This page contains a Flash digital edition of a book.
Viewing and Recording Live Hydrated Specimens with a High-Vacuum SEM

David Scharf Scharf Microscopy , 2100 Loma Vista Place , Los Angeles , CA 90039

Abstract: As a standard preparation technique for the viewing and recording of biological specimens in the scanning electron microscope (SEM), the fi xation, dehydration, and metal deposition of such specimens can have a seriously detrimental effect on their fi nal condition and visual state. In 1973 an alternative technique was developed to make high- quality images of unfi xed, uncoated, live, hydrated insect, plant, and other specimens in their natural state. This technique uses a relatively low SEM beam energy, a rapid vacuum chamber pumpdown, and minimal specimen preparation.


In the SEM there must be a balance between beam parameters (beam voltage, current, and spot size) and a specimen’s ability to dissipate charge in order to prevent beam damage and beam-induced charging. Living specimens, such as insects and plants, have a natural conductivity due to ion mobility in their hydrated tissues.

It has long been asserted in electron microscopy profes- sional and technical literature that it was not possible to image in the SEM live, hydrated specimens. It was thought that such specimens must be fi xed, dehydrated, and conductively coated [ 1 ], or alternatively, imaged with an environmental scanning electron microscope (ESEM) commercially introduced in 1980 by Electroscan, or later, a variable-pressure SEM (VP-SEM). T e other option available was to image hydrated specimens with an SEM outfitted for the cumbersome and expensive technique of cryo-fi xation.

The technique described in this article has been in use successfully since it was developed in 1973. Figure 1 shows the fi rst published image using the techniques described herein [ 2 ].

Materials and Methods Specimen preparation . To simplify mounting of live specimens on an SEM stub, insect and arachnid specimens were first anesthetized with refrigeration of approximately 38°F or with CO 2 and, if necessary, were very gently blown off with compressed air. Interestingly, most live insects and arachnids tend to preen themselves and remove dust from their bodies. T us, these live specimens were usually mounted by adhering the rear legs or the very rear of the body to the stub using denatured alcohol and graphite (DAG), which allowed preening to continue. For botanical specimens, care was taken to keep cut surfaces to a minimum, such as cutting and mounting by the stem only whenever possible. T is slows dehydration and keeps outgassing to a minimum. In many cases insects were found on botanical specimens and leſt there without any interference while the plant specimen was attached to the specimen stub. Instrumentation . An ETEC Autoscan SEM with a tungsten thermionic electron gun, employing an oil diffusion high- vacuum pump, was used for early research and subsequent follow-up work. Most SEMs in the 1970s yielded 4” × 5” images with about 1,000 lines, so the line spacing was just below what the


eye can detect. T e ETEC instrument allowed the user to vary the number of lines per frame up to 4,000 lines, even though the cathode ray recording tube had a resolution of 2,500 lines, allowing acquisition of images that could be enlarged signifi - cantly from the negative with photographic prints having been successfully made and exhibited up to 5 ft × 5 ft. Thus, until about 1992, images were recorded on 4 × 5 fi lm, including Polaroid PN-52, PN-55, Kodak Plus-X, and Ilford FP-4. In 1992 a 4Pi digital acquisition card was used in an Apple Macintosh computer for early experimentation. Beginning in 1993 a Gatan Digiscan with Digital Micrograph soſt ware was used for multi-channel digital image acquisition on an Apple Macintosh computer.

Key to this technique is a vacuum system with a quick pumpdown time. T e ETEC Autoscan’s valved vacuum diff usion pump system, with buff er tank and small specimen chamber, was ideal. T is system could be pumped down to 5 × 10 -5 torr (the system HV turn-on vacuum) in 60–70 seconds. T e fast pump-down was essential for many of the more delicate specimens because they could dehydrate in a few minutes,

Figure 1 : Cannabis sativa fl ower showing resin nodules with glandular multicel- lular capitate triochomes imaged at 5 kV and 15 mm working distance. This was one of the fi rst SEM images to appear in a commercial motion picture (stood in for “snake scale” in Ridley Scott’s 1982 Blade Runner ). Image taken in 1973 [ 2 , 7 ]. Image width = 156 µm.

doi: 10.1017/S1551929516001139 • 2017 January

Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68