Cryo-Planing Biological Specimens
750 (Electron Microscopy Sciences, Hatfield, PA), for overnight
freeze-dry-
ing. Aſter freeze-drying, specimens were gradually warmed to room tempera- ture and removed. Te Workstation was
Figure 1: Protocol steps: (A) Unmodified mPrep/s capsule and screen. (B) mPrep/s capsule with 2–3 mm segment cut off the end (dotted line) and discarded (X). mPrep/s screen opening enlarged to accommodate a ∼2 mm thick specimen (arrow). (C) Mounting specimen using mPrep/s Workstation: back end of specimen is inserted into screen slit that clamps the specimen. mPrep/s capsule with end cut off is slid over screen so that the specimen extends several millimeters beyond the capsule. Capsule and screen with mounted specimen is removed from Workstation. (D) Photo of cactus specimen held in modified mPrep/s capsule. (E) Diagram of plunging specimen into cryogen (LN2 slush). (F) Frozen specimen mounted in cryo-microtome. (G) Photo of 4 similar specimens held upright with mPrep/s screen on SEM stubs.
in Figure 1D. Specimens were kept wet with water throughout mounting and by immersion in water prior to rapid-freezing. Rapid-freezing was performed by plunging the mPrep-
mounted specimen into liquid nitrogen (LN2) slush. Figure 1E shows the capsule was oriented so that the specimen face for cryo-planing was plunged first into the cryogen to ensure rapid-freezing and the best preservation. Aſter freezing, mPrep-mounted specimens were immersed in LN2 while addi- tional specimens were prepared in the same manner. Cryo-planing was performed by transferring the frozen
specimens into the -60 °C chilled cryo-ultramicrotome chuck of an RMC MT 7000 microtome (RMC Boeckeler, Tucson, AZ) equipped with a Boeckeler Cryo System (Figure 1F). Because the polypropylene mPrep capsule is well below its glass transi- tion temperature and because the water in the capsule is solid, the microtome chuck firmly holds the mounted specimen. Te specimen was then cryo-planed with a -60 °C chilled glass knife, with the microtome dissecting scope used to determine when a desired tissue region was in view. Aſter planing, the capsule-mounted specimen was returned to LN2 while addi- tional specimens were cryo-planed. As necessary, fresh LN2 chilled glass knives replaced dulled knives. Once all desired specimens were cryo-planed, they were transferred from LN2 into a pre-chilled freeze-dryer EMS model
22
then used to remove the capsules from the screens in reverse of that shown in Fig- ure 1C, except that the spec- imen remained clamped in the screen. Te screens holding the upright speci- men were picked up from the Workstation using for- ceps and attached to SEM stubs using conductive car- bon tape. Colloidal graphite was then used to cover and ground the non-conductive polymer screens. Specimens were then sputter-coated with 5–10 nm of AuPd with a Denton Desk I sputter- coater (Denton Vacuum, Moorestown, NJ). Figure 1G shows several similar mounted and coated speci- mens. Specimens were then imaged in secondary elec- tron mode with an Hitachi S-4100 FE-SEM at 5 KeV.
Results Multiple Christmas cactus (Figure 2) and canine kidney
(Figure 3) specimens were successfully and easily prepared and imaged. Tese figures show the importance of image plane selection. Cactus leaves. Figure 2, of cactus leaves, shows that image
planes can be selected to predominately reveal the surfaces of intact cells (Figures 2A and 2B) or show cross sections through cells (Figures 2C and 2D). Te boundaries between individual cells are readily observed in the non-sectioned regions (Figure 2A). In combination with a field of view that also shows a few cross-sectioned cells, the image plane selection provides an overall view of the leaf tissue micro-architecture (Figure 2A). In another specimen region, an intact chloroplast with a textured surface is observed (Figure 2B). Te plane of the sectioned face is clearly discerned where the cells are cross-sectioned. Tis is shown in a low-resolution survey cross section through a dif- ferent leaf specimen (Figure 2C). Note the many cells, the cell wall structure, and the chloroplasts and other organelles on the membrane faces. A higher-resolution image of the same region shows a cross-sectioned chloroplast and a wavy textured mem- brane (Figure 2D). Canine kidney. Te images of cryo-planed canine kid- ney (Figure 3) provide an informative view of the overall
www.microscopy-today.com • 2019 March
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