Cryo-Planing Biological Specimens for Scanning Electron Microscopy


William C. Plumley,1 Michael Kostrna,1 * and Steven L. Goodman2 **


1Electron Microscopy Training Program, Madison College, 1701 Wright Street, Madison, WI 53704 USA 2Microscopy Innovations, LLC, 213 Air Park Road, Suite 101, Marshfield, WI 54449 USA


*Current address: Electron Microscopy Sciences, 1560 Industry Road, Hatfield, PA 19440. **steven.goodman@microscopyinnovations.com


Abstract: Cryo-planing SEM provides a powerful 3D view of internal and external tissue and cell features. However, specimen preparation is tedious because it requires custom apparatus and difficult cryogenic manipula- tions. We present an easy method using mPrep/s capsules that provide a “handle” to hold and orient specimens throughout cryo-preparation and imaging. Cryo-facing is done with a cryo-ultramicrotome, requiring no custom equipment while providing accurate control of the image plane. We show this with fresh Christmas cactus leaves and aldehyde-fixed kid- ney tissue imaged by room temperature SEM after freeze-drying, demon- strating the method operating without an expensive cryo-SEM.


Keywords: Cryo-planing, cryogenic, SEM, capsules


freeze-fracture, mPrep


Introduction Cryogenic planing (also called cryo-planing or cryogenic


face-off) is a sample preparation method for scanning electron microscopy (SEM) that provides an intuitive 3D view of tissue and cellular ultrastructure, including internal and external views of organelles, membranes, and cytoskeletal networks [1–4]. Unlike freeze-fracture, where the fracture plane randomly follows a path of least resistance that typically cleaves membranes and goes around cells and organelles [1,3,5], with cryo-planing the image plane can be controlled by the microscopist. With cryo-planing, assuming the microscopist has a means to see the specimen, the image plane can be selected to observe specific structures. Te typical methodology for cryo-planing is to rapidly


freeze a tissue sample, and then cut a cross section though the frozen specimen. Specimen preparation aſter planing may be done in one of two ways: (1) Specimens may be freeze-dried and conductively coated to prepare them for imaging with a conventional non-cryo stage equipped SEM or (2) specimens may be entirely cryogenically prepared for imaging with a cryo-stage equipped SEM. While cryo-planing is a powerful technique, it is tedious


to perform and generally requires specialized custom-built apparatus [1–3]. Te major difficulty is the holding, orienting, sectioning, and transferring of small tissue specimens during cryogenic and ambient temperature manipulations. Te dif- ficulty of “simply” placing a frozen specimen into a suitable chuck for cryo-planing has been documented [1]. In addition, because specimen structures can be invisible in frozen speci- mens, obtaining an accurate orientation when mounting the sample for cryo-planing is oſten problematic. Tis article describes a much easier technique for cryo-


planing small “millimeter-size” specimens, when specimens particularly require orientation and when it is important to con- trol the location of the cross-sectional plane [6]. Tis method is suitable for almost any microscopy lab since it requires no specialized tools other than a standard cryo-ultramicrotome,


20 doi:10.1017/S1551929518001451


a standard mPrep/s Workstation, and mPrep/s capsules [7]. Te mPrep/s capsules are used to hold and orient specimens throughout the entire preparation process, including rapid- freezing, cryo-planing, cryo-storage, freeze-drying, conduc- tive coating, and even SEM imaging. Te cryo-ultramicrotome used for the cryo-planing also enables imaging and positional control of the cross-sectional plane.


Materials and Methods Specimens were prepared from both plant and animal


tissues. Fresh Christmas cactus (genus Schlumbergera) leaves were trimmed into pieces 2–3 mm thick by 5–8 mm long and kept wet until rapid-freezing. Canine kidney was fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phos- phate buffer, based on Karnovsky [8], and then refrigerated. In preparation for cryo-planing, kidney samples were trimmed to about 2 mm thick by 5 mm long, washed in 0.1 M sodium phosphate buffer, post-fixed in buffered 2% OsO4


for one hour,


and rinsed and stored in deionized water. Te specimen preparation protocol is shown in Figure 1.


Te first step was to modify the mPrep/s capsules and the mPrep/s screens that come with the capsules (Microscopy Innovations, LLC, Marshfield, WI, USA). Te mPrep/s screens have a center slit that can be opened and closed to clamp onto the back or non-imaged end of a specimen in order to pro- vide a perpendicular orientation. As received, the screen slit is designed to clamp onto 0.1 to 1 mm thick specimens. Since the specimens in the present study were 2–3 mm thick, the slits were enlarged by cutting with a fine scalpel to make a hole slightly smaller than the specimen thickness (Figure 1A). A biopsy punch can also be used to make a hole in the screen slightly smaller than the specimen. Te second modification was to use a razor blade to cut off about 3 mm of the mPrep/s capsule’s closed end (Figure 1B). Tis is so that the end of the specimen will extend beyond the capsule (Figures 1C and 1D). Specimens were mounted into mPrep/s screens by first


inserting screens into an mPrep/s Workstation, as dia- grammed in Figure 1C. Te Workstation mechanism was then used to open and then close the screen slit onto the back end of the specimen (not the specimen end to be imaged). Once the specimen was clamped in the screen in the desired orientation, the modified capsule was then slid down over the screen so that the specimen extended 1–3 mm beyond the end of the modified capsule (Figure 1D). Te capsule with the screen holding the oriented specimen was then removed from the Workstation. (Workstation operation can be seen at www.MicroscopyInnovations.com). An oriented and mounted cactus specimen ready for plunge-freezing is shown


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