Scanning electron microscopy of plant surfaces - Formatex ...

Microscopy: Science, Technology, Applications and Education 

A. ______________________________________________ 

Méndez-Vilas and J. Díaz (Eds.) 

Scanning electron microscopy of plant surfaces: simple but sophisticated 

methods for preparation and examination 

H. J. Ensikat, P. Ditsche-Kuru and W. Barthlott 

Nees Institute for Biodiversity of Plants, University of Bonn, Meckenheimer Allee 170, 53115 Bonn, Germany 

A repertory of tailored preparation techniques for scanning electron microscopy (SEM) of plant surfaces is introduced in 

this report. Biological specimens for conventional SEM are usually dehydrated by suitable drying methods and coated with 

a thin metal layer to achieve a sufficient electrical conductivity. But many parts of plants, such as leaves and fruits, are 

covered with a cuticle which provides good mechanical strength and low permeability for water. The robustness and the 

low water evaporation rate allow to examine plant samples without drying or to apply simple cryo-procedures which are 

not suitable for other soft biological samples. Some of these methods have been tested in the early times of SEM but seem 

to be almost forgotten. Here we describe advancements of these techniques making an easy application possible. This 

broadens the repertory of practical methods for the examination of plant tissues in a conventional SEM under high vacuum 

conditions without the need for expensive special equipment (such as Environmental SEM or cryo stages). In this article, 

we present the advantages and limitations of these additional methods, which can be used to avoid the artefacts of the 

commonly used preparation techniques, such as the influence of organic solvents or shrinkage. Many undried plant 

samples can be examined successfully in a ‘fresh hydrated’ state, due to the low water permeability of the cuticle. Fresh 

and glycerol-infiltrated samples have a sufficient internal electrical conductivity that they can be examined without metal 

coating. Several preparation techniques are presented which keep the sample surface untapped, without immersion in 

liquids. This is particularly interesting for the study of fragile epicuticular waxes or contaminations on superhydrophobic 

‘self-cleaning’ surfaces (‘Lotus effect’) and air-retaining surfaces (‘Salvinia effect’, [1]). These methods include simplified 

cryo-preparations for low-temperature-SEM and freeze drying. Applications of the methods include ‘material-contrast 

imaging’ of uncoated samples using the backscattered electron (BSE) signal or the study of interactions between liquids 

and plant surfaces by low-temperature SEM. Suggestions for the examination of non-conductive samples complete the 

instructions. 

Keywords Scanning electron microscopy; plant surfaces; cryo-preparation; slow freezing; low-temperature SEM; freeze 

drying; glycerol substitution; fresh-hydrated samples; epicuticular waxes 

1. Introduction 

For conventional scanning electron microscopy (SEM), that means in high vacuum and at ambient temperature, it is a 

widely accepted principle that biological samples must be dehydrated and need a conductive coating. Therefore several 

drying techniques, which avoid or reduce tissue shrinking, such as Critical point drying [2] or Freeze drying, have been 

developed [3]. Mechanically stable samples may be dried simply at the air. In the early days of scanning electron 

microscopy several SEM-pioneers tested various methods and the limitations of the new microscopy [4-6]. For plant 

samples, alternatives to the drying methods were tested with varying success. Undried, fresh-hydrated samples without 

metal coating could be observed without strong charging effects, but some rapidly desiccating samples allowed only 

short observation times and low magnification [7, 6]. Further alternatives, which were developed early, were the 

imaging of glycerol-infiltrated samples [8, 9] and replication techniques [10, 11]. In the meantime, many of the 

experiences of the pioneers seem to be forgotten widely and only few preparation methods for plants are used routinely 

[12, 13]. Only sporadicly, publications present the successful use of simplified methods for the examination of plant 

surfaces. Craig and Beaton [14] successfully used slow cooling for Low-Temperature-SEM. The feasibility to examine 

plant surfaces without metal coating was shown for fresh-hydrated samples [15, 16] and glycerol-infiltrated samples 

[17]. Meanwhile, the focus of the SEM techniques turned towards the development of sophisticated special equipment: 

Low-temperature sample stages with integrated sputter coaters have been developed for the imaging of frozen-hydrated 

samples [18, 19]. The “Environmental Scanning Electron Microscope” (ESEM), which tolerates liquid water in the 

specimen chamber, is useful to view fresh-hydrated samples at ambient temperature, and it can image non-conductive 

samples without disturbing electric chargings [20, 21]. However, these equipments are quite expensive, and many 

biologists do have only the standard equipment. 

During the examination of numerous plant samples we found that unusual preparation methods often gave superior 

results for conventional SEM. The robust and almost impermeable cuticle, which forms many plant surfaces, allows the 

use of some techniques which are not suitable for soft wet biological material. 

Experiments with glycerol-infiltrated samples and with fresh-hydrated plant material had shown that the internal 

liquids provide a sufficient electrical conductivity to avoid charging problems in the SEM, so that the samples can be 

examined without metal coating [17, 22]. The various tasks in the study of plant surfaces (Lotus effect, epicuticular 

waxes, contaminations, droplet-surface interactions) need such a repertory of methods which include the observation of 

untouched and uncoated samples either at ambient temperature or in a frozen state. Our experiences may help other 

248 ©FORMATEX 2010

microscopists to become acquainted with simple cryo-preparation methods and to choose the optimal techniques for 

their sample preparation. 

2. Material and Methods 

2.1 Plant material 

Leaves of 7 plant species are presented in this study. The plants were cultivated in the Botanical Gardens, University of 

Bonn (BG Bonn): Nelumbo nucifera (Lotus), Tropaeolum majus (Garden nasturtium), Urtica dioides (Stinging-nettle), 

Drosera nidiformis, Salvinia molesta, Salvinia oblongifolia, Ruellia devosiana. 

2.2 Equipment 

A Cambridge Stereoscan S200 SEM (Cambridge, UK) equipped with a secondary electron (SE) detector and a 

backscattered electron (BSE) detector (scintillator type) was used at acceleration voltages between 10 and 20 kV. 

Images were recorded with an integrated digital image acquisition system (DISS5, Point Electronic, Germany). A 

home-made cooling holder was used for low-temperature examinations. 

Critical point drying (CP-drying) was made with a Balzers CPD 020 CP-dryer (Bal Tec AG, Liechtenstein). 

Metal coating with gold or gold/palladium was performed with a Balzers SCD 040 sputter coater (Bal Tec AG). 

2.3 Preparation methods 


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A. Méndez-Vilas and J. Díaz (Eds.) 

CP-drying: Samples were fixated in formaldehyde solution (2% formaldehyde, 70% ethanol, 5% acetic acid), 

dehydrated with ethanol (80%, 90%, 99%) and CP-dried with CO2. 

Air-drying: Fresh leaves were mounted on SEM stubs and stored in a desiccator with silica gel until dry. 

Fresh-hydrated material: Pieces of 3 to 10 mm size were cut out of the leaves. The cut faces were sealed with glue 

(conductive carbon glue or two-component epoxy glue). The pieces were mounted on SEM stubs with a conductive 

adhesive tab. The undried samples were examined with or without metal coating. For samples, which run dry rapidly, 

only one piece was mounted on the SEM stub, and the SEM sample holder was cooled with ice close to 0°C prior to 

insertion into the SEM in order to slow down the desiccation. 

Glycerol substitution: The procedure “infiltration from below” was used, which keeps the surface of hydrophobic 

samples dry and untouched [23]. Leaf pieces of 3 to 10 mm size were excised, then the epidermis of the underside of 

the sample was scratched with coarse abrasive paper in order to enable the permeation of the preparation liquids. In a 

petri dish, a piece of fabric was soaked with fixative (2% glutaraldehyde). Then the samples were placed on the fabric, 

so that they were infiltrated by the fixative for ca. 2 h. For the substitution of the water, the liquid soaking the fabric was 

exchanged with glycerol solutions with raising concentrations in 10% steps, or continuously, up to 90%, each step ca 2 

h. Then the samples were stored in a desiccator with silica gel for at least 24 h to remove remnants of water. The 

glycerol-infiltrated samples were mounted on SEM stubs with a conductive glue and examined with or without metal 

coating. 

Simple Cryo-preparations: 

Slow-cooling: The fresh samples were clamped in the cooling holder, which consists of a metal block with a sample 

chamber, a cover, and a thermal isolation (Fig. 1). Then the covered holder was placed in a container with liquid 

nitrogen (LN2) and cooled down without immersing the sample in LN2. The initial cooling rate was ca 2K/sec. 

(Diagram Fig. 1c). The cooled holder was inserted into the SEM for low-temperature examination or into another highvacuum 

chamber for freeze-drying. 

Low-temperature SEM (LT-SEM): After insertion of the cooling holder into the SEM and evacuation of the specimen 

chamber, the cover of the holder was removed and the sample was examined using very low beam intensities. Due to 

the thermal isolation, the temperature of the holder raises slowly (Diagram Fig. 1d). Thus the samples can be examined 

for hours at low temperatures. 

Freeze drying: The cooling holder with the frozen sample was simply left in the SEM or in another high-vacuum 

chamber overnight. Then it has reached room temperature. In the meantime, the tissue water has evaporated at 

temperatures far below 0°C. The dried samples were mounted on normal SEM stubs and sputter-coated. 

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Fig. 1 The self-made cooling holder (a: separate, b: mounted on the SEM stage and closed with a cover) was the only ‘special 

equipment’ for this study. (c): The cooling rate of the holder during ‘slow cooling’ with LN2. (d): The warm-up rate of the holder in 

the SEM during examination. 

3. Results and Discussion 

The cuticles, which cover many parts of terrestrial plants, provide many of them with sufficient mechanical stability and 

resistance to desiccation, that the presented SEM-preparation methods often give superior results. Compared to the 

commonly used methods CP-drying and air-drying with their influence of organic solvents or massive shrinkage, these 

alternative procedures avoid such alterations. The example Lotus leaf (Nelumbo nucifera) is used to demonstrate the 

suitability and differences of these preparation methods (Fig. 2a-f). The advantages and limitations of the methods are 

described in the following paragraph and summarised in Table 1. 

CP-drying (Fig. 2a): The surface of the Lotus leaf shows a good preservation of the papillae, but the epicuticular waxes 

are completely destroyed. It is known that the wax type which occurs on Lotus leaves (secondary-alcohol tubules), is 

easily dissolved in ethanol during the dehydration procedure. Other wax types, such as platelets, on other species are 

less soluble and may be affected only slightly. 

Air-drying (Fig. 2b): The papillae of the Lotus leaf are collapsed, but high-magnification imaging of the wax tubules is 

possible. 

Fresh-hydrated material (Fig. 2c, 3a-c): When we tried to observe fresh leaves in a conventional SEM, we were 

surprised to find that the majority of samples could be examined for at least several minutes before desiccation artefacts 

could be noticed. The durability varied strongly. Many full-grown leaves of native trees and conifer needles resist the 

desiccation for hours. Most thin, still growing leaves of herbs (e.g. Tropaeolum majus, Urtica dioides, Taraxacum 

officinale leaves, Fig. 3) can be examined for several minutes up to an hour, sufficient to acquire some pictures. 

However, leaves with very thin cuticles, such as petals of flowers or leaves from species growing at humid habitats, dry 

out immediately in the SEM before the vacuum is ready. Sometimes, only certain cell types, such as glands or some 

trichomes, shrink, while normal epidermis cells remain turgescent and convex for a longer time. Examination of freshhydrated 

leaves has several advantages: the preparation is fast; the surface remains untouched; the internal electrical 

conductivity allows imaging without metal coating, which is particularly useful for material-contrast imaging using the 

BSE signal. The ‘atomic number contrast’ of the BSE images indicates e.g. the accumulation of Ca and Si in the hairs 

of stinging-nettles (Fig. 3b) or inorganic material in contaminations on leaves (Fig. 3c). 

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Fig. 2 Lotus leaf (Nelumbo nucifera) treated with 6 different preparation methods. (a): CP-drying preserves the papillae, but the 

epicuticular waxes are destroyed. (b): Air-drying causes severe shrinkage of the epidermal cells. (c): The fresh-hydrated sample 

(without metal coating) at ca 5°C shows little shrinkage after 10 minutes observation time. The waxes are intact, but sample drift 

retards high magnifications. (d): Glycerol-infiltration prevents shrinkage of the papillae; the waxes are intact, the surface is dry and 

untouched; sample without metal coating. (e): Low-temperature SEM image of the frozen-hydrated sample without metal coating. 

The waxes and papillae are intact, but the image contrast is poor due to charging of the non-conductive sample. (f): The freeze-dried 

sample with metal coating shows good preservation of the papillae and waxes and is suitable for high magnification (the inset shows 

the wax tubules). 

Fig. 3 Examples of fresh-hydrated leaves at ambient temperature in high vacuum. (a): Tropaeolum majus leaf with turgescent cells 

and the characteristic distribution of wax tubules, with metal coating. The high-magnification inset was acquired after the sample had 

dried. (b):Petiole (leaf stem) of Urtica dioides with stinging-hairs., without metal coating. The SE-image (top) shows the topography; 

the atomic-number contrast of the BSE-image indicates the accumulations of Si- and Ca-compounds as bright structures. (c): Leaf 

surface of Taraxacum officinale, without metal coating. The SE-image (top) shows the topography of the turgescent cells; the BSEimage 

indicates inorganic contaminations. 

Nevertheless the samples may be coated with metal to obtain a better contrast of fine details (e.g. epicuticular wax 

crystals, Fig. 3a, 2f) , if they resist the desiccation in the vacuum of the sputter coater for additional few minutes. A 

severe limitation in the examination of fresh-hydrated material is caused by a drift of the specimen due to continuing 

desiccation, which hinders high magnification imaging. (The high-magnification inset in Fig. 3a was recorded after the 

sample had dried.) Some measures are useful for preparation of rapidly drying samples such as the Lotus leave (Fig. 

2c): Only one small piece should be inserted into the SEM. The cut faces should be sealed with glue. Cooling the 

sample holder simply with ice decelerates the desiccation. For imaging without metal coating, the samples must be 

mounted with a conductive glue. 

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Glycerol substitution (Fig. 2d) is an alternative to drying methods and provides the samples with internal electrical 

conductivity, similar to fresh-hydrated material. Compared to using fresh samples, it allows much longer examination 

times and provides better stability for higher magnifications. Glycerol is a polar, water-miscible liquid with a very low 

vapour pressure, so that it can be used in the high vacuum of the SEM and can be imaged without problems. 

Hydrophobic plant surfaces are not wetted by glycerol during the preparation, thus they remain dry and untouched. The 

samples can be examined with or without metal coating, similar to fresh-hydrated ones. Limitations of the glycerol 

substitution are caused by insufficient infiltration of certain tissues, e.g. hairs; other samples like flowers become too 

soft, so that they collapse. A disadvantage is the long preparation time, but multiple samples can be prepared 

simultaneously. A glycerol infiltration without previous fixation is possible and gives usually satisfying results. Thus 

the use of toxic chemicals can be avoided, which makes the method suitable e.g. for schools. 

Simple cryo-preparations: 

Slow cooling: Although the slow cooling of biological samples causes the formation of coarse ice crystals in the tissue, 

we never observed traces of them on the plant surfaces. Only some minor cracks appeared on some specimens (Fig. 4c, 

arrow). Thus it is not necessary to immerse the samples in a cooling liquid to achieve high cooling rates. Instead, the 

surface remains untouched during the preparation; only the sample holder is in contact with LN2. The frozen samples 

can then be used for LT-SEM and / or for freeze-drying. 

Fig. 4 Examples of LT-SEM images of uncoated samples after slow-cooling. (a, b): Drosera nidiformis. (c, d): Superhydrophobic 

trichomes of Salvinia molesta; a glycerol drop resting on the tips of the trichomes demonstrates the low wettability. 

LT-SEM (Fig. 2e, 4, 5a,c): Using a special thermally-isolated sample holder, the frozen samples can be examined after 

insertion into the SEM. At temperatures below -100°C, the frozen samples without metal coating are non-conductive, 

thus it is difficult and requires some experience to avoid imaging problems caused by electric charging. The main 

advantages of the examination of frozen-hydrated samples are the reliable prevention of desiccation or dehydration 

artefacts and the rapid preparation. The surfaces remain untouched and, without a metal coating, they are suitable for 

material contrast imaging with a BSE-detector. Examination at low magnifications is possible despite the poor 

conductivity (Fig. 2e, Fig. 4) . Charging effects on frozen-hydrated samples appear considerably slighter than on dry 

samples without metal coating. But at higher magnifications the charging problems increase and inhibit successful 

working. During insertion of the sample into the SEM, contact with air moisture should be avoided with a tight cover. 

If, however, ice crystals have grown on the sample, they will evaporate and disappear when the temperature of the 

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sample raises. Then, images of the clean surfaces can be obtained. Further, the conductivity of the sample increases 

with the temperature and reduces charging effects. 

Moderate cooling of the samples to temperatures between -80°C and -40°C combines the advantages of fresh-hydrated 

material and LT-SEM and avoids some difficulties. The cooling reduces the desiccation sufficiently, so that also 

flowers, petals and other delicate samples can be examined. In this temperature range, the internal conductivity is 

sufficient to avoid charging problems (Fig. 5a,c). These temperatures can be generated with peltier elements without the 

risk of freezing problems with the SEM specimen stage; only reliable heat dissipation is necessary. 

Fig. 5 Frozen-hydrated (a, c) and freeze-dried (b, d) samples of Salvinia oblongifolia (a, b) and Ruellia devosiana (c, d). The LT- 

SEM images at moderate cooling without metal coating show perfect preservation and no chargings. The freeze-dried S. oblongifolia 

is well preserved and stable for high magnification imaging of the waxes (inset). The soft leaf of R. devosiana shows shrinkage 

particularly of glands and hairs. 

Freeze drying (Fig. 2f, 5b,d): A simple freeze-drying is achieved when the frozen samples are kept in high vacuum, e.g. 

in the SEM chamber subsequent to low-temperature examination, or in another high-vacuum chamber, until they have 

reached ambient temperature. This may last over night. After warming up, the dried samples need to be sputter-coated 

with metal prior to SEM examination. The preservation quality of the samples depends on their mechanical stability, 

size, and warm-up rate. Usually the results are good or even excellent (Fig. 5b, Salvinia oblongifolia), but a partial 

shrinkage can occur (Fig. 5d, Ruellia devosiana). The structure preservation is definitely far better than for air-dried 

samples. It is advantageous that the surfaces are untouched and the samples are dry, thus they are suitable for high 

magnification imaging (Fig. 2f, wax tubules on the Lotus leaf). The freeze-drying can also be performed with a simple 

massive metal container for the samples; thus the special cryo-SEM-holder is therefore not necessary. 

The presented methods broaden the repertory of preparation techniques for plants, so that the users have a better 

chance to find the right one for their certain tasks particularly for conventional SEM. However, it is not our opinion to 

query the advantages of special equipments such as Cryo-SEMs or ESEMs. On the contrary, these simple methods 

should give the users the oportunity to get experiences with fresh-hydrated, uncoated, or frozen samples, or with the 

examination of liquids in the SEM, prior to purchasing expensive special microscopes. Of course, these are necessary 

and optimal for numerous tasks, since most other biological specimens are not adequate for these special plant 

preparation methods. But every method has its limitations. Users of an ESEM, for example, are sometimes disappointed 

from poor contrast and resolution of certain samples without metal coating [12]. 

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It seems necessary to discuss about the limitations and potential risks in the use of the presented methods. For freshhydrated 

samples, the SEM must tolerate a ‘normal’ (biological grade) high-vacuum of 10 -2 to 10 -3 Pa (10 -4 to 10 -5 

mBar). Rapidly drying samples require short pumping times until the SEM-vacuum is ready, but also for the sputter 

coater in case of metal coating. With our equipment, we had SEM evacuation times of ca 150 sec; for sputter coating, 

the samples spent ca. 3 min. in vacuum. 

Table 1 Comparison of the presented preparation techniques. 

CP-drying Airdrying 

Freshhydrated 

Glycerolsubstitution 

Simple 

LT-SEM 

Suitability for high 

magnification + + - ~ - + 

Untouched surface - + + ± + + 

Shrinkage 

preservation + - ± ± ++ ± 

Internal conductivity - - + + ± - 

SEM of uncoated 

samples - - + + + - 

Fast 

preparation - + ++ - + - 

Suitable for 

epicuticular wax - + ~ + ~ ++ 

Suitable for soft 

samples + - - ± + ± 

Simple 

Freeze-drying 

Table 1: Advantages and disadvantages of the preparation methods and suitability for certain applications. (‘+’ well suitable; ‘-‘ not 

suitable; ‘±’ suitable for many but not for all samples; ‘~’ suitable with limited quality. 

Most of the procedures described in this chapter can be made without special equipment, except for LT-SEM, which 

requires the thermally isolated sample holder. Such an item may be incompatible with certain SEM specimen stages. It 

must be checked whether the SEM can tolerate the deeply cooled holder without disturbing the stage mechanics by too 

low temperatures. A save alternative is a Peltier element for cooling. 

The glycerol substitution has obviously found little distribution, despite its versatility, e.g. for imaging of antibodyconjugated 

gold markers on tissue surfaces [24, 25]. Perhaps the users are concerned about the risk to contaminate the 

vacuum system due to the slight evaporation of glycerol [12]. However, we examine glycerol-infiltrated samples 

routinely and they never caused problems. The continuously running turbomolecular-pump of our SEM reached an age 

of 15 years, which is quite normal. But for ultra-clean SEMs, it may be better to omit this method. 

The examination of non-conducting frozen samples without metal coating is a challenge, because charging effects 

can strongly disturb the images. But, at least for low magnifications, it is possible with satisfying results. Several 

measures can reduce the charging problems. A classical method is to work with very low accelleration voltage below 1 

kV [26, 13] (to reach a charge equilibrium when the secondary electron emission coefficient reaches a value of 1). But 

older SEMs suffer from poor resolution or stronger astigmatism under these conditions. Furthermore, BSE imaging or 

EDX analyses require high accelleration voltages. We used 5 to 20 kV but very low beam intensities (

4. Conclusion 

The results have demonstrated the great potential which arises from the development of tailored preparation methods 

for a specific type of samples. The unique properties of plant samples covered with a robust cuticle ask for adapted 

procedures which are rarely found in the common literature. The main advantages of the presented methods, which are 

essential for many applications, are the following: the sample surfaces remain untouched; plant samples can be 

examined without metal coating for improved material contrast imaging; slow cooling provides good results; LT-SEM 

of frozen plant samples without metal coating is possible particularly at temperatures above -100°C. Most of the 

methods can be carried out with simple and low cost home-made equipment. These experiences may encourage other 

SEM users to query their standard methods and to think about better alternatives for special applications. 

Acknowledgements. We thank Wilfried Assenmacher and Sven-Martin Hühne for their help. Parts of this work were financially 

supported by the German Federal Ministery of Education and Research. 

References 


______________________________________________ 


[1] Barthlott W, Schimmel T, Wiersch S, Koch K, Brede M, Barczewski M, Walheim S, Weis A, Kaltenmaier A, Leder A, Bohn HF. 

The Salvinia paradox: superhydrophobic surfaces with hydrophilic pins for air retention under water. Adv. Mat. 2010;22:1-4. 

[2] Anderson TF. Techniques for the preservation of three dimensional structure in preparing specimens for the electron microscope. 

Transactions of the New York Academy of Sciences. 1951;13:130. 

[3] Boyde A. Pros and cons of critical point drying and freeze drying for SEM. Scanning Electron Microsc. 1978;303-314. 

[4] Pfefferkorn GE, Gruter H, Pfautsch M. Observations on the prevention of specimen charging. Scanning Electron Microsc. 1972; 

147-152. 

[5] Parsons E, Bole B, Hall DJ, Thomas WD. A comparative survey of techniques for preparing plant surfaces for the scanning 

electron microscope. J. Microsc. 1974;101:59-75. 

[6] Falk RH. Preparation of plant tissue for SEM. Scanning Electron Microsc. 1980; II:79-87. 

[7] Ledbetter MC. Practical problems in observation of unfixed, uncoated plant surfaces by SEM. Scanning Electron Microsc. 1976; 

II:453-460. 

[8] Mozingo HN, Klein P, Zeevi Y, Lewis ER. Venus's flytrap observations by scanning electron microscopy. Am. J. Bot. 

1970;57:593-598. 

[9] Pannessa BJ, Gennaro JF. Preparation of fragile botanical tissues and examination of intracellular contents by SEM. Scanning 

Electron Microsc. 1972;327-334. 

[10] Pfefferkorn G, Boyde A. Review of replica techniques for scanning electron microscopy. Scanning Electron Microsc. 1974; 

I:75-82. 

[11] Pameijer CH. Replica technique for scanning electron microscopy - a review. Scanning Electron Microsc. 1978; II:831-836. 

[12] Pathan AK, Bond J, Gaskin RE. Sample preparation for scanning electron microscopy of plant surfaces - Horses for courses. 

Micron. 2008;39:1049-1061. 

[13] Echlin P. Handbook of sample preparation for scanning electron microscopy and X-ray microanalysis.. Springer, New York; 

2009. 

[14] Craig S, Beaton CD. A simple cryo-SEM method for delicate plant tissues. J. Microsc. 1996;182:102-105. 

[15] Kaneko Y, Matsushima H, Wada M, Yamada M. A study of living plant specimens by low-temperature scanning electron 

microscopy. J. Electron Microsc. Technique. 1985; 2:1-6. 

[16] Eveling DW. Examination of the cuticular surfaces of fresh delicate leaves and petals with a scanning electron microscope: a 

control for artefacts. New Phytol. 1984;96:229-237. 

[17] Ensikat HJ, Barthlott W. Liquid substitution: a versatile procedure for SEM preparation of biological materials without drying or 

coating. J. Microsc. 1993;172:195-203. 

[18] Echlin PE. Low temperature scanning electron microscopy: a review. J. Microsc. 1978;112: 47-61. 

[19] Sargent JA. Low temperature scanning electron microscopy: advantages and applications. Scanning Microscopy. 1988;2:835- 

849. 

[20] Shah JS, Beckett A. A preliminary evaluation of moist ambient temperature scanning electron microscopy. Micron. 1979;10:13- 

23. 

[21] Danilatos GD, Postle R. The environmental scanning electron microscope and its application. Scanning Electron Microsc. 

1982;1-16. 

[22] Ensikat HJ, Schulte AJ, Koch K, Barthlott W. Droplets on superhydrophobic surfaces: visualization of the contact area by cryoscanning 

electron microscopy. Langmuir. 2009;25(22):13077-13083. 

[23] Liquid substitution methods. In: Robards AW, Wilson AJ, eds. Procedures in Electron Microscopy. Chichester: John Wiley & 

Sons; 10:2.66-10:2.76. 

[24] Reichelt S, Ensikat HJ, Barthlott W, Volkmann D. Visualization of immunogold-labeled cytosceletal proteins by scanning 

electron microscopy. Euro. J. Cell Biol. 1995;67:89-93. 

[25] Samaj J, Ensikat HJ, Baluska F, Knox JP, Barthlot, W, Volkmann D. Immunogold localization of plant surface arabinogalactanproteins 

using glycerol liquid substitution and scanning electron microscopy. J. Microsc. 1999;193:150-157. 

[26] Flegler SL, Heckman JW, Klomparens KL. Elektronenmikroskopie. Heidelberg: Spektrum Akademischer Verlag; 1995. 

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