Electron Microscopy Optical Microscopy Biology Essay

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As there are two major phases collagen-apatite crystals in bone, chemical extractions were carried out to separate these two phases from each other in order to study the chemical-physical properties of bone. Bovine femur was obtained from local source. The animal age is at ~13 months. The tissue on the surface of the bone and bone marrow were removed. The femoral heads were also removed and only the compact bone was kept and was mechanically sliced at room temperature. The ring shape bone was then further reduced to the size of 5 mm x 5 mm x 1 mm squares by hand sawing and polishing on a series of abrasive paper. The cutting direction towards the bone axis was always marked (see Figure. 3.1). The thickness of the samples was kept at ~1 mm, so that it is easy for the chemicals to penetrate to the centre of the sample. An average weight of a bone square is 0.35 ± 0.05g and the total weight of 10 samples in each batch is 3.3 ± 0.16g. At this stage, the bone samples were ready for the chemical extractions.

Figure. 3.1. Procedure of obtaining bovine bone matrix

These plates were polished with a series of abrasive papers. For comparison, the cut samples were divided into three groups (A, B, C):

Group A - Raw bone;

Group B - Deproteinated bone;

Group C - Demineralised bone.

Many literatures have used the similar extraction methods to obtain the bone matrix. The details of the extraction procedure are as follow:

Bone Deproteination:

Ten bone samples were first rinsed with deionised water. They were put in a Fisher Pyrex coarse-fritted extraction thimble that fitted into the Soxhlet flask. A round-bottom flask with 100 mL of aqueous 80 per cent ethylenediamine (EDA) solution [1] was connected to the bottom of the Soxhlet flask and a water flow system was connected to the top of the Soxhlet flask. The procedure of refluxing took 20 - 30 cycles, ~50 hours at the temperature of 115 - 119°C in the oil bath, which is the boiling point of ethylenediamine. The extracted samples were then cooled to the room temperature and were rinsed with deionised water thoroughly until the reddish-brown amine agent was decanted. Another three times re-rinsing with cold deionised water was required and a continuous flow of water rinsed the samples to ensure the impurities were washed off. Finally, they were dried in an over at 80 °C overnight. Thus, the inorganic matrix of bone (apatite) was obtained (Figure 3.2 a). The average of the weight of the bone apatite from each batch was 2.86 ± 0.07 g.

Bone Demineralisation:

Another ten bone samples were first rinsed with deionised water and were then treated with prolonged agitation in 5% formic acid in an Erlenmeryer flask until the samples became transparent (usually 72 hours) [2]. This procedure was completed in the oil bath to help dissolve out the mineral component. After cooling to the room temperature, the samples were re-rinsed three times with deionised water and were washed with continuous flow of water until the samples were clean. Finally, they were dried in an oven at 80 °C overnight. Thus, the organic matrix of bone (mainly collagen) was obtained (Figure 3.2 b). The average of the weight of the bone collagen from each batch was 2.92± 0.06 g.

Bone Matrix with Orientations

For optimising the extractions, other solutions and procedures were also used in different batches of samples. It was found that the reported methods above were the most suitable methods for this experiment. To study the orientation dependent properties of bone, bone samples were cut with angles, i.e. 0°, 45°, and 90° to the bone axis. After the extraction methods were confirmed, the samples with orientation marks were extracted followed the procedures above. The successful samples were verified with the analytical methods, which will be discussed in the next sections.

Figure 3.2 Chemical extraction procedures of bovine bone (a) deproteination (b) demineralisation

3.3 X-Ray Diffraction

3.3.1 Principles and Theories of XRD

Following the discovery of X-ray by W. C. Röntgen in 1895, this radiation has been widely used in diagnostic methods for medicine and industry. The ability of X-ray to detect and to identify crystal materials is based on the fact that every crystalline substance gives a unique diffraction pattern when interacting with X-ray, even in a mixture of substances each produces its pattern independently of the others [3]. The X-ray diffraction pattern of a pure substance is, therefore, like a fingerprint of the substance. The powder diffraction method is thus ideally suited for characterisation and identification of polycrystalline phases.

Among all solid materials, 95% can be described as crystalline. 50,000 inorganic and 25,000 organic single components, crystalline phases, and diffraction patterns have been collected and stored on magnetic or optical media as standards. The main use of powder diffraction is to identify components in a sample by a search/match procedure. Furthermore, the areas under the peak are related to the amount of each phase present in the sample.

An electron in an alternating electromagnetic field will oscillate with the same frequency as the field. When an X-ray beam hits an atom, the electrons around the atom start to oscillate with the same frequency as the incoming beam. In almost all directions we will have destructive interference, that is, the combining waves are out of phase and there is no resultant energy leaving the solid sample. However the atoms in a crystal are arranged in a regular pattern, and in a very few directions we will have constructive interference. The waves will be in phase and there will be well defined X-ray beams leaving the sample at various directions. Hence, a diffracted beam may be described as a beam composed of a large number of scattered rays mutually reinforcing one another. This model is complex to handle mathematically, and in day to day work we talk about X-ray reflections from a series of parallel planes inside the crystal. The orientation and interplanar spacings of these planes are defined by the three integers h, k, l called indices. A given set of planes with indices h, k, l cut the a-axis of the unit cell in h sections, the b axis in k sections and the c axis in l sections. A zero indicates that the planes are parallel to the corresponding axis. E.g. the 2, 2, 0 planes cut the a- and the b- axes in half, but are parallel to the c- axis. Figure 3.2.1 below shows X-rays being reflected from a crystal. Each layer of atoms acts like a mirror and reflects X-rays strongly at an angle of reflection that equals the angle of incidence. The diagram shows reflection from successive layers. If the path difference between the beams from successive layers of atoms is a whole number of wavelengths, then there is constructive interference.

Figure 3.2.1 Bragg's law

If the three dimensional diffraction grating was used as a mathematical model, the three indices h, k, l become the order of diffraction along the unit cell axes a, b and c respectively. It should now be clear that, depending on what mathematical model, the terms X-ray reflection and X-ray diffraction as synonyms. Considering an X-ray beam incident on a pair of parallel planes P1 and P2, separated by an interplanar spacing d. The two parallel incident rays 1 and 2 make an angle (THETA) with these planes. A reflected beam of maximum intensity will result if the waves represented by 1' and 2' are in phase. The difference in path length between 1 to 1' and 2 to 2' must then be an integral number of wavelengths, (LAMBDA). This relationship can be mathematically expressed in Bragg's law.

The process of reflection is described here in terms of incident and reflected (or diffracted) rays, each making an angle THETA with a fixed crystal plane. Reflections occurs from planes set at angle THETA with respect to the incident beam and generates a reflected beam at an angle 2-THETA from the incident beam. The possible d-spacing defined by the indices h, k, l are determined by the shape of the unit cell. Rewriting Bragg's law is:

Therefore the possible 2-THETA values where reflections are determined by the unit cell dimensions. However, the intensities of the reflections are determined by the distribution of the electrons in the unit cell. The highest electron density is found around atoms. Therefore, the intensities depend on what kind of atoms and where in the unit cell they are located.

Planes going through areas with high electron density will reflect strongly, planes with low electron density will give weak intensities.

3.4 FT-IR Spectroscopy and mapping

3.4.1 Principles and Theories of FT-IR

Infrared (IR) radiation refers broadly to that part of the electromagnetic spectrum between the visible and microwave regions. Infrared spectroscopy is the measurement of the wavelength and intensity of the absorption of mid-infrared light by a sample. Mid-infrared is energetic enough to excite molecular vibrations to higher energy levels [4]. The wavelength of infrared absorption bands is characteristic of specific types of chemical bonds, and infrared spectroscopy finds its greatest utility for identification of organic and organometallic molecules. The high selectivity of the method makes the estimation of an analyte in a complex matrix possible. This method involves examination of the twisting, bending, rotating and vibrational motions of atoms in a molecule. Survey spectra in the mid-infrared region are often measured at a resolution of ~ 4 cm-1. When such spectra between 4000 and 400 cm-1 are measured with a prism or grating monochromator, only one 4-cm-1 resolution element in the 3600-cm-1 wide spectral range of interest is measured at any instant; the remaining 899 resolution elements are not. Thus, the efficiency of the measurement is only about 0.1%. It was typical for survey scans to take several minutes to measure, whereas the measurement of archival-quality spectra (measured at 1 to 2 cm-1 resolution) often took at least 30 minutes [5].

Infrared spectroscopy has been a workhorse technique for materials analysis in the laboratory for over seventy years. An infrared spectrum represents a fingerprint of a sample with absorption peaks which correspond to the frequencies of vibrations between the bonds of the atoms making up the material. Because each different material is a unique combination of atoms, no two compounds produce the exact same infrared spectrum [6]. Therefore, infrared spectroscopy can result in a positive identification (qualitative analysis) of every different kind of material. In addition, the size of the peaks in the spectrum is a direct indication of the amount of material present. With modern software algorithms, infrared is an excellent tool for quantitative analysis [5].

FT-IR stands for Fourier Transform InfraRed, the preferred method of infrared spectroscopy. In infrared spectroscopy, IR radiation is passed through a sample. Some of the infrared radiation is absorbed by the sample and some of it is passed through (transmitted). The resulting spectrum represents the molecular absorption and transmission, creating a molecular fingerprint of the sample. Like a fingerprint no two unique molecular structures produce the same infrared spectrum. This makes infrared spectroscopy useful for several types of analysis. In FT-IR spectrometry, all the resolution elements are measured at all times during the measurement (the multiplex or Fellgett's advantage). In addition, more radiation can be passed between the source and the detector for each resolution element (the throughput or Jacquinot's advantage) [4]. As a result, transmission, reflection, and even emission spectra can be measured significantly faster and with higher sensitivity than ever before.

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Figure 3.3.1 Potential energy of a diatomic molecule as a function of the atomic displacement during a vibration for a harmonic oscillator (dashed line) and an anharmonic oscillator (solid line).

Infrared spectra result from transitions between quantized vibrational energy states. Molecular vibrations can range from the simple coupled motion of the two atoms of a diatomic molecule to the much more complex motion of each atom in a large polyfunctional molecule. Molecules with N atoms have 3N degrees of freedom, three of which represent translational motion in mutually perpendicular directions (the x, y, and z axes) and three represent rotational motion about the x, y, and z axes. The remaining 3N- 6 degrees of freedom give the number of ways that the atoms in a nonlinear molecule can vibrate (i.e., the number of vibrational modes). Each mode involves approximately harmonic displacements of the atoms from their equilibrium positions; for each mode, i, all the atoms vibrate at a certain characteristic frequency, vi. The potential energy, V(r), of a harmonic oscillator is shown by the dashed line in Figure 3.3.1 as a function of the distance between the atoms, r. For any mode in which the atoms vibrate with simple harmonic motion (i.e., obeying Hooke's law), the vibrational energy states, Viv, can be described by the equation [4]

Eq (3.3.1)

where h is Planck's constant, vi the fundamental frequency of the particular mode, and υi the vibrational quantum number of the ith mode (υi = 0, 1, 2, . . .). Note that frequency in units of hertz is usually given the symbol υ. Vibrational frequencies are often given in units of wavenumber, the number of waves per unit length. The most common unit of length is the centimetre, in which case the wavenumber has units of cm-1 and is given the symbol ύ by many chemists and s by many physicists.

The energy difference for transitions between the ground state (vi =0) and the first excited state (vi =1) of most vibrational modes corresponds to the energy of radiation in the mid-infrared spectrum (400 to 4000 cm-1). The motion of the atoms during the vibration is usually described in terms of the normal coordinate, Qi. The molecule is promoted to the excited state only if its dipole moment, μ, changes during the vibration [i.e., provided that ]. For molecules with certain elements of symmetry, some vibrational modes may be degenerate, so that more than one mode has a given vibrational frequency whereas others may be completely forbidden. Thus, because of degeneracy, the number of fundamental absorption bands able to be observed is often less than 3N - 6. Because rotation of a linear molecule about the axis of the bond does not involve the displacement of any of the atoms, one of the rotational degrees of freedom is lost and linear molecules have an additional vibrational mode. Thus, the number of modes of a linear molecule is 3N - 5, so that a diatomic molecule (N = 2) has a single vibrational mode. The actual variation of the potential energy as a function of the displacement of the atoms from their equilibrium positions is shown as a solid line in Figure 3.3.1. From this curve it can be seen that Eq. 3.3.1 is valid only for low values of the vibrational quantum number and is not valid when vi is large. In practice, Vvi must be described using an anharmonic (Morse-type) potential function. This behavior is shown in Figure 3.3.1 as a solid line, and the potential energy is given to a first approximation by the expression [4]

Eq. 3.3.2

where xi is the anharmonicity constant; xi is dimensionless and typically has values between -0.001 and -0.02, depending on the mode.

3.4.2 FT-IR of Bone, Collagen and Apatite

IR spectra have been used extensively to assess mineral crystallinity in synthetic apatites and normal and diseased bones [7-9]. The introduction of computerized Fourier transform (FT) IR spectrometersmade calculation of spectra parameters easier, and there are now numerous papers that rely on FTIR for characterization of bone mineral [10-13]. Coupling an IR spectrometer to a light microscope allowed FTIR spectra to be recorded and mapped at anatomically distinct portions in bone [14-16], as illustrated by the osteon, and its component spectra and spectral properties in Fig. 5.2. More recently, the use of an array detector has allowed IR images of the spectral features seen in Fig. 5.2 to be displayed in three dimensions [17]. The advantage of FTIR microspectroscopy or imaging is that changes in mineral properties can be mapped at a spatial resolution of -20 m. Thus, the variation in mineral quality and quantity across an osteon can be documented [18], and changes across developing bones going from the periosteum to the endosteum or across the trabeculae can be noted [19].

Bone consists of about 45-70 wt% mineral formed mostly of calcium phosphate (hydroxylapatite), 10 wt% water, and the remainder organic materials consisting principally type I collagen with smaller amounts of noncollagenous proteins and lipids [13]. The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and the water within the fibril is replaced with mineral [20]. While bone is a chemical reservoir for phosphorous, which is a life essential element, bone must possess the physical properties required for the functionality of the tissue, such as structural support [21]. Bone strength and fracture risk are generally assessed by measuring bone mineral density (BMD), however; the mechanical properties of bone are determined not only bone mass, but also by architecture= geometry of the bone and by the intrinsic material properties of the tissue. It is considered that bone strength is determined by 70% bone density and 30% bone quality. Bone quality is defined by at least four factors: (1) the rate of bone turnover; (2) properties of the collagen= mineral matrix; (3) microdamage accumulations; (4) architecture= geometry of trabecular and cortical bone [22].

Fourier transform infrared (FTIR) and Raman spectroscopy can provide molecular structure information about mineralized and non-mineralized connective tissue. In recent years, Boskey [23-25] and Morris [26, 27] have succeeded in determination of bone quality with microscopic FTIR, FTIR imaging and Raman spectroscopy without homogenization or stain. The Morris group has characterized micro crack in bone mineral using a newly developed Raman system. In 1997 the Boskey group focused on the state of collagen cross-links in bone and characterized nonreducible = reducible collagen cross-link ratio in disease bone matrix with microscopic FTIR and FTIR imaging. They also studied the changes in mineral and matrix content and composition in replicate biopsies of nonosteoporotic human cortical and trabecular bone and found that changes in osteonal bone in these same samples were reported previously. Spectral maps along and across the lamellae were obtained from iliac crest biopsies of two necropsy cases. Mineral: matrix ratios, calculated from the integrated areas of the phosphate n1, n3 band at 900-1200 cm−1 and the amide I band at ~1585-1725 cm−1, respectively, were relatively constant in both directions of analysis, i.e., along and across the lamellae. Analysis of the components of the υ1, υ3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. Of these, the ratio of the relative areas of the two underlying bands at ~1020 and ~1030 cm−1 has been shown to be a sensitive index of variation in crystal perfection in both human osteonal bone and in synthetic, poorly crystalline apatites. This ratio was calculated in both cortical and trabecular bone from human iliac crest biopsies along and across the lamellae. The protein and mineral constituents produce intense, structure-sensitive IR bands. The protein Amide I (peptide bond C = O stretch) and Amide II (mixed C-N stretch and N-H in-plane bending) modes near 1650 and 1550 wave numbers (cm−1) undergo wave number and intensity changes as a result of changes in protein secondary structure. The apatite phosphate υ3 (P-O asymmetric stretch) and υ4 (in-plane bending) modes have been used to monitor changes in mineral crystallinity [28, 29].

OoI et. al. [30] reported the properties of porous hydroxyapatite (HA) bioceramic produced by heat treatment (annealing) of bovine bone over temperatures between 400°C and 1200°C and revealed the characteristics of a natural bone with the interconnecting pore network being retained in the structure . Figure 3.3.2.shows the FTIR spectra of bovine bone heated from room temperature (RT) to 1200°C.

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Figure 3.3.2. FTIR spectra of bovine bone heated from room temperature (RT) to 1200 8C [30].

Bertazzo et. al. [31] reviewed the effect of of hydrazine deproteination on bone mineral phase. They found that several alterations in the IR spectra of bone after deproteination: all peaks are more narrow, peaks at 2920, 2860 and 1250 cm-1 are absent, and peaks at 870 and 961 cm-1 are more intense. The narrowing of peaks observed in the diffactograms of bones after deprotienation can be connected to the elimination of organic matrix. The presence of organic matrix caused peaks to appear wider in the spectra obtained before deproteination. Peaks at 2920, 2860 and 1250 cm-1, present in the spectrum of bone before deproteination (Figure. 3.3.3a), correspond to bands of organic matrix that are eliminated after deproteination. The peak at 961 cm-1, seen in Figure 3.3.3, is attributed to group HPO2-4 and the peak at 870 cm-1 to group CO2-3 , found in carbonated hydroxyapatites. The presence of such peaks in the spectra of bone both before and after deproteination shows that this process does not significantly eliminate those ions from bone mineral.

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Figure 3.3.3. Infrared spectra of bone, hydroxyapatite, CaHPO4 and CaCO3, before and after deprotienation [31].

Many more FT-IR results of bone have been reported by researchers through the decades. These results are summaries in Table 3.3.1.

Table 3.3.1. FT-IR data of Bone, Collagen and Apatite

Bone

Collagen

Apatite

Rat

Bovine

Bovine

Human

HAp

Rat

Bovine

Human

OH liberation

630[30]

630[30]

630[30]

632[32]

PO4 v4

560[30]

560[30]

650[33]

CO3 v4

870[31] 873[13]

856 wbr [34]

870[31]

CO3 v2

~870[35]

875[30]

PO4 v1

961[31]

962[30]

961.7 s [34]

961[31]

PO4 v3

or

CO3 v1

900-1200 (v1-v3) [13]

1049-1090[30]

1029 vs[34]

1060 sh [34]

1092 vs[34]

1960-2200[30]

500-1100[32]

1000-1060(C-O stretching) [30]

900-1200 (v1-v3) [33]

Amide III

?1250[31] 1240[13]

1231,1248,

1281[32]

CO3 v3

~1420[35]

1410,1450[30]

Amide II

?2860[31] 1550[13]

1554 s [30]

1521,1533,

1543[32]

1650 (mixed C-N stretch and N-H in-plane bending)[33]

Amide I

?2920[31]

1660[13]

2913 s (N-H stretching)[30]

1620,1636,

1685[32]

1585-1725[33]

1650 (C=O stretch)[33]

OH- ions

OH stretching

3570[30]

3567 m [34]

3572 [35]

3570[30]

3570[30]

~3570[35] 3569[32]

Code: vs; very strong, s; strong, m; medium, w; weak, vw; very weak, sh; shoulder, br; broad bands.[34]

3.5 Electron Microscopy

To understand a system, a general experimental approach is a scattering technique: some particles are shot in a well-prepared state on the target and look at particles coming out of the target (which do not have to be the same). The most common particles to scatter from surfaces are electrons, ions, atoms and photons both as probe and response particles. An important issue is the surface sensitivity of an experiment. In general, it is high if the particles were chosen which have a small mean free path in the solid because this means that the detected particles must originate near the surface. The opposite is true, for example, when the scattering of light by a surface is investigated (reflectivity and change of polarization). The photons will penetrate relatively deeply into the crystal. The amount of photons scattered at or near the surface will be very small. Hence, light scattering is not a good tool to study surfaces. In some cases the surface sensitivity can be increased by choosing an experimental set-up where a very grazing angle of incidence or emission is used. In this way the particles travel a long way close to the surface, even if their mean free path is relatively long.

Electron spectroscopy is an analytical technique to study the electronic structure and its dynamics in atoms and molecules. Very many surface science techniques are based on electrons as a probe. Electrons have very useful properties: they are, at certain energies, very surface sensitive. Electrons in this energy range carry also enough momentum to explore the whole surface Brilloin zone of a material (in contrast to light), they also carry a spin and they are easy to generate and to handle. Experimental applications include high-resolution measurements on the intensity and angular distributions of emitted electrons as well as on the total and partial ion yields. Ejected electrons can escape only from a depth of approximately 3 nanometres or less, making electron spectroscopy most useful to study surfaces of solid materials. Depth profiling is accomplished by combining an electron spectroscopy with a sputtering source that removes surface layers.

3.5.1 Optical Microscopy

Optical microscopy is a means of studying the properties of physical objects based on measuring how an object emits and interacts with light. It can be used to measure attributes such as an object's chemical composition, temperature, and velocity. It involves visible, ultraviolet, or infrared light, alone or in combination, and is part of a larger group of spectroscopic techniques called electromagnetic spectroscopy. Optical microscopy is an important technique in modern scientific fields such as chemistry and astronomy.

An object become visible by emitting or reflecting photons, and the wavelengths of these photons depend on the object's composition, along with other attributes such as temperature. The human eye perceives the presence and absence of different wavelengths as different colours. For example, photons with a wavelength of 620 to 750 nanometres are perceived as red, and so an object that primarily emits or reflects photons in that range looks red. Using a device called a spectrometer; light can be analyzed with much greater precision. This precise measurement-combined with an understanding of the different properties of light that different substances produce, reflect, or absorb under various conditions-is the basis of optical spectroscopy. Different chemical elements and compounds vary in how they emit or interact with photons due to quantum mechanical differences in the atoms and molecules that compose them. The light measured by a spectrometer after the light has been reflected from, passed through, or emitted by the object being studied has what are called spectral lines. These lines are sharp discontinuities of light or darkness in the spectrum that indicate unusually high or unusually low numbers of photons of particular wavelengths. Different substances produce distinctive spectral lines that can be used to identify them. These spectral lines are also affected by factors such as the object's temperature and velocity, so spectroscopy can also be used to measure these as well. In addition to wavelength, other characteristics of the light, such as its intensity, can also provide useful information.

Figure. 3.4.1

Optical microscopy can be done in several different ways, depending on what is being studied. Individual spectrometers are specialized devices that focus on precise analysis of specific, narrow parts of the electromagnetic spectrum. They therefore exist in a wide variety of types for different applications. One major type of optical spectroscopy, called absorption spectroscopy, is based on identifying which wavelengths of light a substance absorbs by measuring the photons it allows to pass through. The light can be produced specifically for this purpose with equipment such as lamps or lasers or may come from a natural source, such as starlight. It is most commonly used with gases, which are diffuse enough to interact with light while still allowing it to pass through. Absorption spectroscopy is useful for identifying chemicals and can be used to differentiate elements or compounds in a mixture. This method is also extremely important in modern astronomy and is often used to study the temperature and chemical composition of celestial objects. Astronomical spectroscopy also measures the velocity of distant objects by taking advantage of the Doppler Effect. Light waves from an object that is moving toward the observer appear to have higher frequencies and thus lower wavelengths than light waves from an object at rest relative to the observer, while the waves from an object that is moving away appear to have lower frequencies. These phenomena are called blue shift and red shift, respectively, because raising the frequency of a wave of visible light moves it toward the blue/violet end of the spectrum, while lowering the frequency moves it toward red.

Brightfield microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample. Bright field microscopy is the simplest of a range of techniques used for illumination of samples in light microscopes and its simplicity makes it a popular technique. The typical appearance of a bright field microscopy image is a dark sample on a bright background, hence the name. Bright field microscopy is very simple to use with fewer adjustments needed to be made to view specimens.

Microscopes are used to magnify objects. Through magnification, an image is made to appear larger than the original object. The magnification of an object can be calculated roughly by multiplying the magnification of the objective lens times the magnification of the ocular lens. Objects are magnified to be able to see small details. There is no limit to the magnification that can be achieved; however, there is a magnification beyond which detail does not become clearer. The result is called empty magnification when objects are made bigger but their details do not become clearer. Therefore, not only magnification but resolution is important to the quality of the information in an image.

The resolving power of the microscope is defined as the ability to distinguish two points apart from each other. The resolution of a microscope is dependent on a number of factors in its construction. There is also an inherent theoretical limit to resolution imposed by the wavelength of visible light (400-600nm). The theoretical limit of resolution (the smallest distance able to be seen between two points) is calculated as:

Resolution = 0.61 l/N.A.

where l represents the wavelength of light used and N.A.is the numerical aperture. The student-grade microscopes generally have much lower resolution than the theoretical limit because of lower quality lenses and illumination systems.

Standard brightfield microscopy relies upon light from the lamp source being gathered by the substage condenser and shaped into a cone whose apex is focused at the plane of the specimen. Specimens are seen because of their ability to change the speed and the path of the light passing through them. This ability is dependent upon the refractive index and the opacity of the specimen. To see a specimen in a brightfield microscope, the light rays passing through it must be changed sufficiently to be able to interfere with each other which produces contrast (differences in light intensities) and, thereby, build an image. If the specimen has a refractive index too similar to the surrounding medium between the microscope stage and the objective lens, it will not be seen. To visualize biological materials well, the materials must have this inherent contrast caused by the proper refractive indices or be artificially stained. These limitations require instructors to find naturally high contrast materials or to enhance contrast by staining them which often requires killing them. Adequately visualizing transparent living materials or thin unstained specimens is not possible with a brightfield microscope.

Darkfield microscopy relies on a different illumination system. Rather than illuminating the sample with a filled cone of light, the condenser is designed to form a hollow cone of light. The light at the apex of the cone is focused at the plane of the specimen; as this light moves past the specimen plane it spreads again into a hollow cone. The objective lens sits in the dark hollow of this cone; although the light travels around and past the objective lens, no rays enter it. The entire field appears dark when there is no sample on the microscope stage; thus the name darkfield microscopy. When a sample is on the stage, the light at the apex of the cone strikes it. The image is made only by those rays scattered by the sample and captured in the objective lens. The image appears bright against the dark background. This situation can be compared to the glittery appearance of dust particles in a dark room illuminated by strong shafts of light coming in through a side window. The dust particles are very small, but are easily seen when they scatter the light rays. This is the working principle of darkfield microscopy and explains how the image of low contrast material is created: an object will be seen against a dark background if it scatters light which is captured with the proper device such as an objective lens.

The highest quality darkfield microscopes are equipped with specialized costly condensers constructed only for darkfield application. This darkfield effect can be achieved in a brightfield microscope, however, by the addition of a simple "stop". The stop is a piece of opaque material placed below the substage condenser; it blocks out the center of the beam of light coming from the base of the microscope and forms the hollow cone of light needed for darkfield illumination. Darkfield optics is a low cost alternative to phase contrast optics. The contrast and resolution obtained with inexpensive darkfield equipment may be superior to what you have with student grade phase contrast equipment. It is surprising that few manufacturers and vendors promote the use of dark field optics. To view a specimen in dark field, an opaque disc is placed underneath the condenser lens, so that only light that is scattered by objects on the slide can reach the eye (figure 3.4.2). Instead of coming up through the specimen, the light is reflected by particles on the slide. Everything is visible regardless of colour, usually bright white against a dark background. Pigmented objects are often seen in "false colours," that is, the reflected light is of a colour different than the colour of the object. Better resolution can be obtained using dark field as opposed to bright field viewing. With a compound microscope, dark field is obtained by placing an occulting disk in the light path between source and condenser. A cheap set of occulting disks can be prepared by cutting circular pieces of black electrical tape ranging from dime-size up to a diameter that equals the width of the slide, and sticking them to the slide in a row. The circles should be spaced well apart. A specimen is placed on the microscope stage as usual, and the illumination should be made as uniform as possible. If there is an aperture diaphragm in the condenser (contrast lever), it should be opened up wide. After focusing at low power, the slide with occulting disks is placed in the light path between source and condenser, bringing it as close to the bottom of the condenser as it will go.

Figure. 3.4.3 Dark field optics

Microscopes are used to magnify objects. Through magnification, an image is made to appear larger than the original object. The magnification of an object can be calculated roughly by multiplying the magnification of the objective lens times the magnification of the ocular lens. Objects are magnified to be able to see small details. There is no limit to the magnification that can be achieved; however, there is a magnification beyond which detail does not become clearer. The result is called empty magnification when objects are made bigger but their details do not become clearer. Therefore, not only magnification but resolution is important to the quality of the information in an image.

The resolving power of the microscope is defined as the ability to distinguish two points apart from each other. The resolution of a microscope is dependent on a number of factors in its construction. There is also an inherent theoretical limit to resolution imposed by the wavelength of visible light (400-600nm). The theoretical limit of resolution (the smallest distance able to be seen between two points) is calculated as:

Resolution = 0.611/N. A.

where l represents the wavelength of light used and N.A.is the numerical aperture. The student-grade microscopes generally have much lower resolution than the theoretical limit because of lower quality lenses and illumination systems.

Standard brightfield microscopy relies upon light from the lamp source being gathered by the substage condenser and shaped into a cone whose apex is focused at the plane of the specimen. Specimens are seen because of their ability to change the speed and the path of the light passing through them. This ability is dependent upon the refractive index and the opacity of the specimen. To see a specimen in a brightfield microscope, the light rays passing through it must be changed sufficiently to be able to interfere with each other which produce contrast (differences in light intensities) and, thereby, build an image. If the specimen has a refractive index too similar to the surrounding medium between the microscope stage and the objective lens, it will not be seen. To visualize biological materials well, the materials must have this inherent contrast caused by the proper refractive indices or be artificially stained. These limitations require instructors to find naturally high contrast materials or to enhance contrast by staining them which often requires killing them. Adequately visualizing transparent living materials or thin unstained specimens is not possible with a brightfield microscope.

Darkfield microscopy relies on a different illumination system. Rather than illuminating the sample with a filled cone of light, the condenser is designed to form a hollow cone of light. The light at the apex of the cone is focused at the plane of the specimen; as this light moves past the specimen plane it spreads again into a hollow cone. The objective lens sits in the dark hollow of this cone; although the light travels around and past the objective lens, no rays enter it. The entire field appears dark when there is no sample on the microscope stage; thus the name darkfield microscopy. When a sample is on the stage, the light at the apex of the cone strikes it. The image is made only by those rays scattered by the sample and captured in the objective lens. The image appears bright against the dark background. This situation can be compared to the glittery appearance of dust particles in a dark room illuminated by strong shafts of light coming in through a side window. The dust particles are very small, but are easily seen when they scatter the light rays. This is the working principle of darkfield microscopy and explains how the image of low contrast material is created: an object will be seen against a dark background if it scatters light which is captured with the proper device such as an objective lens.

The highest quality darkfield microscopes are equipped with specialized costly condensers constructed only for darkfield application. This darkfield effect can be achieved in a brightfield microscope, however, by the addition of a simple "stop". The stop is a piece of opaque material placed below the substage condenser; it blocks out the centre of the beam of light coming from the base of the microscope and forms the hollow cone of light needed for darkfield illumination.

3.5.2 Scanning Electron Microscopy

The Scanning Electron Microscope (SEM) is a microscope that uses electrons rather than light to form an image. There are many advantages to using the SEM instead of a light microscope. The SEM has a large depth of field, which allows a large amount of the sample to be in focus at one time. The SEM also produces images of high resolution, which means that closely spaced features can be examined at a high magnification. Preparation of the samples is relatively easy since most SEMs only require the sample to be conductive. The combination of higher magnification, larger depth of focus, greater resolution, and ease of sample observation makes the SEM one of the most heavily used instruments in research areas today.

Accelerated electrons in an SEM carry significant amounts of kinetic energy, and this energy is dissipated as a variety of signals produced by electron-sample interactions when the incident electrons are decelerated in the solid sample. These signals include secondary electrons (that produce SEM images), backscattered electrons (BSE), diffracted backscattered electrons (EBSD that are used to determine crystal structures and orientations of minerals), photons (characteristic X-rays that are used for elemental analysis and continuum X-rays), visible light (cathodoluminescence--CL), and heat. Secondary electrons and backscattered electrons are commonly used for imaging samples: secondary electrons are most valuable for showing morphology and topography on samples and backscattered electrons are most valuable for illustrating contrasts in composition in multiphase samples (i.e. for rapid phase discrimination). X-ray generation is produced by inelastic collisions of the incident electrons with electrons in discrete ortitals (shells) of atoms in the sample. As the excited electrons return to lower energy states, they yield X-rays that are of a fixed wavelength (that is related to the difference in energy levels of electrons in different shells for a given element). Thus, characteristic X-rays are produced for each element in a mineral that is "excited" by the electron beam. SEM analysis is considered to be "non-destructive"; that is, x-rays generated by electron interactions do not lead to volume loss of the sample, so it is possible to analyse the same materials repeatedly.

The SEM is routinely used to generate high-resolution images of shapes of objects (SEI) and to show spatial variations in chemical compositions: 1) acquiring elemental maps or spot chemical analyses using EDS, 2) discrimination of phases based on mean atomic number (commonly related to relative density) using BSE, and 3) compositional maps based on differences in trace element "activators" (typically transition metal and Rare Earth elements) using CL. The SEM is also widely used to identify phases based on qualitative chemical analysis and/or crystalline structure. Precise measurement of very small features and objects down to 50 nm in size is also accomplished using the SEM. Backscattered electron images (BSE) can be used for rapid discrimination of phases in multiphase samples. SEMs equipped with diffracted backscattered electron detectors (EBSD) can be used to examine micro fabric and crystallographic orientation in many materials.

Figure 3.4.2 Schematic drawing of electron and x-ray optics of a combined SEM-EPMA

There is arguably no other instrument with the breadth of applications in the study of solid materials that compares with the SEM. The SEM is critical in all fields that require characterization of solid materials. While this contribution is most concerned with geological applications, it is important to note that these applications are a very small subset of the scientific and industrial applications that exist for this instrumentation. Most SEM's are comparatively easy to operate, with user-friendly "intuitive" interfaces. Many applications require minimal sample preparation. For many applications, data acquisition is rapid (less than 5 minutes/image for SEI, BSE, spot EDS analyses.) Modern SEMs generate data in digital formats, which are highly portable.

Samples must be solid and they must fit into the microscope chamber. Maximum size in horizontal dimensions is usually on the order of 10 cm, vertical dimensions are generally much more limited and rarely exceed 40 mm. For most instruments samples must be stable in a vacuum on the order of 10-5 - 10-6 torr. Samples likely to outgas at low pressures (rocks saturated with hydrocarbons, "wet" samples such as coal, organic materials or swelling clays, and samples likely to decrepitate at low pressure) are unsuitable for examination in conventional SEM's. However, "low vacuum" and "environmental" SEMs also exist, and many of these types of samples can be successfully examined in these specialized instruments. EDS detectors on SEM's cannot detect very light elements (H, He, and Li), and many instruments cannot detect elements with atomic numbers less than 11 (Na). Most SEMs use a solid state x-ray detector (EDS), and while these detectors are very fast and easy to utilize, they have relatively poor energy resolution and sensitivity to elements present in low abundances when compared to wavelength dispersive x-ray detectors (WDS) on most electron probe microanalysers (EPMA). An electrically conductive coating must be applied to electrically insulating samples for study in conventional SEM's, unless the instrument is capable of operation in a low vacuum mode.

3.5.3 Transmission Electron Microscopy

Transmission electron microscopy is an immensely valuable and versatile technique for the characterisation of materials. It exploits the very small wavelengths of high-energy electrons to probe solids at the atomic scale. In addition, information about local structure (by imaging of defects such as dislocations), average structure (using diffraction to identify crystal class and lattice parameter) and chemical composition may be collected almost simultaneously. However, use of the microscope is highly skilled, and along with the interpretation of the information gained requires a good understanding of the processes occurring in the microscope, and the structure of materials. This TLP serves as an introduction to the basic concepts and structure of the transmission electron microscope through a very thin sample to image and analyse the microstructure of materials with atomic scale resolution. The electrons are focused with electromagnetic lenses and the image is observed on a fluorescent screen, or recorded on film or digital camera. The electrons are accelerated at several hundred kV, giving wavelengths much smaller than that of light: 200kV electrons have a wavelength of 0.025Å. However, whereas the resolution of the optical microscope is limited by the wavelength of light, that of the electron microscope is limited by aberrations inherent in electromagnetic lenses, to about 1-2 Å.

Figure 3.4.3 Objective/intermediate lens system of TEM

Because even for very thin samples one is looking through many atoms, one does not usually see individual atoms. Rather the high resolution imaging mode of the microscope images the crystal lattice of a material as an interference pattern between the transmitted and diffracted beams. This allows one to observe planar and line defects, grain boundaries, interfaces, etc. with atomic scale resolution. The brightfield/darkfield imaging modes of the microscope, which operate at intermediate magnification, combined with electron diffraction, are also invaluable for giving information about the morphology, crystal phases, and defects in a material. Finally the microscope is equipped with a special imaging lens allowing for the observation of micromagnetic domain structures in a field-free environment.

The TEM is also capable of forming a focused electron probe, as small as 20 Å, which can be positioned on very fine features in the sample for microdiffraction information or analysis of x-rays for compositional information. The latter is the same signal as that used for EMPA and SEM composition analysis (see EMPA facility), where the resolution is on the order of one micron due to beam spreading in the bulk sample. The spatial resolution for this compositional analysis in TEM is much higher, on the order of the probe size, because the sample is so thin. Conversely the signal is much smaller and therefore less quantitative. The high brightness field-emission gun improves the sensitivity and resolution of x-ray compositional analysis over that available with more traditional thermionic sources.

Sample preparation for TEM generally requires more time and experience than for most other characterization techniques. A TEM specimen must be approximately 1000 Å or less in thickness in the area of interest. The entire specimen must fit into a 3mm diameter cup and be less than about 100 microns in thickness. A thin, disc shaped sample with a hole in the middle, the edges of the hole being thin enough for TEM viewing, is typical. The initial disk is usually formed by cutting and grinding from bulk or thin film/substrate material, and the final thinning done by ion milling. Other specimen preparation possibilities include direct deposition onto a TEM-thin substrate (Si3N4, carbon); direct dispersion of powders on such a substrate; grinding and polishing using special devices (t-tool, tripod); chemical etching and electropolishing; lithographic patterning of walls and pillars for cross-section viewing; and focused ion beam (FIB) sectioning for site specific samples.

Artifacts are common in TEM samples, due both to the thinning process and to changing the form of the original material. For example surface oxide films may be introduced during ion milling and the strain state of a thin film may change if the substrate is removed. Most artifacts can either be minimized by appropriate preparation techniques or be systematically identified and separated from real information.

3.5.3 Confocal Laser Scanning Microscopy

A confocal microscope creates sharp images of a specimen that would otherwise appear blurred when viewed with a conventional microscope. This is achieved by excluding most of the light from the specimen that is not from the microscope's focal plane. The image has less haze and better contrast than that of a conventional microscope and represents a thin cross-section of the specimen. Thus, apart from allowing better observation of fine details it is possible to build three-dimensional (3D) reconstructions of a volume of the specimen by assembling a series of thin slices taken along the vertical axis. Modern confocal microscopes have kept the key elements of Minsky's design: the pinhole apertures and point-by-point illumination of the specimen. Advances in optics and electronics have been incorporated into current designs and provide improvements in speed, image quality, and storage of the generated images. Although there are a number of different confocal microscope designs, this entry will discuss one general type-the other designs are not markedly different [36]. The majority of confocal microscopes image either by reflecting light off the specimen or by stimulating fluorescence from dyes (fluorophores) applied to the specimen. The focus of this entry will be on fluorescence confocal microscopy as it is the mode that is most commonly used in biological applications. The difference between the two techniques is small. There are methods that involve transmission of light through the specimen, but these are much less common.

3.5.1. Fluorescence and Fluorescence Microscopy

If light is incident on a molecule, it may absorb the light and then emit light of a different colour, a process known as fluorescence. At ordinary temperatures most molecules are in their lowest energy state, the ground state. However, they may absorb a photon of light (for example, blue light) that increases their energy causing an electron to jump to a discrete singlet excited state [37]. In Fig. 3.5.1, this is represented by the top black line. Typically, the molecule quickly (within 10-8sec) dissipates some of the absorbed energy through collisions with surrounding molecules causing the electron to drop to a lower energy level (the second black line). If the surrounding molecules are not able to accept the larger energy difference needed to further lower the molecule to its ground state, it may undergo spontaneous emission, thereby losing the remaining energy, by emitting light of a longer wavelength (for example, green light) [38]. Fluorescein is a common fluorophore that acts this way, emitting green light when stimulated with blue excitation light. The wavelengths of the excitation light and the colour of the emitted light are material dependent.

Figure. 3.5.1 Mechanism of fluorescence. The horizontal lines indicate quantum energy levels of the molecule. A fluorescent dye molecule is raised to an excited energy state by a high-energy photon. It loses a little energy to other molecules and drops to a lower excited state. It loses the rest of the energy by emitting light of a lower energy.

Microscopy in the fluorescence mode has several advantages over the reflected or transmitted modes. It can be more sensitive. Often, it is possible to attach fluorescent molecules to specific parts of the specimen, making them the only visible ones in the microscope and it is also possible to use more than one type of fluorophore [39]. Thus, by switching the excitation light different parts of the specimen can be distinguished.

In conventional fluorescence microscopy a dyed specimen is illuminated with light of an appropriate wavelength and an image is formed from the resulting fluorescent light. In Fig. 3.5.2 the excitation light is blue and the emitted light is green. The microscope uses a dichroic mirror (also called a ''dichromatic mirror'') that reflects light shorter than a certain wavelength but transmits light of longer wavelength. Thus, the light from the main source is reflected and passes through the objective to the sample, while the longer-wavelength light from the fluorescing specimen passes through both the objective and the dichroic mirror. This particular type of fluorescence microscopy, in which the objective used by the illuminating light is also used by the fluorescing light in conjunction with a dichroic mirror, is called epifluorescence. In the case of reflected light microscopy, a beam splitter is used in place of the dichroic mirror.

Figure. 3.5.2. Basic setup of a fluorescence microscope. Light from the source is reflected off the dichroic mirror toward the specimen. Returning fluorescence of a longer wavelength is allowed to pass through the dichroic mirror to the eyepiece.

3.5.2 Confocal Microscopy

To understand confocal microscopy it is instructive to imagine a pair of lenses that focuses light from the focal point of one lens to the focal point of the other. This is illustrated by the dark blue rays in Fig. 3.5.3. The light blue rays represent light from another point in the specimen, which is not at the focal point of the left-hand-side lens. (Note that the colours of the rays are purely for purposes of distinguishing the two sets-they do not represent different wavelengths of light.) Clearly, the image of the light blue point is not at the same location as the image of the dark blue point. (Recall from introductory optics that points does not need to be at the focal point of the lens for the system of lenses to form an image.)

Figure.3.5.3 Rejection of light not incident from the focal plane. All light from the focal point that reaches the screen is allowed through. Light away from the focal point is mostly rejected

In confocal microscopy, the aim is to see only the image of the dark blue point [40].

Accordingly, if a screen with a pinhole is placed at the other side of the lens system, then all of the light from the dark point will pass through the pinhole. Note that at the location of the screen the light blue point is out of focus. Moreover, most of the light will get blocked by the screen, resulting in an image of the light blue point that is significantly attenuated compared to the image of the dark blue point.

To further reduce the amount of light emanating from ''light blue'' points, the confocal microscope setup minimises how much of the specimen is illuminated. Normally, in fluorescence microscopy the entire field of view of the specimen is completely illuminated, making the whole region fluoresce at the same time. Of course, the highest intensity of the excitation light is at the focal point of the lens, but the other parts of the specimen do get some of this light and they do fluoresce. Thus, light at a ''dark blue'' point may include light that has been scattered from other ''light blue'' points, thereby obscuring its fluorescence. To reduce this effect the confocal microscope focuses a point of light at the in-focus dark blue point by imaging a pinhole aperture placed in front of the light source [40]. Thus, the only regions that are illuminated are a cone of light above and below the focal (dark blue) point.

Together the confocal microscope's two pinholes significantly reduce the background haze that is typical of a conventional fluorescence image, as shown in Fig. 3.5.4. Because the focal point of the objective lens forms an image where the pinhole/screen is, those two points are known as ''conjugate points'' (or alternatively, the specimen plane and the pinhole/screen are conjugate planes). The pinhole is conjugate to the focal point of the lens, hence the name ''confocal'' pinhole.

Figure. 3.5.4. Basic setup of a confocal microscope. Light from the laser is scanned across the specimen by the scanning mirrors. Optical sectioning occurs as the light passes through a pinhole on its way to the detector.

The confocal microscope incorporates the ideas of point-by-point illumination of the specimen and rejection of out-of-focus light. One drawback with imaging a point onto the specimen is that there are fewer emitted photons to collect at any given instant. Thus, to avoid building a noisy image each point must be illuminated for a long time to collect enough light to make an accurate measurement [40]. In turn, this increases the length of time needed to create a point-by-point image. The solution is to use a light source of very high intensity, which Minsky did with a zirconium arc lamp. The modern choice is a laser light source, which has the additional benefit of being available in a wide range of wavelengths.

In Fig. 3.5.5 (a) the laser provides the intense blue excitation light. The light reflects off a dichroic mirror, which directs it to an assembly of vertically and horizontally scanning mirrors. These motor-driven mirrors scan the laser across the specimen. Recall that Minsky's invention kept the optics stationary and instead scanned the specimen by moving the stage back and forth in the vertical and horizontal directions. As awkward (and slow) as that method seems it does have among others the following two major advantages [41]:

The specimen is everywhere illuminated axially, rather than at different angles as in the case of the scanning mirror configuration, thereby avoiding optical aberrations. Thus, the entire field of view is illuminated uniformly.

The field of view can be made larger than that of the static objective by controlling the amplitude of the stage movements.

In Fig. 3.5.5 the dye in the specimen is excited by the laser light and fluoresces. The fluorescent (green) light is descanned by the same mirrors that are used to scan the excitation light (blue) from the laser and then passes through the dichroic mirror. Thereafter, it is focused onto the pinhole. The light that makes it through the pinhole is measured by a detector such as a photomultiplier tube. In confocal microscopy, there is never a complete image of the specimen because at any instant only one point is observed. Thus, for visualization the detector is attached to a computer, which builds up the image one pixel at a time. For a 512 512-pixel image this is typically done at a frame rate of 0.1-30 Hz. The large range in frame rates depends on a number of factors, the most important of which will be discussed below. The image created by the confocal microscope is of a thin planar region of the specimen-an effect referred to as optical sectioning. Out-of-plane unfocused light has been rejected, resulting in a sharper, better resolved image. Fig. 3.5.6 shows an image created with and without optical sectioning.

Figure 3.5.6. Images of cells of spirogyra generated with and without optical sectioning. The image in (B) was created using a slit rather than a pinhole for out-of focus light rejection. Most of the haze associated with the cell walls of the filamentous algae is absent, allowing clearer distinction of the different parts.

3.6 Scanning Probe Microscopy

3.6.1 Atomic Force Microscopy

3.6.2 Piezo-response Force Microscopy

3.6.3 Force distance curve

3.7 Other Electromechanical Characterisations:

The electromechanical properties of the bone depend on the microstructure and chemical composition of the bone samples. Because they vary with nutrition, age, and disease, it is expected that these biological factors will also affect the electromechanical properties of bone. The methods used in the measurement of the electromechanical properties of bone are important aspects which have neither been given due consideration nor been followed consistently. The concept of biological variability is sometimes invoked to justify the scatter in the experimental data, although the scatter may indicate something important about either the measurement method itself or the tissue.

3.7.1 Permittivity measurements

Propagation of electromagnetic (EM) waves in radiofrequency (RF) and microwave systems is described mathematically by Maxwell's equations with corresponding boundary conditions. Dielectric properties of lossless and lossy materials influence EM field distribution. For a better understanding of the physical processes associated with various RF and microwave devices, it is necessary to know the dielectric properties of media that interact with EM waves. For telecommunication and radar devices, variations of complex dielectric permittivity (referring to the dielectric property) over a wide frequency range are important. For RF and microwave applicators intended for thermal treatments of different materials at ISM (industrial, scientific, medical) frequencies, one needs to study temperature and moisture content dependencies of the permittivity of the treated materials.

Permittivity is a quantity used to describe dielectric properties that influence reflection of electromagnetic waves at interfaces and the attenuation of wave energy within materials. In frequency domain, the complex relative p

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