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A comparison of excimer laser etching and dry etching process for surface fabrication of biomaterials
Journal of Materials Processing Technology 124 (2002) 284±292
A comparison of excimer laser etching and dry etching process for surface fabrication of biomaterials A. Prina Melloa, M.A. Barib, P.J. Prendergasta,* a
Department of Mechanical Engineering, Trinity College, Dublin, Ireland b Department of Physics, Trinity College, Dublin, Ireland Accepted 10 April 2002
Abstract One of the great possibilities for the future of bioengineering is the capability to control the movement and differentiation of individual cells. In this project, we aim to use microtexturing of the surfaces to control the migration of different types of T cell (a speci®c type of cell appearing in the lymphatic system). This paper reports on a part of that work which is concerned with exploring various microfabrication technologies to texture the surface of four biocompatible materials, viz. poly-methyl-methacrylate (PMMA), polystyrene (PS), borosilicate glass (B2O3±SiO2), and silicate glass (SiO2). The microfabrication processes are excimer laser etching and microetching, the latter being divided into microlithography and Ar ion etching. Channels were fabricated in each material. It was found that, using the equipment available, excimer laser etching could not achieve the speci®cations whereas microlithography could. How these conclusions were reached is described in detail. # 2002 Published by Elsevier Science B.V. Keywords: Micromanufacturing; Microtextured; Biomaterials; Nanobiotechnology; Cell biomechanics
1. Introduction In the last 30 years, a great deal of research has been dedicated to the development of microfabrication processes. Today, we can imagine a microchip smaller than the diameter of a hair (25 mm). This has been called the ``microdomain world'' and it is the result of advances in microfabrication technology [1]. Micro technologies like laser ablation and microlithography have developed so much that their involvement is a common aspect of many research innovations and one of the latest bioengineering application areas for micromachining technologies is cellular analysis. Recent applications of laser etching (or laser ablation) and microlithography have been adopted to achieve miniaturised systems for the analysis of biological tissues [2]. Typically, these microanalyses include microchannels and micro chambers in order to control sample ¯ow, mixing and analysis operations [2]. Another application area for micromachining technologies is ``whole cell systems''. Applications of whole cell systems generally fall into one of the
* Corresponding author. Tel.: 353-1-608-1383; fax: 353-1-679-5554. E-mail address: [email protected] (P.J. Prendergast).
0924-0136/02/$ ± see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 9 2 4 - 0 1 3 6 ( 0 2 ) 0 0 1 9 8 - X
following four categories: cell-sorting systems, active cell characterisation, cell growth systems, and micro-systems for cell assays [3]. The objectives of this study are to microtexture a biocompatible substratum for high-contrast microscope cell analysis using commonly available micro technologies. In particular, the uses of excimer laser etching and Ar ion etching techniques have been explored. Various prototypes have been successfully fabricated with both technologies. In particular, a straight channel in an optically transparent, biocompatible material, with speci®c requirements (width of 10 1 mm and depth of 5 1 mm) was requested for cell biomechanics analysis [4]. The quantitative measurements carried out in this paper in terms of micromanufacturing of surfaces for cellular investigation were addressed also to extend the knowledge on biomaterials microfabrication, following Curtis and Wilkinson [5], on the topographical control of cells locomotion. Inspired by their works, we were able to microfabricate biopolymers and bioglasses to mimic a natural biological substratum. The research question to be answered is whether or not it is possible to distinguish between the cell motion behaviour of a normal lymphatic cell and a tumour one and to use microtextured surface to sort the two cell types [4,6].
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Fig. 1. Arrangement used in the excimer laser ablation study; L1 focus lens; L2 anti-aberration lenses; A aperture; S specimen. Lenses: plano-convex, fused silica.
2. Materials and methods 2.1. Manufacturing process: excimer laser etching The excimer laser used in this study was a Lambda Physics LPX 100 Excimer laser source (Lambda Physics, Goettingen, Germany), with a maximum power of 100 W, which produces pulses of 20 ns duration. All experiments were performed at a pulse frequency of 2 Hz, at room temperature in an open air atmosphere. The experimental arrangement is shown in Fig. 1. This simpli®ed arrangement follows the same principle as the focused laser beam of a more sophisticated system [7]. A lens L1 is used to weakly focus the excimer beam onto an aperture A (slit or pinhole usually) controlling the laser ¯uence (radiation). The aperture is then imaged onto the sample S by the lenses system L2, which consists of two plano-convex lenses placed back to back to minimise spherical aberration (of the image projected). A preliminary series of tests have been carried out to determine the etch rate per laser pulse. The materials used in these experiments were polymethyl methacrylate (PMMA) and polystyrene (PS), two materials widely used in cell biology. Care was taken with the calibration and the arrangement, and setup of all the optical lenses. Using different collimators, it was possible to vary the reduction ratio of the image projected on the material surface. Three different optical stages have been used, with three beam reduction ratios 5:1, 4:1 and 2:1. These reduction ratios were achieved by varying the distances between the stages (distance b in Fig. 1). The details of each con®guration are shown in Table 1. Table 1 Excimer lenses specification Size, f (mm)
Focal length (mm)
40.0 50.8 50.8 50.8 50.8
50.0 76.2 101.6 304.8 500.0
The excimer laser was used with two different gas solutions. The two gases were: (a) ArF (l 193 nm); (b) KrF (l 248 nm). The laser beam was projected through a mask and focused on the surface of the material thereby etching (ablating) a thin layer of polymer per pulse. In the beginning, the mask was a simple slit of 100 mm length and 60 mm width. Then, different diameter pinholes (50, 25 and 15 mm) in molybdenum for high power lasers (Coherent Ltd, Leicester, UK) were used to ablate a single spot per pulse. Before starting the laser ablation process, a calibration phase was required to setup all the system. The calibration procedure used was to set the laser frequency to 1 Hz and, through a calorimeter system (Mentor Laser Power, Azusa, USA), to measure and set the intensity of the laser beam, with high accuracy. Using a microscope, as readout, it was possible to focus the beam on top of the material surface; this technique is called ``best image projection'' and can be directly measured. The technique is based on an optical microscope analysis of ablated spot in order to focus the beam on the surface of the materials. Ablated surfaces were subsequently measured using a pro®lometer technique (white light scanning interferometry (WLSI); Zygo Corporation, Middle®eld, USA) in order to have pro®le properties measurement. Roughness, depth, width and shape were measured to a tolerance of 0.1 nm. To complete the investigation, SEM analyses were done on the surfaces of the polymers to obtain more information regarding the surface modi®cation due to ablation. 2.2. Manufacturing processes: microetching The microfabrication techniques used in this work are divided into two processes [8]: (i) microlithography process and (ii) Ar ion etching process.
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Fig. 2. Microlithography process: six-phase scheme. Sequence of steps to prepare the sample to be exposed (cleaning and spinning). Then, UV light exposure and develop to transfer the pattern from the mask to the sample. Finally steps are baking and stripping of the resist excess.
2.2.1. Microlithography process A microlithography process has been used to transfer patterns from a pre-design mask to the substrata used in this work. This process is subdivided into the following steps: (i) resist deposition on substratum, (ii) resist exposure to ultraviolet light (UV), (iii) resist development, and (iv) baking of the resist onto the substratum, and resist removing or stripping, as shown in Fig. 2. All these steps were carried out at room temperature of 19 8C and a humidity rate below 40% to avoid any kind of contamination. All four tasks were carried out in a clean room (class 10 000) in order to run the process under the same environmental conditions. The preparation of the substrata to be spun was done under a bath of acetone for 10 min with gentle stirring. For the polymers we used an ultrasonic bath of distilled water and acetone of ratio 1:1. After that the substrata were dried out with nitrogen. High-resolution positive photoresist Shipley S1818 (Shipley Company L.L.C., Marlborough, USA) was spun at different speeds and times in accordance with the data sheet provided with the resist for semiconductor material. Soft baking followed the resist spin in order to ®x the resist on the substrata and achieve a strong interface bond between the two material layers. Layer thickness measurements were carried out using pro®lometer analysis on ®ve samples for each of the four materials to control the resist thickness deposition varying time and spin speed (data not provided in the Shipley data sheet). Average layer thickness of 2:0 0:2 mm was spun onto the substrata in the analysis. Following that, UV light exposure was used to transfer patterns from mask to substrata (Karl Suss-Contact Photolithography Mask Aligner, Munich, Germany). Patterns were designed
using a speci®c CAD (Kic, Whiteley Research, Inc., Sunnyvale, USA) and manufactured on a quartz mask by Photonics Ltd. (Manchester, UK). After exposure to UV light, it was necessary to develop the exposed samples using a developer from Shipley (MF-319 developer). Development of the exposed substrata was varied in relation to the substrata and the intensity of the exposure light. A series of samples was used to determine the proper developing time in order to avoid over- and under-exposition of the samples. After the etching process, the resist was removed by leaving the sample etched in a bath of Microposit Remover 1165 from Shipley for a minute. The initial requirements for the microetching process were mainly focused on the microlithography aspect. Parameters like resist-spin speed, spin time, baking temperature, UV exposure time, developing temperature, and resistremoving time were required in order to have substrata ready to be etched. Following the basic information given by Shipley, a microlithography standard protocol was developed. Thirty samples were used to optimise the microlithography technique to spin and develop a resist layer of 2 mm circa for: PMMA (15 samples), PS (eight samples) and bioglasses (seven samples). Table 2 lists all the main parameters involved in the microlithography process. 2.2.2. Ar ion etching process Microtexturing of the substrata in this study was done using an Ar ion etching process (Commonwealth Scienti®c, Richmond, USA). Samples were positioned in the vacuum chamber of the machine on the sample holder using special vacuum grease and then etched under different conditions in order to determine the etching rate for the
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Table 2 Microlithography settings for PMMA, PS, borosilicate glass and silicate glass to obtain controlled layer thickness of 2 mm circa Material
Spin speed (rpm)
Spin time (s)
Baking time, temp (s, 8C)
UV exposure (s)
Develop time (s)
Remover time (s)
Resist t hickness (mm)
PMMA PS B2O3±SiO2 SiO2
3000 2800 2000 2000
30 35 30 30
780, 780, 780, 780,
45 50 60 60
45 55 60 60
60 60 60 60
2.0 2.2 1.8 1.7
120 120 150 150
0.5 0.5 0.5 0.5
to maximise the shielding effect made by the resist layer. Furthermore, this shield effect was maximised for a speci®c ion-gun source. Etching curves were determined for the materials in examination using the same procedure as used for the excimer laser ablation, as shown in Fig. 3. Assumption was made on the ion-acceleration voltage output, ®xed to 1200 eV in order to have a constant and homogenous beam. 3. Results 3.1. Excimer laser results
Fig. 3. Relation between ion current density and etching rate of SiO2, PMMA, Si, and S1800 resists. Ion energy 1200 eV (From Miyamoto, Proceedings of the Seventh IPES, 1993, pp. 100±112).
four different materials. The parameters varied during the setting of the machine were ion-gun source pressure, ion-acceleration voltage output, ion-acceleration current density, ion-gun glove, sample table angle, and etching time. Once the samples were ready to be etched, the etching curves for all the materials needed to be determined in order
Two laser settings have been used (ArF: l 193 nm; KrF: l 248 nm) with the same optical arrangement. For these two settings, ablation curves have been determined for PMMA and PS at different values of laser beam intensity and ¯uence (beam intensity per surface area). Notice that the optical arrangement contribution is negligible because not affecting the analysis for the detection of the ablation curve. These tests were done at a constant frequency of 2 Hz to determine the best etch rate for each polymer. Experimental etch curves have been determined and compared at different laser settings for PS and PMMA polymers. The graph in Fig. 4 shows the ablation curve on PS and PMMA with excimer laser set with ArF gas (l 193 nm) and with KrF
Fig. 4. Etch curves for PMMA and PS using an excimer laser beam set to ArF (l 193 nm) and to KrF (l 248 nm) for different fluence intensities (NOTE: Fluence is beam intensity per unit area).
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Fig. 5. Excimer laser ablation technique. Result output of WLSI analysis on PMMA (left) and PS (right) microtextured surfaces. Roughness and cross-section measurements also given (inset boxes on left and right).
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The repeatability of the ablation appeared to be signi®cantly dependent on the setting of each individual experiment. Pinhole size was ®xed to 15 mm to have a high reduction ratio and therefore machine a small channel. SEM surface analyses were conducted to investigate polymer surface modi®cation. Fig. 6 shows a typical phenomenon of thermal shock, which was found in every surface examined. This was due to high beam intensity concentrated in a small area (10 mm). Bioglasses were ablated with the same technique but due to the high beam energy per unit area they were not able to compensate the thermal shock and they broke-up in the early phase of etch rate determination. 3.2. Microetching results Fig. 6. SEM analysis of polymer surface (PMMA). Crack parallel to the channel (left side) is due to thermal shock.
(l 248 nm). Notice that there were no differences on the ablation curves for PS material for the two settings. From these preliminary results, one setting was selected. It was decided to use the excimer laser set to ArF with 193 nm of wavelength in order to achieve smaller pattern than using KrF, l 248 nm setting. In the beginning, only the polymers were ablated on their surfaces with the aim of controlling width and depth of the groove, and limiting the aberration factor (needed for optical transparency). Thus, it was important to control all the parameters involved in this process and a simple ablated pattern has been used of straight channels varying lenses ratio, pinhole diameter, and ¯uence in order to control the geometric dimensions of the channel. The ablated patterns were measured with a pro®lometer (Fig. 5) and SEM analysis (Fig. 6). The following images and tables describe clearly the phenomenon and give an idea of the polymer structure modi®cation. A series of excimer laser ablations was carried out and measured to compare the ablation properties of PMMA and PS. Results of the most signi®cant ablations and WLSI measurements are reported in Table 3.
Once the etching curve for all materials had been determined (see Fig. 3), 12 samples were etched using Ar ion etching process. Pro®le measurements of all the patterns were carried out to improve the testing conditions. Simple and more complex patterns were microfabricated. Table 4 reports measurements of etching time, cross-section, and roughness. Note that the etching time is high because the etch rate of this microetching process is much lower than for laser ablation. The advantage is a sharper, better-de®ned cross-section of the channel, as shown by comparing the WLSI in Fig. 7(a) and (b) with that in Fig. 5. Reproducibility of the etching was found to be signi®cantly dependent on the overall process setup. Silicate and borosilicate glass have shown the same response when etched. Thus, the same etch rate has been used for the two bioglasses. These two glasses have shown high etching rate. Then, since ion etching is not a high beam focussing technique, the glasses were not subjected to thermal shock during etching, in the way they were for laser ablation. PMMA was not easy to prepare and etch because of the reaction to acetone acid and chemicals used to prepare the sample. PS was interacting with the photoresist (based on PMMA). Thus, when etched the pattern pro®les were not as clear as with the other materials.
Table 3 Data measured from excimer ablated surfaces (WLSI measurement) Sample
Etch time (s)
Intensity (mJ/cm2)
Pinhole size (mm)
Width (mm)
Depth (mm)
Roughness (mm)
PMMA
30 30 30 60
1.046 1.123 1.123 0.114
15 15 15 15
13.55 27.83 51.42 36.44
6.40 9.95 11.44 10.20
3.761±4.224 2.257±2.905 2.778±3.434 3.511±3.693
PS
30 60 60
1.046 0.114 0.114
15 15 15
48.31 11.46 38.81
15.82 6.97 11.68
3.761±4.224 2.469±2.761 3.347±3.823
290 A. Prina Mello et al. / Journal of Materials Processing Technology 124 (2002) 284±292 Fig. 7. (a) WLSI cross-section profile of microchannels machined with Ar ion etching on borosilicate (left) and PMMA (right) substrata. Channels width: avg value 36:2 mm (left); avg value 17:1 mm (right). Channels depth: avg value 4:16 mm (left); avg value 1:55 mm (right). (b) WLSI 3D plot of microchannels machined with Ar ion etching on borosilicate (left) and PMMA (right) substrata. Channels roughness: 0.76 mm (left); 0.68 mm (right).
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Table 4 Microetching WLSI results for PMMA, borosilicate and silicate glass: width, depth, and roughness (ion density of 0:75 0:10 A/cm2) Material
Etching time (mins)
Width (mm)
Depth (mm)
Expected depth (mm)
Depth error (mm)
Raa (mm)
sRab (mm)
PMMA
20 35 35 40 40
29.4 10.3 25.2 15.0 32.1
0.53 1.12 1.18 1.26 1.53
0.8 1.4 1.4 1.6 1.6
0.27 0.28 0.22 0.34 0.07
0.08 0.18 0.32 0.27 0.45
0.13 0.06 0.12 0.05 0.20
Borosilicate glass
20 20 40 40
14.6 17.2 25.2 36.2
2.12 1.55 4.43 4.16
2.8 2.8 5.6 5.6
0.68 1.25 1.17 1.44
0.55 0.68 0.85 0.76
0.34 0.22 0.21 0.32
Silicate glass
20 20 40
21.1 23.9 35.3
1.01 0.70 1.13
2.8 2.8 5.6
1.79 2.1 4.47
0.44 0.50 0.5
0.22 0.28 0.24
a b
Ra: average channel roughness. sRa: roughness standard deviation.
SEM analysis was not necessary because the surface cross-section measurements with the WLSI were accurate enough. 4. Discussion The excimer etching system of this work addressed the problem of how to microtexture biomaterial surfaces with good quality and controlled roughness. It was possible to reduce the number of parameters involved, from six to only three: (i) laser beam intensity, (ii) lens ratio against pinhole, and (iii) etching time. From the results shown, the difference in size and shape of the channels measured is extremely clear. Moreover, from the pro®le measured (WLSI) the crosssections of the channels are not constant giving, as a result, channels non-conformant with the requirement for cell engineering analysis. Consequently, the roughness value of the ablated channels shows a high mean value and standard deviation (Raavg 3:353 mm; sRa 0:6 mm). Both materials used for the ablation were thermoplastic and, due to a high temperature gradient, they exceeded the glass temperature (Tg) locally and become viscous. From the SEM analysis, it is evident that due, to the high energy focused in a small area, a high surface modi®cation occurred on the polymer chains structure. Then, when polymers cooled down they shrunk, and, due to that, a series of microcracks propagated parallel to the channel edges of the material (see Fig. 6). For the bioglasses (B2O3±SiO2, SiO2) it was not possible to ablate any samples because of the initial high thermal shock of laser beam focused on the material. Note that our results should be addressed to the particular laser setting system used and not to the technique of laser ablation in general. The microetching system utilised in this work was compatible with the requirements for microtextured surfaces for cellular analysis. In particular, it was possible to microtex-
ture more than one sample at a time with high etching control. Repeatability of the operations was required to control the parameters involved in the process. The microetching results presented for each of the three materials showed: (i) a well-de®ned cross-section, (ii) constant roughness, and (iii) constant depth for the whole length of the channels. This is most evident from the pro®le measurements (WLSI); the roughness value of the channels etched shows a mean value of Raavg 0:465 mm and standard deviation of sRa 0:229 mm. That is a reduction by a factor 10 on the roughness compared to the excimer laser system used although the variability has increased considerably (around 30%). This can only suggest a more accurate analysis of the etching parameters. Another main difference between the two processes was the etching time; for the ablation it was seconds whereas for the microetching it was minutes. This can be explained from the etching curve: ablation showed few micrometers per second whereas etching had nanometers per second. Then, comparing the channel depth with the expected depth calculated from the experimental etch curve (Table 4), it was found that the etch rate per minute was very much reduced. After a more accurate analysis, it was found that the resist layer thickness was not suf®cient to etch a sample longer then 20 min; the shielding effect was not high enough. A thicker resist layer has been considered a solution to overcome this difference on the etching rate and to achieve deeper channels with microetching. Bioglasses were etched with high etch rate and showed good results in terms of surface roughness, biocompatibility (i.e., not toxic when sterile, acid inert, hydrophilic) and microscope optical contrast (i.e., refractive and re¯ective indices unchanged after etching). PMMA and PS were not easy to sterilise and consequently the biocompatibility of the surface was compromised. Therefore, biological tests could be biased by the contamination of the cellular environment.
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5. Conclusions
Acknowledgements
Excimer laser ablation results have shown that the ablation technique reported above can be successfully implemented to create microchannels in biomaterials. However, even if the process is easy and versatile, we could not achieve the requirement to texture biomaterials with high-quality features and without interfering with their optical properties. The results obtained were interesting from the micromanufacturing point of view but they do not respond to the requirements of the research programme, i.e., a microchannel with accurate geometry with high optical contrast for cell investigation. Therefore, a more lengthy and involved speci®c investigation is needed for the laser ablation process in the future if its suitability for this process is to be established. Microetching, divided into microlithography and ion etching process, has been successfully developed to respond to our cell engineering research requirements. Results have shown that the channel optical features are in conformance with the speci®cations given for cell microscopy. Furthermore, the results have shown that a complex pattern can be micromanufactured with the same degree of accuracy as simple patterns; it is unlikely that this could have been achieved with laser ablation. The only elements of doubt were expressed in terms of process complexity and process control; it has been found that each single parameter involved in any process step can play a major role on the overall result. For instance, in this work a very thin resist layer compromised the overall etching process in terms of channel depth and roughness variance. Therefore, future work will address the evaluation of a new resist product called thick resist (Micro-resist Technology GmbH, Berlin, Germany) that is currently used for micro-electro-mechanical systems (MEMSs).
This work was ®nancially supported by a grant in nanobiotechnology from the Higher Education Authority (HEA) under the PTRLI programme. The authors thank Prof. James Lunney, Mr. Tue Hansen and Prof. Michael Coey of the Physics Department in Trinity College, Dublin, for the use of their facilities and the helpful assistance given. References [1] W.R. Runyan, K.E. Bean, Semiconductor Integrated Circuit Processing Technology, Addison-Wesley, Reading, MA, 1990. [2] H. Ayliffe, R. Rabbit, P. Tresco, B. Frazier, Micro-machined cellular analysis characterization systems for studying the biomechanics of individual cells, in: Proceedings of the International Conference on Solid State Sensors and Actuators, 1997, Chicago, IL, pp. 1307±1310. [3] B. Chehroudi, D.M. Brunette, Effects of surface topography on cell behaviour, in: D.L. Wise, et al. (Eds.), Encyclopaedic Handbook of Biomaterials and Bioengineering Materials, New York, 1996, pp. 813±842. [4] A. Prina Mello, P.J. Prendergast, Y. Volkov, M. Bari, J.M.D. Coey, Investigation of T cell locomotion on ion-milled surfaces, in: Proceedings of the 12th Conference of the European Society of Biomechanics, 2000, Dublin, p. 319. http://www.biomechanics.ie/ ESB2000. [5] A.S.G. Curtis, C. Wilkinson, Topographical control of cells, Biomaterials 18 (1997) 1573±1583. [6] A. Prina Mello, M. Moretti, Y. Volkov, P.J. Prendergast, A mechanical analysis of lymphatic cell migration: experimental analysis and modelling, in: Proceedings of the 15th AIMETA Congress of Theoretical and Applied Mechanics, ISSN-1592-8950, (2001), MS_Bio_04. [7] M. Hogan, J.G. Lunney, Laser photoablation of spin-on-glass, Appl. Phys. Lett. 53 (1988) 831±833. [8] M. Madou, Fundamentals of Microfabrication, CRC Press, New York, 1997.
A comparison of excimer laser etching and dry etching process for surface fabrication of biomaterials A. Prina Melloa, M.A. Barib, P.J. Prendergasta,* a
Department of Mechanical Engineering, Trinity College, Dublin, Ireland b Department of Physics, Trinity College, Dublin, Ireland Accepted 10 April 2002
Abstract One of the great possibilities for the future of bioengineering is the capability to control the movement and differentiation of individual cells. In this project, we aim to use microtexturing of the surfaces to control the migration of different types of T cell (a speci®c type of cell appearing in the lymphatic system). This paper reports on a part of that work which is concerned with exploring various microfabrication technologies to texture the surface of four biocompatible materials, viz. poly-methyl-methacrylate (PMMA), polystyrene (PS), borosilicate glass (B2O3±SiO2), and silicate glass (SiO2). The microfabrication processes are excimer laser etching and microetching, the latter being divided into microlithography and Ar ion etching. Channels were fabricated in each material. It was found that, using the equipment available, excimer laser etching could not achieve the speci®cations whereas microlithography could. How these conclusions were reached is described in detail. # 2002 Published by Elsevier Science B.V. Keywords: Micromanufacturing; Microtextured; Biomaterials; Nanobiotechnology; Cell biomechanics
1. Introduction In the last 30 years, a great deal of research has been dedicated to the development of microfabrication processes. Today, we can imagine a microchip smaller than the diameter of a hair (25 mm). This has been called the ``microdomain world'' and it is the result of advances in microfabrication technology [1]. Micro technologies like laser ablation and microlithography have developed so much that their involvement is a common aspect of many research innovations and one of the latest bioengineering application areas for micromachining technologies is cellular analysis. Recent applications of laser etching (or laser ablation) and microlithography have been adopted to achieve miniaturised systems for the analysis of biological tissues [2]. Typically, these microanalyses include microchannels and micro chambers in order to control sample ¯ow, mixing and analysis operations [2]. Another application area for micromachining technologies is ``whole cell systems''. Applications of whole cell systems generally fall into one of the
* Corresponding author. Tel.: 353-1-608-1383; fax: 353-1-679-5554. E-mail address: [email protected] (P.J. Prendergast).
0924-0136/02/$ ± see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 9 2 4 - 0 1 3 6 ( 0 2 ) 0 0 1 9 8 - X
following four categories: cell-sorting systems, active cell characterisation, cell growth systems, and micro-systems for cell assays [3]. The objectives of this study are to microtexture a biocompatible substratum for high-contrast microscope cell analysis using commonly available micro technologies. In particular, the uses of excimer laser etching and Ar ion etching techniques have been explored. Various prototypes have been successfully fabricated with both technologies. In particular, a straight channel in an optically transparent, biocompatible material, with speci®c requirements (width of 10 1 mm and depth of 5 1 mm) was requested for cell biomechanics analysis [4]. The quantitative measurements carried out in this paper in terms of micromanufacturing of surfaces for cellular investigation were addressed also to extend the knowledge on biomaterials microfabrication, following Curtis and Wilkinson [5], on the topographical control of cells locomotion. Inspired by their works, we were able to microfabricate biopolymers and bioglasses to mimic a natural biological substratum. The research question to be answered is whether or not it is possible to distinguish between the cell motion behaviour of a normal lymphatic cell and a tumour one and to use microtextured surface to sort the two cell types [4,6].
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Fig. 1. Arrangement used in the excimer laser ablation study; L1 focus lens; L2 anti-aberration lenses; A aperture; S specimen. Lenses: plano-convex, fused silica.
2. Materials and methods 2.1. Manufacturing process: excimer laser etching The excimer laser used in this study was a Lambda Physics LPX 100 Excimer laser source (Lambda Physics, Goettingen, Germany), with a maximum power of 100 W, which produces pulses of 20 ns duration. All experiments were performed at a pulse frequency of 2 Hz, at room temperature in an open air atmosphere. The experimental arrangement is shown in Fig. 1. This simpli®ed arrangement follows the same principle as the focused laser beam of a more sophisticated system [7]. A lens L1 is used to weakly focus the excimer beam onto an aperture A (slit or pinhole usually) controlling the laser ¯uence (radiation). The aperture is then imaged onto the sample S by the lenses system L2, which consists of two plano-convex lenses placed back to back to minimise spherical aberration (of the image projected). A preliminary series of tests have been carried out to determine the etch rate per laser pulse. The materials used in these experiments were polymethyl methacrylate (PMMA) and polystyrene (PS), two materials widely used in cell biology. Care was taken with the calibration and the arrangement, and setup of all the optical lenses. Using different collimators, it was possible to vary the reduction ratio of the image projected on the material surface. Three different optical stages have been used, with three beam reduction ratios 5:1, 4:1 and 2:1. These reduction ratios were achieved by varying the distances between the stages (distance b in Fig. 1). The details of each con®guration are shown in Table 1. Table 1 Excimer lenses specification Size, f (mm)
Focal length (mm)
40.0 50.8 50.8 50.8 50.8
50.0 76.2 101.6 304.8 500.0
The excimer laser was used with two different gas solutions. The two gases were: (a) ArF (l 193 nm); (b) KrF (l 248 nm). The laser beam was projected through a mask and focused on the surface of the material thereby etching (ablating) a thin layer of polymer per pulse. In the beginning, the mask was a simple slit of 100 mm length and 60 mm width. Then, different diameter pinholes (50, 25 and 15 mm) in molybdenum for high power lasers (Coherent Ltd, Leicester, UK) were used to ablate a single spot per pulse. Before starting the laser ablation process, a calibration phase was required to setup all the system. The calibration procedure used was to set the laser frequency to 1 Hz and, through a calorimeter system (Mentor Laser Power, Azusa, USA), to measure and set the intensity of the laser beam, with high accuracy. Using a microscope, as readout, it was possible to focus the beam on top of the material surface; this technique is called ``best image projection'' and can be directly measured. The technique is based on an optical microscope analysis of ablated spot in order to focus the beam on the surface of the materials. Ablated surfaces were subsequently measured using a pro®lometer technique (white light scanning interferometry (WLSI); Zygo Corporation, Middle®eld, USA) in order to have pro®le properties measurement. Roughness, depth, width and shape were measured to a tolerance of 0.1 nm. To complete the investigation, SEM analyses were done on the surfaces of the polymers to obtain more information regarding the surface modi®cation due to ablation. 2.2. Manufacturing processes: microetching The microfabrication techniques used in this work are divided into two processes [8]: (i) microlithography process and (ii) Ar ion etching process.
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Fig. 2. Microlithography process: six-phase scheme. Sequence of steps to prepare the sample to be exposed (cleaning and spinning). Then, UV light exposure and develop to transfer the pattern from the mask to the sample. Finally steps are baking and stripping of the resist excess.
2.2.1. Microlithography process A microlithography process has been used to transfer patterns from a pre-design mask to the substrata used in this work. This process is subdivided into the following steps: (i) resist deposition on substratum, (ii) resist exposure to ultraviolet light (UV), (iii) resist development, and (iv) baking of the resist onto the substratum, and resist removing or stripping, as shown in Fig. 2. All these steps were carried out at room temperature of 19 8C and a humidity rate below 40% to avoid any kind of contamination. All four tasks were carried out in a clean room (class 10 000) in order to run the process under the same environmental conditions. The preparation of the substrata to be spun was done under a bath of acetone for 10 min with gentle stirring. For the polymers we used an ultrasonic bath of distilled water and acetone of ratio 1:1. After that the substrata were dried out with nitrogen. High-resolution positive photoresist Shipley S1818 (Shipley Company L.L.C., Marlborough, USA) was spun at different speeds and times in accordance with the data sheet provided with the resist for semiconductor material. Soft baking followed the resist spin in order to ®x the resist on the substrata and achieve a strong interface bond between the two material layers. Layer thickness measurements were carried out using pro®lometer analysis on ®ve samples for each of the four materials to control the resist thickness deposition varying time and spin speed (data not provided in the Shipley data sheet). Average layer thickness of 2:0 0:2 mm was spun onto the substrata in the analysis. Following that, UV light exposure was used to transfer patterns from mask to substrata (Karl Suss-Contact Photolithography Mask Aligner, Munich, Germany). Patterns were designed
using a speci®c CAD (Kic, Whiteley Research, Inc., Sunnyvale, USA) and manufactured on a quartz mask by Photonics Ltd. (Manchester, UK). After exposure to UV light, it was necessary to develop the exposed samples using a developer from Shipley (MF-319 developer). Development of the exposed substrata was varied in relation to the substrata and the intensity of the exposure light. A series of samples was used to determine the proper developing time in order to avoid over- and under-exposition of the samples. After the etching process, the resist was removed by leaving the sample etched in a bath of Microposit Remover 1165 from Shipley for a minute. The initial requirements for the microetching process were mainly focused on the microlithography aspect. Parameters like resist-spin speed, spin time, baking temperature, UV exposure time, developing temperature, and resistremoving time were required in order to have substrata ready to be etched. Following the basic information given by Shipley, a microlithography standard protocol was developed. Thirty samples were used to optimise the microlithography technique to spin and develop a resist layer of 2 mm circa for: PMMA (15 samples), PS (eight samples) and bioglasses (seven samples). Table 2 lists all the main parameters involved in the microlithography process. 2.2.2. Ar ion etching process Microtexturing of the substrata in this study was done using an Ar ion etching process (Commonwealth Scienti®c, Richmond, USA). Samples were positioned in the vacuum chamber of the machine on the sample holder using special vacuum grease and then etched under different conditions in order to determine the etching rate for the
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Table 2 Microlithography settings for PMMA, PS, borosilicate glass and silicate glass to obtain controlled layer thickness of 2 mm circa Material
Spin speed (rpm)
Spin time (s)
Baking time, temp (s, 8C)
UV exposure (s)
Develop time (s)
Remover time (s)
Resist t hickness (mm)
PMMA PS B2O3±SiO2 SiO2
3000 2800 2000 2000
30 35 30 30
780, 780, 780, 780,
45 50 60 60
45 55 60 60
60 60 60 60
2.0 2.2 1.8 1.7
120 120 150 150
0.5 0.5 0.5 0.5
to maximise the shielding effect made by the resist layer. Furthermore, this shield effect was maximised for a speci®c ion-gun source. Etching curves were determined for the materials in examination using the same procedure as used for the excimer laser ablation, as shown in Fig. 3. Assumption was made on the ion-acceleration voltage output, ®xed to 1200 eV in order to have a constant and homogenous beam. 3. Results 3.1. Excimer laser results
Fig. 3. Relation between ion current density and etching rate of SiO2, PMMA, Si, and S1800 resists. Ion energy 1200 eV (From Miyamoto, Proceedings of the Seventh IPES, 1993, pp. 100±112).
four different materials. The parameters varied during the setting of the machine were ion-gun source pressure, ion-acceleration voltage output, ion-acceleration current density, ion-gun glove, sample table angle, and etching time. Once the samples were ready to be etched, the etching curves for all the materials needed to be determined in order
Two laser settings have been used (ArF: l 193 nm; KrF: l 248 nm) with the same optical arrangement. For these two settings, ablation curves have been determined for PMMA and PS at different values of laser beam intensity and ¯uence (beam intensity per surface area). Notice that the optical arrangement contribution is negligible because not affecting the analysis for the detection of the ablation curve. These tests were done at a constant frequency of 2 Hz to determine the best etch rate for each polymer. Experimental etch curves have been determined and compared at different laser settings for PS and PMMA polymers. The graph in Fig. 4 shows the ablation curve on PS and PMMA with excimer laser set with ArF gas (l 193 nm) and with KrF
Fig. 4. Etch curves for PMMA and PS using an excimer laser beam set to ArF (l 193 nm) and to KrF (l 248 nm) for different fluence intensities (NOTE: Fluence is beam intensity per unit area).
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Fig. 5. Excimer laser ablation technique. Result output of WLSI analysis on PMMA (left) and PS (right) microtextured surfaces. Roughness and cross-section measurements also given (inset boxes on left and right).
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The repeatability of the ablation appeared to be signi®cantly dependent on the setting of each individual experiment. Pinhole size was ®xed to 15 mm to have a high reduction ratio and therefore machine a small channel. SEM surface analyses were conducted to investigate polymer surface modi®cation. Fig. 6 shows a typical phenomenon of thermal shock, which was found in every surface examined. This was due to high beam intensity concentrated in a small area (10 mm). Bioglasses were ablated with the same technique but due to the high beam energy per unit area they were not able to compensate the thermal shock and they broke-up in the early phase of etch rate determination. 3.2. Microetching results Fig. 6. SEM analysis of polymer surface (PMMA). Crack parallel to the channel (left side) is due to thermal shock.
(l 248 nm). Notice that there were no differences on the ablation curves for PS material for the two settings. From these preliminary results, one setting was selected. It was decided to use the excimer laser set to ArF with 193 nm of wavelength in order to achieve smaller pattern than using KrF, l 248 nm setting. In the beginning, only the polymers were ablated on their surfaces with the aim of controlling width and depth of the groove, and limiting the aberration factor (needed for optical transparency). Thus, it was important to control all the parameters involved in this process and a simple ablated pattern has been used of straight channels varying lenses ratio, pinhole diameter, and ¯uence in order to control the geometric dimensions of the channel. The ablated patterns were measured with a pro®lometer (Fig. 5) and SEM analysis (Fig. 6). The following images and tables describe clearly the phenomenon and give an idea of the polymer structure modi®cation. A series of excimer laser ablations was carried out and measured to compare the ablation properties of PMMA and PS. Results of the most signi®cant ablations and WLSI measurements are reported in Table 3.
Once the etching curve for all materials had been determined (see Fig. 3), 12 samples were etched using Ar ion etching process. Pro®le measurements of all the patterns were carried out to improve the testing conditions. Simple and more complex patterns were microfabricated. Table 4 reports measurements of etching time, cross-section, and roughness. Note that the etching time is high because the etch rate of this microetching process is much lower than for laser ablation. The advantage is a sharper, better-de®ned cross-section of the channel, as shown by comparing the WLSI in Fig. 7(a) and (b) with that in Fig. 5. Reproducibility of the etching was found to be signi®cantly dependent on the overall process setup. Silicate and borosilicate glass have shown the same response when etched. Thus, the same etch rate has been used for the two bioglasses. These two glasses have shown high etching rate. Then, since ion etching is not a high beam focussing technique, the glasses were not subjected to thermal shock during etching, in the way they were for laser ablation. PMMA was not easy to prepare and etch because of the reaction to acetone acid and chemicals used to prepare the sample. PS was interacting with the photoresist (based on PMMA). Thus, when etched the pattern pro®les were not as clear as with the other materials.
Table 3 Data measured from excimer ablated surfaces (WLSI measurement) Sample
Etch time (s)
Intensity (mJ/cm2)
Pinhole size (mm)
Width (mm)
Depth (mm)
Roughness (mm)
PMMA
30 30 30 60
1.046 1.123 1.123 0.114
15 15 15 15
13.55 27.83 51.42 36.44
6.40 9.95 11.44 10.20
3.761±4.224 2.257±2.905 2.778±3.434 3.511±3.693
PS
30 60 60
1.046 0.114 0.114
15 15 15
48.31 11.46 38.81
15.82 6.97 11.68
3.761±4.224 2.469±2.761 3.347±3.823
290 A. Prina Mello et al. / Journal of Materials Processing Technology 124 (2002) 284±292 Fig. 7. (a) WLSI cross-section profile of microchannels machined with Ar ion etching on borosilicate (left) and PMMA (right) substrata. Channels width: avg value 36:2 mm (left); avg value 17:1 mm (right). Channels depth: avg value 4:16 mm (left); avg value 1:55 mm (right). (b) WLSI 3D plot of microchannels machined with Ar ion etching on borosilicate (left) and PMMA (right) substrata. Channels roughness: 0.76 mm (left); 0.68 mm (right).
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Table 4 Microetching WLSI results for PMMA, borosilicate and silicate glass: width, depth, and roughness (ion density of 0:75 0:10 A/cm2) Material
Etching time (mins)
Width (mm)
Depth (mm)
Expected depth (mm)
Depth error (mm)
Raa (mm)
sRab (mm)
PMMA
20 35 35 40 40
29.4 10.3 25.2 15.0 32.1
0.53 1.12 1.18 1.26 1.53
0.8 1.4 1.4 1.6 1.6
0.27 0.28 0.22 0.34 0.07
0.08 0.18 0.32 0.27 0.45
0.13 0.06 0.12 0.05 0.20
Borosilicate glass
20 20 40 40
14.6 17.2 25.2 36.2
2.12 1.55 4.43 4.16
2.8 2.8 5.6 5.6
0.68 1.25 1.17 1.44
0.55 0.68 0.85 0.76
0.34 0.22 0.21 0.32
Silicate glass
20 20 40
21.1 23.9 35.3
1.01 0.70 1.13
2.8 2.8 5.6
1.79 2.1 4.47
0.44 0.50 0.5
0.22 0.28 0.24
a b
Ra: average channel roughness. sRa: roughness standard deviation.
SEM analysis was not necessary because the surface cross-section measurements with the WLSI were accurate enough. 4. Discussion The excimer etching system of this work addressed the problem of how to microtexture biomaterial surfaces with good quality and controlled roughness. It was possible to reduce the number of parameters involved, from six to only three: (i) laser beam intensity, (ii) lens ratio against pinhole, and (iii) etching time. From the results shown, the difference in size and shape of the channels measured is extremely clear. Moreover, from the pro®le measured (WLSI) the crosssections of the channels are not constant giving, as a result, channels non-conformant with the requirement for cell engineering analysis. Consequently, the roughness value of the ablated channels shows a high mean value and standard deviation (Raavg 3:353 mm; sRa 0:6 mm). Both materials used for the ablation were thermoplastic and, due to a high temperature gradient, they exceeded the glass temperature (Tg) locally and become viscous. From the SEM analysis, it is evident that due, to the high energy focused in a small area, a high surface modi®cation occurred on the polymer chains structure. Then, when polymers cooled down they shrunk, and, due to that, a series of microcracks propagated parallel to the channel edges of the material (see Fig. 6). For the bioglasses (B2O3±SiO2, SiO2) it was not possible to ablate any samples because of the initial high thermal shock of laser beam focused on the material. Note that our results should be addressed to the particular laser setting system used and not to the technique of laser ablation in general. The microetching system utilised in this work was compatible with the requirements for microtextured surfaces for cellular analysis. In particular, it was possible to microtex-
ture more than one sample at a time with high etching control. Repeatability of the operations was required to control the parameters involved in the process. The microetching results presented for each of the three materials showed: (i) a well-de®ned cross-section, (ii) constant roughness, and (iii) constant depth for the whole length of the channels. This is most evident from the pro®le measurements (WLSI); the roughness value of the channels etched shows a mean value of Raavg 0:465 mm and standard deviation of sRa 0:229 mm. That is a reduction by a factor 10 on the roughness compared to the excimer laser system used although the variability has increased considerably (around 30%). This can only suggest a more accurate analysis of the etching parameters. Another main difference between the two processes was the etching time; for the ablation it was seconds whereas for the microetching it was minutes. This can be explained from the etching curve: ablation showed few micrometers per second whereas etching had nanometers per second. Then, comparing the channel depth with the expected depth calculated from the experimental etch curve (Table 4), it was found that the etch rate per minute was very much reduced. After a more accurate analysis, it was found that the resist layer thickness was not suf®cient to etch a sample longer then 20 min; the shielding effect was not high enough. A thicker resist layer has been considered a solution to overcome this difference on the etching rate and to achieve deeper channels with microetching. Bioglasses were etched with high etch rate and showed good results in terms of surface roughness, biocompatibility (i.e., not toxic when sterile, acid inert, hydrophilic) and microscope optical contrast (i.e., refractive and re¯ective indices unchanged after etching). PMMA and PS were not easy to sterilise and consequently the biocompatibility of the surface was compromised. Therefore, biological tests could be biased by the contamination of the cellular environment.
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5. Conclusions
Acknowledgements
Excimer laser ablation results have shown that the ablation technique reported above can be successfully implemented to create microchannels in biomaterials. However, even if the process is easy and versatile, we could not achieve the requirement to texture biomaterials with high-quality features and without interfering with their optical properties. The results obtained were interesting from the micromanufacturing point of view but they do not respond to the requirements of the research programme, i.e., a microchannel with accurate geometry with high optical contrast for cell investigation. Therefore, a more lengthy and involved speci®c investigation is needed for the laser ablation process in the future if its suitability for this process is to be established. Microetching, divided into microlithography and ion etching process, has been successfully developed to respond to our cell engineering research requirements. Results have shown that the channel optical features are in conformance with the speci®cations given for cell microscopy. Furthermore, the results have shown that a complex pattern can be micromanufactured with the same degree of accuracy as simple patterns; it is unlikely that this could have been achieved with laser ablation. The only elements of doubt were expressed in terms of process complexity and process control; it has been found that each single parameter involved in any process step can play a major role on the overall result. For instance, in this work a very thin resist layer compromised the overall etching process in terms of channel depth and roughness variance. Therefore, future work will address the evaluation of a new resist product called thick resist (Micro-resist Technology GmbH, Berlin, Germany) that is currently used for micro-electro-mechanical systems (MEMSs).
This work was ®nancially supported by a grant in nanobiotechnology from the Higher Education Authority (HEA) under the PTRLI programme. The authors thank Prof. James Lunney, Mr. Tue Hansen and Prof. Michael Coey of the Physics Department in Trinity College, Dublin, for the use of their facilities and the helpful assistance given. References [1] W.R. Runyan, K.E. Bean, Semiconductor Integrated Circuit Processing Technology, Addison-Wesley, Reading, MA, 1990. [2] H. Ayliffe, R. Rabbit, P. Tresco, B. Frazier, Micro-machined cellular analysis characterization systems for studying the biomechanics of individual cells, in: Proceedings of the International Conference on Solid State Sensors and Actuators, 1997, Chicago, IL, pp. 1307±1310. [3] B. Chehroudi, D.M. Brunette, Effects of surface topography on cell behaviour, in: D.L. Wise, et al. (Eds.), Encyclopaedic Handbook of Biomaterials and Bioengineering Materials, New York, 1996, pp. 813±842. [4] A. Prina Mello, P.J. Prendergast, Y. Volkov, M. Bari, J.M.D. Coey, Investigation of T cell locomotion on ion-milled surfaces, in: Proceedings of the 12th Conference of the European Society of Biomechanics, 2000, Dublin, p. 319. http://www.biomechanics.ie/ ESB2000. [5] A.S.G. Curtis, C. Wilkinson, Topographical control of cells, Biomaterials 18 (1997) 1573±1583. [6] A. Prina Mello, M. Moretti, Y. Volkov, P.J. Prendergast, A mechanical analysis of lymphatic cell migration: experimental analysis and modelling, in: Proceedings of the 15th AIMETA Congress of Theoretical and Applied Mechanics, ISSN-1592-8950, (2001), MS_Bio_04. [7] M. Hogan, J.G. Lunney, Laser photoablation of spin-on-glass, Appl. Phys. Lett. 53 (1988) 831±833. [8] M. Madou, Fundamentals of Microfabrication, CRC Press, New York, 1997.