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Microstructuring of UV-transparent functionalised films on glass by excimer laser irradiation
Materials Science and Engineering C 26 (2006) 1131 – 1135 www.elsevier.com/locate/msec
Microstructuring of UV-transparent functionalised films on glass by excimer laser irradiation T. Rudolph a,*, K. Zimmer a, T. Betz b b
a Leibniz-Institut fu¨r Oberfla¨chenmodifizierung e. V., Permoserstraße 15, 04318 Leipzig, Germany Fakulta¨t fu¨r Physik und Geowissenschaften der Universita¨t Leipzig, Linne´straße 5, 04103 Leipzig, Germany
Available online 27 October 2005
Abstract KrF excimer laser irradiation was used to remove organic moieties from UV-transparent films of organosilanes on borosilicate glass. Highresolution patterns with different functional groups on glass were obtained by a combination of laser modification and silanisation steps. The local material modification near the ablation threshold of glass was investigated by white light interference microscopy. Change in chemical properties of irradiated surface areas were studied by fluorescence microscopy after an appropriate dying of exposed samples. From the results, the domination of thermo-chemical effects induced by the laser irradiation is derived. Finally, an example is given how the patterned organosilane films can be applied to influence cell growth on glass. D 2005 Published by Elsevier B.V. Keywords: Glass; Excimer; Laser; Silanization; Functionalization; Elevated patterns; Patterning; Cell adhesion; Swelling
1. Introduction Progress in the fields of DNA analysis, proteomics, biosensor technology, and related work is accompanied by the requirement of a great number of samples to be analysed or stored systematically and automated as possible. Consequently, solid substrates were introduced into laboratory analytics, and small sample volumes are processed. For most applications, the surface of these substrates must be modified. Since this material is often silicon, glass, or fused silica, organosilanes are frequently used to provide surfaces with versatile chemical groups. Depending on the film thickness and the chemical structure, organosilane films can be removed or modified by excimer laser irradiation. The best results are achieved with at least some micrometer-thick layers [1] or if the silanes contain aromatic hydrocarbon functional groups [2 – 4]. These limitations occur when the structuring process is based on the UV absorption of the organic films. Upon UV irradiation of selfassembled monolayers (SAM) of silanes containing aromatic functional groups, the entire organic fragment is removed. In a repeated silanisation step, another functionality can then be established mainly on the exposed areas [2]. However, the * Corresponding author. Tel.: +49 341 235 2173; fax: +49 341 235 2595. E-mail address: [email protected] (T. Rudolph). 0928-4931/$ - see front matter D 2005 Published by Elsevier B.V. doi:10.1016/j.msec.2005.09.072
second component interpenetrates the first and a film with mixed functionality is obtained, especially when the original film is synthesised from short-chain silane precursors [5]. Therefore, it is desirable to process SAMs from long-chain alkyl silanes like octadecyltrichlorosilane or 1H,1H,2H,2Hperfluorodecyltrichlorosilane which form excellent barriers against the mixing of different functionalities at non-irradiated areas. On the other hand, they are insensitive to extended excimer laser irradiation at wavelengths of 248 nm and 193 nm due to their low UV absorption [5]. In this work, a method is demonstrated to remove such UVinsensitive moieties from borosilicate glass by means of a substrate-mediated laser modification process below the glass ablation threshold. For localised removal of functional groups, various ultra-thin organosilane films on glass were irradiated with 248-nm excimer laser light using mask projection. 2. Materials and methods 2.1. Materials In the presented experiments, standard cover slips (24 60 mm, IDL, Nidderau, Germany) from borosilicate glass and fused silica wafers (Hoya Inc., Japan) were used as substrates. 1H,1H,2H,2H-Perfluorodecyltrichlorosilane (17F), 2-[methoxy-
T. Rudolph et al. / Materials Science and Engineering C 26 (2006) 1131 – 1135
(polyethyleneoxy)propyl]-trimethoxysilane (PEOS), and 3aminopropyltrimethoxysilane (APTMS) were obtained from ABCR GmbH (Karlsruhe, Germany). Fluorescein isothiocyanate (FITC), dried dimethyl sulfoxide (DMSO) and other solvents were purchased from Sigma-Aldrich. 2.2. Methods 2.2.1. Silanisation Prior to silanisation, all substrates were cleaned by immersion in 10% NaOH for 3 – 6 h, washing with Millipore water (deionised water, > 10 MV), neutralisation with 1% HCl, washing with Millipore water again, and blown dry with nitrogen gas. 17F silane SAMs were formed by immersion of cleaned substrates in freshly prepared 0.2 –0.3% (v/v) 17F in isooctane for 30– 60 s and washed three times in isooctane. The success of the procedure was verified by contact angle measurements yielding values of 109– 110-. PEOS and APTMS solutions were prepared from 1% (v/v) silane in methanol. The samples were immersed for 10 min, washed three times with methanol and dried in nitrogen flow. All silanised samples were baked for 45 min at 110 -C in a laboratory oven to complete the surface reactions and anneal the film. 2.2.2. Excimer laser modification A KrF excimer laser (LPX 220i, Lambda Physik) running at k = 248 nm, s å 20 ns was used for the experiments. The laser is incorporated into a workstation (Exitech Ltd.) containing beam shaping and homogenising optics and a computer controlled x – y – z positioning stage (Aerotech Inc.). The homogenised laser beam illuminates a variable rectangular mask which is projected by a 15 demagnifying Schwarzschild objective (NA= 0.28, f = 13 mm) onto the sample. Alternatively, diverse metal or chrome-on-quartz masks can be employed. 2.2.3. Fluorescence labelling and image acquisition For fluorescence microscopy, samples were silanised with APTMS according to the above-described procedure. Afterwards, the generated amino groups on the surface were labelled with the amine-reactive dye FITC which was bound to the surface by immersing the samples in 10 mg FITC/100 ml DMSO for 18 h. To remove unreacted FITC, the samples were washed with DMSO and ethanol in an ultrasonic bath. Fluorescence images were recorded on a Leica DM LM microscope equipped with a 100-W mercury lamp, appropriate filters and a cooled monochrome CCD camera (DFC 350 FX). The data were saved as grey-scale images with 16 bits per pixel and a resolution of 1.4 megapixels. 2.2.4. Surface examination The surface morphology after excimer laser exposure was investigated by white light interference microscopy (MicroXAM, ADE PhaseShift, Tucson, USA).
12317, American Type Culture Collection, Manassas, VA), were employed. The cells were cultured in medium (90% DMEM (Sigma), 10% FBS (ATCC), 10 mM HEPES (Sigma), 100 U/ml penicillin – streptomycin (Sigma)) at 37 -C under 5% CO2 atmosphere. For the experiments, cells were plated on patterned cover slips which were mounted on a customised cell chamber and incubated for 2 days. For observation of the cell growth, the chamber, heated to 37 -C and immersed into a premixed 5% CO2 atmosphere, was mounted on an inverted microscope (DMIRB2, Leica, Bensheim, Germany). Images were recorded with a CCD camera (Cohu), using phase contrast objectives (20, Ph1, NA 0.40 and 63 oil immersion, Ph 3, NA 1.25). 3. Results and discussion 3.1. Excimer laser structuring 3.1.1. Laser modification of the substrates Beside the main component, SiO2, technical glass contains a mixture of many elements and compounds in varying proportions governing its ultraviolet light absorbance and, consequently, its response to excimer laser irradiation. For the investigation of the laser modification characteristics of the cover glass, squares of 40 40 Am were irradiated by mask projection with a KrF excimer laser. The laser fluence and the pulse number were varied between 0.1 and 5 J/cm2 and between 1 and 30 pulses, respectively. The height/depth of generated surface structures determined by interference microscopy is shown in Fig. 1. For fluences between 0.65 J/cm2 and the determined ablation threshold of 1.6 J/cm2, the exposed square areas were elevated with respect to the surrounding unexposed surface. The height of the structures depends on the laser fluence and it increases from 0 to about 40 nm in the mentioned fluence range. The interference microscope picture of a modified area depicted in Fig. 2 shows a well-defined and precisely shaped structure. The top surface is nearly as smooth as the virgin surface of the glass. The described swelling of the material after irradiation is attributed to thermal effects which lead to density changes of the glass. Preliminary secondary ion 50 0
Height [nm]
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1 pulse 3 pulses 10 pulses 30 pulses
1.0
1.5
2.0
Fluence [J/cm2]
2.2.5. Cell culture and image acquisition For the cell behaviour study, NG108-15 cells, a neuronallike mouse neuroblastoma rat glioma hybrid cell line (HB-
Fig. 1. Height of structures generated by excimer laser irradiation of borosilicate glass vs. fluence for pulse numbers of 1, 3, 10, and 30; measured by white light interference microscopy.
Fluorescence Intensity (norm.)
T. Rudolph et al. / Materials Science and Engineering C 26 (2006) 1131 – 1135
Fig. 2. Well-defined swelling of borosilicate glass induced by excimer laser irradiation; interference microscope image.
mass spectrometry (SIMS) data indicate also some alteration of material composition at irradiated sites. With rising laser fluence, the local temperature increases and the swelling is more pronounced. The height of the structures is almost independent of the pulse number as Fig. 1 reveals. Apparently, the material modification is almost complete after one pulse at a given laser fluence regardless of the total fluence (product of fluence and pulse number). Therefore, photochemical effects do not dominate the material modification. Exceeding laser fluences of 1.6 J/cm2, the height of the structures decreases dramatically due to material removal by laser ablation. A similar topology change caused by laser irradiation has also been observed for doped polymers due to photolysis of organic doping compounds causing material expansion [6,7]. For these much more flexible substrates height variations in the micrometer range were reported. 3.1.2. Patterning ultra-thin films of organosilanes SAMs from 17F and PEOS on borosilicate glass were subjected to patterned irradiation as described in the previous section. The change of chemical properties of the surface was studied indirectly by fluorescence microscopy. For this purpose, samples were resilanised with APTMS and labelled with FITC after exposure. In Fig. 3, colourised fluorescence microscopy images of 17F samples are shown which were processed with the denoted pulse numbers at laser fluences of (a) 0.74 J/cm2 (ten pulses), (b) 1.41 J/cm2 (one pulse), and (c) 1.86 J/cm2 (one pulse). Each image shows four adjacent quadratic regions which were irradiated using identical parameters. The fluorescence emission was recorded under the same conditions with a CCD
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1.0 0.9 0.8
1 pulse 3 pulses 10 pulses
0.7 0.6 0.50
0.75
1.00
1.25
Fluence
[J/cm2]
1.50
1.75
Fig. 4. Fluorescence intensity vs. fluence of laser-processed 17F silanised glass after resilanisation with APTMS and labelling with FITC.
exposure time of 2.3 s in all cases. Obviously, the laser-treated surface regions exhibit free silanol groups which react with APTMS leading to patterns where the surface is terminated with amino groups. The density of this functionalisation is visualized through the selective dying with FITC and it depends on the laser processing parameters as shown in Fig. 3. For optimised parameters as in Fig. 3b, a high density of amino groups and sharp boundaries between the regions with very well-defined, homogeneous functionalisation are obtained. In Fig. 3c, the fluorescence signal of the nonirradiated areas is very high compared to unexposed areas at lower laser fluences. But in this case, the fluence was above the ablation threshold and ablated glass components were redeposited in the surrounding of the processed squares. These glass debris are not removed by rinsing with solvents or sonication in solvents and is also amino functionalised and labelled with FITC. Because the debris consist mainly of small particles featuring an increased surface-to-volume ratio, a high fluorescence signal can be observed. Nevertheless, the debris can be wiped away with laboratory tissues. A systematic investigation of the fluorescence intensity depending on laser parameters is shown in Fig. 4. 17F silanised cover glasses were irradiated by one, three, and ten laser pulses at fluences in the same range, where elevated patterns can be observed. For each data point, the mean fluorescence intensity values of four equally processed squares were averaged. Modification of the original SAMs at fluences below 0.6 J/ cm2 could not be detected by fluorescence microscopy. It is observed that the fluorescence intensity increases with the laser
Fig. 3. 17F silanised borosilicate glass, excimer laser irradiated, resilanised with APTMS, dyed with FITC. Fluence and pulse numbers: (a) 0.74 J/cm2 (10 pulses), (b) 1.41 J/cm2 (1 pulse), (c) 1.86 J/cm2 (1 pulse). Fluorescence microscopy images with 50 magnification and 2.3 s CCD exposure.
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T. Rudolph et al. / Materials Science and Engineering C 26 (2006) 1131 – 1135
glass has a minor influence on the high optical resolution of the processing; the thermal diffusion length can be estimated to be less than 1 Am. Because one laser pulse with optimal fluence is sufficient for maximal surface modification, the described procedure is very fast and effective, and the produced patterns are shaped very precisely. Since the surface modification is based on the absorbance of the substrate, probably any organosilane SAM can be patterned in the described way on the employed cover glass. An example is given in Section 3.2. Fig. 5. Borosilicate glass silanised with PEOS, structured by excimer laser direct writing, labelled with APTMS/FITC; fluorescence microscopy image with 10 magnification and 4.8 s CCD exposure.
fluence reaching saturation at ¨ 1.4 J/cm2, whereas the pulse number only slightly affects the results. Because the fluorescence intensity as well as the structure heights increase similar with the laser fluence, exhibiting a little dependence on the pulse number, both effects correlate well. Therefore, the change of chemical properties and topography of the surface originate most likely from the same processes caused by laser irradiation. For photochemical modification, the density of removed moieties and, therefore, the fluorescence intensity should have a strong dependence on the total laser fluence because the fluence is proportional to the density of photons. But in the present study, this is not observed. Although in Fig. 3a the total fluence applied is five times as high as for Fig. 3b, the original SAM is clearly less modified as indicated by the much lower brightness. This gives evidence of the non-photochemical nature of the dominating modification process. Experiments with silanised fused silica samples support this conclusion. After excimer laser irradiation using the same parameters as used for the glass, neither topographical changes nor modification of the organosilane films were observed. In the strong absorbing cover glasses, the energy of the laser pulses is deposited in a narrow surface region resulting in locally high surface temperatures. Nevertheless, for the described photothermal material modification, a fluence is required which is clearly higher than used for photochemical modification where the fluence of single pulses is typically less than 1 –3% of the threshold fluence [2– 4]. However, thermal diffusion due to the raised surface temperatures on borosilicate
3.2. Controlled adherence and guided growth of cells The investigation of artificial neuronal networks is of great interest. To establish neuronal networks according to predetermined arrangements on solid substrates, guided cell adhesion and neurite outgrowth is needed. SAMs containing oligo(ethylene oxide) hydrocarbon chains were shown to inhibit cell adhesion [8]. PEOS on cover glass was structured by excimer laser irradiation to yield a grid of bare glass for cell adhesion and paths for neurite outgrowth in a cell-repelling surrounding. The paths were arranged to constitute a rectangular grid of 15-Am-wide lines with a distance of 200 Am in horizontal and vertical direction. The precise patterning is shown in Fig. 5 for a fluorescently labelled sample. Additionally, squares of 40 Am side length were irradiated at the junctions to promote the adhesion of the cells at this points. All the patterns were generated by direct laser writing on PEOScoated substrates using a variable rectangular mask and a fluence of 1.4 J/cm2. This gives a high degree of flexibility in contrast to photolithographic techniques. Additionally, possible contamination or modification due to chemicals used in a lithographic process are avoided. Fig. 6 shows cells adhering and neurites growing preferentially along the modified grid while hardly adhering on the PEOS silanised areas. Although phase contrast microscopy was applied, the laser patterned areas are hardly visible and do not influence optical investigations of the cells. 4. Conclusions The efficient degradation of UV-transparent organosilanes on borosilicate glass by excimer laser irradiation has been
Fig. 6. HB-12317 hybrid cells plated on patterned cover slips and incubated for 2 days; oil immersion microscopy with phase contrast, 63 magnification.
T. Rudolph et al. / Materials Science and Engineering C 26 (2006) 1131 – 1135
demonstrated and utilised for the fabrication of functionalised patterns. Single laser pulses slightly below the ablation threshold of glass cause both the formation of nanometer-high elevated topographic features and the degradation of the organosilane films. Evidence for the substrate-mediated, thermo-physical nature of the patterning process has been deduced from the comparison of the degradation of SAMs deposited either on borosilicate glass or on fused silica. This conclusion is furthermore supported by the fact that the topography change and SAM degradation occur simultaneously and are independent of the laser pulse number. Interference microscopy reveals well-defined, precisely shaped, up to 40-nm-high patterns with very low surface roughness generated by the laser treatment. By the combination of laser modification and repeated silanisation patterned SAM surfaces with different functionalities can be obtained. At optimised processing conditions, these patterns show a high density of functionalisation with sharp boundaries as visualised by fluorescence microscopy. Consequently, the described procedure enables a fast, reliable, flexible, high-resolution modification of silanised cover glasses for different applications in bio-technology or bio-medicine. As an example, localised cell adhesion and guided neurite outgrowth are demonstrated for patterned PEOS on glass.
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Acknowledgements This work was supported in part by the Sa¨ chsische Aufbaubank (SAB) under contract 7691/1187 and the EFRE support of the European Union. Support from and valuable discussions with C. Elsner are gratefully acknowledged. References [1] S.G. Hansen, T.E. Robitaille, J. Appl. Phys. 62 (1987) 1394. [2] C.S. Dulcey, J.H. Georger Jr., M.S. Chen, S.W. McElvany, C.E. O’Ferrall, V.I. Benezra, J.M. Calvert, Langmuir 12 (1996) 1638. [3] J.M. Calvert, J. Vac. Sci. Technol., B 11 (1993) 2155. [4] A.C. Friedli, R.D. Roberts, C.S. Dulcey, A.R. Hsu, S.W. McElvany, J.M. Calvert, Langmuir 20 (2004) 4295. [5] D.A. Stenger, J.H. Georger, C.S. Dulcey, J.J. Hickman, A.S. Rudolph, T.B. Nielsen, S.M. McCort, J.M. Calvert, J. Am. Chem. Soc. 114 (1992) 8435. [6] H. Fukumura, N. Mibuka, S. Eura, H. Masuhara, Appl. Phys., A 53 (1991) 255. [7] F. Beinhorn, J. Ihlemann, K. Luther, J. Troe, Appl. Phys., A 68 (1999) 709. [8] B. Zhu, T. Eurell, R. Gunawan, D. Leckband, J. Biomed. Mater. Res. 56 (2001) 406.
Microstructuring of UV-transparent functionalised films on glass by excimer laser irradiation T. Rudolph a,*, K. Zimmer a, T. Betz b b
a Leibniz-Institut fu¨r Oberfla¨chenmodifizierung e. V., Permoserstraße 15, 04318 Leipzig, Germany Fakulta¨t fu¨r Physik und Geowissenschaften der Universita¨t Leipzig, Linne´straße 5, 04103 Leipzig, Germany
Available online 27 October 2005
Abstract KrF excimer laser irradiation was used to remove organic moieties from UV-transparent films of organosilanes on borosilicate glass. Highresolution patterns with different functional groups on glass were obtained by a combination of laser modification and silanisation steps. The local material modification near the ablation threshold of glass was investigated by white light interference microscopy. Change in chemical properties of irradiated surface areas were studied by fluorescence microscopy after an appropriate dying of exposed samples. From the results, the domination of thermo-chemical effects induced by the laser irradiation is derived. Finally, an example is given how the patterned organosilane films can be applied to influence cell growth on glass. D 2005 Published by Elsevier B.V. Keywords: Glass; Excimer; Laser; Silanization; Functionalization; Elevated patterns; Patterning; Cell adhesion; Swelling
1. Introduction Progress in the fields of DNA analysis, proteomics, biosensor technology, and related work is accompanied by the requirement of a great number of samples to be analysed or stored systematically and automated as possible. Consequently, solid substrates were introduced into laboratory analytics, and small sample volumes are processed. For most applications, the surface of these substrates must be modified. Since this material is often silicon, glass, or fused silica, organosilanes are frequently used to provide surfaces with versatile chemical groups. Depending on the film thickness and the chemical structure, organosilane films can be removed or modified by excimer laser irradiation. The best results are achieved with at least some micrometer-thick layers [1] or if the silanes contain aromatic hydrocarbon functional groups [2 – 4]. These limitations occur when the structuring process is based on the UV absorption of the organic films. Upon UV irradiation of selfassembled monolayers (SAM) of silanes containing aromatic functional groups, the entire organic fragment is removed. In a repeated silanisation step, another functionality can then be established mainly on the exposed areas [2]. However, the * Corresponding author. Tel.: +49 341 235 2173; fax: +49 341 235 2595. E-mail address: [email protected] (T. Rudolph). 0928-4931/$ - see front matter D 2005 Published by Elsevier B.V. doi:10.1016/j.msec.2005.09.072
second component interpenetrates the first and a film with mixed functionality is obtained, especially when the original film is synthesised from short-chain silane precursors [5]. Therefore, it is desirable to process SAMs from long-chain alkyl silanes like octadecyltrichlorosilane or 1H,1H,2H,2Hperfluorodecyltrichlorosilane which form excellent barriers against the mixing of different functionalities at non-irradiated areas. On the other hand, they are insensitive to extended excimer laser irradiation at wavelengths of 248 nm and 193 nm due to their low UV absorption [5]. In this work, a method is demonstrated to remove such UVinsensitive moieties from borosilicate glass by means of a substrate-mediated laser modification process below the glass ablation threshold. For localised removal of functional groups, various ultra-thin organosilane films on glass were irradiated with 248-nm excimer laser light using mask projection. 2. Materials and methods 2.1. Materials In the presented experiments, standard cover slips (24 60 mm, IDL, Nidderau, Germany) from borosilicate glass and fused silica wafers (Hoya Inc., Japan) were used as substrates. 1H,1H,2H,2H-Perfluorodecyltrichlorosilane (17F), 2-[methoxy-
T. Rudolph et al. / Materials Science and Engineering C 26 (2006) 1131 – 1135
(polyethyleneoxy)propyl]-trimethoxysilane (PEOS), and 3aminopropyltrimethoxysilane (APTMS) were obtained from ABCR GmbH (Karlsruhe, Germany). Fluorescein isothiocyanate (FITC), dried dimethyl sulfoxide (DMSO) and other solvents were purchased from Sigma-Aldrich. 2.2. Methods 2.2.1. Silanisation Prior to silanisation, all substrates were cleaned by immersion in 10% NaOH for 3 – 6 h, washing with Millipore water (deionised water, > 10 MV), neutralisation with 1% HCl, washing with Millipore water again, and blown dry with nitrogen gas. 17F silane SAMs were formed by immersion of cleaned substrates in freshly prepared 0.2 –0.3% (v/v) 17F in isooctane for 30– 60 s and washed three times in isooctane. The success of the procedure was verified by contact angle measurements yielding values of 109– 110-. PEOS and APTMS solutions were prepared from 1% (v/v) silane in methanol. The samples were immersed for 10 min, washed three times with methanol and dried in nitrogen flow. All silanised samples were baked for 45 min at 110 -C in a laboratory oven to complete the surface reactions and anneal the film. 2.2.2. Excimer laser modification A KrF excimer laser (LPX 220i, Lambda Physik) running at k = 248 nm, s å 20 ns was used for the experiments. The laser is incorporated into a workstation (Exitech Ltd.) containing beam shaping and homogenising optics and a computer controlled x – y – z positioning stage (Aerotech Inc.). The homogenised laser beam illuminates a variable rectangular mask which is projected by a 15 demagnifying Schwarzschild objective (NA= 0.28, f = 13 mm) onto the sample. Alternatively, diverse metal or chrome-on-quartz masks can be employed. 2.2.3. Fluorescence labelling and image acquisition For fluorescence microscopy, samples were silanised with APTMS according to the above-described procedure. Afterwards, the generated amino groups on the surface were labelled with the amine-reactive dye FITC which was bound to the surface by immersing the samples in 10 mg FITC/100 ml DMSO for 18 h. To remove unreacted FITC, the samples were washed with DMSO and ethanol in an ultrasonic bath. Fluorescence images were recorded on a Leica DM LM microscope equipped with a 100-W mercury lamp, appropriate filters and a cooled monochrome CCD camera (DFC 350 FX). The data were saved as grey-scale images with 16 bits per pixel and a resolution of 1.4 megapixels. 2.2.4. Surface examination The surface morphology after excimer laser exposure was investigated by white light interference microscopy (MicroXAM, ADE PhaseShift, Tucson, USA).
12317, American Type Culture Collection, Manassas, VA), were employed. The cells were cultured in medium (90% DMEM (Sigma), 10% FBS (ATCC), 10 mM HEPES (Sigma), 100 U/ml penicillin – streptomycin (Sigma)) at 37 -C under 5% CO2 atmosphere. For the experiments, cells were plated on patterned cover slips which were mounted on a customised cell chamber and incubated for 2 days. For observation of the cell growth, the chamber, heated to 37 -C and immersed into a premixed 5% CO2 atmosphere, was mounted on an inverted microscope (DMIRB2, Leica, Bensheim, Germany). Images were recorded with a CCD camera (Cohu), using phase contrast objectives (20, Ph1, NA 0.40 and 63 oil immersion, Ph 3, NA 1.25). 3. Results and discussion 3.1. Excimer laser structuring 3.1.1. Laser modification of the substrates Beside the main component, SiO2, technical glass contains a mixture of many elements and compounds in varying proportions governing its ultraviolet light absorbance and, consequently, its response to excimer laser irradiation. For the investigation of the laser modification characteristics of the cover glass, squares of 40 40 Am were irradiated by mask projection with a KrF excimer laser. The laser fluence and the pulse number were varied between 0.1 and 5 J/cm2 and between 1 and 30 pulses, respectively. The height/depth of generated surface structures determined by interference microscopy is shown in Fig. 1. For fluences between 0.65 J/cm2 and the determined ablation threshold of 1.6 J/cm2, the exposed square areas were elevated with respect to the surrounding unexposed surface. The height of the structures depends on the laser fluence and it increases from 0 to about 40 nm in the mentioned fluence range. The interference microscope picture of a modified area depicted in Fig. 2 shows a well-defined and precisely shaped structure. The top surface is nearly as smooth as the virgin surface of the glass. The described swelling of the material after irradiation is attributed to thermal effects which lead to density changes of the glass. Preliminary secondary ion 50 0
Height [nm]
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1 pulse 3 pulses 10 pulses 30 pulses
1.0
1.5
2.0
Fluence [J/cm2]
2.2.5. Cell culture and image acquisition For the cell behaviour study, NG108-15 cells, a neuronallike mouse neuroblastoma rat glioma hybrid cell line (HB-
Fig. 1. Height of structures generated by excimer laser irradiation of borosilicate glass vs. fluence for pulse numbers of 1, 3, 10, and 30; measured by white light interference microscopy.
Fluorescence Intensity (norm.)
T. Rudolph et al. / Materials Science and Engineering C 26 (2006) 1131 – 1135
Fig. 2. Well-defined swelling of borosilicate glass induced by excimer laser irradiation; interference microscope image.
mass spectrometry (SIMS) data indicate also some alteration of material composition at irradiated sites. With rising laser fluence, the local temperature increases and the swelling is more pronounced. The height of the structures is almost independent of the pulse number as Fig. 1 reveals. Apparently, the material modification is almost complete after one pulse at a given laser fluence regardless of the total fluence (product of fluence and pulse number). Therefore, photochemical effects do not dominate the material modification. Exceeding laser fluences of 1.6 J/cm2, the height of the structures decreases dramatically due to material removal by laser ablation. A similar topology change caused by laser irradiation has also been observed for doped polymers due to photolysis of organic doping compounds causing material expansion [6,7]. For these much more flexible substrates height variations in the micrometer range were reported. 3.1.2. Patterning ultra-thin films of organosilanes SAMs from 17F and PEOS on borosilicate glass were subjected to patterned irradiation as described in the previous section. The change of chemical properties of the surface was studied indirectly by fluorescence microscopy. For this purpose, samples were resilanised with APTMS and labelled with FITC after exposure. In Fig. 3, colourised fluorescence microscopy images of 17F samples are shown which were processed with the denoted pulse numbers at laser fluences of (a) 0.74 J/cm2 (ten pulses), (b) 1.41 J/cm2 (one pulse), and (c) 1.86 J/cm2 (one pulse). Each image shows four adjacent quadratic regions which were irradiated using identical parameters. The fluorescence emission was recorded under the same conditions with a CCD
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1.0 0.9 0.8
1 pulse 3 pulses 10 pulses
0.7 0.6 0.50
0.75
1.00
1.25
Fluence
[J/cm2]
1.50
1.75
Fig. 4. Fluorescence intensity vs. fluence of laser-processed 17F silanised glass after resilanisation with APTMS and labelling with FITC.
exposure time of 2.3 s in all cases. Obviously, the laser-treated surface regions exhibit free silanol groups which react with APTMS leading to patterns where the surface is terminated with amino groups. The density of this functionalisation is visualized through the selective dying with FITC and it depends on the laser processing parameters as shown in Fig. 3. For optimised parameters as in Fig. 3b, a high density of amino groups and sharp boundaries between the regions with very well-defined, homogeneous functionalisation are obtained. In Fig. 3c, the fluorescence signal of the nonirradiated areas is very high compared to unexposed areas at lower laser fluences. But in this case, the fluence was above the ablation threshold and ablated glass components were redeposited in the surrounding of the processed squares. These glass debris are not removed by rinsing with solvents or sonication in solvents and is also amino functionalised and labelled with FITC. Because the debris consist mainly of small particles featuring an increased surface-to-volume ratio, a high fluorescence signal can be observed. Nevertheless, the debris can be wiped away with laboratory tissues. A systematic investigation of the fluorescence intensity depending on laser parameters is shown in Fig. 4. 17F silanised cover glasses were irradiated by one, three, and ten laser pulses at fluences in the same range, where elevated patterns can be observed. For each data point, the mean fluorescence intensity values of four equally processed squares were averaged. Modification of the original SAMs at fluences below 0.6 J/ cm2 could not be detected by fluorescence microscopy. It is observed that the fluorescence intensity increases with the laser
Fig. 3. 17F silanised borosilicate glass, excimer laser irradiated, resilanised with APTMS, dyed with FITC. Fluence and pulse numbers: (a) 0.74 J/cm2 (10 pulses), (b) 1.41 J/cm2 (1 pulse), (c) 1.86 J/cm2 (1 pulse). Fluorescence microscopy images with 50 magnification and 2.3 s CCD exposure.
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glass has a minor influence on the high optical resolution of the processing; the thermal diffusion length can be estimated to be less than 1 Am. Because one laser pulse with optimal fluence is sufficient for maximal surface modification, the described procedure is very fast and effective, and the produced patterns are shaped very precisely. Since the surface modification is based on the absorbance of the substrate, probably any organosilane SAM can be patterned in the described way on the employed cover glass. An example is given in Section 3.2. Fig. 5. Borosilicate glass silanised with PEOS, structured by excimer laser direct writing, labelled with APTMS/FITC; fluorescence microscopy image with 10 magnification and 4.8 s CCD exposure.
fluence reaching saturation at ¨ 1.4 J/cm2, whereas the pulse number only slightly affects the results. Because the fluorescence intensity as well as the structure heights increase similar with the laser fluence, exhibiting a little dependence on the pulse number, both effects correlate well. Therefore, the change of chemical properties and topography of the surface originate most likely from the same processes caused by laser irradiation. For photochemical modification, the density of removed moieties and, therefore, the fluorescence intensity should have a strong dependence on the total laser fluence because the fluence is proportional to the density of photons. But in the present study, this is not observed. Although in Fig. 3a the total fluence applied is five times as high as for Fig. 3b, the original SAM is clearly less modified as indicated by the much lower brightness. This gives evidence of the non-photochemical nature of the dominating modification process. Experiments with silanised fused silica samples support this conclusion. After excimer laser irradiation using the same parameters as used for the glass, neither topographical changes nor modification of the organosilane films were observed. In the strong absorbing cover glasses, the energy of the laser pulses is deposited in a narrow surface region resulting in locally high surface temperatures. Nevertheless, for the described photothermal material modification, a fluence is required which is clearly higher than used for photochemical modification where the fluence of single pulses is typically less than 1 –3% of the threshold fluence [2– 4]. However, thermal diffusion due to the raised surface temperatures on borosilicate
3.2. Controlled adherence and guided growth of cells The investigation of artificial neuronal networks is of great interest. To establish neuronal networks according to predetermined arrangements on solid substrates, guided cell adhesion and neurite outgrowth is needed. SAMs containing oligo(ethylene oxide) hydrocarbon chains were shown to inhibit cell adhesion [8]. PEOS on cover glass was structured by excimer laser irradiation to yield a grid of bare glass for cell adhesion and paths for neurite outgrowth in a cell-repelling surrounding. The paths were arranged to constitute a rectangular grid of 15-Am-wide lines with a distance of 200 Am in horizontal and vertical direction. The precise patterning is shown in Fig. 5 for a fluorescently labelled sample. Additionally, squares of 40 Am side length were irradiated at the junctions to promote the adhesion of the cells at this points. All the patterns were generated by direct laser writing on PEOScoated substrates using a variable rectangular mask and a fluence of 1.4 J/cm2. This gives a high degree of flexibility in contrast to photolithographic techniques. Additionally, possible contamination or modification due to chemicals used in a lithographic process are avoided. Fig. 6 shows cells adhering and neurites growing preferentially along the modified grid while hardly adhering on the PEOS silanised areas. Although phase contrast microscopy was applied, the laser patterned areas are hardly visible and do not influence optical investigations of the cells. 4. Conclusions The efficient degradation of UV-transparent organosilanes on borosilicate glass by excimer laser irradiation has been
Fig. 6. HB-12317 hybrid cells plated on patterned cover slips and incubated for 2 days; oil immersion microscopy with phase contrast, 63 magnification.
T. Rudolph et al. / Materials Science and Engineering C 26 (2006) 1131 – 1135
demonstrated and utilised for the fabrication of functionalised patterns. Single laser pulses slightly below the ablation threshold of glass cause both the formation of nanometer-high elevated topographic features and the degradation of the organosilane films. Evidence for the substrate-mediated, thermo-physical nature of the patterning process has been deduced from the comparison of the degradation of SAMs deposited either on borosilicate glass or on fused silica. This conclusion is furthermore supported by the fact that the topography change and SAM degradation occur simultaneously and are independent of the laser pulse number. Interference microscopy reveals well-defined, precisely shaped, up to 40-nm-high patterns with very low surface roughness generated by the laser treatment. By the combination of laser modification and repeated silanisation patterned SAM surfaces with different functionalities can be obtained. At optimised processing conditions, these patterns show a high density of functionalisation with sharp boundaries as visualised by fluorescence microscopy. Consequently, the described procedure enables a fast, reliable, flexible, high-resolution modification of silanised cover glasses for different applications in bio-technology or bio-medicine. As an example, localised cell adhesion and guided neurite outgrowth are demonstrated for patterned PEOS on glass.
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Acknowledgements This work was supported in part by the Sa¨ chsische Aufbaubank (SAB) under contract 7691/1187 and the EFRE support of the European Union. Support from and valuable discussions with C. Elsner are gratefully acknowledged. References [1] S.G. Hansen, T.E. Robitaille, J. Appl. Phys. 62 (1987) 1394. [2] C.S. Dulcey, J.H. Georger Jr., M.S. Chen, S.W. McElvany, C.E. O’Ferrall, V.I. Benezra, J.M. Calvert, Langmuir 12 (1996) 1638. [3] J.M. Calvert, J. Vac. Sci. Technol., B 11 (1993) 2155. [4] A.C. Friedli, R.D. Roberts, C.S. Dulcey, A.R. Hsu, S.W. McElvany, J.M. Calvert, Langmuir 20 (2004) 4295. [5] D.A. Stenger, J.H. Georger, C.S. Dulcey, J.J. Hickman, A.S. Rudolph, T.B. Nielsen, S.M. McCort, J.M. Calvert, J. Am. Chem. Soc. 114 (1992) 8435. [6] H. Fukumura, N. Mibuka, S. Eura, H. Masuhara, Appl. Phys., A 53 (1991) 255. [7] F. Beinhorn, J. Ihlemann, K. Luther, J. Troe, Appl. Phys., A 68 (1999) 709. [8] B. Zhu, T. Eurell, R. Gunawan, D. Leckband, J. Biomed. Mater. Res. 56 (2001) 406.