High pressure freezing of intact plant tissues. Evaluation and characterization of novel features of the endoplasmic reticulum and associated membrane systems.

We have used plant root tips frozen under high pressure in conjunction with freeze-fracture electron microscopy a) to evaluate the quality of freezing of unfixed, non-cryoprotected tissues obtainable with this method, b) to examine the structure of cells frozen under high pressure, c) to evaluate the usefulness of high pressure freezing to preserve transient membrane events, and d) to look for artifacts caused by the high pressure. A single artifact of high pressure, possibly related to the collapse of air spaces during pressurization before freezing, manifested itself as long tears or folds in the plasma membrane. Excellent freezing, as evidenced by the smooth, turgid appearance of all membrane systems and the lack of aggregated cytosolic materials was observed in 10 to 20% of samples. In the best preserved specimens freezing was uniform throughout the sample volume and all organelles were readily identified. In the remaining ones, a gradient of ice crystal sizes was seen; cells within 50 to 100 microns of the surface being better preserved than those in the interior. Cortical microtubules appeared well preserved as were close associations of endoplasmic reticulum (ER) with nuclear, Golgi and plasma membranes. Junctions between the ER and nuclear membrane were constricted and much thinner (30 nm in diameter) than in chemically-fixed, thin-sectioned tissue, and although no continuities between the ER and Golgi membranes were observed, many Golgi stacks had an adjacent ER cisterna either at the cis or trans face. Both Golgi and ER cisternae exhibited distinct, round dilations indicative of vesicle blebbing or vesicle fusion events. Characteristic disc- and horseshoe-shaped infoldings of the plasma membrane corresponding to fused secretory vesicle and/or membrane recycling structures were also prominent in many cells. Short extensions of the cortical ER cisternae were regularly observed appressed against these plasma membrane infoldings suggesting a functional role for the ER in vesicle-mediated secretion and/or membrane recycling. Many lipid bodies were intimately associated with the ER, some with their surface monolayer fused with the cytoplasmic leaflet of the ER membrane. Our findings demonstrate that high pressure freezing can provide excellent morphological preservation of intact tissues and can preserve fast, transient membrane events such as those associated with vesicle fusion and vesicle blebbing. We conclude that this is the best available method for freezing relatively large (up to 0.6 mm thick) tissue samples for study by electron microscopy.