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By comparing (2) with (3) and (4) it is immediately clear that the two-photon absorption process possesses intrinsic high spatial-localization characteristics, better than the SPA [69]. In fact, it is possible to show that the TPE has a spatial confinement of about 0.1 femto-liter [59].

Two-photon photopolymerization [70, 71] has been hence recognized as a unique nanofabrication tool due to its intrinsic 3D fabrication capability [72,73] and sub-diffraction limited spatial resolution [74, 75]. For its widespread use, a deep understanding on single voxels, the primitive building block for a 3D object [74-77], which can be approximated using following relation:

where S is the voxel size, /diff is the diffraction limit of a laser focal spot and a a constant reflecting characteristics of two-photon materials and exposure schemes and, therefore, describing the fact that diffraction limit is not the only factor to determine the voxel size; Ere and Eh, represent real (actually applied) and threshold laser power (Ph, Pre) at a given exposure time, or real and threshold exposure time (Tth, Tre) at a given exposure power; n = 1, 2 denotes single-photon and two-photon processes, respectively. It is interesting to note that low-numerical aperture (NA) focusing does not necessarily give rise to lateral spatial resolutions worse than high-NA focusing at a medium irradiation level [78]. This is because of threshold effect, and under a linearly polarized laser beam, voxels take shape of ellipsoid with three different axis lengths instead of a spinning ellipsoid. These considerations are important for properly designing and accurately depicting 3D structures with nanoscale features. Another significant merit of two-photon photopolymerization is the possibility of tuning voxel size according to requirements of device structures, conveniently either by adjusting the average irradiation laser power [71] (P scheme) or by changing the exposure time [70, 73] (T scheme). The two processes are equivalent to reach this end according to conventional exposure theory. However, at near-threshold condition, the voxel size scaling abides by different laws under these two approaches [79]. In two-photon absorption (TPA) assisted photopolymerization (solidification), a solidified skeleton is formed by the scanning locus. This solidified skeleton remains after the removal of unsolidified liquid resin [80]. With this method, various micromachines [71, 80] (figure 4.10 and figure 4.11) and photonic crystals [81, 82, 83] have been readily produced with near-diffraction-limit 3-D spatial resolutions. Photopolymerization is a photochemical reaction used for the creation of a polymer through a chain reaction initiated by light. Since most monomers and oligomers commonly employed do not possess initiating species with a sufficient quantum yield upon light exposure, it is necessary to introduce low-molecular-weight molecules called photoinitiators that start polymerization. A photosensitizer is also generally used, which has a large light absorbance and transfers the excitation to a photoinitiator. For many fabrications,

FIGURE 4.10. Micro-gear wheel.

resins consisting of urethane acrylate monomer/oligomer and radical initiators are used. Electronic transition energy for most initiator molecules corresponds to UV spectral range.

In particular, benzoyl chromophor is sensitive to near UV wavelength, and has a good photochemical reactivity, therefore utilized as common UV radical initiators. If the irradiation photon fluence is sufficiently high, e.g., by tightly focusing a fs laser, the probability of an electron simultaneously absorbing two photons is increased, and then TPA becomes practically useful. For producing sufficient photon flux density, it is essential to tightly confine laser pulses in both spatial and time domains. For the work presented hereafter a Titanium Sapphire laser that operated in mode-lock at 76 MHz and 780 nm with a 150-fs pulsewidth was utilized as the exposure source. A two-galvano-mirror set moves the laser beam in the two horizontal dimensions, and a piezo stage moved the laser focus vertically, both controlled by a computer. The laser is focused into the resin by a high numerical aperture (NA ~1.4, oil immersion) objective lens. The single-lens focusing geometry naturally

FIGURE 4.11. Micro-oscillator system.

Two Photon Lithography Bull
FIGURE 4.12. Example of a 3D structure obtained by means of two-photon lithography; (a) using the raster scan method, i.e. all the volume of the structure has been exposed, and (b) using the vector scanning method exposing only the microbull surface.

satisfies the requirement of pulse overlapping in both time and spatial domains. In this system, the utilization of short pulsewidth and tight focusing are critical for exciting sufficient amount of TPA and for achievement of high accuracy of fabrication. An average focal spot power of 1 mW under a 150 fs pulsewidth and 76 MHz repetition rate corresponds to a transient peak power of 20 GW/cm, or a photon flux density of 8- 1020 photon s-1 |j.m-2.

Laser scanning is the step to convert pre-designed CAD pattern to a resist structure. Two basic modes for direct laser scanning has been used, i.e., raster-scan mode and vector-scan mode. In the raster mode, all voxels in a cubic volume that contains the microstructure are scanned by the actual/virtual focal spot, depending on the shutter ON/OFF (actual/ virtual). In the vector mode, the laser focus directly traces the profile to be defined, and requires a smaller number of voxels. Depending on structures, alternations and combination of the these two basic scanning modes could be used. Experimentally we fabricated the same object using the two modes. The microbull in figure 4.12.a was produced using a layer-by-layer raster-scanning scheme, i.e., all voxels consisting of the bull were exposed point-by-point, line-by-line, and layer-by-layer by a two-photon process. As a result, it took 3 hours to complete the manufacturing. If we make a detailed analysis on the bull structure, it is found that the entire bull consists of 2 106 voxels. However, the bull profile can be well defined with only 5% of them. As a test, the bull was written once more by using the vector scanning. Astonishingly, we find it possible to depict the same structure within 13 minutes (figure 4.12.b). In both cases the scanning step in three dimensions was 50 nm, the latter, however, the fabrication time in vector scan was reduced by more than 90%. The TPA produced bull crust was self-supported, standing on glass substrate either in liquid or in air. To avoid possible distortion, the structure has been further solidified under a mercury lamp, which is a single-photon exposure process.

4.3.2. Nanoimprint and Soft Lithography

Nano-Imprint lithography (NIL), introduced by S. Chou et al. [84], is an attractive technique for filling the resolution gap between conventional optical lithography and techniques exploiting the self-assembling capabilities that matter often shows at the nanoscale. This

FIGURE 4.13. Left. Scheme of the nanoiprint process. A hard master is embossed into thin film of a thermoplastics, above the temperature transition Tg. The sample is cooled down below Tg before stamp release in order to avoid deformation of embossed structures. Right: Example of microstructures in a thin termoplasitics (PMMA) film.

FIGURE 4.13. Left. Scheme of the nanoiprint process. A hard master is embossed into thin film of a thermoplastics, above the temperature transition Tg. The sample is cooled down below Tg before stamp release in order to avoid deformation of embossed structures. Right: Example of microstructures in a thin termoplasitics (PMMA) film.

low cost, high-resolution technique is based on a different principle from the main, already established, nanofabrication technologies. The process, depicted in figure 4.13, involves the hot embossing of a thin resist layer with a re-usable master etched in a hard material. In order to ensure low viscosity and easy flow of the resin during the embossing the temperature has to be raised to well above the glass transition temperature Tg of the polymer. The most common hot embossing process is carried out between the hot plates of a press, with a micro or nanostructured silicon substrate as a master and a silicon substrate spun with a thermoplastic resist layer like PolyMethylMethAcrylate (PMMA). Typical temperature and pressure parameters are in the range 150°C-240°C, 40-100 bars and imprinting times from 5 minutes to 1 hour. Nanoimprint is suitable for structuring large areas (8 inch substrates) in a single parallel process, and sub-10 nm resolution has already been demonstrated by several laboratories. Moreover, nanoimprint lithography is capable of replicating very accurately three-dimensional structures, which represents a strong competitive advantage over most of the existing lithographic techniques. Figure 4.14 shows an hexagonal array of micro lenses as an example of the 3D patterning potential of nano-imprint lithography. These arrays of micro lenses might find application to amplify fluorescent signals in the microflu-idic channels when integrated with microfluidic devices for bio-analytical applications. A comprehensive review of the nanoimprint technology is given in [85].

Despite great merits of nanoimprint, few problems are limiting its spreading as an industrial technology for medium to mass volume manufacturing. Main limitations are related to rheology, accurate registration, and short stamp lifetime. Rheological problems have been identified in the flow of high viscosity polymer melt and in the patterning of structures with complex topologies, where the polymer transport occurs in narrow channels and over long distances.

Alignment steps have to be performed whenever the fabrication of a device is obtained in a multi step lithographic process. With the increasing miniaturization trends the registration becomes more and more demanding. Pressure and temperature represent additional difficulties for the accurate registration, and sets special design requirements on tool and

Nanotechnology For Biomedical

stamp characteristics. Finally, the stamp is subject to contamination, and degradation due to the direct contact with a polymer coated substrate. This affects dramatically the number of possible imprinting cycles per stamp. A large international effort has been devoted to the resolution of all the above-mentioned problems.

The improvement of the polymer flow during embossing follows essentially very simple rules: the higher the temperature beyond the glass transition temperature (Tg) of the polymer, and the higher the imprinting pressure, the shorter is the imprinting time. These tendencies unfortunately are not always compatible with the industrial requirement in terms of throughput (due to the increase in the thermal cycling time) and overlay accuracy. Currently, the research has focused on the synthesis of new polymers, specially designed for the nanoimprint process, with the targets of reducing processing temperature (roughly 50-100 degrees above Tg), enhance the resistance to plasma as required for good pattern transfer and improving the stamp release. An alternative approach that overcomes the problems of thermal and pressure budget has gained attention in recent years. This approach consists of imprinting a monomer or a prepolymer fluid precursor of very low viscosity and then cure the film inside the mould cavities by thermal heating at a moderate temperature or using UV illumination.

This latter method is known as Step & Flash Lithography [86]. It requires the use a transparent mould as quartz for the UV curing of the polymer, which brings an additional benefit of greatly simplifies the alignment procedures. The polymer can be easily synthesized and engineered in order to minimize the viscosity of the precursor fluid and to obtain specific mechanical properties after cross-linking, in order to meet the optimal characteristics for subsequent pattern transfer by ion etching, lift-off or electroplating. A very well-known thermally or UV curable material, the polydimethylsiloxane (PDMS) has shown in particular convenient properties for application of nanoimprint and embossing techniques in the field of biomedicine. PDMS is a biocompatible, physiologically inert elastomer that is currently used to produce spare parts in human body (e.g. mammary prosthesis, contact lenses, intra-aortic balloon pumps). At the same time PDMS shows compatibility with classical microfabrication processes, and is a material of choice for micro and nano-fluidics, biochips, and optical components due to its transparency in the UV-visible spectral range. The process for structuring the PDMS, belonging to the class of techniques known as soft lithography [87], is applicable over a large range of dimensions, can be used to fabricate very high structures of resist (100 ^m thick) and is also capable of nanometric resolution. A comprehensive review of the use of PDMS in soft lithography is given in chapters 8 and 10 of reference [85].

4.3.3. Focused Ion Beam Lithography for 3 Dimensional Structures

Focused Ion Beam Lithography FIB is a very powerful technique for writing direct patterns on many substrates, it is a mask-less and resist-less technique that allows a wide range of applications which provide good resolutions (down to 50 nm) [88-90]. FIBs can be used to pattern materials with nanometer dimensions by ion implantation, ion exposure of resist, ion milling, gas-assisted etching, and ion-induced deposition of material. This permits us to apply this technique to a wide range of applications where other techniques are not permitted or are too difficult and complex. If we couple the FIB with a SEM facility (figure 4.15) on the same chamber, it is possible to obtain prototype devices in a simple and fast way. In fact, the possibility to fabricate in the nanometer scale by the FIB and to align it, as well as to inspect in real time the final structure by the SEM,

Focused Ion Beam Milling
FIGURE 4.15. Scheme of a dual beam (focused ion beam and electron beam) lithographic system. The two beams cross at the working point allowing several different operations as the SEM inspection of a sample contemporarily to its milling by the ion beam (courtesy by Carl Zeiss SMT).

Focused ions

Metal atom sputtered away

Gas molecules

Dissociated ^^ species removed

Deposited metal atoms

Spontaneous desorption

Substrate

Metal atom sputtered away

Gas molecules

Spontaneous desorption

Dissociated ^^ species removed

Deposited metal atoms

Substrate

mxif&stit*

Dissociated elements remaining as impurities

FIGURE 4.16. Scheme of focused ion beam induced etch. The milling action of the FIB is enhanced by the presence of ecthing gasses following those main steps: adsorption of the gas molecules on to the substrate surface, activation of a chemical reaction of the gas molecules with the substrate by the ion-beam generation of volatile reaction products (as GaCl3, SiCl4, SiF4, etc.), evaporation of volatile species and sputtering of non volatile species. (courtesy by Carl Zeiss SMT).

allows us to save a lot of time and to optimize all the parameter of the process. One of the most attractive applications is the fast prototyping of 3-dimensional structures for biomedical application. This technology has attracted an increasing interest as a novel tool to control the bio-medical process in the nanometric range. In order to achieve this kind of device, it is necessary to create structures with sub-micron dimensions in 3 dimensionally. Most implementations have been made using direct Electron Beam Lithography (EBL) and carefully optimized reactive ion etching processes (RIE), but they don't provide real 3D structures.

In Focus Ion Beam patterning, a focoused ion beam accelerated to an energy of about 10-100 KeV that is focalized by a magnetic lens on a sample in a vacuum chamber (106 mbar). The diameter of the focused beam spot is about 5-7 nm wide, with which one can write on a substrate sample by many process with almost the same resolution. These high energetic ions (normally Ga+ ions), coming from a liquid source, when interacting with the sample matter produce a complex series of phenomena. Principally the high mass of the particle produces elastic exchange of energy to the nuclei in the bulk. The excitation of those nuclei can cause their release out of the sample. In the vacuum environment they can be removed creating a hole in the substrate. This process is known as Ion Milling. If in the chamber there are some particular gases, they can interact with the same excited surface. These gasses can deposit some molecules and cause the process of FIB Induced Deposition (figure 4.16) or they can enhanced the physical milling effect by a chemical etching process and FIB Gas Assisted Ecthing (FIBGAE) as shown in figure 4.17. We have been applying all such process for 3D fabrication for fabricating wide varies of structures for various

Substrate atoms sputtered away

Focused ions

Volatile species

Incoming gas molecules

Deposited Adsorbed gas metal atoms ^ molecules

Substrate focused ions implanted in the substrate

FIGURE 4.17. Scheme of focused ion beam induced deposition: adsorption of the precursor molecules on the substrate, ion beam / e-beam induced dissociation of the gas molecules, deposition of the material atoms and removal of the organic ligands. (courtesy by Carl Zeiss SMT).

applications including biomedical. Few of the fabricated devices and structure using FIB includes:

• Micro-lens on top of fiber tip

• Bio sensors devices

• Bio actuator devices

• Microfluidic micro channel

• 3-Dimensional structures

4.3.3.1. Micro-lens on Top of Fiber Tip Optical coupling using conventional optical elements placed at the Single Mode Fiber (SMF) end or between the fibre and the waveguide suffers with a poor coupling efficiency primarily due to optical axis alignment mismatch and optical mode mismatch. In order to improve the coupling efficiency between SMF and the optical elements, a large mode active optical device over the whole length of an active device is fabricated. Among such techniques, tapering the exit end of SMF or formation of a micro-ball with higher refractive index melt on fiber end are commonly adopted so as to enhance the numerical aperture of the fiber. However, the coupling losses are still large. In the last few years, various mode matching techniques using spot size transformers have been proposed and demonstrated [91-94]. The function of the mode transformer is to alter the shape and size of the beam from the active device to closely match that of the waveguide. Diffractive Optical Elements (DOEs) with continous relief fabricated as an optical mode

FIGURE 4.18. In some condition is difficult to work with "standard" lithographies. A on-fibre micro-lens for photonics application has been fabricated directly milling the head of an optical fibre. Designed microlens parameters: lens curvature: 28.5 |m, lens diameter: 10 |m; focal length: 58.6 |m, sag height, 1.0 |m; working under wavelength of 1550 nm.

FIGURE 4.18. In some condition is difficult to work with "standard" lithographies. A on-fibre micro-lens for photonics application has been fabricated directly milling the head of an optical fibre. Designed microlens parameters: lens curvature: 28.5 |m, lens diameter: 10 |m; focal length: 58.6 |m, sag height, 1.0 |m; working under wavelength of 1550 nm.

converter to achieve efficient mode matched coupling had been fabricated on top of the fibre tip by e-beam lithography in a polymeric material and was reported by us recently [95]. The role of this element was to focus and shape the beam exiting the fiber into a desired intensity distribution. We adopted FIB technology to derive the required continuous relief in a DOE-lens element for its use as an optical mode converter fabricated on top-of-tip of the cleaved single mode fiber. The most obvious advantage of FIB is the simple procedure, with no need for pattern exchange from resist to substrate and direct milling of the required pattern on the substrate. Therefore, it is easier to control the relief form. In the FIB milling function, the high energy and small spot size of the ion beam (few nanometers) impinging the sample surface and facilitates easy material removal. The details of FIB fabrication of microlens on top of fibre tip by FIB and its characterisation was published recently [96]. FIB fabricated microlens profile on fibre tip is shown in figure 4.18. Designed microlens parameters were as follows: lens curvature: 28.5|im, lens diameter: 10|im; focal length :58.6|im, sag height, 1.0|im; working for wavelength of 1550 nm.

4.3.3.2. Nano-gaps for Molecular Conductivity Measurements The use of biological materials in the field of electronic or in the study of their physical property is a very new filed of application (electrical property like resistivity, thermal conductivity etc.). Sometime it is hard to apply a voltage to the extreme parts of a molecule or even cell. For example, to connect the two extremities of DNA chains in very narrow terminals would be required. In figure 4.19 we see the connection pad, made by electron beam lithography separated by a very small cut made by FIB milling. The dimension of the cut is around 10 nm and it was made by a very weak ion current (5 nA) and in a very short time. This is a very small distance not easy to be obtained by other technique; it is the desired dimension to be used in many experiments. Putting a molecule in between is not easy but possible and many bio-physical properties are now available to study [97].

FIGURE 4.19. Example of device fabrication using a Focused Ion Beam Milling: the electrical circuit and the connection pads, made by electron beam lithography, are separated by a very small cut made by FIB milling.

4.3.3.3. Bio Thermometer Devices Measuring the temperature is a very easy process. We can measure by using many principles applicable in different environment (mercury thermometer, thermocouple, liquid crystal thermometer, resistance thermometer RTD). But to enter inside a cell and check how the heating distribution changes during biological processes requires a special micro-structured device. A micropipette can enter cells without destroying too much; a nano-thermocouple or a nano-RTD detector could be fabricated on the cylindrical extremity of it (the final diameter could be around 1 micron). All those devices are already fabricated in small dimensions but only on a large and flat substrate. As stated before, the main characteristic of the FIB technique is the non required standard substrate. By using Ion milling as well as FIB Assisted Deposition it is possible to pattern a conductive circuit on the glass cylindrical lateral surface of the micro pipette as shown in figure 4.20. Few hundredsof a nanometer are enough to create a sufficient resistor sensitive at the temperature change. In this way, we can detect with precision (of less than 1 micron) where a cell produces heat and how much. In the same way is possible to fabricate a thermo detector and a localized heating system on an Atomic Force Microscope (AFM) tip. In

FIGURE 4.20. By using Ion milling as well as FIB Assisted Deposition it is possible to pattern a conductive circuit on the glass cylindrical lateral surface of the micro pipette. It was used as a intra-molecular thermometer.

FIGURE 4.20. By using Ion milling as well as FIB Assisted Deposition it is possible to pattern a conductive circuit on the glass cylindrical lateral surface of the micro pipette. It was used as a intra-molecular thermometer.

FIGURE 4.21. Example of device fabrication using a Focused Ion Beam Induced Deposition: a platinum induced deposition was used to add a thermometer and an heating devices on an AFM tip.

figure 4.21 shows a modified silicon tip and a resistance which can produce heating when a current passes, in addition a weak resistance at the side which can detect the real temperature; all of these can be used during the normal functioning of the topological imaging process of the AFM. It is a powerful instrument to study the shape of biological samples while obtaining information about heating distribution and deformation.

4.3.3.4. Bio Actuator Devices, Micro Tweezers Another particular application is the fabrication of mechanical tweezers on the top of a micro pipette. That's the case of a real actuator the can act directly at 1 micron range driven by electrical signals. This device is made by using a precursor gas diffused in the vacuum chamber contemporary at the Ion beam interaction with the substrate [98]. Three-dimensional (3D) nanostructures on a glass capillary have a number of useful applications such as manipulators, actuators, and sensors in the various microstructures. It was demonstrated a phenomenon that two diamondlike-carbon pillars on a tip of glass capillary fabricated by 30 keV Ga+ focused-ion-beam-chemical vapor deposition (FIB-CVD) with a precursor of phenanthrene vapor were able to work as a manipulator during FIB irradiation. It clearly works as 3D nanomanipulator and actuator, as it was demonstrated, by applying voltage onto an Au-coated glass capillary. In figure 4.22 is shown the principle of nano actuator and nanosensors and an example of nano tweezers.

4.3.3.5. 3-Dmensional Structures There are many different shapes that are made possible by FIB milling as well as FIB-CVD and FIB-GAE [99]. In figure 4.23 some of them and, particularly, a series of important diamond or amorphous carbon structures are shown, together with an array of metallic pins for capturing particles and SiO2 micro box to hold cells. A complete list of 3D shapes is unlimited. It is only a matter of time and fantasy to find good applications for such a flexible technique. It should be remembered that it is a serial writing system, there is only one beam that writes sequentially all the patterns. Comparing the parallel lithographic system (like the optical lithography) all the serial systems are slower (not good for mass production) but incredibly more flexible (very good for the prototyping). The techniques FIB should represent one of the highest levels

Fib Fabrication

FIGURE 4.22. Example of device fabrication using a Focused Ion Beam Induced Deposition: SIM observation of two pillars moving during FIB irradiation. The surface of glass capillary was not coated by Au sputter. a) Two pillars did not move without FIB irradiation. b) Two pillars opened with FIB irradiation. c) Illustration of moving mechanism due to charge repulsion. (courtesy by Kometani et al. [98]).

FIGURE 4.22. Example of device fabrication using a Focused Ion Beam Induced Deposition: SIM observation of two pillars moving during FIB irradiation. The surface of glass capillary was not coated by Au sputter. a) Two pillars did not move without FIB irradiation. b) Two pillars opened with FIB irradiation. c) Illustration of moving mechanism due to charge repulsion. (courtesy by Kometani et al. [98]).

of flexibility and associate it with a SEM facility, which constitutes the higher level for the fast prototyping of micro and nano structures.

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