Low-temperature Scanning Electron Microscopy of Frozen Hydrated Apple Tissues and Surface Organisms

Frozen hydrated buds and epicarp of ‘Golden Delicious’ apple (Malus domestica Borkh.) were observed with a low-temperature, field emission scanning electron microscope (SEM). In addition to observing surface features of these specimens, holders were modified to observe fractured specimens. A modified hinged holder retained both halves of a fractured specimen for examination of the complementary faces of frozen hydrated tissues. Low-temperature SEM avoided artifacts, such as extraction, solubilization, and shrinkage, which are normally encountered with chemical fixation, dehydration, and drying, respectively. The technique allowed observations of well-preserved frozen hydrated structures, such as the platelets of epicuticular wax; loosely associated organisms on plant surfaces, such as spider-mite eggs; delicate structures, such as fungal hyphae; and partially hydrated tissues, such as fruit epicarp and winter bud scales. During the past 25 years, electron microscopy (EM) has greatly expanded our knowledge about the fine structure of horticultural crops. For example, the transmission electron microscope (TEM) has revealed ultrastructural changes that occurred during fruit ripening (Ben-Arie et al., 1979; Crookes and Grierson, 1983; Laval-Martin, 1974; Masia et al., 1992); the scanning electron microscope (SEM) has provided detailed observations of epicuticular waxes (Bukovac et al., 1981; Faust and Shear, 1972; Glenn et al., 1985) and of the life cycles of several rust fungi (Littlefield and Heath, 1979). Both instruments, the TEM and the SEM, have provided structural information that has helped horticulturists relate ultrastructural changes to physiological processes and understand many of the biological events occurring in horticultural plants. Although EM studies are invaluable, the plant materials examined with these techniques must be subjected to chemical fixation, dehydration, embedding or critical-point drying, and staining or coating. All of these preparation procedures Received for publication 15 June 1993, Accepted for publication 24 Sept. 1993. We are grateful to Robert L. Smiley for helpful discussions and to Christopher Pooley for his photographic assistance in preparing the final plates. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by U.S. Dept. of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. The cost of publishing this paper was defrayed in part by the payment of page charges, Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1Horticultural Crops Quality Laboratory, Building 002, Beltsville Agricultural Research Center–West. 2Electron Microscopy Laboratory, Building 177B, Beltsville Agricultural Research Center–East. HORTSCIENCE , VOL. 29(4), APRIL 1994 Fig. 1. Specimen holders modified and fabricated for observing apple tissues. (a) Modified complementary holder mounted on a flat holder. A segment of the pericarp was mounted in the cavity of the complementary holder. The arrow illustrates how the holder is opened, which fractures the sample but retains the complementary faces. This entire assembly can then be inserted into the microscope to observe the fractured, frozen, hydrated specimen. (b) Flat holder with multiple mounting holes. (c) Flat specimen holder containing a threaded cylindrical ring. The ring maintained the bud in a vertical position and allowed cross fracture of the tissues. Bar = 2 cm. have been associated with structural artifacts (Beckett and Read, 1986; Crang, 1988; Jeffree and Read, 1991; Kellenberger et al., 1992; Wergin and Erbe, 1989; Wergin et al., 1988). For example, penetration of chemical fixatives, which may require several hours, has been associated with material extrusion; dehydration with solvents may extract or dissolve cellular constituents; critical-point drying can shrink and distort tissues; and sputter-coating with 20 to 30 nm of gold-palladium may obliterate fine-structural details. To avoid these problems, examining unfixed hydrated tissues with less coating is highly desirable. However, conventional EM imaging requires high vacuum; therefore, biological samples, which contain as much as 90% water, would quickly collapse. To avoid collapse, Echlin et al. (1970) described a procedure to examine frozen, fully hydrated specimens in a SEM. Specimens observed in this manner did not collapse because they were inserted and observed on a cryostage operating at temperatures below –130C where the vapor pressure of water was not significant. Therefore, sublimation did not occur at a detectable rate. Furthermore, at temperatures below –130C, recrystallization of pure-water ice does not occur. Wergin and Erbe (1992a) have used this procedure to observe frozen samples that remained stable for several hours. Materials and Methods The present study evaluated the advantages of this technique by installing a standard SEM cryostage on a field emission SEM to observe some frozen, hydrated tissues from apple. An Oxford CT 1500 Cryotrans System (Oxford Instruments, Eynsham, England) was mounted on a Hitachi S-4000 field emission SEM (Hitachi Scientific Instruments, Mountain View, Calif.) to perform low-temperature manipulations and observations. Field emission SEM permits observation of samples having less coating than is normally required for a conventional SEM (Wergin et al., 1988). ‘Golden Delicious’ apple specimens consisted of the following: 1) mature healthy fruit harvested from commercial orchards, 2) fruit infected with a fungal pathogen, and 3) vegetative buds removed from orchards during the winter. Several types of specimen holders were either modified or fabricated for the apple specimens (Fig. 1a–c). One-square-centimeter segments of epicarp and underlying parenchyma were removed from mature fruit and mounted in a complementary holder (upper portion of Fig. 1a) that was originally described by Steere and Erbe (1981) to produce platinum/carbon replicas that could be observed in a TEM. This holder was modified so that it could be firmly attached to a flat holder and inserted into the SEM to observe both halves of the frozen fractured tissue. Other segments of epicarp were mounted on a flat