SCANNING VOL. 9999, 1–11 (2014) © Wiley Periodicals, Inc.
The Effects of UV Irradiation on Collagen D-band Revealed by Atomic Force Microscopy STYLIANOS V. KONTOMARIS,1,2 DIDO YOVA,1 ANDREAS STYLIANOU,1 AND GIORGOS BALOGIANNIS1 1
Biomedical Optics & Applied Biophysics Lab, Division of Electromagnetics, Electrooptics & Electronic Materials, School of Electrical and Computer Engineering, National Technical University of Athens, Iroon Polytechniou, Athens, Greece 2 Interuniversity Postgraduate Programme on Biomedical Engineering, Faculty of Medicine of the University of Patras, School of Electrical and Computer Engineering and the School of Mechanical Engineering of the National Technical University of Athens, Athens, Greece
Summary: The objective of this paper was to investigate the influence of UV irradiation on collagen Dband periodicity by using the AFM imaging and nanoindentation methods. It is well known than UV irradiation is one of the main factors inducing destabilization of collagen molecules. Due to the human’s skin chronic exposure to sun light, the research concerning the influence of UV radiation on collagen is of great interest. The impact of UV irradiation on collagen can be studied in nanoscale using Atomic Force Microscopy (AFM). AFM is a powerful tool as far as surface characterization is concerned, due to its ability to relate high resolution imaging with mechanical properties. Hence, high resolution images of individual collagen fibrils and load-displacement curves on the overlapping and gap regions, under various time intervals of UV exposure, were obtained. The results demonstrated that the UV rays affect the height level differences between the overlapping and gap regions. Under various time intervals of UV exposure, the height difference between overlaps and gaps reduced from 3.7 nm to 0.8 nm and the fibril diameters showed an average of 8–10% reduction. In addition, the irradiation influenced the mechanical properties of collagen fibrils. The Young’s modulus values were reduced per 66% (overlaps) and 61% (gaps) compared to their initial values. The observed alterations on the structural and
Address for reprints: Stylianos V. Kontomaris, National Technical University of Athens, School of Electrical and Computer Engineering, Division of Electromagnetics, Electrooptics and Electronic Materials, 9, Iroon Polytechniou, Athens 15780, Greece. E-mail: [email protected] Received 30 April 2014; revised 3 November 2014; Accepted with revision 14 November 2014 DOI: 10.1002/sca.21185 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).
the mechanical properties of collagen fibrils are probably a consequence of the polypeptide chain scission due to the impact of the UV irradiation. SCANNING 9999:XX–XX, 2014. © 2014 Wiley Periodicals, Inc. Key words: AFM, collagen, UV irradiation, nanoindentation, Young’s modulus
Introduction Most parts of the mammalian body, for example tissue, skin, bone, cartilage, or tendons contain collagen in the form of collagen fibrils and especially collagen type I fibrils which provide structural support to the tissues. A characteristic feature of the collagen type I fibrils is their banded structure (Wenger et al., 2007; Minary-Jolandan and Yu, 2009). The diameter of the fibrils changes along the axial direction (the collagen fibril consists of an alternating gap and overlapping regions), with a highly reproducible D-band periodicity of approximately 67 nm (Minary-Jolandan and Yu, 2009). It has been reported that D-band plays a significant role in the mechanical strength of collagen fibrils (Grant et al., 2012; Stylianou and Yova, 2013). Collagen fibrils, depending on the tissue, are aligned laterally to form bundles and fibers, which among other properties offer to the tissue mechanical strength and stability (Kadler et al., 2007; Fratzl, 2008; Stylianou et al., 2012). Thus, the research on collagen under various circumstances is of great importance. A critical issue regarding the collagen research is the influence of UV rays on structural and mechanical properties of collagen fibrils. UV irradiation is one of the most harmful external factors, which results in collagen’s basic properties and leads to photo-degradation (Jariashvili et al., 2012). Photo-degradation of collagen
2
SCANNING VOL. 9999, 9999 (2014)
is responsible for skin wrinkles and probably results in skin cancer (Jariashvili et al., 2012). As a result, UV irradiation is responsible for the acceleration of collagen damage in normal aging (Jariashvili et al., 2012). Moreover, many skin changes like easy bruising which were believed to be a result of aging, are in fact related to prolonged UV exposure (Sionkowska, 2005). In addition, the UV-collagen interaction affects the cell response, since it has been reported that cells that were cultured on UV–irradiated collagen-based materials are influenced by UV irradiation parameters (Rajan et al., 2008; Achilli et al., 2010). Hence, due to the chronic exposure of human skin to sun light the clarification of the UV-collagen interaction mechanisms is crucial. Although the UVcollagen interaction mechanisms have been previously studied, (Anastase-Ravion et al., 2002; Sionkowska and Wess 2004; Kang et al., 2009; Janko et al., 2010; Jariashvili et al., 2012) the influence of UV irradiation on D-band periodicity has not been yet investigated. Hence, the investigation of the influence of UV rays on collagen’s basic nanoscale characteristics, like the Dperiodicity, is of great importance in the fields of medicine, biology and tissue engineering. The investigation of collagen features in nanoscale, under several factors, can be studied using Atomic Force Microscopy (AFM) (Stylianou et al., 2013b). AFM can be potentially used in a great number of investigation areas, related to tissues which contain collagen. It has been reported that AFM is a valuable tool in histology and cytology (Ushiki et al., 1996). Moreover, AFM has been used in the investigation of collagen fibrils structural variations in nanoscale which can be related to pathological situations (e.g diabetes) (Layton et al., 2004). In addition, AFM is increasingly being used in the investigation of the influence of several radiations (arising from nature or from medical activities) in tissues which contain collagen in great amounts. Specifically, Choi et al. investigated the quantitative effects of in vivo medical treatments using radiofrequences in collagen fibrils from rabbit skin (Choi et al., 2012a). Hence, the influence of UV irradiation, on collagen, can be studied in nanoscale using AFM providing sub-molecular imaging and qualitative-quantitative information (Stolz et al., 2004; Minary-Jolandan and Yu, 2009; Kurland et al., 2012; Pramanick et al., 2012). It provides the ability to combine high resolution images with mechanical properties (Stolz et al., 2004; Minary-Jolandan and Yu, 2009; Kurland et al., 2012). Specifically, using AFM nanoindentation method, which is based on recording the elastic response of a material using an indenter (the AFM tip) (Oliver and Pharr, 2004), the Young’s modulus values of the sample of interest in different nanoregions can be determined. The research on the topography alterations and the mechanical properties of the D-band periodicity of single collagen fibrils can be achieved by using AFM methods in collagen thin films (Stylianou et al., 2012; Stylianou
et al., 2013a,b). AFM is a suitable tool for detecting local structural and mechanical variations in nanoregions of a single collagen fibril under several impacts (MinaryJolandan and Yu 2009; Kurland et al., 2012). The ability to perform the indentation procedure in real time and measure localized mechanical properties on the material’s surface has emerged nanoindentation method as one of the most common techniques for the characterization of the mechanical properties of biomaterials including proteins, cells, viruses and bacteria (Sirghi, 2010; Grant et al., 2012; Kurland et al., 2012; Mateu, 2012). Furthermore, collagen thin films can be used to analyze the influence of different parameters and correlating them with other properties like optical ones (Stylianou et al., 2009; Stylianou et al., 2011). The purpose of this paper was to investigate the influence of UV irradiation on collagen D-band periodicity by using the AFM imaging and nanoindentation methods. Alterations on the height level differences between overlapping-gap regions and on the fibrils diameters were investigated, while the percentage change of the Young’s modulus under the influence of various UV irradiation time intervals was recorded. The results indicated that the Young’s modulus values change slightly in the axial direction of collagen fibrils due to the D-band periodicity. In addition, the Young’s modulus values, the fibrils diameters and the height level differences between gap and overlapping regions alter as a result of the UV irradiation.
Materials and Methods Collagen Thin Film Formation
Type I collagen from bovine Achilles tendon (Fluka 27662, by Sigma–Aldrich, Stainheim, Switzerland) was dissolved in acetic acid (CH3COOH 0.5 M) in a final concentration of 8 mg/ml and stored in 4˚C for 24 h. The solution was then homogenized at 24000 rpm (Homogenizer IKA T18 Basic, Staufen, Germany) and stored in 4˚C as the stock solution. For the collagen thin films formation, part of the collagen stock solution (50 ml) was flushed on the substrate, prepared from cleaved mica discs (V1, 9.5 diam., 71856–01 Electron Microscopy Science, Hatfield, PA) and spin coated for 40 s at 6000 rpm (spin coater WS-400B-6NPP/LITE Laurell Technologies North Wales, PA). After each experiment, the collagen films were kept in a desiccator (Nalgene desiccator, Sigma Aldrich).
Samples Ultraviolet Irradiation
Collagen films were irradiated under air using a GL4 germicidal lamp with a maximum at 254 nm (Sankyo Denki Co. Ltd., Japan) 3 cm from the light source for
Kontomaris et al.: The Effects of UV Irradiation on Collagen D-band
various time intervals. The intensity of radiation for this distance was 1813 mW/cm2 i.e. 0.11 J/(cm2.min) and the dose of incident radiation during the 1 h exposure was 6.6 J/cm2. The intensity of the incident light was measured using a GoldiluxTM radiometer-photometer (Model 70234-meter and 70239-probe, Oriel Instruments, Stanford, CA). All measurements were performed in the same conditions of temperature to avoid any influence on the physicochemical and mechanical properties of collagen.
AFM
AFM experiments were carried out using a commercial microscope (CP II, Veeco Bruker, Santa Barbara, CA) in contact mode. For both imaging and indentation tests, pyramidal tips were employed. These tips have a nominal tip radius of approximately () 20 nm on V– shaped cantilevers and a nominal spring constant of 0.5 N/m (type MLCT; Veeco). A cantilever with a low spring constant value was selected due to the fact that the AFM imaging procedure was carried out in contact mode. High spring constant values during the contact mode imaging were avoided since the tip might “move” collagen fibrils or scratch them. The samples were mounted on AFM metal discs with epoxy glue, while locator grids (Copper finder grid, G2761C, Agar Scientific, Essex, United Kinkdom) were used to map the surface (see also paragraph D. “Identification of the same fibril prior and post the irradiation”). All experiments were performed in air, at room temperature (20 2˚C) and relative humidity 40 3%. The image processing was carried out by using the image analysis software that accompanied the AFM system DI SPMLab NT ver.60.2 and IP-Image Processing and Data Analysis ver.2.1.15 (Veeco). In addition, the image analysis software WSxM 5.0 Develop 6.5 (Horcas et al., 2007) was used. All the experiments were carried out on the same collagen fibril each time in order to extract results, independent of possible differences between collagen fibrils.
Identification of the Same Fibril Prior and Post the Irradiation
The samples were mounted on AFM metal discs with epoxy glue, while locator grids (Copper finder grid, G2761C, Agar Scientific) were used to map the surface. (Markiewicz and Goh, 1997; Stylianou et al., 2013b). The locator grids can provide information about general areas on the sample. The main idea was to image every time (i.e. prior and post the irradiation) a specific area which is marked with the locator grid. Firstly, a large image (100 mm–100 mm) was obtained. Subsequently, images
3
with smaller dimensions were acquired. Using the above method one specific fibril or even though only a part of one fibril can be isolated (e.g. an overlapping region). After the irradiation, the same area using the locator grid can be approximately found and images with large dimensions (100 mm–100 mm) which are partly the same with the previous large image can be acquired. Subsequently, images with progressively smaller dimensions are being obtained. Finally, the same single fibril or even a part of a fibril which is exactly the same with the previous one (i.e. prior the irradiation) can be isolated. This procedure can be repeated after each irradiation. Hence, areas with dimensions that can be compared to the tip radius can be found again in order to examine the exact same area after changing controlled parameters.
Calculations
In practice, the indentation procedure is performed by moving the sample up and down toward the tip and measuring the corresponding deflection of the cantilever in order to create a load-displacement curve (Oliver and Pharr, 2004). The load-displacement curve consists of two parts, the loading curve in which the movement of the sample is towards the tip and the unloading curve in which the movement of the sample is the opposite, i.e. away from the tip. The indentation depth in a soft sample’s surface can be easily calculated under the condition that a hard sample is used as a reference. The difference in the sample’s surface movement for the soft and the hard sample in order to achieve the same deflection of the cantilever is the indentation depth (Oliver and Pharr, 2004). Load-indentation curve, can be used for the determination of the material contact stiffness (Oliver and Pharr, 2004; Wenger et al., 2007; Fischer-Cripps, 2009). According to the Oliver-Pharr method the contact stiffness can be calculated from the unloading part of the load-indentation curve (Oliver and Pharr, 2004). It has been reported that the initial data of the unloading curve is almost linear for some materials, and as a result a linear fit is the most obvious solution regarding the upper portion of the curve (FischerCripps, 2006; Choi et al., 2012b). The stiffness value leads to the estimation of the Young’s modulus, under the condition that the shape of the tip and the Poisson’s ratio of the sample are known. The formula that contains both the stiffness and the Young’s modulus is: pffiffiffiffi p S ð1 v2 Þ pffiffiffi E¼ 2 A where, E is the Young’s modulus, n the Poisson’s ratio, S the contact stiffness, and A an area function related to the effective cross-sectional or projecting area of the
SCANNING VOL. 9999, 9999 (2014)
4
indenter (Oliver and Pharr, 2004; Fischer-Cripps, 2009; Sirghi, 2010; Stylianou et al., 2014). Regarding small indentation depths, pyramidal indenters can be analyzed in terms of a paraboloid of revolution which can be approximated by a sphere (Oliver and Pharr, 2004). Thus, the projected area in this case can be derived by the following equation:
A ¼ phc ð2r hc Þ
Statistics
The Young’s modulus values were expressed as the mean standard deviation. An one-way ANOVA was performed to compare differences between fibrils diameters and Young’s modulus values under the impact of UV irradiation. Moreover, post hoc comparisons were carried out using a Student-Newman-Keuls test. The p-values
The Effects of UV Irradiation on Collagen D-band Revealed by Atomic Force Microscopy STYLIANOS V. KONTOMARIS,1,2 DIDO YOVA,1 ANDREAS STYLIANOU,1 AND GIORGOS BALOGIANNIS1 1
Biomedical Optics & Applied Biophysics Lab, Division of Electromagnetics, Electrooptics & Electronic Materials, School of Electrical and Computer Engineering, National Technical University of Athens, Iroon Polytechniou, Athens, Greece 2 Interuniversity Postgraduate Programme on Biomedical Engineering, Faculty of Medicine of the University of Patras, School of Electrical and Computer Engineering and the School of Mechanical Engineering of the National Technical University of Athens, Athens, Greece
Summary: The objective of this paper was to investigate the influence of UV irradiation on collagen Dband periodicity by using the AFM imaging and nanoindentation methods. It is well known than UV irradiation is one of the main factors inducing destabilization of collagen molecules. Due to the human’s skin chronic exposure to sun light, the research concerning the influence of UV radiation on collagen is of great interest. The impact of UV irradiation on collagen can be studied in nanoscale using Atomic Force Microscopy (AFM). AFM is a powerful tool as far as surface characterization is concerned, due to its ability to relate high resolution imaging with mechanical properties. Hence, high resolution images of individual collagen fibrils and load-displacement curves on the overlapping and gap regions, under various time intervals of UV exposure, were obtained. The results demonstrated that the UV rays affect the height level differences between the overlapping and gap regions. Under various time intervals of UV exposure, the height difference between overlaps and gaps reduced from 3.7 nm to 0.8 nm and the fibril diameters showed an average of 8–10% reduction. In addition, the irradiation influenced the mechanical properties of collagen fibrils. The Young’s modulus values were reduced per 66% (overlaps) and 61% (gaps) compared to their initial values. The observed alterations on the structural and
Address for reprints: Stylianos V. Kontomaris, National Technical University of Athens, School of Electrical and Computer Engineering, Division of Electromagnetics, Electrooptics and Electronic Materials, 9, Iroon Polytechniou, Athens 15780, Greece. E-mail: [email protected] Received 30 April 2014; revised 3 November 2014; Accepted with revision 14 November 2014 DOI: 10.1002/sca.21185 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).
the mechanical properties of collagen fibrils are probably a consequence of the polypeptide chain scission due to the impact of the UV irradiation. SCANNING 9999:XX–XX, 2014. © 2014 Wiley Periodicals, Inc. Key words: AFM, collagen, UV irradiation, nanoindentation, Young’s modulus
Introduction Most parts of the mammalian body, for example tissue, skin, bone, cartilage, or tendons contain collagen in the form of collagen fibrils and especially collagen type I fibrils which provide structural support to the tissues. A characteristic feature of the collagen type I fibrils is their banded structure (Wenger et al., 2007; Minary-Jolandan and Yu, 2009). The diameter of the fibrils changes along the axial direction (the collagen fibril consists of an alternating gap and overlapping regions), with a highly reproducible D-band periodicity of approximately 67 nm (Minary-Jolandan and Yu, 2009). It has been reported that D-band plays a significant role in the mechanical strength of collagen fibrils (Grant et al., 2012; Stylianou and Yova, 2013). Collagen fibrils, depending on the tissue, are aligned laterally to form bundles and fibers, which among other properties offer to the tissue mechanical strength and stability (Kadler et al., 2007; Fratzl, 2008; Stylianou et al., 2012). Thus, the research on collagen under various circumstances is of great importance. A critical issue regarding the collagen research is the influence of UV rays on structural and mechanical properties of collagen fibrils. UV irradiation is one of the most harmful external factors, which results in collagen’s basic properties and leads to photo-degradation (Jariashvili et al., 2012). Photo-degradation of collagen
2
SCANNING VOL. 9999, 9999 (2014)
is responsible for skin wrinkles and probably results in skin cancer (Jariashvili et al., 2012). As a result, UV irradiation is responsible for the acceleration of collagen damage in normal aging (Jariashvili et al., 2012). Moreover, many skin changes like easy bruising which were believed to be a result of aging, are in fact related to prolonged UV exposure (Sionkowska, 2005). In addition, the UV-collagen interaction affects the cell response, since it has been reported that cells that were cultured on UV–irradiated collagen-based materials are influenced by UV irradiation parameters (Rajan et al., 2008; Achilli et al., 2010). Hence, due to the chronic exposure of human skin to sun light the clarification of the UV-collagen interaction mechanisms is crucial. Although the UVcollagen interaction mechanisms have been previously studied, (Anastase-Ravion et al., 2002; Sionkowska and Wess 2004; Kang et al., 2009; Janko et al., 2010; Jariashvili et al., 2012) the influence of UV irradiation on D-band periodicity has not been yet investigated. Hence, the investigation of the influence of UV rays on collagen’s basic nanoscale characteristics, like the Dperiodicity, is of great importance in the fields of medicine, biology and tissue engineering. The investigation of collagen features in nanoscale, under several factors, can be studied using Atomic Force Microscopy (AFM) (Stylianou et al., 2013b). AFM can be potentially used in a great number of investigation areas, related to tissues which contain collagen. It has been reported that AFM is a valuable tool in histology and cytology (Ushiki et al., 1996). Moreover, AFM has been used in the investigation of collagen fibrils structural variations in nanoscale which can be related to pathological situations (e.g diabetes) (Layton et al., 2004). In addition, AFM is increasingly being used in the investigation of the influence of several radiations (arising from nature or from medical activities) in tissues which contain collagen in great amounts. Specifically, Choi et al. investigated the quantitative effects of in vivo medical treatments using radiofrequences in collagen fibrils from rabbit skin (Choi et al., 2012a). Hence, the influence of UV irradiation, on collagen, can be studied in nanoscale using AFM providing sub-molecular imaging and qualitative-quantitative information (Stolz et al., 2004; Minary-Jolandan and Yu, 2009; Kurland et al., 2012; Pramanick et al., 2012). It provides the ability to combine high resolution images with mechanical properties (Stolz et al., 2004; Minary-Jolandan and Yu, 2009; Kurland et al., 2012). Specifically, using AFM nanoindentation method, which is based on recording the elastic response of a material using an indenter (the AFM tip) (Oliver and Pharr, 2004), the Young’s modulus values of the sample of interest in different nanoregions can be determined. The research on the topography alterations and the mechanical properties of the D-band periodicity of single collagen fibrils can be achieved by using AFM methods in collagen thin films (Stylianou et al., 2012; Stylianou
et al., 2013a,b). AFM is a suitable tool for detecting local structural and mechanical variations in nanoregions of a single collagen fibril under several impacts (MinaryJolandan and Yu 2009; Kurland et al., 2012). The ability to perform the indentation procedure in real time and measure localized mechanical properties on the material’s surface has emerged nanoindentation method as one of the most common techniques for the characterization of the mechanical properties of biomaterials including proteins, cells, viruses and bacteria (Sirghi, 2010; Grant et al., 2012; Kurland et al., 2012; Mateu, 2012). Furthermore, collagen thin films can be used to analyze the influence of different parameters and correlating them with other properties like optical ones (Stylianou et al., 2009; Stylianou et al., 2011). The purpose of this paper was to investigate the influence of UV irradiation on collagen D-band periodicity by using the AFM imaging and nanoindentation methods. Alterations on the height level differences between overlapping-gap regions and on the fibrils diameters were investigated, while the percentage change of the Young’s modulus under the influence of various UV irradiation time intervals was recorded. The results indicated that the Young’s modulus values change slightly in the axial direction of collagen fibrils due to the D-band periodicity. In addition, the Young’s modulus values, the fibrils diameters and the height level differences between gap and overlapping regions alter as a result of the UV irradiation.
Materials and Methods Collagen Thin Film Formation
Type I collagen from bovine Achilles tendon (Fluka 27662, by Sigma–Aldrich, Stainheim, Switzerland) was dissolved in acetic acid (CH3COOH 0.5 M) in a final concentration of 8 mg/ml and stored in 4˚C for 24 h. The solution was then homogenized at 24000 rpm (Homogenizer IKA T18 Basic, Staufen, Germany) and stored in 4˚C as the stock solution. For the collagen thin films formation, part of the collagen stock solution (50 ml) was flushed on the substrate, prepared from cleaved mica discs (V1, 9.5 diam., 71856–01 Electron Microscopy Science, Hatfield, PA) and spin coated for 40 s at 6000 rpm (spin coater WS-400B-6NPP/LITE Laurell Technologies North Wales, PA). After each experiment, the collagen films were kept in a desiccator (Nalgene desiccator, Sigma Aldrich).
Samples Ultraviolet Irradiation
Collagen films were irradiated under air using a GL4 germicidal lamp with a maximum at 254 nm (Sankyo Denki Co. Ltd., Japan) 3 cm from the light source for
Kontomaris et al.: The Effects of UV Irradiation on Collagen D-band
various time intervals. The intensity of radiation for this distance was 1813 mW/cm2 i.e. 0.11 J/(cm2.min) and the dose of incident radiation during the 1 h exposure was 6.6 J/cm2. The intensity of the incident light was measured using a GoldiluxTM radiometer-photometer (Model 70234-meter and 70239-probe, Oriel Instruments, Stanford, CA). All measurements were performed in the same conditions of temperature to avoid any influence on the physicochemical and mechanical properties of collagen.
AFM
AFM experiments were carried out using a commercial microscope (CP II, Veeco Bruker, Santa Barbara, CA) in contact mode. For both imaging and indentation tests, pyramidal tips were employed. These tips have a nominal tip radius of approximately () 20 nm on V– shaped cantilevers and a nominal spring constant of 0.5 N/m (type MLCT; Veeco). A cantilever with a low spring constant value was selected due to the fact that the AFM imaging procedure was carried out in contact mode. High spring constant values during the contact mode imaging were avoided since the tip might “move” collagen fibrils or scratch them. The samples were mounted on AFM metal discs with epoxy glue, while locator grids (Copper finder grid, G2761C, Agar Scientific, Essex, United Kinkdom) were used to map the surface (see also paragraph D. “Identification of the same fibril prior and post the irradiation”). All experiments were performed in air, at room temperature (20 2˚C) and relative humidity 40 3%. The image processing was carried out by using the image analysis software that accompanied the AFM system DI SPMLab NT ver.60.2 and IP-Image Processing and Data Analysis ver.2.1.15 (Veeco). In addition, the image analysis software WSxM 5.0 Develop 6.5 (Horcas et al., 2007) was used. All the experiments were carried out on the same collagen fibril each time in order to extract results, independent of possible differences between collagen fibrils.
Identification of the Same Fibril Prior and Post the Irradiation
The samples were mounted on AFM metal discs with epoxy glue, while locator grids (Copper finder grid, G2761C, Agar Scientific) were used to map the surface. (Markiewicz and Goh, 1997; Stylianou et al., 2013b). The locator grids can provide information about general areas on the sample. The main idea was to image every time (i.e. prior and post the irradiation) a specific area which is marked with the locator grid. Firstly, a large image (100 mm–100 mm) was obtained. Subsequently, images
3
with smaller dimensions were acquired. Using the above method one specific fibril or even though only a part of one fibril can be isolated (e.g. an overlapping region). After the irradiation, the same area using the locator grid can be approximately found and images with large dimensions (100 mm–100 mm) which are partly the same with the previous large image can be acquired. Subsequently, images with progressively smaller dimensions are being obtained. Finally, the same single fibril or even a part of a fibril which is exactly the same with the previous one (i.e. prior the irradiation) can be isolated. This procedure can be repeated after each irradiation. Hence, areas with dimensions that can be compared to the tip radius can be found again in order to examine the exact same area after changing controlled parameters.
Calculations
In practice, the indentation procedure is performed by moving the sample up and down toward the tip and measuring the corresponding deflection of the cantilever in order to create a load-displacement curve (Oliver and Pharr, 2004). The load-displacement curve consists of two parts, the loading curve in which the movement of the sample is towards the tip and the unloading curve in which the movement of the sample is the opposite, i.e. away from the tip. The indentation depth in a soft sample’s surface can be easily calculated under the condition that a hard sample is used as a reference. The difference in the sample’s surface movement for the soft and the hard sample in order to achieve the same deflection of the cantilever is the indentation depth (Oliver and Pharr, 2004). Load-indentation curve, can be used for the determination of the material contact stiffness (Oliver and Pharr, 2004; Wenger et al., 2007; Fischer-Cripps, 2009). According to the Oliver-Pharr method the contact stiffness can be calculated from the unloading part of the load-indentation curve (Oliver and Pharr, 2004). It has been reported that the initial data of the unloading curve is almost linear for some materials, and as a result a linear fit is the most obvious solution regarding the upper portion of the curve (FischerCripps, 2006; Choi et al., 2012b). The stiffness value leads to the estimation of the Young’s modulus, under the condition that the shape of the tip and the Poisson’s ratio of the sample are known. The formula that contains both the stiffness and the Young’s modulus is: pffiffiffiffi p S ð1 v2 Þ pffiffiffi E¼ 2 A where, E is the Young’s modulus, n the Poisson’s ratio, S the contact stiffness, and A an area function related to the effective cross-sectional or projecting area of the
SCANNING VOL. 9999, 9999 (2014)
4
indenter (Oliver and Pharr, 2004; Fischer-Cripps, 2009; Sirghi, 2010; Stylianou et al., 2014). Regarding small indentation depths, pyramidal indenters can be analyzed in terms of a paraboloid of revolution which can be approximated by a sphere (Oliver and Pharr, 2004). Thus, the projected area in this case can be derived by the following equation:
A ¼ phc ð2r hc Þ
Statistics
The Young’s modulus values were expressed as the mean standard deviation. An one-way ANOVA was performed to compare differences between fibrils diameters and Young’s modulus values under the impact of UV irradiation. Moreover, post hoc comparisons were carried out using a Student-Newman-Keuls test. The p-values
