-long pest in fruit orchards.
Cryo-SEM: mites - from USDA-ARS, Beltsville
The United States Department of Agriculture in Beltsville, Maryland has for many years used cryo-SEM as an essential tool to study a range of mites. For further information please contact: the USDA-ARS Electron & Confocal Microscopy Unit.