Ayache J., Beaunier L., Boumendil J., Ehret G., Laub D. Sample Preparation Handbook for Transmission Electron Microscopy. Methodology

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Bibliography 197

There are 36 sample preparation techniques listed, and their main characteristics

are described. These 36 techniques are developed in “Techniques” of this book. An

interactive image gallery presents the different types of materials prepared using

these techniques and also provides examples of materials and techniques compiled

by the international community of microscopists.

http://temsamprep.in2p3.fr

Bibliography

Anderson, R.M. (1990). Specimen Preparation for Transmission Electron Microscopy of Materials

II, MRS Matérial Research Society Symposium Proceedings, vol. 199, Pittsburgh, PA.

Anderson, R.M., Tracy, B., and Bravman, J. (1992). Specimen Preparation for Transmission

Electron Microscopy of Materials III, MRS Matérial Research Society Symposium Proceedings,

vol. 254, Pittsburgh, PA.

Bousﬁeld, B. (1992). Surface Preparation and Microscopy of Materials. Wiley and Sons, Buehler

Europe Ltd, Coventry.

Bravman, J., Anderson, R.M., and Mcdonald, M.L. (1988). Specimen Preparation for Transmission

Electron Microscopy of Materials, MRS Matérial Research Society Symposium Proceedings,

vol. 115, Pittsburgh, PA.

Delain, E. and Le Cam, E. (1995). The spreading of nucleic acids. In Visualization of Nucleic

Acids, vol. 3 (ed. G. Morel). CRC Press, Boca Raton, London, Tokyo, 35–56.

Pottu-Boumendil, J. (1989). Microscopie Electronique: Principes et méthodes de preparation. Les

Editions INSERM, Paris.

Chapter 8

Comparisons of Techniques

1 Introduction

Given the different nature of the artifacts and drawbacks induced by mechanical,

chemical, or ionic techniques, or even those involving changes in physical state, it

is important to combine several techniques in order to conﬁrm the intrinsic struc-

ture of a given material. The combination of techniques can vary, depending on

the different properties of materials, their physical or chemical state, and their

organization.

This chapter presents comparisons of at least two of the most commonly used

preparation techniques for different types of materials in materials sciences and in

biology.

2 Examples Using Fine Particle Materials

2.1 Comparison of Mechanical Preparations and Replicas

Extractive replica technique and crushing

Fine particle materials: catalyst particles

2.1.1 Crushing Technique (“Techniques” Chapter 4, Section 1) and Extractive

Replica Technique (“Techniques” Chapter 5, Section 3)

Fine particle material: 5% platinum catalyst particles on cerium oxide (CeO

treated with H

at 973 K.

Comparison discussion: The crushing technique enables the conservation of the

substrate and investigating the interactions between it and the material (catalyst par-

ticles or other). In this case, Figs. 8.1 and 8.2 clearly show that platinum particles

199

J. Ayache et al., Sample Preparation Handbook for Transmission Electron Microscopy,

DOI 10.1007/978-0-387-98182-6_8,

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Springer Science+Business Media, LLC 2010

200 8 Comparisons of Techniques

Fig. 8.1 The thin slice is

obtained using the crushing

technique and therefore, the

substrate is maintained. The

arrows indicate the platinum

particles superimposed on the

substrate, cerium oxide. The

platinum particles are

epitaxied with the substrate

(moiré fringes). (M. Abid,

LMPC-ECPM-ULP

Strasbourg)

Fig. 8.2 Same as in Fig. 8.1,

showing the planes of the

cerium oxide, which have

interreticular distances of d =

3.125 Å and moirés of

platinum particles, which

indicate that they are

epitaxied on the cerium

oxide. (M. Abid,

LMPC-ECPM-ULP

Strasbourg)

Fig. 8.3 The material is

prepared using the extractive

replica technique; the

substrate is removed by

means of chemical etching,

only the shadowing of the

cerium oxide is kept. The

platinum particles are

indicated by arrows.The

particle–substrate interaction

is lost and therefore,

observation is more difﬁcult.

(M. Abid, LMPC-ECPM-ULP

Strasbourg)

2 Examples Using Fine Particle Materials 201

are epitaxied on the substrate. With the extractive replica technique, there is

no way to investigate this interactivity since the substrate has to be dissolved

(Fig. 8.3).

2.1.2 Crushing Technique (“Techniques” Chapter 4, Section 1) and Extractive

Replica Technique (“Techniques” Chapter 5, Section 3)

Fine particle material: 0.2% platinum catalyst particles on aluminum oxide

(Al

), treated with H

at 973 K

Comparison discussion: the crushing technique enables keeping the substrate

together with the material. The platinum particle appears in black (high atomic

number) and allows measurements and characterizations in nanodiffraction mode.

The images of platinum in HRTEM do not have good resolution because the thick-

ness of the platinum/substrate is too high. With the extractive replica technique,

the substrate is dissolved and HRTEM observations are both possible and generally

of good quality. Measurements of platinum particle size as well as characteriza-

tions in nanodiffraction mode can also be made. In this case, good resolution can

easily be obtained in HRTEM images, since the substrate is removed (Figs. 8.4

and 8.5).

Fig. 8.4 Pt catalyst on

aluminum oxide Pt/Al

Thin slice obtained using the

crushing technique.

(R. Touroude,

LMPC-ECPM-ULP

Strasbourg)

202 8 Comparisons of Techniques

Fig. 8.5 Identical material,

prepared by the extractive

replica technique.

(R. Touroude,

LMPC-ECPM-ULP

Strasbourg)

2.2 Comparison of “Negative-Staining” Contrast

and Freeze-Fracture Techniques

2.2.1 Negative-Staining (“Techniques” Chapter 7, Section 2) and

Freeze-Fracture Techniques (“Techniques” Chapter 5, Section 4)

Fine particle material: multilayer liposomes

Comparison discussion: The liposomes are spheres made from phospholipids.

Here we have liposomes with multiple layers. Negative staining shows these differ-

ent layers, but liposomes are often coalescent and fuse together (Fig. 8.6). Freeze

fracturing allows us to see them separated from one another, as in the liquid sus-

pension. It is easy to measure their size and evaluate their dispersion. Liposomes

Fig. 8.6 The suspension of

liposomes is treated using

phosphotungstic acid. The

liposomes are viewed using

transparency; the layers are

white on a dark background.

The contrastant also

penetrates inside the

liposomes. (J. Boumendil,

UCB Lyon 1)

2 Examples Using Fine Particle Materials 203

Fig. 8.7 The suspension of

liposomes was frozen in

nitrogen, and then freeze

fractured. Some liposomes

break and open completely

(1), others are half-broken,

between two layers (2), and

others are intact (3), as

viewed from above.

(J. Boumendil, UCB Lyon 1)

fracture, making it possible to highlight the multiple layers (Fig. 8.7). But most

often, the very small and very large liposomes do not fracture. Either their imprint

(1) or their surface is seen (3).

2.3 Comparison of “Negative-Staining”

and Decoration-Shadowing Contrast Techniques

2.3.1 Negative-Staining Contrast (“Techniques” Chapter 7, Section 2) and

Decoration-Shadowing (“Techniques” Chapter 7, Section 1) Techniques

Fine particle material: spread-out chromatin

Comparison discussion: Figs. 8.8, 8.9, and 8.10 show that it is possible to use

the negative-staining and shadowing techniques on comparable samples for cer-

tain biological materials. Figure 8.8 shows chromatin ﬁbers with the classic 30-nm

diameter. Figures 8.9 and 8.10 show a different state of the chromatin, when

the conditions enable decondensing the nucleosomes, and the nucleosomes and

internucleosomal DNA can then be seen.

The negative-staining technique, which is very well adapted to chromatin, is not

as suitable for viewing chromosomes and DNA (although it would still be possi-

ble). Figures 8.9 and 8.10 provide two shadowing variants. Figure 8.9 shows a very

classic shadowing technique using platinum and Fig. 8.10 shows that bidirectional

shadowing using tungsten yields two thinner metallization layers (a few nanome-

ters). This makes possible dark-ﬁeld observation and therefore results in higher

contrast and better resolution. These shadowing methods could also be used for

chromatin, but it may undergo damage due to the placement of the sample under

vacuum for metallization.

204 8 Comparisons of Techniques

Fig. 8.8 Bright-ﬁeld image

of chromatin prepared using

negative staining with 2%

uranyl acetate in water. The

protein complex is not

unwound. (E. Delain,

CNRS-UMR8126-IGR,

Villejuif)

Fig. 8.9 Bright-ﬁeld image

of chromatin prepared using

unidirectional shadowing

with platinum. (E. Delain,

CNRS-UMR8126-IGR,

Villejuif)

2.3.2 Negative-Staining (“Techniques” Chapter 7, Section 2)

and Decoration-Shadowing (“Techniques” Chapter 7, Section 1)

Contrast Techniques

Fine particle material: Escherichia coli bacteria

Comparison discussion: Negative staining is used to quickly obtain information

on the type of bacteria and ﬂagella, but does not identify the pilis. Furthermore, the

2 Examples Using Fine Particle Materials 205

Fig. 8.10 Dark-ﬁeld image

of chromatin prepared using

bidirectional shadowing with

tungsten. (E. Delain,

CNRS-UMR8126-IGR,

Villejuif)

central body of the bacteria appears dehydrated and ﬂattened (Fig. 8.11). Rotary

shadowing shows the pili and ﬂagella, but no information is available on the body

of the bacteria; it would be necessary to make a replica in order to have this kind of

data (Fig. 8.12).

Fig. 8.11 Negative-staining,

bright-ﬁeld observation.

(A. Ryter, Institut Pasteur

Paris)

206 8 Comparisons of Techniques

Fig. 8.12 Metallic rotary

shadowing under a

unidirectional angle of 45

◦

observed in bright-ﬁeld and

inverted image mode (f =

ﬂagella). (A. Ryter, Institut

Pasteur Paris)

2.4 Comparison of “Positive-Staining” and Decoration-Shadowing

Contrast Techniques

2.4.1 Positive-Staining (“Techniques” Chapter 7, Section 2)

and Decoration-Shadowing (“Techniques” Chapter 7, Section 1)

Contrast Techniques

Fine particle material: relaxed circular DNA molecules (pBR 322 plasmid,

4,361 bp)

Comparison discussion: The observation of DNA molecules requires the spread-

ing of the macromolecule, which is negatively charged. Historically, the ﬁrst type

of DNA spreading was done using the cytochrome c technique, which forms a thin

ﬁlm on the surface of an aqueous hypophase and complexes with the DNA. There

are several variants of this method (see ﬁne particle dispersion technique). Next,

the spread DNA can be shadowed or positively stained in order to be viewed. The

contrast depends on these methods and the observation in bright-ﬁeld or dark-ﬁeld

mode. For dark-ﬁeld imaging, we can make a tilted dark ﬁeld, or use the electron

energy loss, either by using elastically transmitted electrons or on the uranium peak

(Figs. 8.13 and 8.14).

The second type of DNA spreading uses the technique of a preliminary coating

of a pentylamine ﬁlm (Dubochet method) on the carbon grid, followed by positive

staining using a uranyl acetate rinse. Positive staining can highlight the conforma-

tion of the DNA. This type of preparation can also be metalized i.e., shadowing as

described above (Fig. 8.14).

Both techniques, “shadowing after spreading of cytochrome c and positive stain-

ing,” yield the same information on the conformation of the DNA, with contrasts

and resolutions depending on the sharpness of the shadowing and on the observation

mode.