Abstract
This chapter briefly describes conventional and microwave-assisted chemical fixation methods, as well as cryo-specimen preparation techniques for studying the cellular and organelle ultrastructure of plant tissues under transmission electron microscopy. The general methods and procedures for the plant specimen preparation (including fixation, dehydration, resin infiltration, and embedding) are similar to those for animal tissues. However, certain special characteristic features of plant tissues such as thick cellulosic cell wall, waxy substance in the cuticle, large amount of gases in the intercellular spaces, and the presence of vacuoles have created fixation and resin filtration difficulties. Specific modifications of the protocols used for animal tissues are therefore required, such as the application of vacuum during the initial fixation and resin infiltration stage to remove gases from the tissues and resin. Microwave-assisted procedure can reduce specimen preparation time, but both conventional and microwave-assisted chemical fixation procedures produce artifacts. Cryo-specimen preparation involves with high-pressure freezing and freeze-substitution can minimize artifact formation, but their application to highly vacuolated, thick-walled plant cells is limited.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Bozzola JJ, Russell LD (1999) Electron microscopy principles and techniques for biologists. Jones and Bartlett Publishers, Sudbury, MA
Hayat MA (2000) Principles and techniques of electron microscopy- biological application, 4th edn. Cambridge University Press, Cambridge, pp 349–471, for Plant tissues
Dashek WV (ed) (2000) Methods in plant electron microscopy and cytochemistry. Humana Press, Totowa, NJ
Dykstra MJ, Ruess LE (2003) Biological electron microscopy: theory, techniques, and troubleshooting. Kluwer, New York
O’Brien TP, Kuo J, McCully ME et al (1973) Coagulant and non-coagulant fixation of plant cells. Aust J Biol Sci 26:1231–1250
Mersey B, McCully ME (1978) Monitoring of the course of fixation of plant cells. J Microsc (Oxford) 114:49–76
Samuels AL, Staehelin LA (1996) Caffeine inhibits cell plate formation by disrupting membrane reorganization just after the vesicle fusion step. Protoplasma 195:144–155
Dong Z, McCully ME, Canny MJ (1994) Retention of vacuoles contents of plant cells during fixation. J Microsc (Oxford) 175:222–228
Mayers CP (1970) Histological fixation by microwave heating. J Clin Pathol 23:273–275
Giberson RT, Demaree RS Jr (1999) Microwave processing techniques for electron microscopy: a four-hour protocol. Methods Mol Biol 117:145–158
Benhamou N, Noel S, Grenier J et al (1991) Microwave energy fixation of plant tissue: an alternative approach that provides excellent preservation of ultrastructure and antigenicity. J Electron Microsc Tech 17:81–94
Heumann HG (1992) Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds. Histochemistry 97:341–347
Russin WA, Trivett CL (2001) Vacuum-microwave combination for processing plant tissues for electron microscopy. In: Giberson RT, Demaree RS Jr (eds) Microwave techniques and protocols. Humana Press, Totowa, NJ, pp 25–35
Lería F, Marco R, Medina FJ (2004) Structural and antigenic preservation of plant samples by microwave-enhanced fixation, using dedicated hardware, minimizing heat-related effects. Microsc Res Tech 65:86–100
Zechmann B, Zellnig G (2009) Microwave-assisted rapid plant sample preparation for transmission electron microscopy. J Microsc (Oxford) 233:258–268. doi:10.1111/j.1365-2818.2009.03116.x
Carpetier A, Abreu S, Trichet M et al (2012) Microwaves and tea: new tools to process plant tissue for transmission electron microscopy. J Microsc (Oxford) 247:94–105. doi:10.1111/j.1365-2818.2012.03626.x
Moor H, Riehle U (1968) Snap-freezing under high pressure: a new fixation technique for freeze-etching. In: Bocciarelli D.S. (ed) Proceedings on 4th Euro Reg Conf Elect Microsc 2, 33–34. Rome
Muelller M, Moor H (1984) Cryofixation of thick specimens by high-pressure freezing. In: Revel J-P, Bamard T, Haggis GH (eds) The science of biological specimen preparation for microscopy and microanalysis. SEM, Inc., AMF O’Hare, Chicago, IL, pp 131–138
Humbel B, Mueller M (1986) Freeze substitution and low temperature embedding. In: Mueller M et al (eds) The science of biological specimen preparation for microscopy and Microanalysis 1985. SEM, Inc., AMF O’Hare, Chicago, IL, pp 175–183
Studer D, Michel M, Meuller M (1989) High pressure freezing comes of age. Scanning Microsc Suppl 3:253–268, discussion 268-269
Dahl R, Staehelin LA (1989) High pressure freezing for the preservation of biological structure: theory and practice. J Elect Microsc Techn 13:165–174
Staehelin LA (1991) High pressure freezing, an advanced cryofixation technique for electron microscopical research. Plant Sci Tomorrow 3:16–17
McDonald K, Schwarz H, Muller-Reichert T et al (2010) “Tips and tricks” for high-pressure freezing of model systems. Methods Cell Biol 96:671–693
Hess MW (2003) Of plants and other pets: practical aspects of freeze-substitution and resin embedding. J Microsc (Oxford) 212:44–52
Hess MW (2007) Cryopreparation methodology for plant cell biology. Methods Cell Biol 79:57–100. doi:10.1016/s0091-679x(06) 79003-3
Kang BH (2010) Electron microscopy and high-pressure freezing of Arabidopsis. Methods Cell Biol 96:259–283
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 146:80–93
Kiss JZ, Giddings TH Jr, Staehelin LA et al (1990) Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis. Protoplasma 157:64–74
Staehelin LA, Giddings TH Jr, Kiss JZ et al (1990) Macromolecular differentiation of Golgi stacks in root tips of Arabidopsis and Nicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples. Proc Natl Acad Sci U S A 157:75–97
Mineyuki Y, Murata T, Giddings TH et al (1998) Observation of meristematic cells in seedlings of higher plants using a high pressure freezing method. Plant Morph 10:30–39
Segui-Simarro JM, Austin JR, White EA et al (2004) Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high pressure freezing. Plant Cell 16:836–856
Studer D, Hennecke H, Müller M (1992) High-pressure freezing of soybean nodules leads to an improved preservation of ultrastructure. Planta 188:155–163
Tiedemann J, Hohenberg H, Kollmann R (1998) High-pressure freezing of plant cells cultured in cellulose microcapillaries. J Microsc (Oxford) 189:163–171
Thijssen MH, Mittempergher F, VanAelst AC et al (1997) Improved ultrastructural preservation of Petunia and Brassica ovules and embryo sacs by high pressure freezing and freeze substitution. Protoplasma 197:199–209
Kaneko Y, Walther P (1995) Comparison of ultrastructure of germinating pea leaves prepared by high-pressure freezing-freeze substitution and conventional chemical fixation. J Electron Microsc 44:104–109
Bourett TM, Czymmek KJ, Howard RJ (1999) Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing. Planta 208:472–479
Pfeiffer S, Krupinska K (2005) Chloroplast ultrastructure in leaves of Urtica dioica L. analyzed after high-pressure freezing and freeze-substitution and compared with conventional fixation followed by room temperature dehydration. Microsc Res Tech 68:368–376
Austin JR II, Staehelin LA (2011) The three-dimensional architecture of grana and stroma thylakoids of higher plants as determined by electron tomography. Plant Physiol 155:1601–1611
Hillmer S, Viotti C, Robinson DG (2012) An improved procedure for low-temperature embedding of high-pressure frozen and freeze-substituted plant tissues resulting in excellent structural preservation and contrast. J Microsc (Oxford) 247:43–47. doi:10.1111/j.1365-2818.2011.03595.x
Ding B, Turgeon R, Parthasarathy MV (1992) Effect of high-pressure freezing on plant microfilament bundles. J Microsc (Oxford) 165:367–376
Lancelle SA, Callaham DA, Hepler PK (1986) A method for rapid freeze fixation of plant cells. Protoplasma 131:153–165
Lancelle SA, Hepler PK (1989) Immunogold labelling of actin on sections of freeze-substituted plant cells. Protoplasma 150:72–74
Ding B, Turgeon R, Parthasarathy MV (1991) Routine cryofixation of plant tissue by propane jet freezing for freeze substitution. J Electron Microsc Tech 19:107–117
Nitta K, Kaneko Y (2004) Simple plunge freezing applied to plant tissues for capturing ultrastructure close to the living state. J Electron Microsc 53:677–680
Dubochet J, Mcdowall AW (1981) Vitrification of pure water for electron-microscopy. J Microsc (Oxford) 124:Rp3–Rp4
Al-Amoudi A, Norlen LPO, Dubochet J (2004) Cryo-electron microscopy of vitreous sections of native biological cells and tissues. J Struct Biol 148:131–135
Michel M, Hillmann T, Mueller M (1991) Cryosectioning of plant material frozen at high pressure. J Microsc (Oxford) 163:3–18
Edwards HH, Yeh YY, Tarnowsky BI et al (1992) Acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: Comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine. Microsc Res Tech 21: 39–50
Mascorro JA (2004) Propylene oxide: to use or not to use in biological tissue processing. Microsc Today 12:45–46
Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43
Ellis EA (2006) Solutions to the problem of ERL 4221 for vinyl cyclohexene dioxide in Spurr low viscosity embedding formulations. Microsc Today 14:32–33
Hohenberg H, Mannweiler K, Muller M (1994) High-pressure freezing of cell-suspensions in cellulose capillary tubes. J Microsc (Oxford) 175:34–43
England WE, McCully ME, Huang CX (1997) Solvent vapour lock: an extreme case of the problems caused by lignified and suberized cell walls during resin filtration. J Miscrosc (Oxford) 185:85–93
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Kuo, J. (2014). Processing Plant Tissues for Ultrastructural Study. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology, vol 1117. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-776-1_3
Download citation
DOI: https://doi.org/10.1007/978-1-62703-776-1_3
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-775-4
Online ISBN: 978-1-62703-776-1
eBook Packages: Springer Protocols