Different microscopy approach for solid surface characterization

Different microscopy approach for solid surface characterization

P. Slepička1, J. Siegel1, Z. Kolská2, V. Hnatowicz3 and V. Švorčík1 1 Department of Solid State Engineering, Institute of Chemical Technology, 166 28 Prague, Czech Republic 2 Department of Chemistry, J.E. Purkyne University, 400 96 Usti nad Labem, Czech Republic 3 Nuclear Physics Institute of Academy of Sciences of the Czech Republic, 25068 Rez near Prague, Czech Republic

The main goal of this study is to describe different microscopic techniques and to demonstrate their usefulness in characterization of the solid surfaces. On several practical examples it is documented that different optical imaging methods combined with the atomic force microscopy provides complex data on the properties of the solid surfaces, which are of interest for potential applications of materials with tailored surfaces, e.g. in bio-medicine. Also some connections between the surface morphology and other surface properties, such are zeta-potential, free carrier volume concentration, electrical sheet resistance, surface wettability are revealed.

Keywords atomic force microscopy, scanning electron microscopy, transmission electron microscopy, laser confocal microscopy, surface roughness, polymer, metal sputtering and evaporation, laser and plasma treatment, some surface properties

1. Introduction

The rapid development of nano-science and nanotechnology in the 20th and 21st centuries brings a lot of questions about the structure and surface properties of nano-materials. Various approaches to characterize material in “nano-scale dimension” have been proposed. Several analytical methods can describe the physico-chemical behaviour of studied nano-materials and their chemical reactivity. Hand in hand with spectroscopy methods such as Raman Spectroscopy, X-ray Diffraction, Atomic Absorption Spectroscopy or Photoelectron Spectroscopy different microscopic methods can be successfully applied for the surface characterization. With the appearance of Scanning Probe Microscopy (SPM) the chance for surface studies of conductive, semi-conductive or non-conductive materials has increased enormously. The visualization of surface structure features down to sub-micro or even atomic scale, provided by the techniques as Scanning Tunneling Microscopy (STM) or Atomic Force Microscopy (AFM), has reached and exceeded the resolution limits of electron based microscopic techniques. Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and optical microscopies have been supplemented not only by SPM, but recently also with new techniques, such as Laser Confocal Microscopy, (LCM), combining high resolution with fast and easy surfaces examination. The combination of various microscopic techniques may give complex information on the properties and structure of different materials and in this respect it is helpful in almost all fields of material science. In this review selected results on pristine and modified synthetic polymers are presented together with the results of few control experiments performed on glass substrates. The polymer modification is accomplished by plasma treatment or irradiation with laser light and in some cases by coating the substrate surface with gold film. Essential part of the presented data refers to the results obtained by optical, atomic force-, scanning electron- and transmission electron microscopies and additional results of electrical and optical measurements or electrokinetical analysis. The authors are engaged in the study of processes of modification of polymer surface properties by different agens for a long time (see e.g. [1-9]). Surface morphology of pristine and modified polymer substrates and deposited metal nano-clusters and nano-layers has been studied with Atomic Force Microscopy (AFM). The “tapping mode” was chosen for the determination of polymer samples and thin metal layers to minimize damage to the sample surface. Contact mode was used for the thickness measurements and the morphology of samples with higher roughness. All surfaces were characterized quantitatively by measuring of the mean roughness (Ra) and semi-qualitatively via surface image inspection. Thickness of metal layers was determined using a combination of Focused Ion Beam (FIB) and Scanning Electron Microscopy (SEM). Surface roughness and morphology of deposited metal nano-structures were examined by Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). Biological properties (e.g. cells adhesion and proliferation) were studied on specific cell cultures in

vitro experiments, the cell proliferation on the polymer samples was characterized by standard methods using optical microscope with digital camera.

The effects of plasma treatment, laser patterning and nano-particle grafting on the surface morphology, roughness and other physico-chemical properties have been studied on different, polymer based systems and it has been shown that, as a result of the modification, the polymers acquires new surface properties which can be of interest for various applications. For example it has been shown that Au nano-particles deposited on the modified polymer surface increase adhesion and proliferation of living cells; the finding could be important for tissue engineering. Gold deposition onto the surface of a polymer modified by the light of a KrF laser led to creation of metal “nano-wires”. Most important

Microscopy: Science, Technology, Applications and Education A. Méndez-Vilas and J. Díaz (Eds.)

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results of these studies are summarized in this review. Relations between the surface morphology determined by microscopic methods and physico-chemical properties of the prepared structures obtained by e.g. goniometry, electrical measurements, electrokinetical analyses, and X-ray photoelectron spectroscopy (XPS) are discussed too.

2. Selected surface characteristics

The study of surface properties of polymers, pristine or modified by different processes and agents, is very important not only for understanding of basic processes taking place during the modification but also for subsequent usage of the modified polymers. It is known that the modification may affect significantly the surface morphology and various physico-chemical properties such as surface wettability, sheet resistance, free carrier volume concentration and ζ-potential. The modification may influence the process of polymer metal coating, adhesion of the deposited metal films, grafting of the polymer surface with different chemical agents and living cell adhesion and proliferation.

2.1 Free carrier volume concentration and Hall mobility

Free carrier volume concentration and their Hall mobility significantly affect the electrical conductance of materials [8]. Dependence of the free carrier concentration and the mobility on the thickness of the gold layer sputtered onto PET and PTFE substrates is shown in Figs 1A and 1B. As can be seen from Fig. 1A with increasing layer thickness the carrier concentration increases dramatically and the layers become conductive. The change of the free carrier concentration can be explained by different types of conductivity for continuous and very thin, island structured layers. In the later case a tunneling mechanism of the conductivity is supposed. Threshold thicknesses for the transition between both types of conductivity (3.7 nm for PET and 4.9 nm for PTFE) are in a good correspondence with the measurement of sheet resistance. For electrically continuous gold layers, the carrier concentration remains constant regardless of the layer thickness. The carrier mobility changes dramatically with increasing layer thickness (Fig. 1B). The mobility first declines, achieves a minimum and then increases slowly for electrically continuous layers. For sufficiently thick, electrically continuous gold layers on both polymers, the mobility is a slowly increasing function of the layer thickness. In this region, the carrier mobility in the gold/PTFE system is significantly lower than that in gold/PET system.

Fig. 1 Dependence of surface free carrier volume concentration (A) and surface free carrier Hall mobility (B) on Au layer thickness sputtered on PET and PTFE measured by the Van der Pauw method [8].

The roughness of the PET and PTFE substrates influences significantly the properties of the sputtered Au layer. In gold/PTFE system the gold continuous layer is formed after longer deposition time thanks to the higher initial roughness of the PTFE substrate. According to AFM measurements, the sputtering of electrically continuous Au layer onto PET increases the surface roughness of the gold/PET system dramatically [8].

The electrical properties of the sputtered gold layers on PET and PTFE have been studied in dependence on the average layer thickness. It has been found that the concentration and mobility of the charge carriers change rapidly for lower thicknesses of the Au layer. This is mainly valid for the electrically discontinuous Au layers. The reason is probably the different mechanism of the charge carrier transport, with quantum effects such as tunnelling mechanism playing a significant role in discontinuous, island structured layers. For the electrically continuous layer, the carrier concentration declines slowly and the carrier mobility increases monotonously. The transition from discontinuous to an electrically continuous layer was proven by the Van der Pauw electrical measurements.

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2.2 Electrokinetical analysis (Zeta potential)

Electrokinetic analyses make possible to characterize surface properties by measuring the streaming potential or streaming current and by investigation of electrokinetic effects at the solid/liquid interface. Zeta potential (ζ-potential) was determined for pristine polymers and polymers treated in Ar plasma discharge for 120 s (see Fig. 2). Zeta potential of the pristine polymers increases in the following order PTFE < PP ≈ PET < PE < PS. Its value is affected by several factors, such as surface morphology, chemical composition and polarity of the polymer surface. It is therefore little bit surprising that the ζ-potential of polar PET and non-polar PP are comparable. Possible explanation was suggested in our previous study [3] in which we observed that in pristine PET most of oxygen containing molecular segments are oriented toward the polymer bulk and the first atomic layer is effectively depleted of oxygen. Due to this phenomenon, the pristine PET, with low surface concentration of oxygen and PP behave in the same manner as can be seen also from similar values of the contact angle (see Fig. 3). Plasma treatment results in a dramatic increase of the ζ-potential, the value of the increase being ordered as follows PTFE < PP < PS < PET < PE [10].

Fig. 2 ζ-potential determined by SurPASS for pristine PP, PET, PS, PE, PTFE (empty columns) and those treated in plasma discharge for 120 s (full columns) [10].

2.3 Contact angle and chemical structure of polymers after plasma treatment (goniometry and XPS)

In Fig. 3, dependence of the contact angle on the plasma exposure time for plasma treated polymers is shown. The measurement was accomplished 5 min after the plasma exposure. Plasma treatment results in a dramatic decline of the contact angle and corresponding increase of the polymer wettability. The wettability increase is caused by creation of polar groups, exhibiting enhanced hydrophilicity, at the polymer surface [6]. After the plasma treatment the highest contact angle is observed on PTFE. Aging of the plasma treated polymers is accompanied by an increase of the contact angle which is due to a reorientation of the molecular segments, produced by the plasma treatment, thanks to their interaction with ambient atmosphere [11]. Major role in this process has an orientation of the polar groups toward the polymer bulk. It is well known that the contact angle is mostly affected by the chemical structure and morphology of the polymer surface layer, especially by the presence of oxidized degradation products [11].

Fig. 3. Dependence of the contact angle on the plasma exposure time for plasma treated polymers (PP, PET, PS, PE, PTFE). The measurement was accomplished 5 min after the exposure. [10].

Presence of oxygen (in the first 8-10 atomic layers) was also proved by XPS method. Results of these measurements are summarized in Fig. 4. After the plasma treatment the highest oxygen concentration was found on PS, lower on PP and PE and lowest one on PTFE. Also in PET, the molecule of which contains oxygen, a low increase of the oxygen



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content is observed. Low oxygen content in PTFE may be due to its high ablation rate, which is well known [11]. High contact angle (see Fig. 3) and low wettability of PTFE is probably due to the low oxygen content.

Fig. 4. Oxygen concentrations measured by XPS on pristine PET (empty column) and PP, PET, PS, PE, PTFE plasma treated for 120 s (full columns). Plasma modified samples were aged in air (RT) for 2 weeks [10].

2.4 Sheet resistance and optical band gap

The dependence of the electrical sheet resistance (Rs) of sputtered gold layers on the sputtering time before and after annealing is shown in Fig. 5A. The sheet resistance decreases rapidly for the sputtering times up to 50 s, when a continuous gold coverage is formed. After annealing a dramatic change in the Rs is observed. The annealed layers are electrically discontinuous up to the sputtering time of 250 s when the continuous coverage is created and a percolation limit is overcome. However, even for longer sputtering times (up to 500 s) the sheet resistance changes slowly and it does not achieve a saturation which is observed on the as sputtered layers [12]. The UV-Vis spectra from the gold layers were interpreted in the frame of well known Tauc’s model [13] and the optical band gap (Eg

opt) was calculated as a function of the sputtering time for sputtered and annealed samples. This dependence is shown in Fig. 5B. Also the thickness of the gold layer, measured by AFM method on the scratch step, as a function of the sputtering time is presented. It is seen from Fig. 5B that the layers sputtered for the times below 150 s exhibit non-zero Eg

opt. Rather dramatic change in the Egopt is observed after annealing. The values of the Eg

opt become much higher in comparison with those of the as sputtered samples. For the samples sputtered for the times up to 300 s a non-zero Eg

opt is observed. For behaviour of ultra-thin metal structures the surface-size and quantum-size effects must be considered [14]. This quantum-size effect in small structures leads e.g. to semi-conducting character, which is accompanied by non-zero Eg (band gap) or Eg

opt. This effect was observed in the present case [12].

Fig. 5A Dependence of the electrical sheet resistance (Rs) of gold structures on the sputtering time before (RT) and after annealing (300°C) (-□- for RT and -■- for 300°C) for 300°C) [12].

Fig. 5B Dependence of the optical band gap on the sputtering time of gold structures before (RT) and after annealing (300°C) (-□- for RT and -■- for 300°C) and thickness (-○-) [12].

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3. Selected microscopic methods of solid surface characterization

3.1 Atomic Force Microscopy (AFM)

The systems combining metals and polymers are attractive for many applications, especially in microelectronics. Mutual interaction between metal and polymers is generally very weak but can be increased if the polymer surface is modified prior to metal deposition. In this way metal adhesion to polymer substrate can be increased substantially. Several modification techniques, physical, chemical or their combination, have been suggested for enhancing metal to polymer adhesion [3]. It was shown [4] that the sole plasma modification of PE and PET does not enhance adhesion of the sputtered Au layers. Scratch tests were performed on the pristine PE and the PE plasma treated and subsequently grafted with –SH + Au-nano-particles. It was observed that the grafting with the nano-particles leads to the dramatic changes of the adhesion of the subsequently deposited Au layer. The surface morphology and roughness of the PE samples was examined by the AFM method in order to disclose relations between the surface morphology and adhesion of the sputtered gold layers. Typical AFM images are shown in Fig.6. Plasma ablation results in appearance of a lamellar structure on the PE surface [2,6]. After the plasma treatment the roughness Ra increases and the surface morphology changes because of an etching process [6]. Surface roughness decreases after additional treatment in dithiol solution. As can be seen from Fig. 6, subsequent grafting with Au nanoparticles leads to an increase of the surface roughness and to morphology changes [4].

Fig. 6 AFM images of pristine PE (PE), PE treated with plasma (PE/plasma), grafted with dithiol (PE/plasma/SH) and grafted with Au nanoparticles (PE/plasma/SH/Au). Ra is surface roughness in nm [15]. Exposure to plasma discharge results in significant changes of HDPE and LDPE surface morphology which can be seen from Fig.7, where AFM images of the plasma treated samples are compared with pristine ones. For the sake of clarity only the images of samples exposed to the plasma discharge for 400 s are shown. It is evident that both LDPE and HDPE pristine samples exhibit similar surface morphology. After 400 s treatment in the plasma, however, the surface morphology of the LDPE and HDPE samples differ significantly. On the surface of the plasma modified HDPE a lamellar structure of the macromolecular chains is visible. In contrast, on the surface of the plasma modified LDPE formations are created which may reflect spherulitic arrangement of the macromolecular chains. Modified LDPE exhibits smoother surface in comparison with HDPE. It can therefore be concluded, in accordance with the data reported earlier [16], that during the plasma treatment PE amorphous regions are degraded preferentially and crystalline ones later. Surface roughness is an increasing function of the scan dimension and the plasma exposure time. [2] Analogous results were reported in [17]. Plasma treatment for 400 s results in a mild increase of the surface roughness for LDPE but in much larger increase for HDPE.

Fig. 7 The AFM images of PE before (HDPE and LDPE) and after 400 s (HDPE/400, LDPE/400) modification in Ar plasma [2]. Gold in the form of thin films is nowadays used in a vast range of applications such as e.g. micro and nano-electromechanical systems (MEMS and NEMS) [18], sensors [19], bioengineering [20] or devices for Surface-enhanced Raman scattering [21]. For a long time we have studied many physical and physico-chemical properties of thin gold



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nano-layers and nano-clusters sputtered or evaporated on different substrates, e.g. glass, silicon and some polymers [4,8,22-24]. The AFM images that illustrate the surface morphology and average roughness (Ra), determined from the AFM measurements of gold coated glass before and after annealing are shown in Fig. 8. For the sake of clarity the images with identical z-scale were chosen. From Fig. 8 it is clear that the gold structures before and after annealing exhibit different surface morphology and roughness. Surface morphology of the as deposited samples does not differ significantly. The annealing changes the surface morphology and roughness dramatically. Similar changes in the surface morphology were also observed on thin gold structures annealed at 200°C for 20 hours [25] and at the temperature above 450°C for 2 hours [26]. After the annealing (see Fig. 8), „spherulitic and hummoc-like“ Au structures are formed as a result of the surface diffusion of Au atoms. It is known, that melting temperature of Au nano-structures dramatically decreases with their dimension [26]. It can be assumed, that the surface diffusion is influenced by e.g. lower wettability of Au on the glass substrate and “low“ thermal stability of the Au nano-structures. In the sputtered gold layers preferential (111) orientation of the Au micro-crystals was determined by XRD method [22,26]. It is known, that the gold crystals with this orientation are not thermodynamically stable and their melting and cracking starts from the edge parts that should be bounded by Au (110) surface [26].

Fig. 8 AFM scans of gold structures sputtered for 75 and 400 s on glass substrate before (RT) and after annealing (300°C). Ra is average surface roughness (in nm) [12].

Surface „writing“ was performed by scanning of PMMA surface with a linearly polarized laser beam (405 nm wavelength and 0.25 mW laser power) from confocal microscope Olympus Lext. The laser beam was focused to a spot with a diameter of about 0.5 µm. Fig. 9A shows a schematic of the experimental setup. The PMMA film was exposed to periodical laser scanning under ambient conditions. Scanning was performed line by line. Mechanical movement was applied in the direction parallel to the scanned lines. The RSG (Regular Surface Grating) formation was monitored by confocal microscope from evaluation of the reflected laser light or in usual optical regime. Surface morphology of prepared samples was examined by atomic force microscopy (AFM), too. AFM confirms that the simple sinusoidal pattern was obtained. Fig. 9B shows the modified surface patterned with different velocity of mechanical movement.

Fig. 9A Schematic of the experimental arrangement, PMMA was deposited by spin coating onto Si wafer, PMMA surface layer was doped with porphyrin by deep coating. Then the surface was scanned by focused laser beam [27]. Fig. 9B The AFM images showing the surface morphology of the modified PMMA surface taken over 10 x 10 µm area. Numbers give the velocity of the mechanical movement in µm/sec [27].

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The interaction with the scanned laser beam results in a “redistribution” of PMMA mass. The resulting profile consists of periodical array of lines where the PMMA mass was pulled or pushed above the initial surface. In this way periodical grating was produced. Important fact is that the grating constant can be varied by changing the movement velocity. At the same time the grating amplitude seems to decrease with decreasing velocity of movement [27].

3.2 Scanning Electron Microscopy (SEM)

Irradiation with laser light affects physical and biological properties of polymers [28]. Modified polymer surface may facilitate adhesion, proliferation, morphology, viability or migration of cells seeded at such modified samples [29, 30]. Linearly polarized light from pulsed KrF and F2 lasers, working at 248 nm and 157 nm wavelengths respectively, was used for irradiation of PET surface, which subsequently was coated with a sputtered gold film. For a certain choice irradiation parameters [30], the irradiation resulted in the formation of coherent ripples patterns with a lateral periodicity in the order of the wavelength of the laser light and with a corrugation height of several 10 nm. The modified and sputtered samples were cut by FIB (focused ion beam) technique. The cuts were then investigated with the scanning electron microscope (SEM) (Fig. 10). The gold sputtered onto the laser-induced ripples is deposited in the form of “nano-wires”, which grow on the ridges of the ripples Images of the FIB cuts indicate that there exist gaps between the individual wires and that the metal layer is not continuous. In addition a granularity along the wires is visible on the FIB cuts with the grains being interconnected. The morphology of the gold layer deposited on the ripples produced by the F2 laser is distinctly different. The gold is also deposited in the valleys of the ripple structure (Fig. 10B). The gold layer partially eradicates the initial ripple profile. On the continuous gold layers cracks are observed which, however, are not directly connected with the ripple pattern.

Fig. 10 SEM images of FIB cuts of lasers irradiated and Au sputtered PET: (A) PET irradiated by KrF laser (6.6 mJ/cm2) and sputtered with 200 nm of Au, (B) PET irradiated by F2 laser (4.4 mJ/cm2) and sputtered with 50 nm of Au [9].

For various applications of PE it is desirable to modify its surface properties. The irradiation (e.g. with laser light, ion or electron beams, UV light, X- and γ-rays) or treatment in the plasma discharge lead to degradation of polymeric chains, chemical bond cleavage, creation of free radicals and release of gaseous degradation products. Subsequent chemical reactions of transient, highly reactive species result in creation of multiply double bonds, production of low mass stable degradation products, large cross-linked structures and eventually oxidized structures [31]. The irradiation of non-polar polyolefins (PE, PP, PS and fluoropolymers) leads to creation of polar groups on the polymer surface and in this way to an enhancement of printability, wettability, adhesion with inorganic materials (e.g. metals) or with biologically active components [32].

Fig. 11 The SEM images of PE foils before (HDPE and LDPE) and after 400 s (HDPE/400, LDPE/400) treatment in Ar plasma with power 1.7 W [2]. With SEM method it is possible to study either pristine or modified polymers without any metal coverage. Exposure to plasma discharge results in significant changes of HDPE and LDPE surface morphology which are illustrated by SEM images shown in Fig. 11. For the sake of clarity only the images of samples exposed to plasma discharge for 400 s are shown. On the surface of the plasma modified HDPE a lamellar structure of macromolecular chains is visible which is similar to that shown in Fig. 7. In contrast, on the surface of plasma modified LDPE formations are created which may reflect spherulitic arrangement of macromolecular chains. Since SEM results indicate different surface roughness



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of plasma modified LDPE and HDPE, AFM examination of the same samples was accomplished too. The AFM results for selected samples were shown in Fig. 7. Scratch tests were performed on pristine PE, plasma treated and -SH+Au grafted PE (Fig. 12). Elastic and plastic deformations are observed along the scratches. Dramatic changes in the form and character of the scratch appear on the PE sample grafted with Au nano-particles and then covered with Au film (Fig. 12). In this case the mechanical damage of the surface is more pronounced than in the case of non-grafted surface. It was shown that the plasma treatment does not positively affect adhesion between Au and PE [4]. It can therefore be concluded that the adhesion of the gold film is higher on the PE surface grafted with the gold nano-particles. Positive effect of the grafted gold nanoparticles on adhesion of subsequently deposited Au layer was demonstrated in this way.

Fig. 12 SEM images of the scratch path produced in Au layer (50 nm) sputtered on pristine PE (PE/Au) and the plasma treated PE grafted with dithiol and Au nano-particles (PE/plasma/SH/Au) [15].

Thin carbon layers are considered as a prospective material for a wide range of biomedical application [33,34], e.g. tissue regeneration [35], controlled drug delivery, surface coating for bone-related implants, increase of resistance to microbial adherence [36], blood interfacing implants applications or neuronal growth [37]. Carbon monolayer (graphene) has a great potential in development of FET transistors [38] We deposited carbon layers by flash evaporation onto PET and PTFE substrates. A scratch test on carbon coated polymers was performed using NanoIntender device. The results obtained for C2/PET sample (carbon evaporated from the distance of 2 cm) are shown in Fig. 13. The test shows a semicrystalline structure of the carbon layer. Dependence of the surface hardness and elastic modulus of on the tip displacement into the surface proved that the hardness and elastic modulus decline with increasing depth, the decline being much slower at the depths of about 75–80 nm. This depth coincides well with the thickness of the carbon layer [39]. It may therefore be concluded that the carbon layer has higher hardness and elastic modulus than underlying, pristine PET.

Fig. 13 Optical (A) and SEM (B) images of the scratch path produced in carbon layer, deposited from 2 cm distance on PET. During movement the tip gradually was loaded from 0 to 20 mN [39].

Gold nano-layers were deposited by sputtering on PTFE and PET films. The FIB method (focused ion beam) was chosen as an alternative method for the determination of the layer thickness. The structure of the FIB cross-sections taken from a gold/PET sample is seen from the SEM image shown in Figs. 14 A and B). Two samples were examined with the gold layers deposited for 500 and 30 s, corresponding to layer thicknesses of 68 and 8 nm, respectively.

Fig. 14 SEM scan of Au layer deposited on PET with deposition time 500 s (A) and 30 s (B). The cut was done with the FIB (focused ion beam) method [8].

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The thickness determined from the SEM image for the 500 s sputtering time is in excellent agreement with that determined by the AAS method (atomic absorption spectroscopy) [8]. For the deposition time of 30 s, however, the SEM thickness (8 nm) is twice of that determined indirectly from the AAS data (about 4 nm). The difference is obviously due to the fact that the layer deposited for 30 s is not continuous and homogeneous (see Fig. 14B), and by the AAS method only a thickness averaged over large surface area is determined.

3.3 Transmission Electron Microscopy (TEM)

Gold, silver, palladium and platinum layers were sputtered on PET films [40]. Transmission electron microscopy (TEM) shows different thicknesses of the continuous metal layers sputtered under same deposition conditions (Fig. 15). Specimens for TEM were cut from the centre of the PET foils with metal layers and fixed in epoxy resin (Durcupan). Ultrathin cross-sections (ca 60 nm) were prepared with ultramicrotome Ultracut UCT (Leica) at room temperature. TEM images of continuous metal layers, sputtered for 300 s, revealed that the Pd layer exhibits best homogeneity without any anomalies or internal structure [40]. In Au and Pt layers a worm-like, heterogeneous structures are observed. For Au layer the heterogeneities are even larger but less frequent along the layer. In Ag layer large spherical clusters are observed. The thickness of the layers was determined from undamaged part of the cut to avoid an error due to possible mechanical deformation taking place in the cut preparation. It was observed that Au and Pt form the layers of similar thickness on PET and Si. The thickness of Ag layer is lower and Pd creates the thinnest layers on both substrates for the same deposition conditions.

Fig. 15 TEM scans of Au, Pt, Pd and Ag structures sputtered for 300 s on PET substrate [40].

3.4 Optical Microscopy

Material application

Besides of interesting catalytic and electronic properties, nano-particles of noble metals exhibit also distinctive, shape-dependent optical properties which are of great technological interest [26]. Thin gold layers were prepared by sputtering onto glass substrate. In a next step the samples were annealed at the temperature of 3000C. Optical images of the as sputtered and sputtered/annealed samples prepared for different sputtering times are shown in Fig. 16. With increasing sputtering time the samples become darker, due to increasing thickness of the gold layer. Also a gradual change of the sample color from blue to green is seen. After annealing all structures exhibit red colour, regardless of the sputtering time or layer thickness. The change in the layer color indicates pronounced change of the layer structure caused by the annealing. It is in accord with previously published data and a known fact that small gold nano-particles (about 10 nm in diameter) absorb green light and thus appear red [14].

Fig. 16 Images of the glass samples with gold layers sputtered for increasing times (in s). The as sputtered (RT) and annealed samples (300°C) are compared [12].

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Biological application

Polyethylene (PE) was exposed to inert Ar plasma, and then the chemically active PE surface was grafted with Au nano-particles. The modified PE samples were seeded with rat vascular smooth muscle cells (VSMC) or mouse NIH 3T3 fibroblasts, and their adhesion and proliferation were studied. AFM examination of the sample surface shows that the plasma discharge and the gold grafting lead to dramatic changes in the surface morphology and roughness. The Au nano-particles were found not only on the sample surface, but also in the sample interior up to the depth of about 100 nm. In addition, plasma modification of the PE surface, followed with grafting with Au nano-particles, significantly increases the attractiveness of the PE surface for VSMC growth and adhesion, and also for mouse embryonic 3T3 fibroblasts [5]. The higher proliferation activity of VSMC on the modified PE samples can be explained by the oxidation of the polymer surface after the plasma treatment, resulting in the increase in the surface wettability (at least for samples plasma treated for 50 s). The increase is pronounced especially on the samples grafted with Au nano-particles. All these samples show increased wettability regardless of the exposure time to plasma discharge. It is believed that hydrophilic surfaces adsorb the cell adhesion-mediating molecules (such as vitronectin or fibronectin present in the serum supplement of cell culture media) in a more physiological conformation, which supports the accessibility of specific amino acid sequences (e.g. RGD) in these molecules to cell adhesion receptors (e.g. integrins) [41,42]. Receptor mediated adhesion and appropriate cell spreading on the adhesion substrate are prerequisites for proliferation capability in all types of anchorage-dependent cells, including VSMC (for a review, see [43]). Other factors influencing the growth activity of the cells is the surface roughness and morphology of the substrate. Both plasma treatment and gold grafting create nano-scale irregularities on the polymer surface. The surface nano-roughness acts synergetically with the surface wettability, i.e. by improving the geometrical conformation of adsorbed ECM molecules, and thus a better exposure of active sites on these molecules to the cell adhesion receptors [44]. Also the shape of the irregularities on the material surface is important for its colonization with cells [43]. After grafting with the Au nano-particles, the surface irregularities on plasma-treated PE lost their conical shape and sharpness and become rounded and filiform, which support better spreading of cells (Fig. 17) and their subsequent proliferation activity. As suggested above, not only the number of cells but also their spreading, shape and the homogeneity of their distribution on the material surface are important indicators of the cell growth activity. Although the numbers of VSMC on day 1 after seeding on pristine and modified PE were similar, these cells markedly differed in their morphology. As shown in

Fig. 17 The photographs of VSMC adhered (1st day) on pristine PE (PE), PE treated for 150 seconds with plasma (PE/plasma) and subsequently grafted with Au nano-particles (PE/plasma/Au) [5].

Fig. 17, the cells on the pristine PE were mostly rounded, while the cells on plasma treated and especially on Au-grafted samples were spread by a significantly larger cell-material contact area, which most probably stimulated their subsequent proliferation activity. In addition, these cells were more homogeneously distributed on the material surface. On 5th day after seeding, the entire surface of the plasma-irradiated and Au-grafted PE is homogeneously covered with a confluent and dense VSMC layer, whereas the cells on the pristine PE formed islands irregularly distributed on the material surface [5].

3.5 Laser-Confocal Microscopy

The differences in the surface morphology between pristine and plasma modified HDPE were studied with laser confocal microscope (Fig.18A and B). This device works with the maximum magnification 2400x in optical mode. In laser confocal mode, where the laser light (λ = 405 nm) is used, it is possible to achieve magnification up to 14400x.



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Plasma modification leads to the creation of low molecular fragments on the HDPE surface and the ablation of the polymer surface layers [6]. As a result the polymer chains become clearly visible on the polymer surface (Fig. 18B).

Fig. 18 The photographs from scanning laser confocal microscope Olympus Lext of pristine high-density polyethylene (HDPE) (A) and HDPE modified with argon plasma (modification power 8W and exposure time 480s) (B). Dimension of the picture is 100x100 µm.

4. Conclusion

An overview of experiments performed by authors during recent years on synthetic polymers and polymers modified by the plasma discharge or laser light was presented. Attention was also given to the results obtained on the thin metallic layers and metal nano-particles deposited onto different non-conductive substrates (polymers, glasses). In accord with several previous studies it was found that the exposure of polymers to the plasma discharge or to the laser light leads to deep structural and compositional changes on the polymer surface. Significant changes in the polymer surface morphology and roughness are also observed. As a result the modified polymers exhibit increased wettability, electrical conductivity and bio-compatibility which may increase their application potential in different technologies. Other techniques of the polymer modification, namely grafting with metal nano-particles, coating with thin metal layers are also discussed and illustrated on several examples. The effect of the plasma treatment and the metal deposition on the polymer bio-compatibility is demonstrated too. Very interesting, original results were obtained on patterning of the polymer surface by laser beam. It was shown that regular, periodic structures with controllable parameters can be produced in this way which may find application e.g. in opto-electronics. By gold deposition onto the patterned surface metal nano-wires could be prepared too. The experiments and the results described in this overview should demonstrate how simultaneous use of different diagnostic techniques including various kinds of electron and optical microscopes and AFM is necessary to obtain complete information on the properties of materials and structures and underlying physico-chemical processes as well. The authors believe that the results summarized in this overview may be of interest not only for specialists in the polymer science but also for researches working in related fields, such as micro-electronics, opto-electronics, and bio-medicine.

Acknowledgements The work was supported by the Grant Agency of the Czech Republic under the projects 106/09/0125, 106/09/P046 and 108/10/1106 and Ministry of Education of the CR under Research program LC 06041 and GAAS CR under the projects KAN400480701 and KAN200100801.

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