Journal of Microscopy, Vol. 247, Pt 1 2012, pp. 43–47
doi: 10.1111/j.1365-2818.2011.03595.x
Received 15 December 2011; accepted 4 January 2012
An improved procedure for low-temperature embedding of high-pressure frozen and freeze-substituted plant tissues resulting in excellent structural preservation and contrast S . H I L L M E R ∗, C . V I O T T I † & D . G . R O B I N S O N ∗
∗ Department of Plant Cell Biology, Centre for Organismal Studies, University of Heidelberg,
Heidelberg, Germany †Department of Plant Developmental Biology, Centre for Organismal Studies, University of Heidelberg, Heidelberg, Germany
Key words. Freeze substitution, high-pressure freezing, low-temperature embedding.
Summary Here we describe refinements in the processing of highpressure frozen samples of delicate plant tissues for immunoelectron microscopy. These involve: shortened freezesubstitution schedules, lower temperatures during processing and polymerisation, the avoidance of temperature fluctuations and the optimisation of heat transfer from the specimens using small disposable aluminium containers. The application of these modifications leads to very good structural preservation and selective membrane contrast. As a result, the versatility of the method is increased since not only immuno-electron microscopical studies can be performed but often the quality is also quite suitable for structural investigations.
Introduction High-pressure freezing (HPF) is a well-established technique to prepare samples for electron microscopy of plant specimens (Kiss et al., 1990). Due to the rapid cooling process the samples are immobilised instantly leaving no time for artefacts inside the cell to develop. After rapid freezing, cellular water is replaced with solvents in a process termed freeze substitution before infiltrating with resin prior to polymerisation. Numerous freeze-substitution schedules have been described in the literature (see Humbel, 2009 for a review) but the theory behind this process is still poorly understood (see Heuser, 2011). Recent developments show that the time needed for this process can be reduced from a few days, as was common in the past (Browning
and Gunning, 1977), to a few hours for many specimens (McDonald and Webb, 2011). Nevertheless, all of the processing steps for low-temperature embedding have to be performed at temperatures far below 0◦ C. It is therefore common sense that avoiding temperature fluctuations should be important so as to minimise distortion and extraction, while hopefully maintaining excellent structural preservation and antigenicity of the biological material. To minimise loss of antigenicity due to fixation effects, we have adopted a strategy which entails the inclusion of only a small amount of uranyl acetate in the freeze-substitution medium (Tse et al., 2004; Hawes et al., 2007). In our laboratory over the last few years root tips of the model plant Arabidopsis thaliana became the most frequently used specimen. Here visual control and easy manipulation of the tiny, nearly translucent root tips as well as effective heat transfer from and to the material during the processing are essential to reduce temperature fluctuations. We have therefore developed a processing scheme relying on disposable aluminium containers that meet all requirements with respect to handling and heat transfer throughout the whole process. In addition, aluminium provides a shiny surface insuring optimal light reflection during polymerisation and the closed bottom of the mould makes sure that even tiny samples are not lost during polymerisation, which is often a problem with the Leica flatt embedding system based on metal blocks with plastic insets that is sold as accessories for the Leica freeze-substitution machines (AFS2; Leica, Wetzlar, Germany).
Materials and methods Correspondence to: Dr. Stefan Hillmer, COS Plant Cell Biology, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg. Tel: +49-622-1545610; fax: +49-622-154-6404; e-mail:
[email protected] C 2012 The Authors C 2012 Royal Microscopical Society Journal of Microscopy
Arabidopsis seedlings ecotype Columbia-0 were grown on Murashige and Skoog (MS) medium supplemented with 1% sucrose and 0.6% Phyto Agar (Duchefa Biochemie, Harlem,
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Fig. 1. Setting up the Leica AFS2 for prolonged work in the open chamber. During manual processing of the specimens the regular lid is replaced with a custom one that extends the chamber by 4.5 cm and improves temperature stability. In addition it prevents moisture uptake during long opening times since the volume containing cold nitrogen gas is increased.
NL) at 22◦ C, with cycles of 16 h light for 4–5 days. Four- to five-day-old Arabidopsis root tips were cut from the seedling, submerged in 200 mM sucrose, 10 mM trehalose, and 10 mM Tris buffer, pH 6.6, transferred into planchettes (Wohlwend GmbH, Sennwald, Switzerland; type 241 and 242), and frozen in a high-pressure freezer (Viotti et al., 2010; Scheuring et al., 2011).
Machines and modifications A high-pressure freezer (Baltec, HPM010; Kaech, 2009) was used in all cases and the subsequent freeze substitution was carried out in an AFS1 or AFS2 base unit (Leica, Wetzlar, Germany) supplemented with an external binocular (Zeiss, Jena, Germany) with long working distance (achieved with a 0.4× reduction lens) and illumination through the objective. In cases where extensive work inside the chamber of the AFS is required (many samples or samples difficult to transfer or to release) an extension tube for the chamber of the AFS (built by our workshop) was used to increase temperature stability and prevent moisture uptake during these long opening times (Fig. 1).
Fig. 2. A series of aluminium containers as used for sample processing. Container A holds the samples during freeze substitution and infiltration. B and C are used for embedding and polymerisation. All containers allow for easy manipulation of the plant material with needles and pipetts and ensures an effective heat transfer to and from the specimen during substitution and infiltration as well as effective removal of heat during polymerisation.
12 mm × 4 mm × 2.7 mm, Hafenstrasse 3, D-77694 Kehl/Rhein, www.novodirect.de). Freeze substitution, resin infiltration and embedding Freeze-substitution medium (0.4% uranyl acetate in acetone (stock solution 20% uranyl acetate in methanol) was pipetted into Alu-caps [Fig. 2(A)] and frozen with liquid nitrogen immediately before transferring the samples. Specimen carriers containing samples were transferred directly from the HPM010 or after storage in liquid nitrogen onto the surface of the frozen freeze-substitution medium that is still covered with liquid nitrogen and placed inside the AFS previously equilibrated to –85◦ C. During the next 15 min the freeze-substitution medium melts and planchets on the surface of this medium slowly reach the temperature of – 85◦ C. Usually some planchets float on the surface of the freeze-substitution medium and have to be submerged with
Small tools Alu-caps for freeze substitution under visual control that allow manipulation of the material with needles were from Diagonal ¨ (# 3621313, Havixbecker Straße 62, D-48161 Munster, www.diagonal.de), Alu-weighing dishes as outer containers for polymerisation were from VWR (# 611–1362, 28 mm × 10 mm, VWR, Hilpertstraße 20a, D-64295 Darmstadt, www.vwr.com) and Alu-micro-weighing dishes as molds for the samples were obtained from Novodirect (#46719,
Fig. 3. Aluminium containers hold the specimen after polymerisation. In panel A the outer container is still present whereas it has been peeled of in panel B. Panel C shows that the almost translucent root tips have been aligned for longitudinal sectioning prior to polymerisation. Lowicryl HM20 has a very low viscosity and it is relatively hard to keep the roots in perfect alignment. C 2012 The Authors C 2012 Royal Microscopical Society, 247, 43–47 Journal of Microscopy
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Fig. 4. Golgi apparatus and endoplasmic reticulum of an Arabidopsis root cell. The section has been poststained after sectioning for 2 min with aqueous uranyl acetate (3%, w:v) and lead citrate. Note the trilamellar structure of membranes and the tear between cell wall and cytoplasm (asterisk).
a needle to ensure proper substitution. Our freeze-substitution protocol for Arabidopsis root tips and other thin walled tissues included 16 h in freeze-substitution medium at –85◦ C, a 5 h linear warm up to –60◦ C and a 1 h wash with dry ethanol at –60◦ C before the specimens are released from the carriers using precooled needles. Infiltration with resin was done stepwise from 30%–50%–70% Lowicryl HM20 in ethanol to 100% Lowicryl HM20. All steps were done at –60◦ C and each lasted 1 h.
C 2012 The Authors C 2012 Royal Microscopical Society, 247, 43–47 Journal of Microscopy
For embedding, Alu-micro-weighing dishes [Fig. 2(C)] were placed inside a larger round Alu-weighing dish [Fig. 2(B)], prefilled to about 50% with HM20 inside and outside the small containers. Specimens were then transferred using precooled plastic pipettes with a wide opening at the tip into the small moulds for polymerisation. After filling the small moulds up to the rim, the specimen was oriented for longitudinal or cross sectioning with the precooled needles. Without covering the surface of the resin, the lid of the AFS was then replaced with
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Fig. 5. Part of a section of an Arabidopsis root cell which was labelled with antibodies against vacuolar pyrophosphatase. Golgi apparatus and ER are still visible but due to long incubation on aqueous solutions (PBS) structural detail is lost and cannot be regained by poststaining. Moreover poststaining reduces the visibility of gold particles and is therefore omitted.
the UV lamp (Leica, Wetzlar, Germany). Polymerisation was started at –60◦ C and continued for at least two days. After 24 h the temperature was slowly raised and reached room temperature at the end of the second day. To compensate for loss of HM20 during the process, additional HM20 was added after about one day. The final level should be as high as the rim of the small moulds to produce blocks with optimal stability for sectioning. Figure 3 shows the samples after polymerization.
avoids most of the common fixatives but nevertheless provides excellent membrane contrast. Sections can be poststained with aqueous uranyl acetate and lead citrate immediately after sectioning to be used for structural investigations. This procedure provides an even better basis for immunogold studies, although with somewhat reduced quality due to the long incubation of the section on aqueous media (compare Fig. 4 and Fig. 5). In contrast to the recently published protocol for a 3 h freeze substitution of McDonald and Webb (2011), this procedure is somewhat more time consuming but the samples are not agitated during the freeze-substitution process simply because freeze substitution is done inside the Leica AFS2. The AFS2 here is not only important for maintaining the low temperature during freeze substitution but also for effectively excluding oxygen from the resin during low-temperature infiltration, embedding and polymerisation. Oxygen is known to inhibit polymerisation (Carlemalm et al., 1985) and therefore reduces the section quality of the final block. Since the main drawback compared to other protocols remains the reduced stability of Lowicryl HM20 sections compared to epoxy sections, it is essential to maintain the samples in a constant nitrogen atmosphere and we therefore routinely use the Leica AFS2. Nevertheless the transition zone between plasma membrane and cell wall tended to break during sectioning even under optimal conditions (Fig. 4, asterisk). This is a major problem if cell wall or plasma membrane antigens are of interest but it is probably due to the inability of methacrylates to form covalent bonds with the biological
Immunolabelling and imaging Immunolabelling of longitudinal sections of Arabidopsis root tips was done as described in Niemes et al. (2010) using primary antibodies against vacuolar pyrophosphatase (CosmoBio Co. Ltd, Japan) at a dilution of 1:1500 in PBS. Micrographs were obtained with a JEM1400 transmission electron microscope (JEOL, http://www.jeol.com) operating at 80 kV using a TVIPS F214 digital camera (http://www. tvips.com). Micrographs were taken as 16-bit grayscale images and optimised in brightness and contrast with EMMENU 4.0 (http://www.tvips.com) before being exported as 8-bit tiff files. Results and Discussion Here we describe a relatively simple and short protocol for freeze substitution and low-temperature embedding of plant material after HPF with aqueous freezing media, that not only
Fig. 6. Golgi apparatus of a BY-2 cell processed in the same way as the root tips. Note the low contrast of the cytoplasm but the excellent selective contrast of the Golgi membranes. C 2012 The Authors C 2012 Royal Microscopical Society, 247, 43–47 Journal of Microscopy
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material whereas epoxy resins can even replace fixatives during freeze substitution (Matsko and Mueller, 2005). The protocol described here is not restricted to Arabidopsis root tips but works equally well on BY-2 suspension culture cells. Figure 6 shows that, although the general electron density of the cytoplasm appears to be relatively low, the selective contrast of membranes (especially of the Golgi apparatus) was surprisingly high. In general our observations show that a number of small refinements during processing leads to specimens that are valuable for structural as well as immuno work. It also emphasises that great care has to be taken during processing of cryo samples.
Acknowledgements We wish to thank Steffi Gold and Barbara Jesenofsky for technical help. The financial support of the German Research Council (RO 440/11–3/14–1) is gratefully acknowledged.
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Hawes, P., Netherton, C.L., Mueller, M., Wileman, T. & Monaghan, P. (2007) Rapid freeze-substitution preserves membranes in highpressure frozen tissue culture cells. J. Microsc. 226, 182–189. Heuser, J.E. (2011) The origins and evolution of freeze-etch electron microscopy. J. Electron Microsc. 60, S3–S29. Humbel, B. (2009) Freeze substitution. Handbook of Cryo-Preparation Methods for Electron Microscopy (ed. by A. Cavalier, D. Spehner & B.M. Humbel), pp. 319–341. CRC Press, Boca Raton, Florida. Kaech, A. (2009) BAL-TEC HPM010 High pressure freezing machine. Handbook of Cryo-Preparation Methods for Electron Microscopy (ed. by A. Cavalier, D. Spehner & B.M. Humbel), pp. 105–128. CRC Press, Boca Raton, Florida. Kiss, J.Z., Giddings, T.H., Staehelin, L.A. & Sack, F.D. (1990) Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis. Protoplasma. 157, 64–74. Matsko, N. & Mueller, M. (2005) Epoxy resin as fixative during freezesubstitution. J. Struct. Biol. 152, 92–103. McDonald, K.L. & Webb, R.I. (2011) Freeze substitution in 3 hours or less. J. Microsc. 243, 227–233. Niemes, S., Langhans, M., Viotti, C., et al. (2010) Retromer recycles vacuolar sorting receptors from the trans-Golgi network. Plant J. 61, 107–121. ¨ Scheuring, D., Viotti, C., Kruger, F., et al. (2011) Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell 23, 3463–3481. Tse, Y.C., Mo, B., Hillmer, S., Zhao, M., Lo, S.W., Robinson, D.G. & Jiang, L. (2004) Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell. 16, 672–693. Viotti, C., Bubeck, J., Stierhof, Y.D., et al. (2010) Endocytic and secretory traffic in Arabidopsis merge in the Trans-Golgi Network/Early Endosome, an independent and highly dynamic organelle. Plant Cell. 22, 1344–1357.
