dismissed. It appears that adapting the particle geometry in DDA calculations does improve the agreement between theory and experiment but still does not fully explain the difference between Gans theory and experimental results (Prescott & Mulvaney 2006). A different dielectric environment around the nanowires could explain this discrepancy. Therefore, we believe that our results show that Gans theory is a useful tool in the determination of the aspect ratios of Au nanowires and offers a good alternative to the

Our implementation of Gans theory provides a quick, easy, and nondestructive method to accurately obtain the aspect-ratio distributions of nanowire solutions from UV-vis

calculations, we obtain a good agreement with TEM measurements for low-aspect-ratio gold nanowires. We find that the dielectric constant of the medium is an important factor in the characterization of gold nanowires. A comparison of Gans theory with other experimental results also shows that a higher dielectric constant of the local environment of the nanowires improves the absorption spectrum calculations for a larger range of aspect ratios. The earlier work by Eustis and El-Sayed reported mixed results in applying Gans theory to inhomogeneously broadened absorption spectra (Eustis & El-Sayed 2006). We have improved on their methods and shown that their results can be explained better under the assumption that the dielectric properties of the local environment around the nanowires have a profound effect on the gold nanowire absorption characteristics (Sprünken et al.

We thank Mr. Daan Sprünken, Dr. Kazuaki Furukawa, Dr. Hiroshi Nakashima, Dr. Ilya Sychugov, Prof. Yoshihiro Kobayashi, Dr. Keiichi Torimitsu, Dr. Koji Sumitomo and Dr. Katsuhiro Ajito, Mr. Seiichiro Mizuno, Mrs. Hiroko Takahata for their comments, insightful discussions, and technical assistances. D.S. is supported through a Vulcanus in Japan

Agarwal, A.; Huang, S.-W.; Day, K.; O'Donnell, M.; Day, M.; Kotov, N. & Ashkenazi, S.

Amendola, V & Meneghetti (2009), Size Evaluation of Gold Nanoparticles by UV-vis

Brioude, A.; Jiang, X.; Pileni, M. (2005). Optical Properties of Gold Nanorods: DDA

(2007). Targeted Gold Nanorod Contrast Agent for Prostate Cancer Detection by Photoacoustic Imaging, *Journal of Applied Physics,* Vol. 102, No. 6, (September 2007),

Spectroscopy, *Journal of Physical Chemistry C* Vol. 113, No. 11, (February 2009),

Simulations Supported by Experiments. *Journal of Physical Chemistry B* Vol. 109, No.

scholarship by the EU-Japan Centre for Industrial Cooperation.

ε

*m* , in our

absorption spectra. By optimizing the dielectric constant of the medium,

#### **6. Discussion**

Using the dielectric constant of the medium as a fitting parameter, it is possible for one to obtain the aspect-ratio distribution of nanowires in solution from the absorption spectrum by fitting with Gans theory. To obtain a good match between the calculated aspect-ratio distributions and TEM measurements, we are required to set the dielectric constant of the medium to a significantly higher value than the dielectric constant of the pure solvent. For our gold nanowires with aspect ratios *R* ≤ 2.5, we find that a value of ε *m* = 2.14 ± 0.05 gives a reasonable fit. However, the fitting has limited accuracy for very-low aspect ratios and thus it is hard to quantify the presence of spherical particles accurately. This could be the result of a limitation of Gans theory, but it can also be caused by errors in the size measurements from TEM images. We believe that the optical properties of the local nanowire environment are strongly affected by the presence of the surfactant as well as possible residue from the fabrication process. In analyzing the aspect ratios of nanowiresolutions, it has proven to be of great importance to incorporate a dependence on the dielectric properties of the local environment. We have shown that with Gans theory it is also possible to do this by optimizing the fit of the absorption spectrum for ε *m* . In this way, one can not only determine the aspect-ratio distribution but also obtain insight into the local environment of the nanowires. Inversely, we have shown that Gans theory can be used to reproduce an absorption spectrum from a known particle size distribution. Here too, we find an optimal fit for ε *m* = 2.1 ± 0.1, which is significantly higher than the dielectric constant of water ( ε *m* = 1.77). This value for ε *m* = 2.1 ± 0.1 lies between the two values of ε *m* = 1.93 and ε *m* = 2.36 that we derived from the experimental results of Eustis and El-Sayed (Figure 5b and c in Eustis & El-Sayed 2006). The difference between the dielectric constants that we find for our particles and the experimental results of Pérez-Juste et al. could possibly be explained by differences in the nanowire synthesis procedures (see Pérez-Juste et al. 2005 and chapter 3).

We have analyzed particle sizes for a very small range of aspect ratios. Most contemporary research is done using particles with *R* > 2, whereas we have used particles with *R* ≤ 2.5. The nanowires of Eustis and El-Sayed (Eustis & El-Sayed 2006) span a much larger range of aspect ratios (1.5 ≤ *R* ≤ 9), and when we apply our method to their results we also find a higher dielectric constant than expected for the solvent, as described in chapter 5. In fact, we need to define two different ε *m* values in order to fit all of their experimental results. From their TEM measurements, it can be concluded that there is an additional variable besides the aspect ratio that influences the absorption spectrum. Three possible factors have been mentioned: end-cap geometry, particle size, and the dielectric constant of the medium. We have shown that Gans theory fits the results of Eustis and El-Sayed if we assume that not all samples have the same dielectric constant. A different dielectric constant could be the result of impurities in the solvent or different surfactant coverage densities of the nanowires. This could be the result of variations in the fabrication and growth process.

The results presented in this paper can probably be duplicated by using DDA calculations with varying particle geometries. We deem it likely that in reality a combination of these two effects occurs. In any case, the possibility that the dielectric constant of the medium in the vicinity of the nanowires is different from that of the pure solvent should not be easily dismissed. It appears that adapting the particle geometry in DDA calculations does improve the agreement between theory and experiment but still does not fully explain the difference between Gans theory and experimental results (Prescott & Mulvaney 2006). A different dielectric environment around the nanowires could explain this discrepancy. Therefore, we believe that our results show that Gans theory is a useful tool in the determination of the aspect ratios of Au nanowires and offers a good alternative to the DDA method.

## **7. Conclusions**

142 The Transmission Electron Microscope

Using the dielectric constant of the medium as a fitting parameter, it is possible for one to obtain the aspect-ratio distribution of nanowires in solution from the absorption spectrum by fitting with Gans theory. To obtain a good match between the calculated aspect-ratio distributions and TEM measurements, we are required to set the dielectric constant of the medium to a significantly higher value than the dielectric constant of the pure solvent. For

a reasonable fit. However, the fitting has limited accuracy for very-low aspect ratios and thus it is hard to quantify the presence of spherical particles accurately. This could be the result of a limitation of Gans theory, but it can also be caused by errors in the size measurements from TEM images. We believe that the optical properties of the local nanowire environment are strongly affected by the presence of the surfactant as well as possible residue from the fabrication process. In analyzing the aspect ratios of nanowiresolutions, it has proven to be of great importance to incorporate a dependence on the dielectric properties of the local environment. We have shown that with Gans theory it is

one can not only determine the aspect-ratio distribution but also obtain insight into the local environment of the nanowires. Inversely, we have shown that Gans theory can be used to reproduce an absorption spectrum from a known particle size distribution. Here too, we

ε

(Figure 5b and c in Eustis & El-Sayed 2006). The difference between the dielectric constants that we find for our particles and the experimental results of Pérez-Juste et al. could possibly be explained by differences in the nanowire synthesis procedures (see Pérez-Juste et al. 2005

We have analyzed particle sizes for a very small range of aspect ratios. Most contemporary research is done using particles with *R* > 2, whereas we have used particles with *R* ≤ 2.5. The nanowires of Eustis and El-Sayed (Eustis & El-Sayed 2006) span a much larger range of aspect ratios (1.5 ≤ *R* ≤ 9), and when we apply our method to their results we also find a higher dielectric constant than expected for the solvent, as described in

experimental results. From their TEM measurements, it can be concluded that there is an additional variable besides the aspect ratio that influences the absorption spectrum. Three possible factors have been mentioned: end-cap geometry, particle size, and the dielectric constant of the medium. We have shown that Gans theory fits the results of Eustis and El-Sayed if we assume that not all samples have the same dielectric constant. A different dielectric constant could be the result of impurities in the solvent or different surfactant coverage densities of the nanowires. This could be the result of variations in the

The results presented in this paper can probably be duplicated by using DDA calculations with varying particle geometries. We deem it likely that in reality a combination of these two effects occurs. In any case, the possibility that the dielectric constant of the medium in the vicinity of the nanowires is different from that of the pure solvent should not be easily

*m* = 2.36 that we derived from the experimental results of Eustis and El-Sayed

ε

*m* = 2.1 ± 0.1, which is significantly higher than the dielectric

*m* = 2.1 ± 0.1 lies between the two values of

ε

*m* = 2.14 ± 0.05 gives

ε

*m* values in order to fit all of their

*m* . In this way,

ε*m*

our gold nanowires with aspect ratios *R* ≤ 2.5, we find that a value of

also possible to do this by optimizing the fit of the absorption spectrum for

**6. Discussion**

find an optimal fit for

ε

constant of water (

= 1.93 and

and chapter 3).

ε

chapter 5. In fact, we need to define two different

*m* = 1.77). This value for

ε

fabrication and growth process.

Our implementation of Gans theory provides a quick, easy, and nondestructive method to accurately obtain the aspect-ratio distributions of nanowire solutions from UV-vis absorption spectra. By optimizing the dielectric constant of the medium, ε *m* , in our calculations, we obtain a good agreement with TEM measurements for low-aspect-ratio gold nanowires. We find that the dielectric constant of the medium is an important factor in the characterization of gold nanowires. A comparison of Gans theory with other experimental results also shows that a higher dielectric constant of the local environment of the nanowires improves the absorption spectrum calculations for a larger range of aspect ratios. The earlier work by Eustis and El-Sayed reported mixed results in applying Gans theory to inhomogeneously broadened absorption spectra (Eustis & El-Sayed 2006). We have improved on their methods and shown that their results can be explained better under the assumption that the dielectric properties of the local environment around the nanowires have a profound effect on the gold nanowire absorption characteristics (Sprünken et al. 2007).

## **8. Acknowledgment**

We thank Mr. Daan Sprünken, Dr. Kazuaki Furukawa, Dr. Hiroshi Nakashima, Dr. Ilya Sychugov, Prof. Yoshihiro Kobayashi, Dr. Keiichi Torimitsu, Dr. Koji Sumitomo and Dr. Katsuhiro Ajito, Mr. Seiichiro Mizuno, Mrs. Hiroko Takahata for their comments, insightful discussions, and technical assistances. D.S. is supported through a Vulcanus in Japan scholarship by the EU-Japan Centre for Industrial Cooperation.

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**9**

*Russia*

**The Cell Ultrastructure of Diatoms -**

Yekaterina D. Bedoshvili and Yelena V. Likhoshway

*Limnological Institute of the Siberian Branch of the Russian Academy of Sciences,*

Diatoms (BACILLARIOPHYTA) are unicell eucaryotes with species-specific silica external skeleton forming intracellularly. The main part of ultrastructural studies are devoted to siliceous valve morphogenesis (Reimann, 1964; Drum & Pankratz, 1964; Schmid & Schultz, 1979; Pickett-Heaps et al., 1990; Pickett-Heaps, 1998; Van de Meene & Pickett-Heaps, 2002) and involvement of cytoskeleton elements in the control of nuclear division and formation large structures on the silica frustule (Pickett-Heaps & Kowalski, 1981; Pickett-Heaps et al.,

According to Round et al. (1990), BACILLARIOPHYTA consists of three classes: centric diatoms with radial and bipolar symmetry of frustules (class Coscinodiscophyceae Round & Crawford) and two classes of pennate diatoms differing in presence (Bacillariophyceae

For the last decade systematics of diatoms based on the morphological data (Round et al., 1990) has been subject to revision (http:\\www.algaebase.org) due to the appearance of molecular phylogeny and understanding of its data (Medlin et al., 2000; Medlin, 2009).

Molecular-phylogenetic analysis revealed that BACILLARIOPHYTA are divided into three clades. Clade 1 includes centric diatoms with radial symmetry of frustules, clade 2a consists of centric diatoms with bi- and multipolar symmetry of frustules and the order Thalassiosirales

Despite the limited number of papers describing diatom cell structure and lack of all major taxa, researchers attempted to detect characteristic features of cell organelle structure for different taxonomic groups of diatoms several times. For example, Mann (1996) and Schmid (2001) considered pyrenoid structure to be as a source of taxonomic and phylogenetic information. They discovered that pyrenoids of diatoms of phylogenetic clade 2 were more diverse than pyrenoids of clade 1. Lately, data on thin structure of chloroplasts and pyrenoids were enlarged and character sets were specified for different diatom phylogenetic

Medlin and Kaczmarska (2004) systematized data on arrangement of Golgi bodies in diatom cells and defined three types. Type 1 is typical of centric diatoms with radial symmetry (except the order Aulacoseirales Crawford). In this case, dictyosomes are associated with ER cistern and mitochondrion and form the so-called G-ER-M unit (*Coscinodiscus* Ehrenberg – Schmid,

1988; Tippit & Pickett-Heaps, 1977; Tippit et al., 1980; Pickett-Heaps, 1983).

Glezer & Makarova, and clade 2b includes all pennate diatoms (Medlin, 1997).

Haeckel) or absence of a raphe (Fragilariophyceae Round).

**1. Introduction**

clades (Bedoshvili et al., 2009).

**Implications for Phylogeny?**

## **The Cell Ultrastructure of Diatoms - Implications for Phylogeny?**

Yekaterina D. Bedoshvili and Yelena V. Likhoshway *Limnological Institute of the Siberian Branch of the Russian Academy of Sciences, Russia*

## **1. Introduction**

146 The Transmission Electron Microscope

Yu, C. & Irudayaraj, J. (2007). Multiplex Biosensor Using Gold Nanorods, *Journal of*

Zweifel, D. & Wei, A. (2005). Sulfide-Arrested Growth of Gold Nanorods, *Chemistry of*

*Analytical Chemistry,* Vol. 79, No. 2, (December 2007), pp. 572-579.

*Materials,* Vol. 17, No. 16, (June 2005), pp. 4256-4261.

Diatoms (BACILLARIOPHYTA) are unicell eucaryotes with species-specific silica external skeleton forming intracellularly. The main part of ultrastructural studies are devoted to siliceous valve morphogenesis (Reimann, 1964; Drum & Pankratz, 1964; Schmid & Schultz, 1979; Pickett-Heaps et al., 1990; Pickett-Heaps, 1998; Van de Meene & Pickett-Heaps, 2002) and involvement of cytoskeleton elements in the control of nuclear division and formation large structures on the silica frustule (Pickett-Heaps & Kowalski, 1981; Pickett-Heaps et al., 1988; Tippit & Pickett-Heaps, 1977; Tippit et al., 1980; Pickett-Heaps, 1983).

According to Round et al. (1990), BACILLARIOPHYTA consists of three classes: centric diatoms with radial and bipolar symmetry of frustules (class Coscinodiscophyceae Round & Crawford) and two classes of pennate diatoms differing in presence (Bacillariophyceae Haeckel) or absence of a raphe (Fragilariophyceae Round).

For the last decade systematics of diatoms based on the morphological data (Round et al., 1990) has been subject to revision (http:\\www.algaebase.org) due to the appearance of molecular phylogeny and understanding of its data (Medlin et al., 2000; Medlin, 2009).

Molecular-phylogenetic analysis revealed that BACILLARIOPHYTA are divided into three clades. Clade 1 includes centric diatoms with radial symmetry of frustules, clade 2a consists of centric diatoms with bi- and multipolar symmetry of frustules and the order Thalassiosirales Glezer & Makarova, and clade 2b includes all pennate diatoms (Medlin, 1997).

Despite the limited number of papers describing diatom cell structure and lack of all major taxa, researchers attempted to detect characteristic features of cell organelle structure for different taxonomic groups of diatoms several times. For example, Mann (1996) and Schmid (2001) considered pyrenoid structure to be as a source of taxonomic and phylogenetic information. They discovered that pyrenoids of diatoms of phylogenetic clade 2 were more diverse than pyrenoids of clade 1. Lately, data on thin structure of chloroplasts and pyrenoids were enlarged and character sets were specified for different diatom phylogenetic clades (Bedoshvili et al., 2009).

Medlin and Kaczmarska (2004) systematized data on arrangement of Golgi bodies in diatom cells and defined three types. Type 1 is typical of centric diatoms with radial symmetry (except the order Aulacoseirales Crawford). In this case, dictyosomes are associated with ER cistern and mitochondrion and form the so-called G-ER-M unit (*Coscinodiscus* Ehrenberg – Schmid,

The Cell Ultrastructure of Diatoms - Implications for Phylogeny? 149

accelerating voltage 80 kV. Microphotographs were made by CCD camera MegaView II

All organelles in the cell of *Thalassiosira proschkinae* are tightly pressed to one another. Large nucleus is located in the cell centre between the lobes of the only chloroplast (fig. 1A). Poorlyexpressed nucleolus is displaced on the nucleus periphery and pressed to the nucleus envelope (fig. 1B). Nuclear pores are turned to the cytoplasm free of chloroplast (fig. 1B). Golgi arrangement is perinuclear (fig. 1C). Large mitochondria (one or two per cell) are of diverse

A big vacuole occupies the main volume of *Aulacoseira baicalensis* cell and the nucleus and cell organelles are pressed to the cell wall (fig. 2A). There is a well-defined nucleolus in the centre of the nucleus (fig. 2B); Golgi bodies (it can be seen no more than 4; fig. 2B) are situated on the side of the nucleus directed to the cell periphery. Nuclear pores are directed to Golgi bodies (fig. 2C). There are vesicles budding from the outer nuclear membrane (fig. 2С). The analysis of serial sections of mitochondria revealed that they have either an intricate branchy form (fig. 2D) or a length up to 4.5 µm (fig. 2E). Mitochondria are frequently situated near the forming

The nucleus of *Chaetoceros muelleri* is shifted to girdle bands and the Golgi bodies are pressed to nuclear membrane (fig. 3A, B). Nuclear pores are directed to either Golgi bodies (fig. 3A) or cell periphery (fig. 3C). Nucleolus is poorly expressed pressing to the nuclear envelope (fig. 3C). The analysis of serial sections showed that the only mitochondrion in the cell can be of diverse shape: complicated (fig. 3D), elongate, sometimes branchy or roundish

A nucleus with a well-defined nucleolus is seen in the cell centre of *Attheya ussurensis* (fig. 4A). Nuclear pores are directed to the Golgi bodies on most of the sections (fig. 4B). Up to four Golgi bodies are located near the nucleus (fig. 4С). A great number of small vesicles around Golgi bodies attest to their activity (fig. 4D). Mitochondria on some sections (fig. 4E)

The nucleus of *Synedra acus* subsp. *radians* of a complicated form with lobes and invaginations (fig. 5A) is located diagonally, ER tanks being inside the invaginations (fig. 5B). There is a roundish or oval nucleolus in the nucleus centre (fig. 5B, C). Multiple Golgi vesicles are often observed budding from the outer nuclear membrane (fig. 5D). Six Golgi bodies are located along the nucleus; nuclear pores turn to them (fig. 5E). Multiple mitochondria of up to 2.7 μm long and to 0.4 μm wide are on the cell periphery (fig. 5F).

The nucleus of *Cymbella ventricosa* is located in the wide part of the cell and pressed to girdle bands (fig. 6A). There is a nucleolus in the nucleus centre (fig. 6B). Up to five Golgi bodies are around the nucleus (fig. 6A, C). A great number of vesicles and ER tanks are detected in the cytoplasm (fig. 6D). Long and narrow mitochondria reach 5 μm on the cell periphery (fig. 6E). *Ditylum brightwellii* has a roundish nucleus in the cell centre (fig. 7А). There is a nucleolus in the nucleus centre (fig. 7B), multiple nuclear pores are aligned regularly on the nuclear envelope (fig. 7С). Multiple Golgi bodies are near the nucleus (fig. 7C). The G-ER-M units

shape on the sections: ring-shaped (fig. 1D), complicated (fig. 1E) or oblong (fig. 1F).

(Zeiss, Germany) and processed with the MegaVision programme.

silica valves (fig. 2F) or lipid drops tightly pressed to them (fig. 2G).

have a commissure of outer and inner membrane looking like a pore.

(fig. 3E). Invaginations were frequently observed (fig. 3F).

are also observed (fig. 7D).

**3. Results**

1987; *Stephanopyxis* (Ehrenberg) Ehrenberg – Medlin et al., 2000; *Ellerbeckia* Crawford – Schmid & Crawford, 2001). Thalassiosirales and Aulacoseirales, most of pennates and bipolar centrics have Golgi arrangement of type 2. Dictyosomes encircle the nucleus and form a perinuclear shell around it (Medlin & Kaczmarska, 2004). *Stephanodiscus niagare* Ehrenberg is a pattern (Drum et al., 1966). Modifications of this type of Golgi arrangement were revealed in cells of *Synedra ulna* (Nitzsch) Ehrenberg (Schmid, 1989) and *Pinnularia nobilis* (Ehrenberg) Ehrenberg (Drum, 1966) where dictyosomes are located along the long nuclear extension towards the poles or periphery of cell. In the cell of *Pinnularia nobilis*, dictyosomes are paired. Type 3 was found in *Biddulphiopsis titiana* (Grunow) von Stosch & Simonsen (Coscinodiscophycidae Round *et* Crawford, clade 1). In this species only outer nuclear envelope forms filose tentacles along which Golgi bodies are paired (Medlin & Kaczmarska, 2004).

This work was aimed at studying cell ultrastructure of seven diatom species from different orders and phylogenetic clades. Ultrathin cell sections were analysed paying attention to a nucleus form, Golgi arrangement and it number, form and number of mitochondria. An attempt was made to reveal specific features of diatom cell ultrastructure of different taxa.

## **2. Materials and methods**

#### **2.1 Cultures**

The cell culture of *Synedra acus* subsp. *radians* (Kützing) Skabitschevsky (Fragilariophyceae Round, Fragilariales Round, clade 2b) was isolated from Listvennichnyi Bay of Lake Baikal and was grown by T.A. Safonova on the medium DM1 (Thompson et al., 1988). *Cymbella ventricosa* Agardh (Bacillariophyceae Haeckel, Cymbellales Mann, clade 2b) was isolated from Irkutsk Reservoir and was grown by T.N. Basharina on the same medium. Cultures of *Thalassiosira proschkinae* Makarova (Coscinodiscophyceae, Thalassiosirales Glezer *et* Makarova, clade 2a), *Attheya ussurensis* Stonik, Orlova *et* Crawford (Coscinodiscophyceae, Chaetocerotales Round *et* Crawford, clade 2a), *Ditylum brightwellii* (West) Grunow (Coscinodiscophyceae, clade 2a), and *Chaetoceros muelleri* Lemmermann (Coscinodiscophyceae, Chaetocerotales Round *et* Crawford, clade 2a) were kindly provided by N.A. Aizdaicher from A.V. Zhirmunsky Institute of Marine Biology FEB RAS (Vladivostok). Sampling of *Aulacoseira baicalensis* (K. Meyer) Simonsen (Coscinodiscophyceae, Aulacoseirales Crawford, clade 1) was carried out in Listvennichnyi Bay of Lake Baikal during its vegetation in spring.

#### **2.2 Transmission electron microscopy**

For transmission microscopy cells in their logarithmic phase were fixed by 2,5 % glutar aldehyde (Sigma-Aldrich, Germany) on PBS (pH 7.0) for 12 h and then by 1% osmium tetroxide (Merck KGaA, Germany) on the same buffer (2 h). Then the cells were placed in 1 % agarose (Helicon, Moskow, Russia). Dehydration was made in ethanol solution series (Reahimkomplekt, Angarsk, Russia) and then in ethanol and acetone dehydrated with copper sulphate (Reahimkomplekt, Angarsk, Russia) for 5 min in each solution. Dehydrated material was soaked in three mixtures with resin Araldite 502 Kit (SPI, USA) and acetone and in pure resin for 12 h. After this, the material was placed in resin with catalyst DMP-30 (SPI, USA) and polymerised in a thermostat at 60°С (for 3 days). Ultrathin sections were cut with a diamond knife ULTRA 35° (Diatom, Switzerland) on the ultramicrotome Ultracut R (Leica, Austria). Sections were stained with Reinolds's lead citrate (Reynolds, 1963). Samples were analysed under a transmission electron microscope Leo 906 Е (Zeiss, Germany) at accelerating voltage 80 kV. Microphotographs were made by CCD camera MegaView II (Zeiss, Germany) and processed with the MegaVision programme.

## **3. Results**

148 The Transmission Electron Microscope

1987; *Stephanopyxis* (Ehrenberg) Ehrenberg – Medlin et al., 2000; *Ellerbeckia* Crawford – Schmid & Crawford, 2001). Thalassiosirales and Aulacoseirales, most of pennates and bipolar centrics have Golgi arrangement of type 2. Dictyosomes encircle the nucleus and form a perinuclear shell around it (Medlin & Kaczmarska, 2004). *Stephanodiscus niagare* Ehrenberg is a pattern (Drum et al., 1966). Modifications of this type of Golgi arrangement were revealed in cells of *Synedra ulna* (Nitzsch) Ehrenberg (Schmid, 1989) and *Pinnularia nobilis* (Ehrenberg) Ehrenberg (Drum, 1966) where dictyosomes are located along the long nuclear extension towards the poles or periphery of cell. In the cell of *Pinnularia nobilis*, dictyosomes are paired. Type 3 was found in *Biddulphiopsis titiana* (Grunow) von Stosch & Simonsen (Coscinodiscophycidae Round *et* Crawford, clade 1). In this species only outer nuclear envelope forms filose tentacles

This work was aimed at studying cell ultrastructure of seven diatom species from different orders and phylogenetic clades. Ultrathin cell sections were analysed paying attention to a nucleus form, Golgi arrangement and it number, form and number of mitochondria. An attempt was made to reveal specific features of diatom cell ultrastructure of different taxa.

The cell culture of *Synedra acus* subsp. *radians* (Kützing) Skabitschevsky (Fragilariophyceae Round, Fragilariales Round, clade 2b) was isolated from Listvennichnyi Bay of Lake Baikal and was grown by T.A. Safonova on the medium DM1 (Thompson et al., 1988). *Cymbella ventricosa* Agardh (Bacillariophyceae Haeckel, Cymbellales Mann, clade 2b) was isolated from Irkutsk Reservoir and was grown by T.N. Basharina on the same medium. Cultures of *Thalassiosira proschkinae* Makarova (Coscinodiscophyceae, Thalassiosirales Glezer *et* Makarova, clade 2a), *Attheya ussurensis* Stonik, Orlova *et* Crawford (Coscinodiscophyceae, Chaetocerotales Round *et* Crawford, clade 2a), *Ditylum brightwellii* (West) Grunow (Coscinodiscophyceae, clade 2a), and *Chaetoceros muelleri* Lemmermann (Coscinodiscophyceae, Chaetocerotales Round *et* Crawford, clade 2a) were kindly provided by N.A. Aizdaicher from A.V. Zhirmunsky Institute of Marine Biology FEB RAS (Vladivostok). Sampling of *Aulacoseira baicalensis* (K. Meyer) Simonsen (Coscinodiscophyceae, Aulacoseirales Crawford, clade 1) was

For transmission microscopy cells in their logarithmic phase were fixed by 2,5 % glutar aldehyde (Sigma-Aldrich, Germany) on PBS (pH 7.0) for 12 h and then by 1% osmium tetroxide (Merck KGaA, Germany) on the same buffer (2 h). Then the cells were placed in 1 % agarose (Helicon, Moskow, Russia). Dehydration was made in ethanol solution series (Reahimkomplekt, Angarsk, Russia) and then in ethanol and acetone dehydrated with copper sulphate (Reahimkomplekt, Angarsk, Russia) for 5 min in each solution. Dehydrated material was soaked in three mixtures with resin Araldite 502 Kit (SPI, USA) and acetone and in pure resin for 12 h. After this, the material was placed in resin with catalyst DMP-30 (SPI, USA) and polymerised in a thermostat at 60°С (for 3 days). Ultrathin sections were cut with a diamond knife ULTRA 35° (Diatom, Switzerland) on the ultramicrotome Ultracut R (Leica, Austria). Sections were stained with Reinolds's lead citrate (Reynolds, 1963). Samples were analysed under a transmission electron microscope Leo 906 Е (Zeiss, Germany) at

carried out in Listvennichnyi Bay of Lake Baikal during its vegetation in spring.

along which Golgi bodies are paired (Medlin & Kaczmarska, 2004).

**2. Materials and methods**

**2.2 Transmission electron microscopy**

**2.1 Cultures**

All organelles in the cell of *Thalassiosira proschkinae* are tightly pressed to one another. Large nucleus is located in the cell centre between the lobes of the only chloroplast (fig. 1A). Poorlyexpressed nucleolus is displaced on the nucleus periphery and pressed to the nucleus envelope (fig. 1B). Nuclear pores are turned to the cytoplasm free of chloroplast (fig. 1B). Golgi arrangement is perinuclear (fig. 1C). Large mitochondria (one or two per cell) are of diverse shape on the sections: ring-shaped (fig. 1D), complicated (fig. 1E) or oblong (fig. 1F).

A big vacuole occupies the main volume of *Aulacoseira baicalensis* cell and the nucleus and cell organelles are pressed to the cell wall (fig. 2A). There is a well-defined nucleolus in the centre of the nucleus (fig. 2B); Golgi bodies (it can be seen no more than 4; fig. 2B) are situated on the side of the nucleus directed to the cell periphery. Nuclear pores are directed to Golgi bodies (fig. 2C). There are vesicles budding from the outer nuclear membrane (fig. 2С). The analysis of serial sections of mitochondria revealed that they have either an intricate branchy form (fig. 2D) or a length up to 4.5 µm (fig. 2E). Mitochondria are frequently situated near the forming silica valves (fig. 2F) or lipid drops tightly pressed to them (fig. 2G).

The nucleus of *Chaetoceros muelleri* is shifted to girdle bands and the Golgi bodies are pressed to nuclear membrane (fig. 3A, B). Nuclear pores are directed to either Golgi bodies (fig. 3A) or cell periphery (fig. 3C). Nucleolus is poorly expressed pressing to the nuclear envelope (fig. 3C). The analysis of serial sections showed that the only mitochondrion in the cell can be of diverse shape: complicated (fig. 3D), elongate, sometimes branchy or roundish (fig. 3E). Invaginations were frequently observed (fig. 3F).

A nucleus with a well-defined nucleolus is seen in the cell centre of *Attheya ussurensis* (fig. 4A). Nuclear pores are directed to the Golgi bodies on most of the sections (fig. 4B). Up to four Golgi bodies are located near the nucleus (fig. 4С). A great number of small vesicles around Golgi bodies attest to their activity (fig. 4D). Mitochondria on some sections (fig. 4E) have a commissure of outer and inner membrane looking like a pore.

The nucleus of *Synedra acus* subsp. *radians* of a complicated form with lobes and invaginations (fig. 5A) is located diagonally, ER tanks being inside the invaginations (fig. 5B). There is a roundish or oval nucleolus in the nucleus centre (fig. 5B, C). Multiple Golgi vesicles are often observed budding from the outer nuclear membrane (fig. 5D). Six Golgi bodies are located along the nucleus; nuclear pores turn to them (fig. 5E). Multiple mitochondria of up to 2.7 μm long and to 0.4 μm wide are on the cell periphery (fig. 5F).

The nucleus of *Cymbella ventricosa* is located in the wide part of the cell and pressed to girdle bands (fig. 6A). There is a nucleolus in the nucleus centre (fig. 6B). Up to five Golgi bodies are around the nucleus (fig. 6A, C). A great number of vesicles and ER tanks are detected in the cytoplasm (fig. 6D). Long and narrow mitochondria reach 5 μm on the cell periphery (fig. 6E).

*Ditylum brightwellii* has a roundish nucleus in the cell centre (fig. 7А). There is a nucleolus in the nucleus centre (fig. 7B), multiple nuclear pores are aligned regularly on the nuclear envelope (fig. 7С). Multiple Golgi bodies are near the nucleus (fig. 7C). The G-ER-M units are also observed (fig. 7D).

The Cell Ultrastructure of Diatoms - Implications for Phylogeny? 151

Fig. 2. The cell structure of *Aulacoseira baicalensis*. TEM. А – cell cross section; B – nucleus and Golgi bodies section; С –Golgi bodies section; D-G – mitochondria sections; D –

Legend: G – Golgi bodies; Li –lipid drop; m – mitochondrion; N – nucleus.

mitochondrion longitudinal section; F – section of the mitochondrion near the forming valve (indicated by the black arrowhead); G – section of the mitochondrion near the lipid drop.

Fig. 1. The cell structure of *Thalassiosira proschkinae*. TEM. А – cell section; В – nucleus section, the nucleus is indicated by black arrowhead, nuclear pore – white arrowhead; С – Golgi bodies section; D-F – sections of mitochondria of different shape. Legend: Chl – chloroplast; G – Golgi bodies; m – mitochondrion; N – nucleus.

Fig. 1. The cell structure of *Thalassiosira proschkinae*. TEM. А – cell section; В – nucleus section, the nucleus is indicated by black arrowhead, nuclear pore – white arrowhead; С – Golgi bodies section; D-F – sections of mitochondria of different shape. Legend: Chl –

chloroplast; G – Golgi bodies; m – mitochondrion; N – nucleus.

Fig. 2. The cell structure of *Aulacoseira baicalensis*. TEM. А – cell cross section; B – nucleus and Golgi bodies section; С –Golgi bodies section; D-G – mitochondria sections; D – mitochondrion longitudinal section; F – section of the mitochondrion near the forming valve (indicated by the black arrowhead); G – section of the mitochondrion near the lipid drop. Legend: G – Golgi bodies; Li –lipid drop; m – mitochondrion; N – nucleus.

The Cell Ultrastructure of Diatoms - Implications for Phylogeny? 153

Fig. 4. The cell structure of *Attheya ussurensis*. TEM. А – cell cross section (the nucleolus is indicated by arrow); B –nucleus section (the arrows show the nuclear pores); C – nucleus and Golgi bodies section (arrows show the Golgi bodies); D – Golgi bodies section (black

arrowheads – the nuclear pores); E – mitochondrion longitudinal section; F – mitochondrion cross section (arrows indicate the comissures of outer and inner mitochondrion membranes). Legend: Chl – chloroplast; G – Golgi bodies; m –

mitochondrion; N – nucleus.

Fig. 3. The cell structure of *Chaetoceros muelleri*. TEM. А – cell section (the nuclear pore is showed by the arrowhead); B –Golgi bodies section; С – nucleus section (black arrowhead – the nucleolus; white arrowhead – the nuclear pores); D-F –sections of mitochondria of different shape. Legend: Chl – chloroplast; G – Golgi bodies; m – mitochondrion; N – nucleus.

Fig. 3. The cell structure of *Chaetoceros muelleri*. TEM. А – cell section (the nuclear pore is showed by the arrowhead); B –Golgi bodies section; С – nucleus section (black arrowhead – the nucleolus; white arrowhead – the nuclear pores); D-F –sections of mitochondria of different

shape. Legend: Chl – chloroplast; G – Golgi bodies; m – mitochondrion; N – nucleus.

Fig. 4. The cell structure of *Attheya ussurensis*. TEM. А – cell cross section (the nucleolus is indicated by arrow); B –nucleus section (the arrows show the nuclear pores); C – nucleus and Golgi bodies section (arrows show the Golgi bodies); D – Golgi bodies section (black arrowheads – the nuclear pores); E – mitochondrion longitudinal section; F – mitochondrion cross section (arrows indicate the comissures of outer and inner mitochondrion membranes). Legend: Chl – chloroplast; G – Golgi bodies; m – mitochondrion; N – nucleus.

The Cell Ultrastructure of Diatoms - Implications for Phylogeny? 155

Fig. 6. The cell structure of *Cymbella ventricosa*. TEM. A – cell cross section; B – nucleus cross section (the nucleolus is shown by the arrow); C – cross section of nucleus and Golgi bodies;

longitudinal section. Legend: Chl – chloroplast; G – Golgi bodies; m – mitochondrion; N –

D – cytoplasm section (the ER tanks are shown by the arrows); E – mitochondrion

nucleus; P – pyrenoid.

Fig. 5. The cell structure of *Synedra acus* subsp. *radians*. TEM. А – longitudinal section of the nucleus with the invagination (dotted line), the nucleolus is shown by the arrowhead; B – cross section of the nucleus invagination with the smooth ER tank inside (arrow); the arrowhead indicates the nuclear pore; C – the nucleus longitudinal section (the nucleolus is shown by the arrow); D – cross section of nucleus and Golgi bodies, the vesicles budded from the nuclear outer membrane are indicated by the arrows; E – nucleus and Golgi bodies longitudinal section, the arrowheads indicate the nuclear pores; F – mitochondrion longitudinal section. Legend: Chl – chloroplast; G – Golgi bodies; m – mitochondrion; N – nucleus.

Fig. 5. The cell structure of *Synedra acus* subsp. *radians*. TEM. А – longitudinal section of the nucleus with the invagination (dotted line), the nucleolus is shown by the arrowhead; B – cross section of the nucleus invagination with the smooth ER tank inside (arrow); the arrowhead indicates the nuclear pore; C – the nucleus longitudinal section (the nucleolus is shown by the arrow); D – cross section of nucleus and Golgi bodies, the vesicles budded from the nuclear outer membrane are indicated by the arrows; E – nucleus and Golgi bodies longitudinal section, the arrowheads indicate the nuclear pores; F – mitochondrion longitudinal section.

Legend: Chl – chloroplast; G – Golgi bodies; m – mitochondrion; N – nucleus.

Fig. 6. The cell structure of *Cymbella ventricosa*. TEM. A – cell cross section; B – nucleus cross section (the nucleolus is shown by the arrow); C – cross section of nucleus and Golgi bodies; D – cytoplasm section (the ER tanks are shown by the arrows); E – mitochondrion longitudinal section. Legend: Chl – chloroplast; G – Golgi bodies; m – mitochondrion; N – nucleus; P – pyrenoid.

The Cell Ultrastructure of Diatoms - Implications for Phylogeny? 157

Golgi bodies of all examined species are located perinuclearly, with some exception. For example, we found two types of Golgi arrangement (G-ER-M unit and perinuclear

Golgi complexes in *S. acus*. subsp. *radians* are located along the nucleus according to type 2 contrary to *S. ulna* whose Golgi arrangement is referred to modification of this type (Medlin & Kacmarska 2004). The nucleus shape differs in these two species: long tubular extension is absent in the nucleus of *S. acus*. subsp. *radians*, whereas in *S. ulna* it is present. Our data support the conclusion of Medlin and Kacmarska (2004) – this is the same perinuclear arrangement of Golgi bodies (type 2) as well as type 3 and it is not worth marking out

Golgi bodies in *A. baicalensis* are located only on one side of the nucleus which turns to the cell periphery. The same arrangement of Golgi bodies is described for *Melosira varians* Agardh (Crawford, 1973) from the closely-related order of the same class

According to the literature data, most of diatoms have a pillow-like or spherical nucleus. However, there are some exceptions. For example, the nucleus of *Lauderia annulata* Cleve consists of two lobes situated on different valves and connected in the cell centre with a thin filament (Holmes, 1977). The nucleus in some species of *Surirella* Turpin can be of H-like shape (Drum & Pankratz, 1964). We revealed that the nucleus shape can differ in cells of species of the same genus. Both *S. ulna* and *S. acus* subsp. *radians* have an oblong nucleus but the latter can form invaginations instead of long extensions. Such nucleus shape increases its surface providing better contact with other cell organelles through the nuclear pores. All examined species have polarization of the nuclear pores which turn to Golgi bodies (figs 1C, 2C, 3A, 4B, D, 5E, 6C). This polarization is clearly expresses in *A. baicalensis* (fig. 2C). The increase of the nucleus surface and the polarization of nuclear pores make it possible to accelerate metabolism of nucleus and cytoplasm. They are not characters of any taxon or

Numerous studies of eukaryotes revealed that mitochondrion morphology is complicated and plastic (Hoffmann & Avers, 1973; Bereiter-Hahn & Voth, 1994; Nunnari et al., 1997; Boldogh et al., 2001). According to the literature data, long mitochondria with tubular cristae localized in the peripheral cytoplasm layer are characteristic of diatoms (Round et al., 1990, p. 60). Comparative studies of mitochondrion structure have not been performed

The shape of diatom mitochondrion appeared to be extremely changeable even in the cells of the same species. Small-celled *C. muelleri* and *T. proschkinae,* belonging to clade 2a, possess one large mitochondrion of various shapes in different cells (figs 1D-F, 3D-F). Lager-celled *A*. *ussurensis* from the same clade have many long mitochondria. The longest mitochondria (up to 5 μm) are detected in the cells (approximately 6 μm) of the pennate *C. ventricosa*. Multiple long and often branched mitochondria (figs 2 D-E, 5F) are recorded in the cells of two species from different clades having the largest cells – *A. baicalensis* (clade 2a) and *S.*

phylogenetic clade but reflect physiological state of the cell.

**4.3 The number and structure of mitochondrion**

dictyosomes) in the same cell of *D. brightwellii* (a member of clade 2a; fig. 7).

individual modifications in it.

Coscinodiscophyceae.

**4.2 The nucleus shape**

before.

Fig. 7. The cell structure of *Ditylum brightwellii*. TEM. A – cell cross section; B – nucleus cross section (the nucleolus is indicated by the arrow); C – cross section of nucleus and Golgi bodies (the nuclear pores are shown by the arrowheads); D – cross section of G-ER-M unit. Legend: Chl – chloroplast; G – Golgi bodies; m – mitochondrion; N – nucleus.

## **4. Discussion**

### **4.1 The Golgi bodies number and their arrangement relative to the nucleus**

Among the examined species the relationship between the cell size and the number of Golgi bodies is observed. The only Golgi complex is recorded in small cells of *C. muelleri* and *T. proschkinae* from different orders and phylogenetic clades. In this study, *A. baicalensis* and *S. acus* subsp. *radians* have the largest cell size (140 and 290 μm long, respectively). Six Golgi complexes are located in the cells of *S. acus* subsp. *radians*. *Synedra ulna*, a larger cell species of the same genus, possesses a higher number of Golgi complexes (7) located along the filamentous extensions of the nucleus (Schmid, 1989, fig. 17). There are four Golgi bodies in *A. baicalensis* as well as in small-celled (up to 6 μm) centric *A. ussurensis* and pennate *C. ventricosa*. It is attributed to small cytoplasm volume in the *A. baicalensis* cells and to a large vacuole occupying the major part of the cell.

Golgi bodies of all examined species are located perinuclearly, with some exception. For example, we found two types of Golgi arrangement (G-ER-M unit and perinuclear dictyosomes) in the same cell of *D. brightwellii* (a member of clade 2a; fig. 7).

Golgi complexes in *S. acus*. subsp. *radians* are located along the nucleus according to type 2 contrary to *S. ulna* whose Golgi arrangement is referred to modification of this type (Medlin & Kacmarska 2004). The nucleus shape differs in these two species: long tubular extension is absent in the nucleus of *S. acus*. subsp. *radians*, whereas in *S. ulna* it is present. Our data support the conclusion of Medlin and Kacmarska (2004) – this is the same perinuclear arrangement of Golgi bodies (type 2) as well as type 3 and it is not worth marking out individual modifications in it.

Golgi bodies in *A. baicalensis* are located only on one side of the nucleus which turns to the cell periphery. The same arrangement of Golgi bodies is described for *Melosira varians* Agardh (Crawford, 1973) from the closely-related order of the same class Coscinodiscophyceae.

#### **4.2 The nucleus shape**

156 The Transmission Electron Microscope

Fig. 7. The cell structure of *Ditylum brightwellii*. TEM. A – cell cross section; B – nucleus cross section (the nucleolus is indicated by the arrow); C – cross section of nucleus and Golgi bodies (the nuclear pores are shown by the arrowheads); D – cross section of G-ER-M unit.

Among the examined species the relationship between the cell size and the number of Golgi bodies is observed. The only Golgi complex is recorded in small cells of *C. muelleri* and *T. proschkinae* from different orders and phylogenetic clades. In this study, *A. baicalensis* and *S. acus* subsp. *radians* have the largest cell size (140 and 290 μm long, respectively). Six Golgi complexes are located in the cells of *S. acus* subsp. *radians*. *Synedra ulna*, a larger cell species of the same genus, possesses a higher number of Golgi complexes (7) located along the filamentous extensions of the nucleus (Schmid, 1989, fig. 17). There are four Golgi bodies in *A. baicalensis* as well as in small-celled (up to 6 μm) centric *A. ussurensis* and pennate *C. ventricosa*. It is attributed to small cytoplasm volume in the *A. baicalensis* cells and to a large

Legend: Chl – chloroplast; G – Golgi bodies; m – mitochondrion; N – nucleus.

**4.1 The Golgi bodies number and their arrangement relative to the nucleus**

**4. Discussion**

vacuole occupying the major part of the cell.

According to the literature data, most of diatoms have a pillow-like or spherical nucleus. However, there are some exceptions. For example, the nucleus of *Lauderia annulata* Cleve consists of two lobes situated on different valves and connected in the cell centre with a thin filament (Holmes, 1977). The nucleus in some species of *Surirella* Turpin can be of H-like shape (Drum & Pankratz, 1964). We revealed that the nucleus shape can differ in cells of species of the same genus. Both *S. ulna* and *S. acus* subsp. *radians* have an oblong nucleus but the latter can form invaginations instead of long extensions. Such nucleus shape increases its surface providing better contact with other cell organelles through the nuclear pores. All examined species have polarization of the nuclear pores which turn to Golgi bodies (figs 1C, 2C, 3A, 4B, D, 5E, 6C). This polarization is clearly expresses in *A. baicalensis* (fig. 2C). The increase of the nucleus surface and the polarization of nuclear pores make it possible to accelerate metabolism of nucleus and cytoplasm. They are not characters of any taxon or phylogenetic clade but reflect physiological state of the cell.

#### **4.3 The number and structure of mitochondrion**

Numerous studies of eukaryotes revealed that mitochondrion morphology is complicated and plastic (Hoffmann & Avers, 1973; Bereiter-Hahn & Voth, 1994; Nunnari et al., 1997; Boldogh et al., 2001). According to the literature data, long mitochondria with tubular cristae localized in the peripheral cytoplasm layer are characteristic of diatoms (Round et al., 1990, p. 60). Comparative studies of mitochondrion structure have not been performed before.

The shape of diatom mitochondrion appeared to be extremely changeable even in the cells of the same species. Small-celled *C. muelleri* and *T. proschkinae,* belonging to clade 2a, possess one large mitochondrion of various shapes in different cells (figs 1D-F, 3D-F). Lager-celled *A*. *ussurensis* from the same clade have many long mitochondria. The longest mitochondria (up to 5 μm) are detected in the cells (approximately 6 μm) of the pennate *C. ventricosa*. Multiple long and often branched mitochondria (figs 2 D-E, 5F) are recorded in the cells of two species from different clades having the largest cells – *A. baicalensis* (clade 2a) and *S.*

The Cell Ultrastructure of Diatoms - Implications for Phylogeny? 159

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*acus* subsp*. radians* (clade 2b). Projections, invaginations and intricate branched shape of mitochondria are similar to complex mitochondrial networks which were detected in other eukaryotes, particularly in yeast (Rizzuto et al., 1998; Mannella, 2000; Rutter & Rizzuto, 2000).

#### **4.4 ER and vesicles**

ER is poorly developed in diatoms like in other heterokont algae (Gibbs, 1981; Round et al., 1990; Van den Hoek et al., 1997) (fig. 6D). It is of an intricate shape when it is located near the nucleus (fig. 5B, D). The outer nuclear membrane performs the functions of ER in synthesis and substance transport (fig. 5D). We believe that vesicles near the outer nuclear membrane are budded from it (instead of coalescence). Synthesized nutrients in the vesicles and transported to the Golgi complex (fig. 5D). Perinuclear location of Golgi bodies promotes rapid transport of vesicles and is observed in all cases especially in the cells with oblong nuclei (fig. 5E).

## **5. Conclusion**

Based on the results of analysis of some diatom species, we tried to apply specific features of cell ultrastructure to systematics of diatoms. Golgi arrangement relative to the nucleus is consistent with characters of phylogenetic clades proposed by Medlin & Kacmarska (2004), i.e., perinuclear Golgi arrangement (type 2) is characteristic of the members of phylogenetic clades 2a and 2b, except *D. brightwellii* with G-ER-M units present in the cell. Both *Aulacoseira* and *Melosira* have perinuclear Golgi arrangement.

Other characteristics of cell ultrastructure are not associated with the systematic position of species. The number of Golgi bodies in species from the same clade and order can differ and depends on the cell size and/or cytoplasm volume. The number and size of mitochondria can be similar in species from different orders and also depend on cell size, whereas the shape of mitochondria can differ even in representatives of the same species.

Thus, the application of cell ultrastructure characteristics in phylogenetic reconstructions is limited except pyrenoids: their structure defines certain taxa and phylogenetic clades (Schmid, 2001; Bedoshvili et al., 2009).

#### **6. Acknowledgements**

The work was supported by the RAS Presidium Programme "Molecular and Cell Biology", Grant 6.3.

#### **7. References**

Bedoshvili, Ye.; Popkova, T. & Likhoshway, Ye. (2009). Chloroplast structure of diatoms of different classes. *Cell and Tissue Biology*, Vol. 3, № 3, pp. 297-310, ISSN *1990-519X*

Bereiter-Hahn, J., & Voth, M. (1994). Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria. *Microsc. Res. Tech.*, Vol. 27, № 3, pp. 198-219

*acus* subsp*. radians* (clade 2b). Projections, invaginations and intricate branched shape of mitochondria are similar to complex mitochondrial networks which were detected in other eukaryotes, particularly in yeast (Rizzuto et al., 1998; Mannella, 2000; Rutter & Rizzuto,

ER is poorly developed in diatoms like in other heterokont algae (Gibbs, 1981; Round et al., 1990; Van den Hoek et al., 1997) (fig. 6D). It is of an intricate shape when it is located near the nucleus (fig. 5B, D). The outer nuclear membrane performs the functions of ER in synthesis and substance transport (fig. 5D). We believe that vesicles near the outer nuclear membrane are budded from it (instead of coalescence). Synthesized nutrients in the vesicles and transported to the Golgi complex (fig. 5D). Perinuclear location of Golgi bodies promotes rapid transport of vesicles and is observed in all cases especially in the cells with

Based on the results of analysis of some diatom species, we tried to apply specific features of cell ultrastructure to systematics of diatoms. Golgi arrangement relative to the nucleus is consistent with characters of phylogenetic clades proposed by Medlin & Kacmarska (2004), i.e., perinuclear Golgi arrangement (type 2) is characteristic of the members of phylogenetic clades 2a and 2b, except *D. brightwellii* with G-ER-M units present in the cell. Both

Other characteristics of cell ultrastructure are not associated with the systematic position of species. The number of Golgi bodies in species from the same clade and order can differ and depends on the cell size and/or cytoplasm volume. The number and size of mitochondria can be similar in species from different orders and also depend on cell size, whereas the

Thus, the application of cell ultrastructure characteristics in phylogenetic reconstructions is limited except pyrenoids: their structure defines certain taxa and phylogenetic clades

The work was supported by the RAS Presidium Programme "Molecular and Cell Biology",

Bedoshvili, Ye.; Popkova, T. & Likhoshway, Ye. (2009). Chloroplast structure of diatoms of different classes. *Cell and Tissue Biology*, Vol. 3, № 3, pp. 297-310, ISSN *1990-519X* Bereiter-Hahn, J., & Voth, M. (1994). Dynamics of mitochondria in living cells: shape

changes, dislocations, fusion, and fission of mitochondria. *Microsc. Res. Tech.*, Vol.

shape of mitochondria can differ even in representatives of the same species.

*Aulacoseira* and *Melosira* have perinuclear Golgi arrangement.

(Schmid, 2001; Bedoshvili et al., 2009).

27, № 3, pp. 198-219

**6. Acknowledgements**

Grant 6.3.

**7. References**

2000).

**4.4 ER and vesicles**

oblong nuclei (fig. 5E).

**5. Conclusion**

**10**

**Influence of Pulse-Impact on Microstructure of**

**Liquid-Phase-Pulse-Impact Diffusion Welding**

The high specific strength, good wear-ability and corrosion resistance of Aluminum Matrix Composites (AMCs) attract substantial industrial applications. Typically, AMCs are currently used widely in automobile and aerospace industries, structural components, and heat resistant-wearable parts in engines, *etc*.  The particles of reinforcement elements in AMCs may be either in form of particulates or as short fibers, whiskers and so forth (Loyd DJ, 1994; Maity J et al., 2009). These discontinuous natures create several problems to their joining techniques for acquiring their high strength and good quality weld-joints.

2. The interface between the particulate reinforcements and aluminum matrix

As properties of welded joints are usually influenced directly by the distribution of particulate reinforcements in the weld, their uniform distribution in the weld is likely to give tensile strength higher than 70~80 % of the parent AMCs. Conglomeration distribution or absence (viz. no-reinforcements-zone) of the particulate reinforcements in the weld generally degrades markedly the joint properties and subsequently resulted in the failure of

High welding temperature in the fusion welding methods (typically: TIG, laser welding, electron beam etc.) is likely to yield pernicious Al4C3 phase in the interface. Long welding time (e.g. several days in certain occasions) in the solid-state welding methods (such as diffusion welding) normally leads to (i) low efficiency and (ii) formation of harmful and

1. The distribution of particulate reinforcements in the weld

brittle intermetallic compounds in the interface.

**1. Introduction**

welding.

**Welded Joints at Various Temperatures in**

**Particle Reinforcement Aluminum**

**Matrix Composites**

*MBE, City University of Hong Kong,*

Kelvii Wei Guo

*Hong Kong*

## **Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites**

Kelvii Wei Guo *MBE, City University of Hong Kong, Hong Kong*

#### **1. Introduction**

160 The Transmission Electron Microscope

Rizzuto, R.; Pinton, P.; Carrington, W.; Fay, F.; Kevin, E.; Lifshitz, L.; Tuft & Pozzan, T.

Round, F.; Crawford, R. & Mann, D. (1990). *The diatoms*, Cambridge University Press, Bristol Rutter, G. & Rizzuto, R. (2000). Regulation of mitochondrial metabolism by ER Ca21 release:

Schmid, A.-M. & Schultz, D. (1979). Wall morphogenesis in diatoms: deposition of silica by

Schmid, A.-M. & Crawford, R. (2001). *Ellerbeckia arenaria* (Bacillariophyceae): formation of

Schmid, A.-M. (1989). Geitler's Plattenband in *Synedra ulna* in the light of TEM

Schmid, A.-M. (2001). Value of pyrenoids in the systematics of the diatoms: their

Schmid, A.-M. (1987). Morphogenetic forces in diatom cell wall formation, In: *Cytomechanics*, J. Bereiter-Hahn, O. Anderson & W. Reif (Eds), 1983-199, Springer, Berlin Thompson, A., Rhodes J., Pettman, I. (Eds). (1988). *Culture collection of algae and protozoa:*

Tippit, D. & Pickett-Heaps, J.D. (1977). Mitosis in the pennate diatom *Surirella ovalis*. *J. Cell*

Tippit, D., Pickett-Heaps, J.D. & Leslie R. (1980). Cell division in two large pennate diatoms

Van de Meene, A.M.L. & Pickett-Heaps, J.D. (2002). Valve morphogenesis in the centric

Medlin, L. (1997). Evolution of the diatoms – a total approach using morphology, molecules and geology. Report of the Workshop June 1-2, 1997, *Diatom Res*., pp. 371-379 Medlin, L. (2009). Diatoms (Bacillariophyta) In: *The timetree of life*, S.B. Hedges & S. Kumar,

diatom *Proboscia alata* Sundstrom. *J. Phycol.*, Vol. 38, pp. 351-363

*Hantzschia* and *Nitzschia*. III. A new proposal for kinetochore function during

morphology and ultrastructure, *Proceedings of the 16th International Diatom*

cytoplasmic vesicles. *Protoplasma*, Vol. 100, pp. 267-288

investigations. *Pl. Syst. Evol*., Vol. 164, pp. 239-252

*Symposium*, Athens, August, 25 – September, 1, 2000

*Catalogue of strains*, Titus: Wilson and Son, Kendal

prometaphase. *J. Cell Biol*., Vol. 86, pp. 402-416

(Eds), 127-130, Oxford University Press

*Biol*., Vol. 73, pp. 705-727

http:\\www.algaebase.org

auxospores and initial cells. *Eur. J. Phycol*., Vol. 36, pp. 307-320

8075

0004

(1998). Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca21 responses. *Science*, Vol. 280, № 5370, pp. 1763-1766, ISSN 0036-

an intimate connection. *Trends Biochem*. *Sci*., Vol. 25, № 5, pp. 215-221, ISSN 0968-

The high specific strength, good wear-ability and corrosion resistance of Aluminum Matrix Composites (AMCs) attract substantial industrial applications. Typically, AMCs are currently used widely in automobile and aerospace industries, structural components, and heat resistant-wearable parts in engines, *etc*.  The particles of reinforcement elements in AMCs may be either in form of particulates or as short fibers, whiskers and so forth (Loyd DJ, 1994; Maity J et al., 2009). These discontinuous natures create several problems to their joining techniques for acquiring their high strength and good quality weld-joints.

1. The distribution of particulate reinforcements in the weld

As properties of welded joints are usually influenced directly by the distribution of particulate reinforcements in the weld, their uniform distribution in the weld is likely to give tensile strength higher than 70~80 % of the parent AMCs. Conglomeration distribution or absence (viz. no-reinforcements-zone) of the particulate reinforcements in the weld generally degrades markedly the joint properties and subsequently resulted in the failure of welding.

2. The interface between the particulate reinforcements and aluminum matrix

High welding temperature in the fusion welding methods (typically: TIG, laser welding, electron beam etc.) is likely to yield pernicious Al4C3 phase in the interface. Long welding time (e.g. several days in certain occasions) in the solid-state welding methods (such as diffusion welding) normally leads to (i) low efficiency and (ii) formation of harmful and brittle intermetallic compounds in the interface.

Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in

Fig. 2. Microstructure of aluminum matrix composite SiCp/6061Al

Fig. 3. Microstructure of aluminum matrix composite Al2O3p/6061Al

Figure 4 illustrates a typical temperature and welding time cycle of a LPPIDW. It basically involved with: (i) an initially rapid increase of weld specimens, within a time of *ta*, to an

**2.3 Operation of LPPIDW**

Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites 163

To alleviate these problems incurred by the available welding processes for welding AMCs, a liquid-phase-pulse-impact diffusion welding (LPPIDW) technique has been developed (Guo W et al., 2007; Guo W et al., 2008; Guo W et al., 2008). This paper aims at providing some specifically studies the influence of pulse-impact on the microstructures of welded joints. Analysis by means of scanning electron microscope (SEM), transmission electron microscope (TEM) and X-Ray Diffraction (XRD) allows the micro-viewpoint of the effect of pulse-impact on LPPIDW to be explored in more detail.

## **2. Experimental material and process**

#### **2.1 Specimens**

Stir-cast SiCp/A356, P/M SiCp/6061Al and Al2O3p/6061Al aluminum matrix composite, reinforced with 20 %, 15 % volume fraction SiC, Al2O3 particulate of 12 μm, 5 μm mean size, are illustrated in Figs. 1~ 3.

### **2.2 Experiment**

Fig. 1. Microstructure of aluminum matrix composite SiCp/A356

The quench-hardened layer and oxides, as induced by wire-cut process, on the surfaces of aluminum matrix composite specimens were removed by careful polishing using 400 # grinding paper. The polished specimens were then properly cleaned by acetone and pure ethyl alcohol so as to remove any contaminants off its surfaces. A DSI Gleeble®-1500D thermal/mechanical simulator with a 4×10-1 Pa vacuum chamber was subsequently used to perform the welding.

The microstructures and the interface between the reinforcement particle and the matrix of the welded joints were analyzed by SEM and TEM.

Fig. 2. Microstructure of aluminum matrix composite SiCp/6061Al

#### **2.3 Operation of LPPIDW**

162 The Transmission Electron Microscope

To alleviate these problems incurred by the available welding processes for welding AMCs, a liquid-phase-pulse-impact diffusion welding (LPPIDW) technique has been developed (Guo W et al., 2007; Guo W et al., 2008; Guo W et al., 2008). This paper aims at providing some specifically studies the influence of pulse-impact on the microstructures of welded joints. Analysis by means of scanning electron microscope (SEM), transmission electron microscope (TEM) and X-Ray Diffraction (XRD) allows the micro-viewpoint of the effect of

Stir-cast SiCp/A356, P/M SiCp/6061Al and Al2O3p/6061Al aluminum matrix composite, reinforced with 20 %, 15 % volume fraction SiC, Al2O3 particulate of 12 μm, 5 μm mean size,

pulse-impact on LPPIDW to be explored in more detail.

Fig. 1. Microstructure of aluminum matrix composite SiCp/A356

the welded joints were analyzed by SEM and TEM.

The quench-hardened layer and oxides, as induced by wire-cut process, on the surfaces of aluminum matrix composite specimens were removed by careful polishing using 400 # grinding paper. The polished specimens were then properly cleaned by acetone and pure ethyl alcohol so as to remove any contaminants off its surfaces. A DSI Gleeble®-1500D thermal/mechanical simulator with a 4×10-1 Pa vacuum chamber was subsequently used to

The microstructures and the interface between the reinforcement particle and the matrix of

**2. Experimental material and process**

**2.1 Specimens**

**2.2 Experiment**

perform the welding.

are illustrated in Figs. 1~ 3.

Figure 4 illustrates a typical temperature and welding time cycle of a LPPIDW. It basically involved with: (i) an initially rapid increase of weld specimens, within a time of *ta*, to an

Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in

strength of parent aluminum matrix composite.

Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites 165

When the temperature is up to 570 ºC, the formed liquid phase matrix alloy was enough and suitable for wetting reinforcements effectively, and the rate of the atom diffusion was more active. As a result, for reinforcements the welding mode in the joint region changed from reinforcement – reinforcement to reinforcement – matrix – reinforcement. Consequently, the joint was welded successfully (Fig. 5(c)). The average strength of 179 MPa for the welded joints produced at welding temperature of 570 oC was about 74.6 % of the 240 MPa for the

(a) 563 ºC

400 μm

400 μm

400 μm

(b) 565 ºC

(c) 570 ºC Fig. 5. Microstructures of welded joints of SiCp/A356 at various temperatures by LPPIDW

optimal temperature *Ta* at which heat was preserved constantly at *Ta* for a period of (*tb* - *ta*), (ii) at time *tc*, a quick application of pulse impact to compress the welding specimens so as to accomplish an anticipated deformation *δ* within a glimpse of 10-4~10-2 s, whilst the heat preservation was still maintained at the operational temperature *Ta*; and (iii) a period of natural cooling to room temperature after time *tb*.

Fig. 4. Schematic diagram of liquid-phase-pulse-impact diffusion welding

### **3. Results and discussion**

#### **3.1 Microstructure of welded joint**

Figure 5 shows the microstructures of welded joints of SiCp/A356 at various temperatures with *VI*=560 mm/s, *tI=*10-2~10-4 s, *t*=30 s, *P0*=5 MPa, *δ*=1 mm, where *VI* was velocity of pulseimpact, *tI* was the impacting time, *t* was holding time for heat preservation, *P0* was holding pressure during the welding, *δ* was the horizontal deformation. It elucidated that when the welding temperature was 563 ºC, under the effect of pulse-impact, the liquid phase matrix alloy wasn't formed enough to wet the particle reinforcements. In addition, at this temperature, the diffusion capability of the atoms within the matrix was relatively low. As a result, the welding interface between two specimens could be observed obviously as shown in Fig. 5(a) and followed by the unsuitable strength (about 118 MPa). Moreover, because of lower welding temperature, the area of the formed solid-liquid phase was smaller, which led to some streamlines scattered in the matrix (Fig. 5(a)) after the pulse-impact acting on the substrates. When the temperature reached 565 ºC, the rate of the atom diffusion in the joint region within the matrix was accelerated (Fig. 5(b)). At the same time, more liquid phase matrix alloy was formed to wet reinforcements (SiC). Therefore, the interface state of reinforcement and reinforcement was improved and the reinforcements were distributed uniformly to some extent. Also, the streamlines scattered in the matrix were disappeared, and the tensile strength of welded joints was about 134 MPa higher than that of 563 ºC.

optimal temperature *Ta* at which heat was preserved constantly at *Ta* for a period of (*tb* - *ta*), (ii) at time *tc*, a quick application of pulse impact to compress the welding specimens so as to accomplish an anticipated deformation *δ* within a glimpse of 10-4~10-2 s, whilst the heat preservation was still maintained at the operational temperature *Ta*; and (iii) a period of

Fig. 4. Schematic diagram of liquid-phase-pulse-impact diffusion welding

Figure 5 shows the microstructures of welded joints of SiCp/A356 at various temperatures with *VI*=560 mm/s, *tI=*10-2~10-4 s, *t*=30 s, *P0*=5 MPa, *δ*=1 mm, where *VI* was velocity of pulseimpact, *tI* was the impacting time, *t* was holding time for heat preservation, *P0* was holding pressure during the welding, *δ* was the horizontal deformation. It elucidated that when the welding temperature was 563 ºC, under the effect of pulse-impact, the liquid phase matrix alloy wasn't formed enough to wet the particle reinforcements. In addition, at this temperature, the diffusion capability of the atoms within the matrix was relatively low. As a result, the welding interface between two specimens could be observed obviously as shown in Fig. 5(a) and followed by the unsuitable strength (about 118 MPa). Moreover, because of lower welding temperature, the area of the formed solid-liquid phase was smaller, which led to some streamlines scattered in the matrix (Fig. 5(a)) after the pulse-impact acting on the substrates. When the temperature reached 565 ºC, the rate of the atom diffusion in the joint region within the matrix was accelerated (Fig. 5(b)). At the same time, more liquid phase matrix alloy was formed to wet reinforcements (SiC). Therefore, the interface state of reinforcement and reinforcement was improved and the reinforcements were distributed uniformly to some extent. Also, the streamlines scattered in the matrix were disappeared, and the tensile strength of welded joints was about 134 MPa higher than that of 563 ºC.

natural cooling to room temperature after time *tb*.

**3. Results and discussion**

**3.1 Microstructure of welded joint**

When the temperature is up to 570 ºC, the formed liquid phase matrix alloy was enough and suitable for wetting reinforcements effectively, and the rate of the atom diffusion was more active. As a result, for reinforcements the welding mode in the joint region changed from reinforcement – reinforcement to reinforcement – matrix – reinforcement. Consequently, the joint was welded successfully (Fig. 5(c)). The average strength of 179 MPa for the welded joints produced at welding temperature of 570 oC was about 74.6 % of the 240 MPa for the strength of parent aluminum matrix composite.

(a) 563 ºC

(b) 565 ºC

Fig. 5. Microstructures of welded joints of SiCp/A356 at various temperatures by LPPIDW

Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in

reinforcement/matrix /reinforcement.

the effect of pulse-impact are similar to that of SiCp/A356.

microstructures well.

shown in Fig. 7 to Fig. 10.

the joint strength (Figs. 7(c) and 9(c)).

Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites 167

The relevant fractographs are shown in Fig. 6. It illustrated that when the welding temperature was 563 oC, the initial morphology of substrate could be detected obviously, and some sporadic welded locations appeared together with some rather densely scattering bare reinforcement particles as shown in Fig. 6(a). With the welding temperature was increased to 565 oC, more liquid phase was formed. Under the effect of pulse-impact, some wet locations in the joint had been excellently welded and the aggregated solid reinforcement particles were improved. However, the bare reinforcement particles were still distributed on the fractographic surface. It indicated that substrates did not weld ideally the pieces together and it consequently resulted in a low strength joint (Fig. 6(b)). Figure 6(c) shows the fractograph of welded joint at 570 oC. It illustrated that the fracture was dimple fracture. Moreover, SEM of the fracture surface showed some reinforcement particles (SiC) in the dimple. In order to confirm the state of these reinforcement particles, particles itself and matrix neighboring to these particles were analyzed by energy dispersive X-ray analysis (EDX) respectively. It indicated that reinforcement particles (SiC) were wet by matrix alloy successfully suggesting that the reinforcement particles had been perfectly wet and the composite structure of reinforcement/reinforcement had been changed to the state of

As welding temperature increasing to 575 oC, it led to more and more liquid phase matrix alloy distributing in the welded interface, meanwhile, more liquid phase matrix alloy reduced the effect of impact on the interface of the welded joints, subsequently the application of transient pulse-impacting would cause the relative sliding of the weldpieces that jeopardized ultimately the formation of proper joint as shown in Fig. 6(d). It demonstrated that results of fractographs were agreed with the corresponding

The relevant results of SiCp/6061Al and Al2O3p/6061Al at various welding temperatures are

It showed that the microstructure evolutions and its corresponding fracture surfaces under

Figures 7(a) and 9(a) show when the welding temperature was too low to form enough liquid phase matrix alloy to wet the reinforcement particles, and the diffusion capability of the atoms within the matrix was relatively low. Therefore, the indistinct welding interface between two specimens could be observed resulted in low tensile strength (about 240 MPa for SiCp/6061Al and 270MPa for Al2O3p/6061Al). When the temperature is higher (623 ºC for SiCp/6061Al and 644 ºC for Al2O3p/6061Al), the liquid phase matrix alloy was formed enough to wet the reinforcement particles (SiC), together with higher rate of the atom diffusion in the joint region (Figs. 7(b) and 9(b)). Consequently, the joints could be welded successfully with the average strength of 260 MPa for SiCp/6061Al (about 72.2 % of the 360 MPa for the strength of parent aluminum matrix composite) and 282 MPa for Al2O3p/6061Al (about 70.5 % of the 400 MPa for the strength of parent aluminum matrix composite). As welding temperature increasing further (such as 625 oC for SiCp/6061Al and 647 oC for Al2O3p/6061Al), more and more liquid phase matrix alloy would be distributed in the welded interface, at the same time, more liquid phase matrix alloy reduced the effect of impact on the interface of the welded joints, subsequently prompted for the descending of

Fig. 6. Fractographs of SiCp/A356 at various temperatures

(a) 563 ºC

(b) 565 ºC

(c) 570 ºC

(d) 575 ºC

Fig. 6. Fractographs of SiCp/A356 at various temperatures

The relevant fractographs are shown in Fig. 6. It illustrated that when the welding temperature was 563 oC, the initial morphology of substrate could be detected obviously, and some sporadic welded locations appeared together with some rather densely scattering bare reinforcement particles as shown in Fig. 6(a). With the welding temperature was increased to 565 oC, more liquid phase was formed. Under the effect of pulse-impact, some wet locations in the joint had been excellently welded and the aggregated solid reinforcement particles were improved. However, the bare reinforcement particles were still distributed on the fractographic surface. It indicated that substrates did not weld ideally the pieces together and it consequently resulted in a low strength joint (Fig. 6(b)). Figure 6(c) shows the fractograph of welded joint at 570 oC. It illustrated that the fracture was dimple fracture. Moreover, SEM of the fracture surface showed some reinforcement particles (SiC) in the dimple. In order to confirm the state of these reinforcement particles, particles itself and matrix neighboring to these particles were analyzed by energy dispersive X-ray analysis (EDX) respectively. It indicated that reinforcement particles (SiC) were wet by matrix alloy successfully suggesting that the reinforcement particles had been perfectly wet and the composite structure of reinforcement/reinforcement had been changed to the state of reinforcement/matrix /reinforcement.

As welding temperature increasing to 575 oC, it led to more and more liquid phase matrix alloy distributing in the welded interface, meanwhile, more liquid phase matrix alloy reduced the effect of impact on the interface of the welded joints, subsequently the application of transient pulse-impacting would cause the relative sliding of the weldpieces that jeopardized ultimately the formation of proper joint as shown in Fig. 6(d). It demonstrated that results of fractographs were agreed with the corresponding microstructures well.

The relevant results of SiCp/6061Al and Al2O3p/6061Al at various welding temperatures are shown in Fig. 7 to Fig. 10.

It showed that the microstructure evolutions and its corresponding fracture surfaces under the effect of pulse-impact are similar to that of SiCp/A356.

Figures 7(a) and 9(a) show when the welding temperature was too low to form enough liquid phase matrix alloy to wet the reinforcement particles, and the diffusion capability of the atoms within the matrix was relatively low. Therefore, the indistinct welding interface between two specimens could be observed resulted in low tensile strength (about 240 MPa for SiCp/6061Al and 270MPa for Al2O3p/6061Al). When the temperature is higher (623 ºC for SiCp/6061Al and 644 ºC for Al2O3p/6061Al), the liquid phase matrix alloy was formed enough to wet the reinforcement particles (SiC), together with higher rate of the atom diffusion in the joint region (Figs. 7(b) and 9(b)). Consequently, the joints could be welded successfully with the average strength of 260 MPa for SiCp/6061Al (about 72.2 % of the 360 MPa for the strength of parent aluminum matrix composite) and 282 MPa for Al2O3p/6061Al (about 70.5 % of the 400 MPa for the strength of parent aluminum matrix composite). As welding temperature increasing further (such as 625 oC for SiCp/6061Al and 647 oC for Al2O3p/6061Al), more and more liquid phase matrix alloy would be distributed in the welded interface, at the same time, more liquid phase matrix alloy reduced the effect of impact on the interface of the welded joints, subsequently prompted for the descending of the joint strength (Figs. 7(c) and 9(c)).

Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in

Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites 169

(a) 620 oC

(b) 623 oC

(c) 625 oC

Fig. 8. Fractographs of SiCp/6061Al at various temperatures

(c) 625 ºC

Fig. 7. SEM micrographs of SiCp/6061Al welded joints at various welding temperatures

Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites 169

(a) 620 ºC

(b) 623 ºC

(c) 625 ºC Fig. 7. SEM micrographs of SiCp/6061Al welded joints at various welding temperatures

(a) 620 oC

(b) 623 oC

(c) 625 oC

Fig. 8. Fractographs of SiCp/6061Al at various temperatures

Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in

Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites 171

(a) 641 oC

(b) 644 oC

(c) 647 oC

Fig. 10. Fractographs of Al2O3p/6061Al at various temperatures

(c) 647 oC

Fig. 9. SEM micrographs of Al2O3p/6061Al welded joints at various welding temperatures

(a) 641 oC

(b) 644 oC

(c) 647 oC Fig. 9. SEM micrographs of Al2O3p/6061Al welded joints at various welding temperatures

(a) 641 oC

(b) 644 oC

(c) 647 oC

Fig. 10. Fractographs of Al2O3p/6061Al at various temperatures

Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in

favorable welding strength (Guo W et al., 2007; Guo W et al., 2008; Guo W et al., 2008).

**3.2 Distribution of dislocation in the welded joint**

SiC

Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites 173

any harmful phase or brittle phase of Al4C3. This suggested the effective interface transfers between reinforcement particles and matrix in the welded joint that subsequently provided

> (i) welded joint (ii) parent composite (a) SiCp/A356

0.25 μm

0.25 μm

0.25 μm

SiC

Matrix

Matrix

Matrix

SiC

0.25 μm

SiC

0.25 μm

Matrix

Matrix

(i) welded joint (ii) parent composite (b) SiCp/6061Al

Al2O3

Al2O3

(i) welded joint (ii) parent composite (c) Al2O3p/6061Al Fig. 12. Distribution of dislocation in the matrix neighboring to the interface of the welded

0.25 μm

joint and parent composite respectively

Matrix

Moreover, according to the fractures of welded joints at various temperatures shown in Figs. 8 and 10, it showed that it agreed with Figs. 7 and 9 very well, and the fractures were all dimple fractures with some reinforcement particles (SiC, Al2O3) in the dimple. Also, the results of SiCp/6061Al were better than that of Al2O3p/6061Al due to a mild interfacial reaction between the reinforcement and matrix, which released the thermal mismatch stress to an acceptable extent between the reinforcement and matrix to allow load transfer from the matrix to reinforcement successfully. As a result, it had advantageous effect of improving the strength of welded joints further (Guo W et al., 2008).

Fig. 11. XRD pattern of the fracture surfaces

Based on microstructures of the welded joints with the optimal parameters (i.e., *T*SiCp/A356=570 C, *T*SiCp/6061Al=623 C, *T*Al2O3p=644 C, *VI*=560 mm/s, *tI=*10-2~10-4 s, *δ*=1 mm, *t*=30 s, *P*0=5 MPa) and its corresponding fracture surfaces as shown in Figs. 5, 6, 7-10, the welded joint displayed with uniformly distributing reinforcement particles and microstructure almost similar to that of its parent composite (Figs. 1, 2 and 3). SEM of the fracture surface showed that the reinforcement particles had been perfectly wet and the composite structure of reinforcement/reinforcement had been changed to the state of reinforcement/matrix /reinforcement. XRD pattern of the fracture surfaces (Fig. 11) did not illustrate the existence of any harmful phase or brittle phase of Al4C3. This suggested the effective interface transfers between reinforcement particles and matrix in the welded joint that subsequently provided favorable welding strength (Guo W et al., 2007; Guo W et al., 2008; Guo W et al., 2008).

## **3.2 Distribution of dislocation in the welded joint**

172 The Transmission Electron Microscope

Moreover, according to the fractures of welded joints at various temperatures shown in Figs. 8 and 10, it showed that it agreed with Figs. 7 and 9 very well, and the fractures were all dimple fractures with some reinforcement particles (SiC, Al2O3) in the dimple. Also, the results of SiCp/6061Al were better than that of Al2O3p/6061Al due to a mild interfacial reaction between the reinforcement and matrix, which released the thermal mismatch stress to an acceptable extent between the reinforcement and matrix to allow load transfer from the matrix to reinforcement successfully. As a result, it had advantageous effect of

(a) SiCp/A356

Al SiC Si

× SiC

Al

Al × Al2O3

× × × ×

×

(b) SiCp/6061Al

2theta (deg.)

× × × ×

(c) Al2O3p/6061Al

2theta (deg.)

×

Based on microstructures of the welded joints with the optimal parameters (i.e., *T*SiCp/A356=570 C, *T*SiCp/6061Al=623 C, *T*Al2O3p=644 C, *VI*=560 mm/s, *tI=*10-2~10-4 s, *δ*=1 mm, *t*=30 s, *P*0=5 MPa) and its corresponding fracture surfaces as shown in Figs. 5, 6, 7-10, the welded joint displayed with uniformly distributing reinforcement particles and microstructure almost similar to that of its parent composite (Figs. 1, 2 and 3). SEM of the fracture surface showed that the reinforcement particles had been perfectly wet and the composite structure of reinforcement/reinforcement had been changed to the state of reinforcement/matrix /reinforcement. XRD pattern of the fracture surfaces (Fig. 11) did not illustrate the existence of

Fig. 11. XRD pattern of the fracture surfaces

× × × × ×

× ×

improving the strength of welded joints further (Guo W et al., 2008).

Intensity (cps)

Intensity (cps)

Intensity (cps)

Fig. 12. Distribution of dislocation in the matrix neighboring to the interface of the welded joint and parent composite respectively

Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in

diffusion welding (Guo W et al., 2007; Guo W et al., 2008; Guo W et al., 2008).

**3.3 Formation of nano-grains in the weld**

composites during the LPPIDW.

Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites 175

The distribution of dislocation in the matrix neighboring to the interface of the welded joint by LPPIDW in comparison with its parent composite is shown in Fig. 12. The clearly distinctive interface between reinforcement particle and matrix indicated that the integration between the reinforcement particle and matrix was prominent. The effect of pulse-impact subsequently led to dislocation in the matrix lattices and showed sign of mutually entwisting to give higher welded strength. Comparatively, its dislocation distribution in the matrix neighboring to the interface was relatively denser than that in its parent composite (cf. Figs. 12(i) and 12(ii)). Similarly, the density of dislocation and dislocation entwisting in the matrix away from the welded interface was also higher than that of its parent composite (cf. Figs. 13(i) and 13(ii)). Such favorable characteristics ultimately gave relatively superior strength of the welded joint to that of conventional

(a) SiCp/A356

(b) SiCp/6061Al

Fig. 14. (a,b) Nano-grains formed in the weld of particle reinforcement aluminum matrix

(c) Al2O3p/6061Al

Fig. 13. Distribution of dislocation in the matrix away from the interface of the welded joint and parent composite respectively

Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites 175

The distribution of dislocation in the matrix neighboring to the interface of the welded joint by LPPIDW in comparison with its parent composite is shown in Fig. 12. The clearly distinctive interface between reinforcement particle and matrix indicated that the integration between the reinforcement particle and matrix was prominent. The effect of pulse-impact subsequently led to dislocation in the matrix lattices and showed sign of mutually entwisting to give higher welded strength. Comparatively, its dislocation distribution in the matrix neighboring to the interface was relatively denser than that in its parent composite (cf. Figs. 12(i) and 12(ii)). Similarly, the density of dislocation and dislocation entwisting in the matrix away from the welded interface was also higher than that of its parent composite (cf. Figs. 13(i) and 13(ii)). Such favorable characteristics ultimately gave relatively superior strength of the welded joint to that of conventional diffusion welding (Guo W et al., 2007; Guo W et al., 2008; Guo W et al., 2008).

#### **3.3 Formation of nano-grains in the weld**

174 The Transmission Electron Microscope

(i) welded joint (ii) parent composite (a) SiCp/A356

(i) welded joint (ii) parent composite (b) SiCp/6061Al

(i) welded joint (ii) parent composite (c) Al2O3p/6061Al Fig. 13. Distribution of dislocation in the matrix away from the interface of the welded joint

and parent composite respectively

(b) SiCp/6061Al

Fig. 14. (a,b) Nano-grains formed in the weld of particle reinforcement aluminum matrix composites during the LPPIDW.

Influence of Pulse-Impact on Microstructure of Welded Joints at Various Temperatures in

American Welding Society (1996). *Welding Handbook*. Miami.

Materials Characterization, Vol. 52, No. 1, pp. 65-75.

Science and Engineering A, Vol. 352, No. 1-2, pp. 162-168.

68, No. 9, pp. 2141-2148.

DOI:10.1007/s00170-008-1411-y.

Reviews, Vol. 39, No. 1, pp. 1-22.

Technology, Vol. 66, No. 2, pp. 363-371.

Vol. 31, No. 1, pp. 111-121.

1-2, pp. 427-437.

3568-3580.

**5. Acknowledgement**

No. 7002582.

**6. References**

Liquid-Phase-Pulse-Impact Diffusion Welding Particle Reinforcement Aluminum Matrix Composites 177

This work is supported by City University of Hong Kong Strategic Research Grant (SRG)

Arik H, Aydin M, Kurt A, Turker M (2005). *Weldability of Al4C3-Al composites via diffusion*

Feng AH, Xiao BL, Ma ZY (2008). *Effect of microstructural evolution on mechanical properties of*

Fernandez GJ & Murr LE (2004). *Characterization of Tool Wear and Weld Optimization in the*

Go´mez de Salazar JM, Barrena MI (2003). *Dissimilar Fusion Welding of AA7020/MMC*

Guo W, Hua M & Ho JKL (2007). *Study on liquid-phase-impact diffusion welding SiCp/ZL101.*

Guo W, Hua M, Ho JKL, Law HW (2008). *Mechanism and Influence of Pulse-Impact on*

Guo W, Hua M, Law HW, Ho JKL (2008). *Liquid Phase Impact Diffusion Welding of*

Hsu CJ, Kao PW & Ho NJ (2005). *Ultrafine-Grained Al–Al2Cu Composite Produced In-Situ by Friction Stir Processing*. Scripta Materialia, Vol. 53, No. 3, pp. 341-345. Loyd DJ (1994). *Particle Reinforced Aluminum Magnesium Composites*. International Materials

Maity J, Pal TK, Maiti R (2009). *Transient liquid phase diffusion bonding of 6061-15 wt% SiCp in*

Marzoli LM, Strombeck AV, Dos Santos JF, Gambaro C, Volpone LM (2006). *Friction Stir*

Nair SV, Tien JK & Bates RC (1995). *SiC-Reinforced Aluminium Metal Matrix Composites*.

Pirondi A, Collini L (2009). *Analysis of crack propagation resistance of Al-Al2O3 particulate-*

Rotundo F, Ceschini L, Morri A, Jun TS & Korsunsky AM (2010). *Mechanical and*

International Metals Reviews, Vol. 30, No. 6, pp. 275-288.

Composites Science and Technology, Vol. 67, No. 6, pp. 1041-1046.

*friction stir welded AA2009/SiCp composite*. Composites Science and Technology, Vol.

*Friction-Stir Welding of Cast Aluminum 359+20% SiC Metal-Matrix Composite*.

*Reinforced with Al2O3 Particles. Microstructure and mechanical properties*. Materials

*Properties of Liquid Phase Pulse-Impact Diffusion Welded SiCp/A356*. The International Journal of Advanced Manufacturing Technology.

*SiCp/6061Al and Its Mechanism.* Materials Science and Engineering: A. Vol. 490, No.

*argon environment*. Journal of Materials Processing Technology, Vol. 209, No. 7, pp.

*Welding of an AA6061/Al2O3/20p Reinforced Alloy*. Composites Science and

*reinforced composite friction stir welded butt joints*. International Journal of Fatigue,

*microstructural characterization of 2124Al/25vol.%SiCp joints obtained by linear friction*

*welding technique*. Materials & Design, Vol. 26, No. 6, pp. 555-560.

(c) Al2O3p/6061Al

Fig. 14. (c) Nano-grains formed in the weld of particle reinforcement aluminum matrix composites during the LPPIDW. (Continuation)

TEM micrograph (Fig. 14) of a weld by LPPIDW displayed some newly-formed nano-grains in the lattices of the joint. These nano-grains would seat in the interstices of crystal lattices and create new grain boundary in hindering the movement of neighbouring grains and subsequently improved obviously the properties of the welded joints. The formation of new nano-grains was the advantageous effect of pulse-impact in LPPIDW. In addition, XRD pattern of the fracture surface (Fig. 11) did not illustrate the existence of any harmful phase or brittle phase of Al4C3. This suggested the effective interface transfers between reinforcement particles and matrix in the welded joint that subsequently provided favorable welding strength (Guo W et al., 2007; Guo W et al., 2008; Guo W et al., 2008).

#### **4. Conclusions**

Results of this study on the microstructures of welded joints of particle reinforcement aluminum matrix composites (SiCp/A356, SiCp/6061Al, Al2O3p/6061Al) using liquid-phasepulse-impact diffusion welding process show that:

## **5. Acknowledgement**

This work is supported by City University of Hong Kong Strategic Research Grant (SRG) No. 7002582.

## **6. References**

176 The Transmission Electron Microscope

(c) Al2O3p/6061Al Fig. 14. (c) Nano-grains formed in the weld of particle reinforcement aluminum matrix

TEM micrograph (Fig. 14) of a weld by LPPIDW displayed some newly-formed nano-grains in the lattices of the joint. These nano-grains would seat in the interstices of crystal lattices and create new grain boundary in hindering the movement of neighbouring grains and subsequently improved obviously the properties of the welded joints. The formation of new nano-grains was the advantageous effect of pulse-impact in LPPIDW. In addition, XRD pattern of the fracture surface (Fig. 11) did not illustrate the existence of any harmful phase or brittle phase of Al4C3. This suggested the effective interface transfers between reinforcement particles and matrix in the welded joint that subsequently provided favorable

Results of this study on the microstructures of welded joints of particle reinforcement aluminum matrix composites (SiCp/A356, SiCp/6061Al, Al2O3p/6061Al) using liquid-phase-

1. Pulse-impact in liquid-phase-pulse-impact diffusion welding in joining particle reinforcement aluminum matrix composites (SiCp/A356, SiCp/6061Al, Al2O3p/6061Al) resulted in higher density of dislocation in the matrix neighboring to and away from the interface than their parent composite. Simultaneously, the dislocation entwisted mutually and intensively in the welded joint propitious to improve the strength of

2. There was distinctly clear interface between reinforcement particle and matrix. It overcame some diffusion problems normally encountered in conventional diffusion welding, and prevented the formation of harmful microstructure or brittle phase in the

3. The joint by LPPIDW process would form nano-grains. The newly-formed nanograins would improve the properties of welded joints resulted in higher tensile

welding strength (Guo W et al., 2007; Guo W et al., 2008; Guo W et al., 2008).

composites during the LPPIDW. (Continuation)

pulse-impact diffusion welding process show that:

**4. Conclusions**

welded joints.

welded joint.

strength.

American Welding Society (1996). *Welding Handbook*. Miami.

**11**

*1Poland 2Austria*

**TEM Investigations of Wear Mechanisms of**

**Single and Multilayer Coatings**

*2Joanneum Research Forschungsges. m. b. H., Institute for Surface Technologies and Photonics,*

Łukasz Major1, Jürgen M. Lackner2 and Jerzy Morgiel1

*1Institute of Metallurgy and Materials Science Polish Academy of Sciences,*

Wear resistant ceramic coatings deposited by physical vapour deposition (PVD) techniques, like magnetron sputtering, ion beam assisted deposition, arc evaporation and pulsed laser deposition (PLD), are successfully used to protect surfaces of mechanical components

The titanium nitride (TiN) is of special interest due to its corrosion resistance and high hardness (R. F. Bunshah, 2001, D. S. Rickerby & A. Matthews, 1991). The other promising material for wear resistant applications is amorphous, hydrogenated carbon (a-C:H). The a-C:H coatings are characterised by very low friction and biological inertness (V. Kumar et al., 2011). The tribology- related engineering applications for highly- stressed components require the development of new multifunctional thin films materials providing superior mechanical, tribological, chemical and high- temperature performance. It could be achieved by connecting the properties of different type of materials in multilayer coatings (Li Chen et al., 2008, M. Stueber et al., 2009, E. Martinez et al., 2003, J. Smolik et al., 1999, Y. L. Su et al.,

The multilayer properties could be controlled by appropriate chemical composition, microstructure and thickness of individual layers. In many tribological applications, coatings with a stack of soft and hard layers can offer much improved mechanical properties compared to single- layered hard coatings (Ł. Major et al., 2010, Ł. Major & J. Morgiel, 2009, M. Bromark et al., 1997, A. Leyland & A. Matthews, 1994, A. Matthews & S. S. Eskilden, 1994, K. Holmberg et al., 1998). The multilayered titanium/titanium nitride (Ti/TiN) coatings display much improved fracture resistance as compared to single- layered TiN. The presence of plastically deformable metallic layers play an important role in cancelling cracks

The coatings for medical application and especially those remaining in contact with human body fluid should both pre- sent high mechanical properties and contain as limited metallic material as possible. Otherwise such coatings may start the metalosis (the metal ions adverse interaction with human organism). The carbon, being part of most tissue of plants

working under high wear loads for nearly half century.

1998, N. Dück et al., 2001, M. Nordin et al., 1999).

propagation mechanisms.

**1. Introduction**

*welding (LFW)*. Composite Part A: Applied Science and Manufacturing, Vol. 41, No. 9, pp. 1028-1037.

## **TEM Investigations of Wear Mechanisms of Single and Multilayer Coatings**

Łukasz Major1, Jürgen M. Lackner2 and Jerzy Morgiel1 *1Institute of Metallurgy and Materials Science Polish Academy of Sciences, 2Joanneum Research Forschungsges. m. b. H., Institute for Surface Technologies and Photonics,*

*1Poland 2Austria*

#### **1. Introduction**

178 The Transmission Electron Microscope

Schell JSU, Guilleminot J, Binetruy C, Krawczak P (2009). *Computational and experimental*

Journal of Materials Processing Technology, Vol. 209, No. 11, pp. 5211-5219. Shanmuga Sundaram N, Murugan N (2010). *Tensile behavior of dissimilar friction stir welded joints of aluminum alloys*. Materials & Design, Vol. 31, No. 9, pp. 4184-4193. Wert JA (2003). *Microstructures of Friction Stir Weld Joints between an Aluminium-Base Metal*

9, pp. 1028-1037.

6, pp. 607-612.

*welding (LFW)*. Composite Part A: Applied Science and Manufacturing, Vol. 41, No.

*analysis of fusion bonding in thermoplastic composites: Influence of process parameters*.

*Matrix Composite and a Monolithic Aluminium Alloy*. Scripta Materialia, Vol. 49, No.

Wear resistant ceramic coatings deposited by physical vapour deposition (PVD) techniques, like magnetron sputtering, ion beam assisted deposition, arc evaporation and pulsed laser deposition (PLD), are successfully used to protect surfaces of mechanical components working under high wear loads for nearly half century.

The titanium nitride (TiN) is of special interest due to its corrosion resistance and high hardness (R. F. Bunshah, 2001, D. S. Rickerby & A. Matthews, 1991). The other promising material for wear resistant applications is amorphous, hydrogenated carbon (a-C:H). The a-C:H coatings are characterised by very low friction and biological inertness (V. Kumar et al., 2011). The tribology- related engineering applications for highly- stressed components require the development of new multifunctional thin films materials providing superior mechanical, tribological, chemical and high- temperature performance. It could be achieved by connecting the properties of different type of materials in multilayer coatings (Li Chen et al., 2008, M. Stueber et al., 2009, E. Martinez et al., 2003, J. Smolik et al., 1999, Y. L. Su et al., 1998, N. Dück et al., 2001, M. Nordin et al., 1999).

The multilayer properties could be controlled by appropriate chemical composition, microstructure and thickness of individual layers. In many tribological applications, coatings with a stack of soft and hard layers can offer much improved mechanical properties compared to single- layered hard coatings (Ł. Major et al., 2010, Ł. Major & J. Morgiel, 2009, M. Bromark et al., 1997, A. Leyland & A. Matthews, 1994, A. Matthews & S. S. Eskilden, 1994, K. Holmberg et al., 1998). The multilayered titanium/titanium nitride (Ti/TiN) coatings display much improved fracture resistance as compared to single- layered TiN. The presence of plastically deformable metallic layers play an important role in cancelling cracks propagation mechanisms.

The coatings for medical application and especially those remaining in contact with human body fluid should both pre- sent high mechanical properties and contain as limited metallic material as possible. Otherwise such coatings may start the metalosis (the metal ions adverse interaction with human organism). The carbon, being part of most tissue of plants

TEM Investigations of Wear Mechanisms of Single and Multilayer Coatings 181

prepared from a section of mechanically deformed place by the Focused Ion Beam technique (FIB). The FIB technique together with in-situ OmniProbe micro- manipulator allowed to receive foil directly from the place of interest, in this particular case from deformed part of

The as deposited single layered a-C:H coatings was characterized by totally amorphous structure (Fig. 1). The atom arrangements in amorphous materials are still relatively poorly understood. An amorphous material is one where the locations of the neighboring atoms are defined by a probability function, so they average inter- atomic distance vary significantly.

Fig. 1. Microstructure analysis of single layered amorphous carbon coating done on a cross-

As a result a diffraction pattern from an amorphous material looks similar to that from polycrystalline material, but rings are broad and diffused. Aside from first ring representing distribution of "nearest neighbours" usually also similar but fainter second ring

In the case of crystalline materials, like TiN, thin ceramic PVD coatings deposited at low temperatures are generally characterized by columnar structure with microcracks, pinholes and transient grain boundaries (Fig. 2a). Due to high compressive residual stress in the single layer TiN coating, microcrack-like features are forming along the column boundaries. (Fig. 2b) They consist of accumulation and pile-up of dislocations between the columns, as

section by TEM technique; a). TEM Bright Field; b). electron diffraction pattern.

1µm

representing "next nearest neighbours" is present.

**3.1 TEM analysis of microstructure of single layered coatings**

a b

coating after mechanical test.

**3. Results and discussion**

**3.1.1 As deposited coatings**

and animals is definitely the best material for such coatings. Carbon provides the framework for all tissues of plants and animals. These tissues are built of elements grouped around in chains and rings made of carbon atoms (J. Grabczyk et al., 2007). This is why in bio- application a development of multilayered coatings with carbon spacers are also of great importance.

The aim of the present paper was to describe the microstructure changes of single (ceramic) layered coating, multilayer ceramic/metallic (with different ratio) coating, and multilayer ceramic/ceramic coating after static mechanical tests by application of transmission electron microscopy techniques. The coatings, were subjected to static mechanical test because it simulates the uploading in their real application (as a coatings for pumps elements which support artificial heart chambers).

## **2. Experimental**

Thin and ultra- thin coatings are preferably produced by deposition from the gaseous phase (PVD-Physical Vapour Deposition and CVD- Chemical Vapour Deposition). PVD coating requires as the first step the vaporization of the basic material- the target. CVD coating starts from mixtures of reactive gases. In contrast to CVD techniques, the PVD processes are generally characterized by lower coating temperatures, a wide range of possible coating and substrate materials, and the higher purity of the deposited coatings. Besides an optimized microstructure, the adhesion of a coating on a substrate is the most crucial property for a coating defining its durability in industrial applications. One of the techniques combining classical evaporation with plasma activation is the Pulsed Laser Deposition (PLD) technique. The target evaporation (ablation) and vapor activation take place by means of a highenergetic focused pulsed laser beam. The high- energetic vapour (plasma) flux allows deposition of highly adhesive coatings even at room temperature. In addition the industrially- scaled room- temperature PLD deposition process was linked with other vacuum coating technique- magnetron sputtering and ion- assisted deposition to combine the advantages of these techniques.

The hybrid PLD with titanium and carbon targets (99, 9at. %) was used for coatings deposition also in presented work. Single layered TiN coatings were deposited using Ti target in nitrogen atmosphere. Multilayer Ti/TiN coatings were deposited using Ti target and sequential gas atmosphere change in between argon for Ti phase deposition to nitrogen for TiN phase deposition. Multilayer TiN/Ti/a-C:H coatings were deposited using both sequential atmosphere change (nitrogen for TiN and argon for a-C:H) and sequential target change (Ti for TiN and C for a-C:H). The details of deposition process is described elsewhere (J. M. Lackner, 2006).

Analyzed coatings were deformed by pushing a diamond indenter with 20μm tip radius under 1N load into it. The test simulates the mechanical interaction by which elements are uploaded in their real application (as a coatings for pumps elements which support artificial heart chambers). The microstructure of as deposited coatings as well as coatings after mechanical tests was characterized using TECNAI G2 F20 FEG (200kV) transmission electron microscope (TEM). Phase analysis was performed by electron diffraction pattern and confirmed by identification of high resolution images (HRTEM). Energy Dispersive X-Ray technique (EDS) was done for chemical analysis. Thin foils for TEM analysis have been prepared from a section of mechanically deformed place by the Focused Ion Beam technique (FIB). The FIB technique together with in-situ OmniProbe micro- manipulator allowed to receive foil directly from the place of interest, in this particular case from deformed part of coating after mechanical test.

## **3. Results and discussion**

180 The Transmission Electron Microscope

and animals is definitely the best material for such coatings. Carbon provides the framework for all tissues of plants and animals. These tissues are built of elements grouped around in chains and rings made of carbon atoms (J. Grabczyk et al., 2007). This is why in bio- application a development of multilayered coatings with carbon spacers are also of

The aim of the present paper was to describe the microstructure changes of single (ceramic) layered coating, multilayer ceramic/metallic (with different ratio) coating, and multilayer ceramic/ceramic coating after static mechanical tests by application of transmission electron microscopy techniques. The coatings, were subjected to static mechanical test because it simulates the uploading in their real application (as a coatings for pumps elements which

Thin and ultra- thin coatings are preferably produced by deposition from the gaseous phase (PVD-Physical Vapour Deposition and CVD- Chemical Vapour Deposition). PVD coating requires as the first step the vaporization of the basic material- the target. CVD coating starts from mixtures of reactive gases. In contrast to CVD techniques, the PVD processes are generally characterized by lower coating temperatures, a wide range of possible coating and substrate materials, and the higher purity of the deposited coatings. Besides an optimized microstructure, the adhesion of a coating on a substrate is the most crucial property for a coating defining its durability in industrial applications. One of the techniques combining classical evaporation with plasma activation is the Pulsed Laser Deposition (PLD) technique. The target evaporation (ablation) and vapor activation take place by means of a highenergetic focused pulsed laser beam. The high- energetic vapour (plasma) flux allows deposition of highly adhesive coatings even at room temperature. In addition the industrially- scaled room- temperature PLD deposition process was linked with other vacuum coating technique- magnetron sputtering and ion- assisted deposition to combine

The hybrid PLD with titanium and carbon targets (99, 9at. %) was used for coatings deposition also in presented work. Single layered TiN coatings were deposited using Ti target in nitrogen atmosphere. Multilayer Ti/TiN coatings were deposited using Ti target and sequential gas atmosphere change in between argon for Ti phase deposition to nitrogen for TiN phase deposition. Multilayer TiN/Ti/a-C:H coatings were deposited using both sequential atmosphere change (nitrogen for TiN and argon for a-C:H) and sequential target change (Ti for TiN and C for a-C:H). The details of deposition process is described elsewhere

Analyzed coatings were deformed by pushing a diamond indenter with 20μm tip radius under 1N load into it. The test simulates the mechanical interaction by which elements are uploaded in their real application (as a coatings for pumps elements which support artificial heart chambers). The microstructure of as deposited coatings as well as coatings after mechanical tests was characterized using TECNAI G2 F20 FEG (200kV) transmission electron microscope (TEM). Phase analysis was performed by electron diffraction pattern and confirmed by identification of high resolution images (HRTEM). Energy Dispersive X-Ray technique (EDS) was done for chemical analysis. Thin foils for TEM analysis have been

great importance.

**2. Experimental**

support artificial heart chambers).

the advantages of these techniques.

(J. M. Lackner, 2006).

#### **3.1 TEM analysis of microstructure of single layered coatings**

#### **3.1.1 As deposited coatings**

The as deposited single layered a-C:H coatings was characterized by totally amorphous structure (Fig. 1). The atom arrangements in amorphous materials are still relatively poorly understood. An amorphous material is one where the locations of the neighboring atoms are defined by a probability function, so they average inter- atomic distance vary significantly.

Fig. 1. Microstructure analysis of single layered amorphous carbon coating done on a crosssection by TEM technique; a). TEM Bright Field; b). electron diffraction pattern.

As a result a diffraction pattern from an amorphous material looks similar to that from polycrystalline material, but rings are broad and diffused. Aside from first ring representing distribution of "nearest neighbours" usually also similar but fainter second ring representing "next nearest neighbours" is present.

In the case of crystalline materials, like TiN, thin ceramic PVD coatings deposited at low temperatures are generally characterized by columnar structure with microcracks, pinholes and transient grain boundaries (Fig. 2a). Due to high compressive residual stress in the single layer TiN coating, microcrack-like features are forming along the column boundaries. (Fig. 2b) They consist of accumulation and pile-up of dislocations between the columns, as

TEM Investigations of Wear Mechanisms of Single and Multilayer Coatings 183

The ring patterns of selected area electron diffraction patterns confirmed that it belong to cubic (TiN) and hexagonal (Ti) systems. The presence of large number of fine spots in these rings indicate that layers are formed from extremely fine crystallites. Metallic buffer layer may prevent contact of substrate with environment by sealing cracks. Energy of brittle cracking in ceramic layer may be compensated by energy of plastic deformation of

Above described coatings were subjected to mechanical test. The test simulated the condition by which coatings are uploaded in their real application. The test was static and based on pushing diamond indenter into the coating with 1N of the applied load

Fig. 4. Theoretical scheme of mechanical test by which analyzed coatings were subjected (R.

Reaction of coatings to the mechanical load during application is as follows: at the beginning elastic deformation dominates and next plastic deformation takes over. Because of ionic or covalent bonding, which characterize ceramic materials, plastic deformation is limited. As

First described material was amorphous carbon single layer coating after static mechanical

Carbon, single layered coating turned out very brittle, as expected. During static mechanical test, substrate deformed plastically, while coating deformation was limited. It deformed elastically and just after that brittle cracks occured. The test caused coating delamination

the dislocations are locked, brittle cracking is dominating.

from the substrate. The adhesion was relatively weak.

buffer.

(Fig. 4).

F. Bunshah, 2001).

test (Fig. 5).

**3.1.2 Coatings after mechanical test**

proved by high resolution imaging. According to literature, microcracks are nucleated in crystalline materials by three different mechanisms: dislocation pile-ups, twin intersections, and strain incompatibility (Gwidon & W. Stachowiak, 2005). In present material all these mechanisms are probably operating simultaneously.

Fig. 2. Microstructure analysis of single layered TiN coating done on a cross- section by TEM technique; a). TEM Bright Field; b). high resolution image (HRTEM) of microcrack at columnar boundary.

The increase TiN single layered coating/substrate adhesion is achieved by inserting, a thin metallic Ti buffer layer (~150nm) between substrate and the coatig (Fig. 3).

Fig. 3. Microstructure analysis of single layered TiN coating with Ti additional buffer layer done on a cross- section by TEM technique; a). TEM Bright Field; b). phase analysis done by electron diffraction pattern (SAEDP).

The ring patterns of selected area electron diffraction patterns confirmed that it belong to cubic (TiN) and hexagonal (Ti) systems. The presence of large number of fine spots in these rings indicate that layers are formed from extremely fine crystallites. Metallic buffer layer may prevent contact of substrate with environment by sealing cracks. Energy of brittle cracking in ceramic layer may be compensated by energy of plastic deformation of buffer.

## **3.1.2 Coatings after mechanical test**

182 The Transmission Electron Microscope

proved by high resolution imaging. According to literature, microcracks are nucleated in crystalline materials by three different mechanisms: dislocation pile-ups, twin intersections, and strain incompatibility (Gwidon & W. Stachowiak, 2005). In present material all these

Micro -cracks / Defects accumulation area

Fig. 2. Microstructure analysis of single layered TiN coating done on a cross- section by TEM

5 nm

The increase TiN single layered coating/substrate adhesion is achieved by inserting, a thin

Fig. 3. Microstructure analysis of single layered TiN coating with Ti additional buffer layer done on a cross- section by TEM technique; a). TEM Bright Field; b). phase analysis done by

500 nm

**pod łoże**

**substrate**

**Ti**

**TiN**

technique; a). TEM Bright Field; b). high resolution image (HRTEM) of microcrack at

metallic Ti buffer layer (~150nm) between substrate and the coatig (Fig. 3).

a b

100 nm

mechanisms are probably operating simultaneously.

Coating surface

a b

columnar boundary.

**residual-stress caused micro-cracks**

Austenite steel substrate **100 nm**

electron diffraction pattern (SAEDP).

Above described coatings were subjected to mechanical test. The test simulated the condition by which coatings are uploaded in their real application. The test was static and based on pushing diamond indenter into the coating with 1N of the applied load (Fig. 4).

Fig. 4. Theoretical scheme of mechanical test by which analyzed coatings were subjected (R. F. Bunshah, 2001).

Reaction of coatings to the mechanical load during application is as follows: at the beginning elastic deformation dominates and next plastic deformation takes over. Because of ionic or covalent bonding, which characterize ceramic materials, plastic deformation is limited. As the dislocations are locked, brittle cracking is dominating.

First described material was amorphous carbon single layer coating after static mechanical test (Fig. 5).

Carbon, single layered coating turned out very brittle, as expected. During static mechanical test, substrate deformed plastically, while coating deformation was limited. It deformed elastically and just after that brittle cracks occured. The test caused coating delamination from the substrate. The adhesion was relatively weak.

TEM Investigations of Wear Mechanisms of Single and Multilayer Coatings 185

TiN layer brittle cracked (Fig. 6). Cracking in ceramic crystalline materials mainly depends on the crystallographic plane which is the most responsible for cracking propagation as

Fig. 7. Schemes of cracking character in dependence on the crystallographic plane on which

Normally the process occurs on the most packed planes. If cracking is propagating along {110} planes the cleavage like cracking is visible. In case of cracks on {111} planes, steps like line is formed. Finally on {001} planes, branches like pattern is visible. Titanium nitride is characterized by face center cubic (fcc) structure. The most packed plane for fcc is {111} on

High resolution analysis of crack path performed through the TiN layer revealed that

presents on the scheme (Fig. 7).

it may be realized (C. L. Rountree et al. 2002).

cracking was propagating along {111} plane (Fig. 8).

Fig. 8. HRTEM image of crack propagating through the TiN layer.

which cracking is the most probable.

Coating/ substrate delamination

Fig. 5. Microstructure analysis of single layered a-C:H coating on a cross- section, after mechanical test, done by TEM technique.