Abstract
The progress of plant biology, as well as many other biological subdisciplines, has been based on the microscopical observation of tissues, cells, and subcellular elements. For conventional electron microscopy, the specimens have to be fixed and processed, before they can be imaged. The preservation of subcellular structures during different processing steps has been a major concern of electron microscopists. For decades, structural preservation has relied on the use of aldehyde-based chemical fixatives. They are relatively inexpensive, easy to use, and very efficient for the preservation of certain subcellular elements. However, they present important limitations, including the induction of damages and artifacts that may alter significantly the cellular ultrastructure. To overcome this, one of the best options is to process specimens by high-pressure freezing (HPF) followed by freeze substitution (FS) procedures. Here I describe combined HPF and FS methods currently used in our laboratory to process different plant cells and tissues, grown in vivo and in vitro, for ultrastructural analyses and in situ localization studies.
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References
Ruska E (1980) The early development of electron lenses and electron microscopy. Hirzel Verlag, Stuttgart
Craig S, Staehelin LA (1988) High-pressure freezing of intact plant tissues—Evaluation and characterization of novel features of the endoplasmic reticulum and associated membrane systems. Eur J Cell Biol 46:80–93
Gilkey JC, Staehelin LA (1986) Advances in ultrarapid freezing for the preservation of cellular ultrastructure. J Electron Microsc Tech 3:177–210
McDonald KL, Auer M (2006) High-pressure freezing, cellular tomography, and structural cell biology. Biotechniques 41:137–143
Seguí-Simarro JM, Austin JR, White EA, Staehelin LA (2004) Electron tomographic analysis of somatic cell plate formation in meristematic cells of arabidopsis preserved by high-pressure freezing. Plant Cell 16:836–856
Otegui M, Austin JR (2007) Visualization of membrane-Cytoskeletal Interactions during Plant Cytokinesis. In: McIntosh JR (ed) Cellular electron microscopy. Methods Cell Biol 79:221–240
Kang BH, Nielsen E, Preuss ML, Mastronarde D, Staehelin LA (2011) Electron tomography of RabA4b- and PI-4Kbeta1-labeled trans Golgi network compartments in Arabidopsis. Traffic 12:313–329
Otegui MS, Staehelin LA (2004) Electron tomographic analysis of post-meiotic cytokinesis during pollen development in Arabidopsis thaliana. Planta 218:501–515
Kang B-H (2010) Electron microscopy and high-pressure freezing of Arabidopsis. Methods Cell Biol 96:259–283
Karahara I, Kang B-H (2014) High-pressure freezing and low-temperature processing of plant tissue samples for electron microscopy. In: Žárský V, Cvrčková F (eds) Plant cell morphogenesis, vol 1080. Methods in molecular biology. Humana Press, New York, pp 147–157
Daghma DS, Kumlehn J, Melzer M (2011) The use of cyanobacteria as filler in nitrocellulose capillaries improves ultrastructural preservation of immature barley pollen upon high pressure freezing. J Microsc 244:79–84
Seguí-Simarro JM, Nuez F (2008) How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis. Physiol Plant 134:1–12
Corral-Martínez P, Parra-Vega V, Seguí-Simarro JM (2013) Novel features of Brassica napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning. J Exp Bot 64:3061–3075
Reynolds ES (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208–212
McDonald K, Schwarz H, Müller-Reichert T, Webb R et al (2010) “Tips and tricks” for high-pressure freezing of model systems. Methods Cell Biol 96:671–693
Hayat MA (2000) Principles and techniques of electron microscopy: biological applications, 4th edn. Cambridge University Press, Cambridge
Robards AW, Wilson AJ (1993) Procedures in electron microscopy. Wiley, Chichester
Merighi A (1992) Post-embedding electron microscopic immunocytochemistry. In: Polak JM, Priestley JV (eds) Electron microscopic immunocytochemistry. Principles and practice. Oxford University Press, Oxford, pp 51–85
Wilson SM, Bacic A (2012) Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes. Nat Protoc 7:1716–1727
Thiry M, Thiry-Blaise L (1989) In situ hybridization at the electron microscope level: an improved method for precise localization of ribosomal DNA and RNA. Eur J Cell Biol 50:235–243
Thiry M, Thiry-Blaise L (1991) Locating transcribed and non-transcribed rDNA spacer sequences within the nucleolus by in situ hybridization and immunoelectron microscopy. Nucl Acids Res 19:11–15
Lichter R (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z Pflanzenphysiol 105:427–434
Bergman L (2001) Growth and maintenance of yeast. In: MacDonald P (ed) Two-hybrid systems, vol 177. Methods in molecular biology. Humana Press, Totowa, pp 9–14
Acknowledgements
I want to acknowledge Dr. Patricia Corral-Martínez and Mrs. Verónica Parra-Vega for providing some of the images used in this manuscript. Thanks are also due to the staff of the Electron Microscopy Service of Universitat Politècnica de València.
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Seguí-Simarro, J. (2015). High-Pressure Freezing and Freeze Substitution of In Vivo and In Vitro Cultured Plant Samples. In: Yeung, E., Stasolla, C., Sumner, M., Huang, B. (eds) Plant Microtechniques and Protocols. Springer, Cham. https://doi.org/10.1007/978-3-319-19944-3_7
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DOI: https://doi.org/10.1007/978-3-319-19944-3_7
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