**5. Characterization of contact lenses**

Introduction of effective improvements in production quality as well as therapy efficiency requires the application of non-destructive surface analysis methods on the nanometer scale with minimal sample preparation. Many interesting studies have been performed on CL (Lim et al., 2001), (Kim et al., 2002), (Bruinsma et al., 2003), so that two research approaches can be perceived from the literature: one aimed at surface modification and the other that was concerned with varying or simulating exploitation parameters. In both cases morphology of CL surface served as the main parameter used to describe behavior and quality of CL material under deterioration influences.

**Production engineering aspects**. Conformation states of polymers constituting CL surface are changed during final stages of manufacturing process (polishing) which presents a complex problem because surface molecules and their orientation influence the final state of surface quality. In general, over-polished internal surface renders a dysfunctional product since it prevents adequate adherence to corneal tissue. CL with over-polished surface slides over the cornea and cannot maintain its initial position therefore disrupting the geometry and function of the optical system (human eye + CL). However, too rough a surface will eventually lead to irritation and possibly damage of corneal tissue.

**Exploitation aspects**. During CL exploitation a lacrymal film is formed between inner CL surface and cornea. A good fit of CL and cornea depends on the adequacy of CL geometry and surface roughness. Moreover, every single RGP CL wearer provides a unique ambient conditions in which these CL biosurfaces have to function properly. Since CL surfaces become significantly rougher after prolonged wear, they become more prone to bacterial adhesion and protein and lipid deposits. CL can lose functionality due to accumulated proteins, lipids, and other tear components on CL surface, despite routine cleaning activities. The loss of RGP CL functionality needs to be investigated and related to measurable parameters in order to recommend replacement based on significant changes in surface properties.

### **5.1 Multimodal, complementary and non-invasive approach**

Answering the questions of optimal processing parameters directly influences surface quality of CLs which in turn reaches all aspects of its functionality: optical, medical and patient-comfort. Since our measurement confirmed that surface roughness of end-product ranges in the order of several tens of nanometers, the adequate tools for such samples must be derived from applied nanotechnology.

Experimental design, aimed at quantifying surface quality on the level of nano scale, utilized three methods of characterization that are meant to complement each other in interpreting and quantifying the measurement results. Moreover, we have applied a new method that is concerned with obtaining magnetic properties of samples from the interaction of material with visible light, named Opto-Magnetic Fingerprint of matter (OMF), with the aim to improve the speed and accuracy of analysis. Testing the product quality, reliabely and accurately, in production environment is an essential characteristic of maintaining high level of product quality control.

We have chosen to investigate near-surface magnetic and optical properties of chosen CL materials since they are the physical quantities that most closely correlate with subtle modifications in material structure and composition. Therefore, we investigated five types of CL materials (two conventional and three nanophotonic materials) by selected three approaches (techniques):


The experiment was designed to provide proofs for three interrelated phenomena:


All three methods possess very high sensitivity (nanonewton for forces, nanotesla for magnetization), which is a necessity for this type of measurements.

### **5.2 Ultraviolet-visible spectroscopy**

6 Will-be-set-by-IN-TECH

be perceived from the literature: one aimed at surface modification and the other that was concerned with varying or simulating exploitation parameters. In both cases morphology of CL surface served as the main parameter used to describe behavior and quality of CL material

**Production engineering aspects**. Conformation states of polymers constituting CL surface are changed during final stages of manufacturing process (polishing) which presents a complex problem because surface molecules and their orientation influence the final state of surface quality. In general, over-polished internal surface renders a dysfunctional product since it prevents adequate adherence to corneal tissue. CL with over-polished surface slides over the cornea and cannot maintain its initial position therefore disrupting the geometry and function of the optical system (human eye + CL). However, too rough a surface will eventually lead to

**Exploitation aspects**. During CL exploitation a lacrymal film is formed between inner CL surface and cornea. A good fit of CL and cornea depends on the adequacy of CL geometry and surface roughness. Moreover, every single RGP CL wearer provides a unique ambient conditions in which these CL biosurfaces have to function properly. Since CL surfaces become significantly rougher after prolonged wear, they become more prone to bacterial adhesion and protein and lipid deposits. CL can lose functionality due to accumulated proteins, lipids, and other tear components on CL surface, despite routine cleaning activities. The loss of RGP CL functionality needs to be investigated and related to measurable parameters in order to

Answering the questions of optimal processing parameters directly influences surface quality of CLs which in turn reaches all aspects of its functionality: optical, medical and patient-comfort. Since our measurement confirmed that surface roughness of end-product ranges in the order of several tens of nanometers, the adequate tools for such samples must

Experimental design, aimed at quantifying surface quality on the level of nano scale, utilized three methods of characterization that are meant to complement each other in interpreting and quantifying the measurement results. Moreover, we have applied a new method that is concerned with obtaining magnetic properties of samples from the interaction of material with visible light, named Opto-Magnetic Fingerprint of matter (OMF), with the aim to improve the speed and accuracy of analysis. Testing the product quality, reliabely and accurately, in production environment is an essential characteristic of maintaining high level of product

We have chosen to investigate near-surface magnetic and optical properties of chosen CL materials since they are the physical quantities that most closely correlate with subtle modifications in material structure and composition. Therefore, we investigated five types of CL materials (two conventional and three nanophotonic materials) by selected three

1. **Classical methods**: spectroscopic examination in the region of ultraviolet and visible light

2. **Nanotechnology based methods**: Phase-Contrasted Atomic Force Microscopy (PC-AFM)

3. **Nanomedicine based methods**: Novel method named Opto-Magnetic Fingerprint (OMF) that obtains magnetic properties of materials on the basis of interaction with visible light.

The experiment was designed to provide proofs for three interrelated phenomena:

with extended mode of Magnetic Force Microscopy (MFM).

recommend replacement based on significant changes in surface properties.

**5.1 Multimodal, complementary and non-invasive approach**

be derived from applied nanotechnology.

quality control.

approaches (techniques):

(UV-Vis spectroscoscopy).

under deterioration influences.

irritation and possibly damage of corneal tissue.

In order to investigate optical properties of RGP CLs, we have performed spectroscopic measurements in the range of ultraviolet-visible (UV-Vis) light in the range of wavelengths between 280–800 nm, using the UV-Vis scanner produced by Horiba JobinYvon, USA. The measurements were conducted in diffuse reflectance regime and the result is displayed as a graph of reflected energy vs. wavelength of emitted radiation. The spectroscopic measurements are used as a reference guide and were performed on two locations: the central point on the outer surface and the same point measured from inner surface in order to determine the difference and hence the degree of eye protection to different ranges of wavelengths and radiation intensities.

### **5.3 Phase-contrasted atomic force microscopy – PC-AFM**

Morphology of CL surfaces was obtained by atomic force microscope (PC-AFM) that can measure sample surface roughness with high precision (less than 10−12*<sup>m</sup>* or <sup>≈</sup> <sup>1</sup>*pm* and confirm sample surface state as belonging to either group adequate or inadequate roughness. Topography of lens surface is important for determining the connection between surface morphology (conformational states of surface layer polymers) and corresponding optical properties that are influenced by the processing parameters.

Basically, AFM is a scanning probe microscopy technique based on point-to-point examination of the specimen made by a sharpened tip probe (Binnig et al., 1986). AFM probe is a micro-cantilever with sharpened conical or pyramidal tip whose radius can range from 2 – 90 nm, depending on the application. All samples were imaged using phase-contrast technique in tapping mode and in ambient air. The AFM system used in this study was JSPM-5200, JEOL, Japan. The cantilever were type PPP-MFMR, produced by Nanosensors, Switzerland.

The principle of operation of AFM is shown in figure 3. In AFM, the probe (cantilever tip) is vibrated at near-resonant frequency and brought ino interaction with the sample by the mechanism of intermolecular attractive/repulsive forces that are distance-dependent. The cantilever is maintained in the close proximity from the sample so that probe tip is within reach of attractive/repulsive forces. A typical AFM system is able to detect intermolecular forces in the order of 10−<sup>11</sup> <sup>−</sup> <sup>10</sup>−13*<sup>N</sup>* which makes it an extremely sensitive device. The intermolecular interactions belong to the class of van der Waals type forces, usually modeled by Lennard-Jones potentials. These forces cause the cantilever to deflect from the initial equilibrium position making it possible to derive the distance from the sample on the basis of force field gradient change that modulates the vibration of the cantilever.

A diode laser is directed to the back sided surface of the tip and is reflected to photo-diode detector. During the scanning movement, the angle of reflected beam is changed due to deflection of the cantilever (that is, in turn, due to interactions with the sample) and this movement is precisely recorded via photo-diode detector. A feedback loop is used to adjust the z-position of the sample so that a constant interaction is maintained as the tip is scanned across the sample in x and y directions. The tip-sample interaction equilibrium is constantly disrupted by changes in sample profile height which generates the control signal applied

Fig. 3. Principle of operation of atomic force microscope.

to the z-scanner stage. This disturbance represents the changes in surface topography and the action of the control system is recorded and represents the actual data represented on topography image of the sample.

The PC-AFM imaging technique is based on the fact that intermolecular force gradients have certain physico-chemical specificity. The variations in the sample generate variations in the slopes of Lennard-Jones potential curves because of the different intermolecular forces acting on the AFM sensor tip. These differences modulate the vibration amplitude of the AFM cantilever, creating a higher-harmonics in the feedback signal of AFM system. By recording higher harmonics in the oscillation signal (see 3) we can detect different force-distance relationships, hence, different components of a material can be visualized. However, the PC-AFM signal correlation to separate chemical species is still unknown, which disables the AFM from making exact chemical characterization of the sample. The data obtained by PC-AFM are rather of a qualitative nature and are suitable for two or three component systems.

### **5.4 Magnetic Force Microscopy – MFM**

Magnetic Force Microscopy is an extended operation mode of AFM that was used to obtain the distribution of magnetic properties of the surface that are previously imaged with topography mode. This tecnique was used to measure the magnetic properties in para- and diamagnetic range because we are also probing the magnetic properties in the same range with a novel technique that is introduced in the next section (Opto-Magnetic Fingerprint). MFM will is used as a comparative method for OMF. The MFM technique utilizes special cantilever sensors that are coated with a thin film of cobalt (≈ 50*nm*) that renders its ferromagnetic properties and ensures magnetic interaction with the sample.

8 Will-be-set-by-IN-TECH

to the z-scanner stage. This disturbance represents the changes in surface topography and the action of the control system is recorded and represents the actual data represented on

The PC-AFM imaging technique is based on the fact that intermolecular force gradients have certain physico-chemical specificity. The variations in the sample generate variations in the slopes of Lennard-Jones potential curves because of the different intermolecular forces acting on the AFM sensor tip. These differences modulate the vibration amplitude of the AFM cantilever, creating a higher-harmonics in the feedback signal of AFM system. By recording higher harmonics in the oscillation signal (see 3) we can detect different force-distance relationships, hence, different components of a material can be visualized. However, the PC-AFM signal correlation to separate chemical species is still unknown, which disables the AFM from making exact chemical characterization of the sample. The data obtained by PC-AFM are rather of a qualitative nature and are suitable for two or three component

Magnetic Force Microscopy is an extended operation mode of AFM that was used to obtain the distribution of magnetic properties of the surface that are previously imaged with topography mode. This tecnique was used to measure the magnetic properties in para- and diamagnetic range because we are also probing the magnetic properties in the same range with a novel technique that is introduced in the next section (Opto-Magnetic Fingerprint). MFM will is used as a comparative method for OMF. The MFM technique utilizes special cantilever sensors that are coated with a thin film of cobalt (≈ 50*nm*) that renders its ferromagnetic properties

Fig. 3. Principle of operation of atomic force microscope.

topography image of the sample.

**5.4 Magnetic Force Microscopy – MFM**

and ensures magnetic interaction with the sample.

systems.

Fig. 4. Principle of operation of atomic force microscope.

This thin film creates magnetic interaction with the sample that is recorded via two-pass technique or "lift scanning"(see 4) during which the sample is scanned twice, with second scan being performed with a gap distance in order to physically filter the slowly decaying magnetic from all other more rapidly decaying intermolecular forces. The sensitivity of magnetic force measurements goes around 10−11*N* which is able to detect very weak magnetic interaction that encompasses para- and diamagnetic range. The cantilevers which we used in this study are produced by Nanosensors (Switzerland), with a lateral resolution under 50 nm and coercivity of 300 Oe.

The cantilever has been magnetized previously to the measurement in order to ensure its magnetic interaction with the sample. Although MFM, in its current stage of development, still represents a qualitative measurement of magnetic properties, we attempted to improve the accuracy of this analysis by measuring the remnant magnetization of the cantilever, using JR-5 spinner magnetometer (AGICO, Brno, Czech Republic), and identified that our series of measurements fitted within the range of 72 ± 2*nT*, with a standard deviation of 0.3%. The measured axis of cantilever remnant magnetization was positioned in the vertical direction, so it was perpendicular to sample surface. The data on components of vectors of remnant magnetization enabled us to confirm that we have measured the magnetic field component that is perpendicular to the sample surface.

### **5.5 Opto-Magnetic Fingerprint – OMF**

OptoMagnetic Fingerprint (OMF) is a novel method in investigating optical and magnetic properties of materials that is based on electron properties of matter (covalent bonds, hydrogen bonds, ion-electron interaction, van der Walls interaction) and its interaction with light (Koruga et al., 2008). The method was originally developed for early skin cancer and melanoma detection by MySkin, Inc., USA (Bandi´c et al., 2002). Bearing in mind that the orbital velocity of valence electron in atoms is about 106*m*/*s*, this gives the ratio between magnetic force (*FM*) and electrical force (*FE*) of matter, of around *FM FE* <sup>≈</sup> <sup>10</sup>−4. Since force (F) is directly related to quantum action – Planck's action – defined as:

$$h = F \times d \times t = 6.626 \times 10^{-34} \text{Js} \tag{1}$$

where F is force, d is displacement and t is time of action) this means that the action of magnetic forces is four orders of magnitude closer to quantum action than the electrical ones. Since quantum state of matter is primarily responsible for conformational changes on the molecular level, this means that detecting differences between matter states is by far more likely to give greater sensitivity on the level of magnetic forces than it would be on the level of measurement of electrical forces (Koruga et al., 2002).

Picture of surface that is taken by classical optical microscope is based on electromagnetic property of light, while OMF is based on difference between diffuse white light (like that of daily light) and reflected polarized light. Reflected polarized light is produced when source of diffuse light irradiates the surface of matter under certain angle (Brewster's angle, see figure 5). Each type of matter has different angle value of light polarization. Since reflected polarized light contains electrical component of light-matter interaction, taking the difference between white light (electromagnetic) and reflected polarized light (electrical) yields magnetic properties of matter based on light-matter interaction. Because such measurement can identify the conformational state and change in tissue on molecular level we named this method the opto-magnetic fingerprint of matter (OMF).

Fig. 5. Incident white light can give different information about thin layer of matter (surface) properties of sample depending on the angle of light incidence. When the incident white light is diffuse, the reflected white light is then composed of electrical and magnetic components, whereas diffuse incident light that is inclined under certain angle will produce reflected light which contains only electrical component of light. For each type of matter there is a characteristic angle of incidence (Bandi´c et al., 2002) for obtaining the appropriate reflected polarized light.

We used digital images in RGB (R-red, G-green, B-blue) system in our analysis, therefore we chose basic pixel data in red and blue channels for white diffuse light (W) and reflected polarized white light (P). Algorithm for data analysis is based on chromaticity diagram called "Maxwell's triangle"and spectral convolution operation according to ratio of (R-B)& (W-P) (Koruga et al., 2008). The abbreviated designation means that Red minus Blue wavelength of White light and reflected Polarized light are used in spectral convolution algorithm to calculate data for opto-magnetic fingerprint of matter. Therefore, method and algorithm for creating a unique spectral fingerprint are based on the convolution of RGB color channel spectral plots generated from digital images that have captured single and multi-wavelength light-matter interaction (Koruga et al., 2008). Preparation of digital pictures for OMF was made by usage of dermoscopic imaging device (MySkin, USA) that has previously been successfully used in biophysical skin characterization (skin photo type, moisture, conductivity, etc) (Bandi´c et al., 2002).

The final purpose of our research in applying OMF is the construction of quality control method which would be purely optical and able to, on the basis of digital image analysis and processing, detect both the morphology and functionality parameters in a quicker and more accurate manner. In order to do so we need to construct quantification parameters but, in this stage of research, primarily integrate results from morphological research and opto-magnetic properties.
