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Live Hydrated Specimens


Figure 2 : Gladiola flower (Gladiolus, family Iriaceae) petal surface showing individual cells at 5 kV and 14 mm working distance. Image taken in 1974 [ 7 ]. Image width = 437 µm.


and fast pumping gives the operator more work time. Many specimens could be observed and imaged for tens of minutes at magnifi cations from 10× to approximately 5,000×. Imaging . Because insects sit higher than the specimen stub by several millimeters, imaging with a large depth of fi eld is usually desirable. Because of the electron optical column design and the position of the objective aperture in the fi nal lens, the most-used aperture size for imaging in the ETEC was 150 µm. T is aperture was a good compromise between beam intensity and depth of fi eld for most users. Larger apertures were used


Figure 3 : Black ant ( Tapinoma sessile ) at 5 kV and 24 mm working distance. These ants eat honeydew, which is made by aphids and scale insects, and other sugary foods. These ants are commonly found in the home. A 5 ft × 5 ft photographic print was made of this image for a museum exhibition. Image taken in 1974. Image width = 581 µm.


occasionally for imaging but more often for x-ray analysis. However, by employing a smaller aperture of 100 µm or so, a relatively long working distance (up to 40 mm between the specimen and the bottom of the fi nal lens), and careful use of electronic dynamic focus, an entire specimen can be brought into focus while displaying fine surface detail. It should be noted that very long working distances tend to visually fl atten images and slightly degrade resolution, therefore moderate working distances of 10 mm to 20 mm were more routinely used, preserving the perception of depth in the images. This advantage of the SEM is particularly useful in imaging insects and larger botanical specimens. A high specimen tilt, 90° or so, was used in many situations in order to have a good portrait perspective and have a darkened, non-distracting background. Through trial and error, a beam accelerating voltage of approximately 5 kV was found to give the best results, producing just the right amount of surface detail and beam penetration on live specimens for acceptable photographic image quality. Accelerating voltages of 2–3 kV were useful in some situations. A moderate condenser lens setting (for spot size/beam current) was used as a starting point. Optimum condenser settings were found for particular specimens and magnifi cation ranges by trial and error. Fast scan rates (short beam dwell per picture point) allowed the use of larger beam currents for good quality images (high signal to noise [S/N]) or just observation. Slower scans (longer beam dwell) required lower beam current to avoid beam damage and specimen charging.


Figure 4 : Compound eye of a house fl y ( Fannia canniculartis ) at 5 kV and 20 mm working distance. Each facet is an individual eye with its own lens and retina. Image taken in 1974 [ 7 ]. Image width = 508 µm.


2017 January • www.microscopy-today.com


Examining live insects, and hydrated specimens in general, requires keeping observation time to a minimum. Standard practice was to perform critical focusing on images at a magnification at least ten times higher than that to be used for recording. T is focusing would be accomplished


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