A simple freeze-substitution method for electron microscopy

doi:10.1016/S0022-5320(66)80119-3

Journal of Ultrastructure Research

Volume 15, Issues 5–6, August 1966, Pages 469-479

https://doi.org/10.1016/S0022-5320(66)80119-3 Get rights and content

A method has been developed for freeze-substitution fixation of thin specimens. A special device shoots the specimen into propane cooled by liquid nitrogen. The substitution liquid (1.5 ml absolute methanol containing 20% uranyl acetate and 0.5 ml acrolein) is frozen in a tube below propane. After freezing of the specimen, the tube is transferred into a dry-ice acetone bath, where the substitution liquid thaws and the specimen sinks into it. Propane is then removed by suction and the preparation is kept in dry ice for 2 days and in a refrigerator for 2 more days. The combination of substituent and fixative preserves cellular structures without distortions. Neurospora hyphae thus fixed show regularly shaped mitochondria, vacuoles, and nuclei. Mitochondria are surrounded by a layer of ribosomes. Salivary glands of Drosophila show extensive ergastoplasmic lamellae with ribosomes of diffuse appearance. Older glands have amorphous looking droplets of secretion product, reduced ergastoplasm, and numerous mitochondria. Preparations can be stained with lead citrate to increase the contrast.

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Strategies to improve the antigenicity, ultrastructure preservation and visibility of trafficking compartments in Arabidopsis tissue
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Immunolabelling of (ultra)thin thawed cryosections according to Tokuyasu is one of the most reliable and efficient immunolocalisation techniques for cells and tissues. However, chemical fixation at ambient temperature, a prerequisite of this technique, can cause problems for samples, like plant tissue, because cell walls, hydrophobic surfaces and intercellular air slow down diffusion of fixative molecules into the sample. We show that a hybrid technique, based on a combination of cryofixation/freeze-substitution and Tokuyasu cryosection immunolabelling, circumvents the disadvantages associated with chemical fixation and results in an improved ultrastructure and antigenicity preservation of Tokuyasu cryosections used for light and electron microscopic immunolabelling (as shown for Myc- or mRFP-tagged proteins, KNOLLE and carbohydrate epitopes). In combination with the most sensitive particulate marker systems, like 1-nm gold or quantum dot markers, we were able to obtain a differentiated labelling pattern which allows a more detailed evaluation of plant Golgi, trans-Golgi network and multivesicular body/prevacuolar compartment markers (COPI-specific γCOP, the ADP-ribosylation factor GTPase ARF1, ARA7/RabF2b and the vacuolar sorting receptor VSR). We also discuss possibilities to improve membrane contrast, e.g., of transport vesicles like COPI, COPII and clathrin-coated vesicles, and of compartments of endosomal trafficking like the trans-Golgi network.
Insects as model systems in cell biology
2010, Methods in Cell Biology
Citation Excerpt :
Transfer by hand, if done quickly (<1 s), is usually without deleterious effects. With particularly sensitive specimens the method of Zalokar (1966) can be applied, in which the substitution medium and the cryogen for freezing are contained in the same vessel. A convenient storage vial for keeping specimens under LN2 can be made from Eppendorf centrifuge tubes (1.5 ml) by screwing their conical end into metal nuts.
For almost 100 years, insects have been favorable “model systems” in biology. Just to mention a few examples: fruit flies in genetics and developmental biology; bugs and caterpillars in hormone research; houseflies, blowflies, and locusts in neurobiology; silk moths in pheromone research; honeybees and crickets in neuroethology. For more than 50 years the electron microscope (EM) has been a valuable tool in analyzing the structure of cells and organs of these creatures. However, progress in specimen preparation was relatively slow compared with mammalian material and, in 1970, it was taken for granted that insects were much more difficult to fix than mammals. Since then, methods have dramatically improved, and satisfactory results can now be obtained routinely with chemical as well as cryofixation. In this chapter we briefly demonstrate what can be achieved with insect material, and help the researcher to find the most appropriate method for her/his systems and scientific questions.
Cryopreparation of biological specimens for immunoelectron microscopy
2009, Annals of Anatomy
Cryopreparation methods for transmission electron microscopy comprise a variety of different low temperature approaches. For any particular specimen a careful comparison of preparation methods should be made with respect to the preservation of structure and antigenicity. Here an overview of cryomethods is provided especially suitable for immunoelectron microscopy. Replacing chemical fixation by cryofixation, which immobilizes a sample physically by rapid freezing, is the basis of several technical lines of ultrastructure research. Cryofixed samples can be cryosectioned and directly observed by cryo-electron microscopy in their most native, fully hydrated state. For the purpose of immunoelectron microscopy, cryofixed specimens are freeze substituted and embedded for room temperature sectioning or freeze fractured for replica cytochemistry. Since the invention of high-pressure-freezing (HPF) cryofixation is routinely applicable for larger specimens of up to 200 μm thickness. Cryosectioning of chemically fixed and cryoprotected samples according to Tokuyasu results in sections which are ideal substrates for immunogold labelling. Recently, rehydration protocols have been developed to combine cryofixation with routine Tokuyasu cryosectioning. Progressive lowering of temperature (PLT) protocols are also applied to chemically fixed samples. Here dehydration and embedding are carried out accompanied by a stepwise lowering of the temperature to minimize negative effects of dehydration on the distribution and antigenicity of proteins. Conclusions drawn from ultrastructural data should always be based on the best possible technique of sample preparation. Cryofixation is the state-of-the-art method for ultrastructural preservation, but for unstable and anoxia sensitive tissues, a combination of chemical prefixation with HPF and freeze substitution might be a valuable compromise.
Cryopreparation Methodology for Plant Cell Biology*
2007, Methods in Cell Biology
Citation Excerpt :
Rapid freezing at ambient pressure is technically less demanding, but adequate freezing is usually confined to the outermost 10–15 μm of a specimen [for a review on slam, plunge, jet, and spray freezing, see the work by Sitte et al. (1987)]. Some of these freezing techniques prove to be as useful for thin fungal hyphae (Ashford et al., 1999; Howard and Aist, 1980; Muller et al., 1980; Rupes et al., 1995; Zalokar, 1966) whereas higher plants are less often well preserved by these methods [for good examples, see the work by Ding et al. (1991a,b, 1992) and Huang et al. (1993); for further details on rapid freezing and especially HPF, see Chapter 1 by Dubochet, this volume]. Air replacement around the specimens is generally accepted to be a prerequisite for successful HPF (Craig and Staehelin, 1988; Craig et al., 1987; Müller and Moor, 1984).
This chapter reviews literature on cryoimmobilization, freeze-substitution, and resin embedding for ultrastructural and immunocytochemical transmission electron microscopy (TEM) studies of plants and fungi. It illustrates the reasons behind the difficulty to fix plants and fungi usually, while they are rather easy to freeze. It mentions the favorable features of plants and fungi, which make cryo-immobilization of these organisms quite easy. The chapter details step-by-step cryo-immobilization of plant and fungal bulk specimens by means of high pressure freezing. The following aspects are discussed in detail: (1) sampling for high pressure freezing; (2) specimen mounting and space fillers; (3) high-pressure freezing, specimen storage, and transport; and (4) freeze-substitution and embedding with epoxy or (meth) acrylate resins for morphology, immunolabeling, microanalysis, and autoradiography. In addition, it also discusses other cryo-based EM-preparation techniques. It is essential to take meticulous care with the pre- and post-freezing manipulations and to aim at a thorough understanding of the whole chain of procedures used for preparation and analysis.
Introduction to Electron Microscopy of Cells
2007, Methods in Cell Biology
Electron microscopy (EM) has helped to close the gap between optical microscopy of cell architecture and biochemical studies of macromolecules. It is now possible for cell biologists to see macromolecules fixed in situ, presumably in the act of doing their biological job. Despite such progress, the concern persisted that the methods required for getting cells ready for EM, such as fixation, dehydration, embedding in plastic for sectioning and staining with heavy metals, were altering cellular structure. However, alternative preparative methods provided independent evidence for the validity of at least some of the cellular EM that was being done on fixed material. This chapter provides three important kinds of information: (1) authoritative accounts of the ways world's best practitioners of cellular EM currently ply their structural trade, (2) an accurate sense of the limitations imposed by current methods and instruments, and (3) a clear-eyed view of what may plausibly be achieved in the near future.
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The most important goal of structural cell biology is to elucidate the mechanisms of the processes of life. The structure of a membrane system or fibrous array, the changes in such structures over time or the localizations of enzymes relative to organelle boundaries can often illuminate associated cellular functions. To be of maximal value, structural studies should provide isotropic, 3D information about well-preserved samples at the highest possible resolution. Electron tomography can provide such information about many kinds of cells and organelles.

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¹: This research was supported by the National Institutes of Health, USPHS-GM-12027.

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A simple freeze-substitution method for electron microscopy1

J. Ultrastruct. Res.

J. Ultrastruct. Res.

J. Biophys. Biochem. Cytol.

J. Biophys. Biochem. Cytol.

Ann. N.Y. Acad. Sci.

J. Ultrastruct. Res.

Histochimie et cytochimie animales