Low‐temperature scanning electron microscopy of frozen hydrated mouse lung

Abstract

A method is presented by which water is preserved as ice during examination of the lung in the scanning electron microscope (SEM). The lung need only be inflated, frozen, transferred to the microscope and examined with the electron beam. Chemical fixation, solvent dehydration, and drying are not necessary. The low‐temperature SEM of Pawley and Norton [11] maintains lung at −180° C, nearly liquid nitrogen temperature, for extended periods with a Joule‐Thomson refrigerator built into the stage. It has an integral high‐vacuum preparation chamber attached to the microscope column which allows serial fracture, low‐magnification stereo light microscopy, radiant etching, and evaporative coating with gold or carbon. The stage can be tilted from 0° to 45° and rotated a full 360°. It is demonstrated that the air‐liquid interface in the lung can be examined and that low‐temperature SEM can be used to investigate the shape of alveoli, the patency of the pores of Kohn in the hydrated state, and the shrinkage and distortion of lung with drying.

Cryo‐planing for cryo‐scanning electron microscopy

“…Several methods have been used to reduce the sizes of frozen samples. Bastacky (personal communication) initially used a carbide-tipped circular saw to reduce the size of frozen lung tissues and then a fine diamond circular dental saw for the final specimens (Bastacky et al 1987, Wu et al 1996. Investigators working with plant tissues have used fracturing (Tyree et al 1999), cutting (Dong et al 1997), trimming (Canny 1997), splitting (Canny 1997), and probably also sawing (Utsumi et al 1996) to reduce sample sizes.…”

A specimen holder for low‐temperature scanning electron microscopy

“…The frozen sample is cut into a rectilinear block, preferably the width of the trough (1 1 mm), 1-2 mm taller than the trough (4-5 mrn) and 1-5 mm thick. The block is cut with a 2 cm diameter circular saw blade, which turns under liquid nitrogen, and is driven by a modified dental handpiece (Bastacky, et al, 1987). The sample is tranferred with curved forceps under liquid nitrogen to the trough in the specimen holder.…”

“…The low-temperature scanning electron microscope (LTSEM) is utilized for examining frozen specimens with water present as ice, thus avoiding chemical fixation and dehydration (Echlin et al, 1970;Hayes and Koch, 1975;Pawley and Norton, 1970;Hook et al, 1980;Bastacky et al, 1987). During fixation by freezing, liquid water in biological systems is converted to ice, the size of ice crystals being inversely proportional to the rate of freezing.…”

Ultrastructure of Tracheal Surface Liquid: Low‐Temperature Scanning Electron Microscopy

“…However, the low-temperature scanning electron microscope (LTSEM) (Echlin 1990) permits the maintenance of lung tissue below -1 80 "C during the entire procedure from fixation by rapid cooling to viewing (Bastacky et al 1987). Thus, ASL can be preserved with its water present and viewed without significant change in its dimensions or composition.…”

“…Though two layers have been seen in microscopic studies of dried material (Puchelle et al 1991, Reissig et al 1978, Sanderson and Sleigh 1981, Sturgess 1977, Yoneda 1976, the depths of these layers will have been altered due to shrinkage during processing (Bastacky et al 1985). However, the low-temperature scanning electron microscope (LTSEM) (Echlin 1990) permits the maintenance of lung tissue below -1 80 "C during the entire procedure from fixation by rapid cooling to viewing (Bastacky et al 1987). Thus, ASL can be preserved with its water present and viewed without significant change in its dimensions or composition.…”