Protoplasma (2003) 222: 129137 DOI 10.

1007/s00709-003-0014-6

PROTOPLASMA
Printed in Austria

Combined use of confocal laser scanning microscopy and transmission electron microscopy for visualisation of identical cells processed by cryotechniques
S. Pfeiffer1,2,*, M. Beese1, M. Boettcher 2, K. Kawaschinski 3, and K. Krupinska 2
1 2

Central Microscopy, Center of Biology, University of Kiel, Kiel Institute of Botany, University of Kiel, Kiel 3 Beiersdorf AG, Hamburg Received February 11, 2003; accepted May 9, 2003; published online October 29, 2003 Springer-Verlag 2003

Summary. Successive visualisation of identical plant cells by light and electron microscopy is reported. For this purpose segments of pea and barley leaves were prepared by high-pressure freezing, freeze-substitution, and low-temperature embedding. The use of Safranin O during low-temperature dehydration allowed, on one hand, staining of all cellular components as investigated by confocal laser scanning microscopy and, on the other hand, excellent ultrastructural and antigenic preservation. A newly constructed specimen holder enabled precise relocation of the target cells for electron microscopic investigations. Transmission electron microscopy and immunohistochemistry revealed that during the whole procedure the ultrastructure of the cells as well as the antigenicity of cell constituents were preserved. Keywords: Correlative microscopy; High-pressure freezing; Freezesubstitution; Plant ultrastructure; Immunohistochemistry; Safranin O. Abbreviations: CLSM confocal laser scanning microscopy; TEM transmission electron microscopy.

Introduction Confocal laser scanning microscopy (CLSM) is a widely used technique to investigate the three-dimensional (3-D) structure of plant material. A prerequisite for the investigation of plant tissue and cell compartments is their uorescence. It is well known that some structures, especially cell walls and chloroplasts, possess the ability to uoresce without pretreatment by special staining agents (Buschmann and Lichtenthaler 1998, Hepler and Gunning 1998, Blancaor and Gilroy 2000, Roos 2000). Visualisation of other cellular constituents, however, requires stain* Correspondence and reprints: Central Microscopy, Center of Biology, University of Kiel, Am Botanischen Garten 5, 24098 Kiel, Federal Republic of Germany.

ing with specic dyes or, alternatively, decoration with antibodies. Generally, such investigations require manipulations, e.g., sectioning by hand or vibratome and the storage of the sections in buffer solutions or tap water, that might induce artifactual changes in the morphology of the cells. Another common problem associated with investigations by CLSM is the irradiation by laser light which causes photobleaching, e.g., of chlorophylls, and thereby induces changes in the native state of the cellular structures. The optical resolution of the CLSM technique is limited to about 250 nm. In recent years, new CLSM techniques like uorescence resonance energy transfer microscopy (FRETM) were developed in order to overcome this limitation. Unfortunately, FRETM signals are restricted to the detection of single specic molecules (Reddy et al. 1998) and do not allow a concomitant visualisation of cellular constituents or compartments which interact with these molecules. For the analysis of structures at higher resolution levels, electron microscopy still is the method of choice and has to be employed as a supplement to CLSM analysis. However, the application of transmission electron microscopy (TEM) techniques (e.g., ultrathin sectioning) is not feasible for investigations on large areas of tissue. Often, trimming of the preselected area for sectioning results in a loss of the surrounding parts of the specimen. For 3-D reconstructions of cells analysed by TEM, a large number of serial sections have to be used and the 3-D image has to be subsequently reconstructed by use of an appropriate software. One way to circumvent this time-consuming procedure is the use of

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S. Pfeiffer et al.: Correlative microscopy based on cryopreparation methods inltrated with nonpolar Lowicryl HM20 (Polysciences, Eppelheim, Federal Republic of Germany) at 50 C according to the following protocol. Specimens were washed in pure acetone for 30 min, inltrated with 30% (v/v) HM20 and 70% (v/v) acetone for 2 h, and then inltrated with 70% HM20 and 30% acetone for 2 h, before nally three incubation steps for 2 h each were performed in pure HM20. Polymerisation was carried out under UV-light at 50 C for 48 h and thereafter continued at room temperature for at least 24 h. Blockface preparation and CLSM investigation After polymerisation, the surface of the block, referred to as blockface, was prepared by trimming and presectioning with an ultramicrotome (UCT; Leica) using a specially designed small specimen holder. For the investigation with the CLSM (TCS SP; Leica) we used a 63 oil immersion plan apochromat objective lens with a numerical aperture of 1.32 and a working distance of 170 m. An argon laser with 458 and 476 nm light was used for excitation. Safranin O emissions were detected with two channels, one ranging between 460 and 550 nm and the second between 590 and 610 nm. A sequence of 52 x,y optical slices, each with a thickness of 1 m, was collected. Images of 1024 by 1024 pixels were captured as computer les. The intensity was digitally coded in 256 levels of grey, with 0 representing the lowest intensity and 255 the highest. To allow detection of weak uorescence signals emitted from deeper parts of the specimen, a dramatically enhanced uorescence intensity of some structures in the rst images had to be tolerated. The image stacks were coloured and reconstructed to 3-D images with the Leica software (version 2.585). Ultramicrotomy and TEM investigation Ultrathin sections from the target area, which was preselected from the CLSM image stack, were cut with an ultramicrotome (UCT; Leica), then counterstained with uranyl acetate and lead citrate (Reynolds 1963), and nally analysed with a Philips EM 208 S transmission electron microscope (Philips, Eindhoven, The Netherlands.). Digital image acquisition was performed with a charge-coupled-device camera (Mega-View; Soft Imaging Systems, Mnster, Federal Republic of Germany). Image processing was performed with the AnalySIS 2.01 software (Soft Imaging System). Immunohistochemistry

an en bloc optical-sectioning method (Prior et al. 1999) employing resin-embedded specimens after conventional chemical xation and dehydration at room temperature. It is known that conventional preparation techniques of plant tissue usually involve long xation times and the use of poisonous osmium tetroxide (Mersey and McCully 1978). In combination with dehydration at ambient temperatures multiple structural artefacts were obtained (Kiss et al. 1990, Kaneko and Walther 1995). Conventional TEM preparation techniques applied to barley aleurone cells lead to the extraction of macromolecules from the cytosol, protein storage vacuoles, and cytoplasmic organelles. Furthermore, the antigenicity is reduced dramatically (Bourett et al. 1999). In contrast, cryopreparation techniques which combine high-pressure freezing and freeze-substitution are known to preserve the cellular ultrastructure close to the native state (Craig and Staehelin 1988, Studer et al. 1989, Galway et al. 1993, Tiedemann et al. 1998). In order to circumvent the disadvantages of conventional EM preparation techniques, we applied a new strategy for visualisation of plant cells. Cryotechniques, freeze-substitution in combination with low-temperature embedding, were employed to achieve both structural preservation of the plant tissue close to the native state and immunological detection of specic molecules. The use of Safranin O as new xative simultaneously allowed a uorescent staining of the cell compartments. After preselection by CLSM and ultrathin sectioning, the cells were nally analysed by TEM at high resolution. Material and methods
Plant material Barley (Hordeum vulgare L. cv. Stef) and pea plants (Pisum sativum L.) were grown as described by Krupinska (1992) and Morin and Soll (1997). Cryoxation, freeze-substitution, and low temperature embedding For cryoimmobilisation of plant leaves, punch biopsies (2 mm in diameter, biopsy punch; Stiefel Laboratorium, Offenbach am Main, Federal Republic of Germany) were taken. In order to inltrate the intercellular space, the leaf discs were incubated for 10 min in low vacuum (80 mbar) in 8% methanol diluted with tap water (Lee et al. 2000). Subsequently the samples were transferred into the cavity of a high-pressure-freezing aluminium platelet which was 100 m in depth and prelled with 1hexadecene. This platelet was covered by a second one, inserted into a high-pressure-freezing specimen holder and high-pressure frozen in the HPM 010 (Bal-Tec, Balzers, Liechtenstein) (Mller and Moor 1984). Prior to freeze-substitution, the frozen 1-hexadecene was carefully removed under liquid nitrogen (Hohenberg et al. 1994) and the specimens were then transferred into precooled test tubes, lled with acetone containing 0.01% Safranin O. Dehydration was carried out in a conventional freeze-substitution unit (AFS; Leica, Bensheim, Federal Republic of Germany) at 90 C for 20 h, followed by two substitution steps at 70 and 50 C, each for 8 h. After freeze-substitution the leaf samples were

All following preparation steps were performed at room temperature and all antibodies were diluted with washing buffer (phosphate-buffered saline, 0.5% bovine serum albumin, 0.2% sh gelatin). Immunogold staining was performed on ultrathin sections (50 nm) of HM20-embedded pea leaves. Sections were treated with blocking buffer (phosphate-buffered saline, 2% bovine serum albumin, 2% sh gelatin) for 1 h, washed twice, and incubated with an anti-DNA antiserum (1: 50) (Boehringer, Ingelheim, Federal Republic of Germany) or with an antitubulin antiserum (1:100) (Sigma, Deisenhofen, Federal Republic of Germany) for 1 h. Subsequently, the sections were washed eight times and incubated with a goat anti-mouse immunoglobulin G F(ab )2 conjugated with 10 nm diameter gold particles (Dianova, Hamburg, Federal Republic of Germany) for 1 h. Finally the sections were poststained and investigated by TEM as described above.

Results Blockface investigation of leaf tissue by CLSM In order to guarantee precise orientation of the blockface in the z-direction during investigation by CLSM and relo-

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Fig. 1. Image of the newly constructed specimen holder made of stainless steel. The arrow indicates the clamped specimen

calisation of the target area for ultrathin sectioning, parallel orientation of the blockface and the bottom side of the holder platform is required. A newly designed specimen holder allows both CLSM investigation and ultramicrotomy without a change in the position of the block xed to the holder. For CLSM investigation the specimen holder (Fig.1) was glued onto a coverslip, for the subsequent ultramicrotomy the specimen holder was directly inserted into the ultramicrotome. After processing by high-pressure freezing and freezesubstitution as described above, pea leaf segments were investigated by CLSM. An image of the blockface of an embedded young pea leaf (cross section) is shown in Fig. 2. The starch grains appear in an extremely bright yellow colour, the cell walls emit a green-yellow uorescence. The colouration of cellular components depends on the sample depth in z-orientation which differed when the orientation of the embedded specimen slightly deviated

Fig. 2. CLSM image of the blockface of an embedded young pea leaf (cross section). The nuclei within the epidermal cells on the left side of the leaf appear in red colour. On the right leaf side, the uorescence of nuclei in the mesophyll and epidermal cells is shifted to orange or green colour. The nucleoli in the mesophyll cells on the left image side are uorescing in green-yellow (arrow). The starch grains appear in an extremely bright yellow, and the cell walls are visualised in green-yellow colour

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from being in parallel to the surface. Due to their different orientation, the nuclei of the mesophyll cells and of the epidermal cells on the right side appear in orange or green colour, while nuclei of the mesophyll cells on the left side show a reddish uorescence. Autouorescence induced by the embedding media was not detectable. To visualise the structural context, a 3-D image was generated from an image stack of 52 optical sections, whereby each image represents a sample slice with a thickness of 1 m. Cellular constituents, e.g., brightly shining starch grains, orange stained nuclei, and dark vacuoles as well as yellow cell walls are clearly distinguishable (Fig. 3a). The target area for electron microscopy was selected in one of the optical sections of the 3-D image stack (Fig. 3a). Due to the precise orientation of the specimen in x,y-direction and the exact thickness and numbers of sections, it was possible to remove the material above the target area precisely with the ultramicrotome. Thereby phenotypically different cells, e.g., conducting element cells or stomata, served as markers. Close to the target area, the right positioning of the blockface was reinvestigated by CLSM and the cells in the target area were analysed by TEM after ultrathin sectioning. The pores of the sieve plate elements and the companion cell are well visible. The sieve elements and the adjacent phloem cells which are surrounded by a red line in the optical section in Fig. 3b are also labelled in red in the section analysed by TEM (Fig. 3c). Analysis of ultrathin sections by TEM TEM investigation revealed that the ultrastructure of the pea leaf was well preserved. As an example, the ultrastructure of a parenchyma cell in the neighbourhood of sieve elements was analysed in detail (Fig. 4a). The nucleus with the darkly stained heterochromatin inside, chloroplasts with starch grains and thylakoid membranes, cell walls and plasmodesmata as well as the vacuole with the tonoplast are clearly distinguishable (Fig. 4ac). The plasmalemma is perfectly attached to the cell wall surface. Clusters of ribosomes and ribosome-free areas are distinguishable in the cytoplasm (Fig. 4b, c). A plasmodesmal connection between two plant cells is clearly visualised in Fig. 4c. Preservation of the antigenicity of cellular components was demonstrated by immunogold labelling of the tubulin laments in pea leaves and of the nuclear DNA in barley leaves. Tubulin laments were immunogold labelled by an anti-tubulin antiserum and a goat anti-mouse immunoglobulin G gold-conjugated secondary antibody. As typical for plant cells, the tubulin laments are arranged close to the cell walls in parallel to the plas-

malemma (Fig. 5). For DNA labelling an anti-DNA antiserum and a goat anti-mouse immunoglobulin G goldconjugated secondary antibody were employed (Fig. 6). Gold labelling of DNA was detected in the heterochromatic parts of the nucleus, demonstrating nicely the high concentration of the DNA in these areas (Fig. 6). Discussion To visualise the ne structure of plant material, CLSM investigation is often used. Due to the autouorescence of distinct cellular components, e.g., the blue light emitted from the cell walls or the red uorescence of chloroplasts, the corresponding structures can be easily visualised in situ by CLSM after thin hand sectioning. Nevertheless, CLSM-based investigations of plant material have striking disadvantages. Firstly, hand sectioning may impose a severe stress on the plant tissue, which often results in changes in ultrastructure. Photobleaching and other phototoxic processes are further problems associated with CLSM investigations (Sandison et al. 1995) and may result in reduced signal intensity (Herman 1998) or in a shift of the wavelength of the emitted light (data not shown). In order to visualise the cellular ultrastructure at higher resolution, TEM still is the technique of choice. By TEM, however, only small areas of the specimen can be investigated without any information about their structural context. To visualize the same cells both in detail and in their structural context, semithin sections of chemically xed plant material were used for subsequent light and transmission electron microscopy (Drr 1968, Prior et al. 1999). By this approach, however, the disadvantages associated with hand sectioning and conventional xation could not be avoided. With the preparation technique used here, it is possible to screen large specimens, up to 2 mm in diameter by successive analysis of 160 m steps, and to preselect by CLSM the target regions for subsequent detailed investigation by TEM. For the precise orientation of the specimen in the z-direction during trimming, microscopical investigation, and preparation of the target area on the ultramicrotome, a new specimen holder was invented. Thereby it is possible to adjust the surface of the blockface, the z-plane, exactly in parallel to the bottom of the specimen holder. An exact parallel orientation of the z-plane and the surface of the blockface is a prerequisite for the proper preparation of the target area. One severe disadvantage of investigations by TEM is the use of chemical xatives and the dehydration at room temperature. The long xation times of conventional

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protocols, which sometimes exceed 48 h, in combination with dehydration at ambient temperatures were shown to induce structural artefacts (Mersey and McCully 1978, Kiss et al. 1990, Kaneko and Walther 1995, Thijssen et al. 1997, Tiedemann et al. 1998) and a loss of the antigenic-

ity of cell constituents (Kellenberger et al. 1987). Osmium tetroxide is often used as a xative during the freezesubstitution. While osmium tetroxide binds covalently to the unsaturated linkage of lipid components at room temperature, at low temperature it reduces the extraction of

Fig. 3ac. Combination of CLSM and TEM techniques. a A 3-D reconstruction of CLSM images from a blockface of a cross-sectioned pea leaf processed by high-pressure freezing and freeze-substitution embedding. The red line indicates the slice of the target area inside the 3-D image. b and c Red lines label the same cells in the target area inside the optical slice, which are visualised in the TEM after ultrathin sectioning

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Fig. 4ac. TEM images from a cryoprocessed parenchyma cell located adjacent to the sieve elements. The nucleus with darkly stained heterochromatin (a), the chloroplasts with starch grains (b), the vacuole (V) and the tonoplast and cell walls (CW) and plasmodesmata (c) are clearly visible. The plasmalemma is located adjacent to the cell wall surface (arrows). Clusters of ribosomes (arrowhead in b) and ribosomefree areas (asterisk in b) are depicted

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Fig. 5ae. Ultrathin sections of a parenchyma cell within a pea leaf. Tubulin laments, longitudinal and cross-sectioned (indicated by arrows in d), were labelled with anti-tubulin antisera and goat anti-mouse immunoglobulin conjugated to gold

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Fig. 6. Nucleus of a parenchyma cell of a barley leaf. DNA was labelled with anti-DNA antiserum and a goat anti-mouse immunoglobulin G secondary antibody conjugated to gold (10 nm in diameter)

lipids during dehydration (Pfeiffer et al. 2000) and results in a good ultrastructural preservation. Nevertheless, the less toxic uranylic acid is preferred during dehydration at low temperatures. In this case, loss of lipids is reduced (Humbel and Schwarz 1989) and ultrastructure as well as the antigenicity are well preserved. A disadvantage of uranylic acid, however, is its radioactivity. To avoid the disadvantages of both common xatives, we have used instead Safranin O, which is normally used as a staining agent in light microscopy, as a xative during freeze-substitution. Safranin O is soluble in acetone as well as in ethanol, is less toxic, and may be used at very low concentrations ( 0.01%). The application of the high-pressure freezing and freeze-substitution preparation technique as described above provides several advantages compared to conventional preparation techniques. On one hand, the specimens retain their uorescence properties, which makes it possible to investigate them en bloc by CLSM. Secondly, no articial autouorescence, which is usally caused by the reaction of aldehyde xatives with cellular aromatic components (Herman 1998), can be detected. Moreover, the

effect of photobleaching is minimised by using the uorochrome Safranin O during freeze-substitution (Gray et al. 1999). Finally, plasmolytic effects, which often occur during the conventional preparation of plant material for TEM investigation, were not detectable. The attachment of the plasmalemma to the cell wall is one of the features clearly showing the preservation of the structure close to its native state (Fig. 4b). Furthermore, the organisation of ribosomes into microdomains is indicative of the high preservation of the structural organisation within the cytoplasm (Fig. 4c). Membranes, e.g., the thylakoid membranes of chloroplasts (Fig. 4b) and plasmodesmata (Fig. 4c), are clearly visible. In this study we have shown that the uorochrome Safranin O is well suited for very good ultrastructural preservation if used as supplement during freezesubstitution, rendering the use of further xatives like osmium tetroxide or glutaraldehyde unnecessary. Successful immunolabelling of tubulin laments and of DNA demonstrated that the use of Safranin O during the lowtemperature dehydration does not interfere with the preservation of biomolecule epitopes.

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137 Humbel B-M, Schwarz H (1989) Freeze-substitution for immunochemistry. In: Verkleij A (ed) Immunogold labelling in cell biology. CRC Press, Boca Raton, Fla, pp 115134 Kaneko Y, Walther P (1995) Comparison of ultrastructure of germinating pea leaves prepared by high-pressure freezing/freeze-substitution and conventional chemical xation. J Microsc 44: 104109 Kellenberger E, Drrenberger M, Villiger W, Carlemalm E, Wurtz M (1987) The efciency of immunolabel on Lowicryl sections compared to theoretical predictions. J Histochem Cytochem 35: 959969 Kiss JZ, Giddings TH, Staehelin LA, Sack FD (1990) Comparison of the ultrastructure of conventionally xed and high-pressure frozen/freezesubstituted root tips of Nicotiana and Arabidopsis. Protoplasma 157: 6474 Krupinska K (1992) Transcriptional control of plastid gene expression during development of primary foliage leaves of barley grown under a daily light-dark regime. Planta 186: 294303 Lee YK, Hippe-Sanwald S, Lee SC, Hohenberg H, Hwang BK (2000) In situ localization of PR-1 mRNA and PR-1 protein in compatible and incompatible interactions of pepper stems with Phytophthora capsici. Protoplasma 211: 6475 Mersey B, McCully ME (1978) Monitoring of the course of xation of plant cells. J Microsc 114: 4976 Morin X-K, Soll J (1997) Immunogold labelling of cryosectioned pea chloroplasts and initial localization of the proteins associated with the protein import machinery. Planta 201: 119127 Mller M, Moor H (1984) Cryoxation of thick specimens by high pressure freezing. Scanning Electron Microsc 1984: 131138 Pfeiffer S, Vielhaber G, Vietzke J-P, Wittern K-P, Hintze U, Wepf R (2000) High-pressure freezing provides new information on human epidermis: simultaneous protein antigen and lamellar lipid structure preservation. Study on human epidermis by cryoimmobilization. J Invest Dermatol 114: 10301038 Prior DAM, Oparka KJ, Roberts IM (1999) En bloc optical sectioning of resin embedded specimen using a confocal laser scanning microscope. J Microsc 193: 2027 Reddy LG, Jones LR, Thomas DD (1998) Depolymerization of phospholamban in the presence of calcium pump: a uorescence energy transfer study. Biochemistry 38: 39543962 Reynolds EW (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17: 208212 Roos W (2000) Ion mapping in plant cells: methods and applications in signal transduction research. Planta 210: 247270 Sandison DR, Williams RM, Wells KS, Strickler J, Webb WW (1995) Quantitative uorescence confocal laser scanning microscopy (CLSM). In: Pawley JB (ed) Handbook of biological confocal microscopy. Plenum, New York, pp 3954 Studer D, Michel M, Mller M (1989) High-pressure freezing comes of age. Scanning Microsc 3: 253269 Thijssen MH, Mittempergher F, Van Aelst AC, Van Went JL (1997) Improved ultrastructural preservation of Petunia and Brassica ovules and embryo sacs by high-pressure freezing and freeze-substitution. Protoplasma 197: 199209 Tiedemann J, Hohenberg H, Kollmann R (1998) High-pressure freezing of plant cells cultured in cellulose microcapillaries. J Microsc 189: 163171

In conclusion, the new procedure here presented allows the investigation of identical cells by light and electron microscopy. Due to the cryopreparation, the structure of the specimen was preserved close to the native state. The new specimen holder allows exact orientation in z-direction in the specimen which is necessary for precise relocalisation of cells. By employing Safranin O during the low-temperature dehydration, the use of highly toxic or radioactive xatives can be avoided without loss of ultrastructural information and antigenic preservation. Acknowledgments
We thank Dorothee Dhnhardt, Inge Drr, Kirsten Krause (Institute of Botany, University of Kiel, Federal Republic of Germany), Gabriele Vielhaber (Haarman und Reimer AG, Holzminden, Federal Republic of Germany), and Heinrich Hohenberg (Heinrich-Pette-Institut, Hamburg, Federal Republic of Germany) for helpful motivating discussions and for critical reading of the manuscript.

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