Unit 5 Biomaterial Processing and Surface Properties PDF

UBM008: Biomaterials B.Tech. BME 2nd year Odd semester 2024-25 Faculty: Debasmita Mondal Assistant Professor, DEIE, TIET, Patiala [email protected] 9004008796 Unit 5: Syllabus Biomaterial Proc...

UBM008: Biomaterials B.Tech. BME 2nd year Odd semester 2024-25 Faculty: Debasmita Mondal Assistant Professor, DEIE, TIET, Patiala [email protected] 9004008796 Unit 5: Syllabus Biomaterial Processing and Surface Properties: Processing of metals, ceramics and polymers to improve bulk properties, processing techniques for improving biocompatibility, Chemical, biological and physical modifications of biomaterial surfaces, contact angle analysis, surface characterization techniques: light and electron microscopy. Introduction Processing of biomaterials is usually done to change the bulk or surface properties of the material, obtain a desired shape, sterilize or otherwise improve the biocompatibility of the material. The bulk property generally targeted in material processing is mechanical strength. To create stronger or harder materials, dislocation motion should be reduced so that more energy is needed before plastic deformation can occur. Processing methods to improve bulk properties of metals Alloying Strain Hardening Grain size refinement Annealing Precipitation Hardening Alloying Alloy is a solid solution which is composed of two or more elements in solid solution form. Alloying is adding many substitutional point defects to the base metal. (a) Tensile strain Generally, alloys have greater corrosion resistance and are stronger than metals. The point defects produce localized lattice strain on the surrounding host atoms (b) Compressive strain depending on its size. Fig: Localized lattice strain Larger the size difference, larger is the strain caused by alloying. (a) If the impurity atom is smaller field. than those in the base metal, a tensile strain will be created Results in lattice strain field interactions on the surrounding environment. between already existing dislocations and (b) Conversely, a substitute atom that is larger than the host these impurity atoms. atoms will cause localized compressive strains. Alloying Apart from lattice strain, there is crystal distortion due to impurities around a dislocation (either tensile or compressive depending on which side of the dislocation the impurity atoms are located). Impurity atoms can cluster around the area of a dislocation to help cancel the lattice strains. It would be more stable for large atoms to lie on the “tensile side” of an edge dislocation where the atoms are already slightly farther apart due to the presence of the dislocation. If the dislocation is moved away from the impurity, the Fig: Crystal overall lattice strain in the system would increase. distortion around a dislocation. Smaller atoms tend to cluster on the compressive side. Strain Hardening Adding point defects improves the strength of a metal. Additional line defects (dislocations) also create a stronger material. Strain hardening is a process to make a metal harder and stronger due to plastic deformation. The metal is strained beyond the yield point. More number of dislocations are generated when plastic deformation occurs in the metal. The dislocations will interact and become pinned or tangled. This can block the further movement of the dislocations and promote the strength of the material i.e., higher and higher forces will be required to continue deformation. Strain Hardening Strain hardening is also called cold working since it is carried out at low temperatures relative to the melting point of metals. As the dislocation density increases, the movement of any one dislocation becomes difficult due to the interfering effect of the stress fields of the surrounding dislocations. Result of increased dislocation density is an increased strength, but reduced ductility. Grain Size Refinement Grain size refinement is a process that reduces the size of grains in a metal casting to improve its quality and properties (mechanical strength, response to heat, etc.). Metals are polycrystalline, consisting of grains with different orientations sharing a common boundary. Dislocations can not easily cross each other, as they apply a stress on one another. When they intersect they can no longer move through normal slip. For plastic deformation to occur, dislocations must cross between grains. If two neighboring grains are of different orientations, a dislocation passing into grain B will have to change its direction of motion at the grain boundary. The atomic disorder at grain boundary leads to discontinuity of slip planes from one grain into the other. Grain Size Refinement Grain boundaries act as barriers to the movement of dislocations, as these boundaries are intersections between different crystal orientations, where the slip plane for dislocation movement is effectively ended. The dislocations “pile up” at the grain boundaries, until sufficient stress is built up that the dislocations either change orientation to slip on the adjoining grain boundary, or dislocations are created in the neighboring grain. If the grain size is finer, there are more boundaries to dislocation movement. However, the number of dislocations that pile-up on the boundary is also smaller. More the dislocation pileup  More stress is generated on the dislocations by themselves  More stress is applied on the dislocation pileup at the boundary  Dislocations easily cross into the neighboring grain for plastic deformation. With a small grain size only a small number of dislocations pileup so the stress on the boundary is very small which leads to an increase in the yield stress. A material with many smaller grains (fine-grained material) is usually stronger than the same material with larger grains (coarse grained materials). Annealing Cold working (strain hardening) improves the strength of metals, but decreases material ductility and can negatively affect corrosion resistance. Annealing is a form of heat treatment in which the material is exposed to high temperatures for relatively long periods of time in order to increase the ductility or toughness of the material and reduce internal stresses. Annealing occurs in three distinct stages 1. Heating to the required temperature 2. Maintaining or “soaking” the material at that temperature 3. Controlled cooling (quenching) Annealing Factors affecting the microstructural and mechanical properties of annealed material Materials composition Quenching rate Quenching rate refers to the rate of heat extraction from the material. Outside of the specimen will always cool more quickly than the inside  Formation of temperature gradient can affect the final grain structure and the overall properties of the metal. Heat extraction also depends on the media in which the material is quenched. Movement of the quenching media over the surface also increases heat extraction and causes a more rapid quench. Since cooling occurs primarily at the surface, pieces with a higher surface area to volume ratio will experience faster quenching. Precipitation Hardening Volume defects (i.e., extremely small uniformly dispersed particles of a second phase within the original phase matrix) may be added to a material via precipitate hardening. Precipitate hardening, also known as age hardening or particle hardening, is a heat treatment process that increases the strength and hardness of materials. Precipitates are included within the host crystal, which causes local lattice strains. These strains then provide a barrier to dislocation motion in the area, thereby strengthening the material. Dislocations moving in the matrix are hindered by closely spaced precipitate particles. Strengthening effect is inversely proportional to the particle spacing. Processing to Improve Bulk Properties In Ceramics Due to presence of ionic bonds in ceramics, dislocation movement is very difficult. Techniques to further reduce dislocation movement is of little use. Increasing the number of slip systems to improve ductility of ceramics is more important. In Polymers Dislocation motion is also difficult in covalent crystals, but many polymers are semi crystalline. It is possible to improve the strength of polymeric materials by increasing their percent crystallinity. Thermal processing – Cooling a polymer slowly after being heated will allow time for the chains to become more fully aligned, thus increasing the crystallinity of the material. Pre-drawing procedures – Analogous to strain hardening in metals. Processing techniques for improving biocompatibility Biocompatibility is a broad term suggesting that a material produces an acceptable host response in a given application i.e., the material does not induce infection or a deleterious immune response (rejection). Sterilization To avoid infection from innocuous pathogens, sterilization of biomedical implants prior to implantation is extremely important. Pre-sterilization decreases the possibility of the transfer of viruses or bacteria associated with naturally derived materials. It is impossible to remove all pathogens from an implant; hence, the concept of a sterility assurance level (SAL) was developed. The SAL for a given device is measured by culturing implants in nutrient media after various sterilization times and determining how many bacterial colonies remain. From these data, manufacturers can choose the time and dose of sterilization to maintain a certain SAL. Steam Sterilization or Autoclaving Steam sterilization or autoclaving is achieved by exposing the items to be sterilized with saturated steam under high pressure at a temperature of at least 121°C. Steam enhances the ability of heat to kill microorganisms by reducing the time and temperature required to denature protein and lipid constituents important to microorganism survival. Advantages Very effective Relatively quick and simple Leaves no toxic residues within the sample Disadvantages Can not be used for low melting point materials due to high temperatures employed Not suitable for sterilization of hydrolytically cleavable materials which are susceptible to degradation in such an environment Ethylene Oxide Sterilization Ethylene Oxide Sterilization A specialized sterilization apparatus is required to expose implants to ethylene oxide (EtO) gas. Since EtO is toxic (may cause cancer and is potentially flammable), it is often mixed with an inert gas within the machine. Sterilization occurs when a chamber holding the implants at 30°C to 50°C is first evacuated, then flooded with EtO, and finally purged several times with air. Following this, further aeration outside the sterilizer helps in removal of any remaining EtO. EtO causes pathogen death primarily through permanent, chemical alteration of nucleic acids (DNA, RNA). Ethylene Oxide Sterilization Advantages Very effective, even deep within crevices or pores Since it is carried out at a low temperature, it is can be used for a wide variety of materials. Disadvantages Toxic residues of EtO that may be left in the device Due to the reactivity and possible carcinogenic nature of the gas, personnel must be properly protected while performing the sterilization procedure. Radiation Sterilization Radiation Sterilization Gamma rays or an electron beam is used in radiation sterilization. Samples are placed in an irradiator and monitored to assure that they have received the proper dosage of radiation. Gamma ray source is a 𝟔𝟎 𝑪𝒐 isotope. Electron beam is created by an accelerator. Radiation acts by ionizing important cellular elements, including nucleic acids, thus killing adherent microorganisms. Advantages of radiation sterilization Rapid, effective and compatible with many materials. Disadvantages Requires a large capital investment to install an irradiator Certain polymers, e.g. poly(tetrafluoroethylene) are susceptible to radiation degradation. Radiation Sterilization Radiation Sterilization Electron beam method When the accelerator is turned off, no radiation is produced. Short treatment time + High dose rate No waste products Has a very small penetration depth, so only thin devices may be sterilized in this manner. X-ray High depth penetration Good dose uniformity ratio Gamma ray method Most popular Simple, easily controlled 60 𝐶𝑜 source is constantly decaying and releasing radiation, which can be detrimental to the operator’s health. Concepts in Surface Chemistry and Biology Biomaterial surface is very important to determine the biological response and the success of implant. Protein Adsorption and Biocompatibility Surface of a material can be considered a type of planar defect. Since atoms at the surface are not bonded on all sides to other atoms, there is extra energy associated with this region due to unfilled valence shells. This excess energy is called the surface tension (𝜸). This state is thermodynamically unstable  There is a driving force to minimize the surface tension by the adsorption of atoms or molecules, which satisfy the unfilled bonds at the material surface. Concepts in Surface Chemistry and Biology Protein Adsorption and Biocompatibility Adsorption is the adhesion of molecules to a solid surface. Absorption is the penetration of molecules into the bulk of another material, such as water is absorbed by a sponge. At physiological conditions, the adsorbate on the biomaterial surface is composed primarily of ions, water, and proteins. The body reacts to this coated surface, rather than to the “pure” biomaterial. Controlling protein adsorption to biomaterial surfaces is a key aspect to assuring biocompatibility. Surface Properties Governing Protein Adsorption Surface properties having largest effect on favorable adsorption 1. Surface hydrophobicity 2. Surface charge 3. Steric concerns (spatial arrangement of atoms in a molecule) 4. Surface roughness Surface hydrophobicity Hydrophobicity of a material describes how it responds to water. Hydrophobic (water-fearing, or water-repelling) - Most of the synthetic polymers Hydrophilic (water-loving) - Ceramic and metallic biomaterials are often more hydrophilic than these polymers in their unmodified state. Protein adsorption generally increases with increasing hydrophobicity of the surface and the protein. Surface Properties Governing Protein Adsorption Surface charge A significant surface charge can have the additional effect of attracting or repelling charged areas of proteins. Surface charge occurs via dissociation of ionizable surface groups or though specific adsorption of ions from the solution. Surface roughness A surface with a high degree of roughness may promote protein adsorption in certain areas by physically “trapping” the proteins in the valleys on the surface. Surface Properties Governing Protein Adsorption Steric concerns Adding large, flexible hydrophilic polymer chains (poly(ethylene glycol) (PEG)) to a biomaterial surface result in decreased protein adsorption. A large volume at the surface is taken up by these bulky chains that are in constant motion. Since they move too quickly to allow the proteins to adsorb to them and are too large for the proteins to move through, they form a type of wall, using steric repulsion to prevent adsorption to the surface. Physicochemical Surface Modification Techniques Surface modification possesses the advantage that bulk characteristics such as mechanical properties are not altered. An ideal technique would produce a surface treatment with the following characteristics: Thin film (to minimize effects on bulk properties) Resistant to delamination (a process where a material breaks or separates into layers) Simple and robust (to promote commercialization) Discourage surface rearrangement Generally surface modification techniques are grouped into physicochemical and biological modifications. Physicochemical Surface Coatings Physicochemical surface treatments use physical principles or chemical reactions to alter the surface composition of the sample. These modifications do not involve the attachment of active biological molecules. Surface coatings can be covalently or non-covalently attached. Covalent Surface Coatings: Plasma Treatment Plasma Treatment Plasma refers to an assembly of species in an atomically/ molecularly dissociated gaseous environment. The gaseous species present can include positive and negative ions, free radicals, electrons, atoms, molecules, and photons. Plasma discharge can occur at a range of temperatures (usually 25°C and higher) and is most often created under vacuum. A plasma environment is obtained by applying an electric potential across a gas using two electrodes i.e., cathode and anode. Covalent Surface Coatings: Plasma Treatment Plasma Treatment Cathode is the surface to be treated and has a negative potential relative to the anode. Electrons must traverse the gas in the chamber to travel from cathode to anode. During this stage, they collide with molecules in the gaseous environment to form gaseous ions and radicals. These species can then interact with the sample and cause a variety of surface reactions. The plasma is sustained because electrons flow from the sample while positive ions flow toward the sample. Covalent Surface Coatings: Plasma Treatment Plasma Treatment At the sample surface, two processes can happen. Deposition and Ablation/etching Plasma discharge is often used for either cleaning or addition of hydroxyl (– OH) or amine groups (– NH2) to biomaterials as a precursor to further modification. Advantages of plasma discharge treatments 1. Depositions are conformal, free of voids/pinhole defects. 2. Are easily prepared. 3. Are sterile when removed from the reactor. 4. Produce a low amount of leachable substances. 5. Demonstrate good adhesion to substrate. 6. Allow unique film chemistries to be produced. 7. Can be characterized relatively easily. Covalent Surface Coatings: Plasma Treatment Plasma Treatment Disadvantages of plasma discharge treatments 1. The chemistry within the reactor can be ill defined. 2. The equipment is often expensive. 3. Uniform reaction within long, narrow pores may be difficult. 4. Particular care must be taken in sample preparation to prevent contamination during or after processing. Covalent Surface Coatings: CVD Chemical Vapor Deposition (CVD) It is a surface treatment in which a mixture of gases is exposed to a sample at a high temperature. Plasma-enhanced chemical vapor deposition (PECVD) – Plasma environments facilitate increased reactivity of the gaseous species and therefore, reduced reaction temperature. CVD techniques are most commonly used in biomaterials applications to deposit pyrolytic carbon coatings on substrates such as molybdenum/rhenium or graphite. In this case, the gases are hydrocarbons and they undergo thermal decomposition, or pyrolysis, within the reaction chamber, allowing carbon deposition on the surface of the material. Covalent Surface Coatings: CVD Steps in Chemical Vapor Deposition (CVD) 1. Transport – Reactive gases are transported in the gas phase to the reaction zone, often with a carrier gas. 2. Diffusion – The gas diffuses through the boundary layer and onto the surface of the material. 3. Adsorption – The gas is adsorbed onto the surface of the material. 4. Reaction – A chemical reaction occurs on the surface of the material, forming a solid film and by-products. 5. Desorption – The by-products are desorbed from the surface and transported out of the reactor. Covalent Surface Coatings: PVD Physical Vapor Deposition (PVD) PVD results in surface coating via deposition of atoms generated through physical processes on the sample. PVD techniques are used for orthopedic implants, surgical tools, orthodontic appliances. E.g., Metal alloy coatings deposited using PVD to increase wear resistance of metallic hip implants. PVD includes sputtering and thermal evaporation. Covalent Surface Coatings: PVD Sputter deposition Sputtering is a PVD technique that involves the removal of atoms from a solid target material and their deposition onto a substrate. Sputter deposition is a two-step process. Step 1: Energetic ions or atoms (inert gas ions) bombard a target material and transfer their momentum to atoms within the target. This causes the ejection of a certain number of target surface atoms. Step 2: The released target atoms strike the sample surface and condense to form a thin film. Both covalent and non-covalent coatings are possible via this method. Covalent Surface Coatings: PVD Plasma-assisted PVD The formation of plasma is used to create high-energy species which collide with the target in plasma-assisted PVD. The target is held at a large negative potential compared to the sample to be coated. An environment of sufficient vacuum will initiate formation of plasma near the target. Species from the plasma then strike the target to release atoms that can be deposited on the substrate surface. Covalent Surface Coatings: PVD Thermal evaporation Thermal evaporation, also known as vacuum evaporation, involves the sublimation of a material and its condensation onto a substrate. Typically uses a resistive heat source to evaporate a solid coating material in a vacuum environment to form a thin film. The material is heated in a high vacuum chamber until vapor pressure is produced. The vaporized material condenses onto the substrate, forming a thin film. Covalent Surface Coatings: SAM Self assembled monolayer (SAM) These molecules are amphiphilic i.e., possess both hydrophilic (polar) and hydrophobic (non-polar) areas and have three key regions: Attachment group Long hydrocarbon (alkyl) chain (non-polar) Functional (polar) head group Once the SAM molecules begin to gather on the surface, the van der Waals forces between the non-polar regions of the alkyl chains in each molecule will cause crystallization once they are sufficiently close. The functional group can be used to alter the hydrophobicity of the substrate material. Covalent Surface Coatings: SAM Self assembled monolayer (SAM) Advantages of SAMs Ease of formation Chemical stability of the coating Variety of chemical moieties can be included either in the attachment or the functional groups. Form molecularly smooth surfaces, thus altering the physical properties of the substrate. Non-Covalent Surface Coatings Solution coatings In this technique, the substrate is dipped in a solution containing the dissolved coating material (usually a polymer dissolved in an organic solvent). The substrate is then left to dry and, as the solvent evaporates, the coating is deposited on the surface. This method can also be employed as a simple means to coat substrates with bioactive molecules. The solvent is often aqueous rather than organic. Non-Covalent Surface Coatings: LB film Langmuir-Blodgett Films The coating molecules are amphiphilic. Coating molecules are transferred to a biomaterial surface using a Langmuir trough. The substrate to be coated is placed into aqueous media and the amphiphilic molecules are added so that the polar head groups interact with the water and the remainder of the molecule rests in the air. By changing the position of moveable barrier, the coating is slowly compressed until all the molecules are orientated as standing on end. At this point, the area per molecule reaches a minimum and is nearly constant. This value is called the critical area and is a function of the size and type of hydrophobic tail on the molecule. By maintaining a surface pressure corresponding to the critical area, as the material to be coated is slowly removed from the trough, a homogenous, well-orientated coating can be deposited. Non-Covalent Surface Coatings: LB film Langmuir-Blodgett Films A major disadvantage is the relative instability of the coating, due in large part to the fact that it is not chemically bonded to the surface. Biological Surface Modification Techniques Biological surface modification techniques involve attachment of biologically active molecules to a substrate by a variety of means, including many of the physicochemical methods. The attached molecules then interact with specific target areas on cells or other tissue components. A primary concern with these techniques is maintaining the biological activity of the molecule of interest while remain attached. Biological Surface Modification Techniques Covalent Biological Coatings Covalently linked coatings may impart additional stability. Covalent coatings require the presence of a reactive substrate surface, often containing hydroxyl (OH), carboxyl (COOH), or amine (NH2) groups. If these are not found on the chosen biomaterial surface, it may be modified (via plasma treatment, etc.) to add appropriate functionality before proceeding with the reaction. Fig. (a-c): Attachment via post-fabrication methods Biological Surface Modification Techniques Covalent Biological Coatings (d-e) Attachment during synthesis Surface characterization of biomaterials Contact angle analysis Used to provide overall information about the hydrophobicity of a surface. The surface free energy or surface tension (𝜸) of a material can be defined thermodynamically as the work required to create a unit area of surface at constant temperature and pressure. In most contact angle experiments, a system with three interfaces is created Liquid-vapor surface (𝜸𝑳𝑽 ), solid-liquid surface (𝜸𝑺𝑳) and solid-vapor surface (𝜸𝑺𝑽 ) In most cases, the liquid chosen for testing of biomedical materials is water. Change in wettability via surface Schematic of contact angle testing modification. Surface characterization of biomaterials Contact angle analysis The energies at each of the interfaces causes the water droplet to assume a particular shape (different degree of spreading). By accurately measuring the angle between the drop and the solid surface (the contact angle, 𝜽), the surface tension can be calculated. Young’s equation which represents a force balance between the horizontal components of three surface tensions is given by 𝜸𝑺𝑽 − 𝜸𝑺𝑳 − 𝜸𝑳𝑽 𝐜𝐨𝐬 𝜽 = 𝟎 𝜸𝑺𝑳 = 𝜸𝑺𝑽 − 𝜸𝑳𝑽 𝐜𝐨𝐬 𝜽 Both 𝛾𝑆𝑉 and 𝛾𝑆𝐿 are unknown. The water droplet spreads more on the modified surface because the modification decreases the surface tension of the liquid/solid interface, thus reducing the contact angle as calculated using Young’s equation. Surface characterization of biomaterials Contact angle analysis 𝜸𝑺𝑽 is often approximated using the critical surface tension (𝜸𝑪). Diagram shows how the critical surface tension (𝛾𝐶 ) is determined. The contact angle of various liquids is measured for a specific material, and a plot of contact angle vs. 𝛾𝐿𝑉 is generated. The extrapolated value of 𝜸𝑳𝑽 at 𝜽 = 𝟎 is 𝜸𝑪. Contact angle analysis The contact angle of a liquid on a solid surface can range from 0° to 180°. The highest possible contact angle is 180°, which is considered completely non-wetting. A contact angle of 180° is achieved when a surface is ultra- hydrophobic, i.e., it repels liquids completely. This property is also known as the lotus effect. Contact angle analysis Instrumentation Components Holder for solid sample Holder for liquid Means to determine contact angle (may be automated) Output of contact angle analysis is a single number (𝜃 or 𝛾𝐶 ) This technique cannot provide detailed information about the chemical composition of the surface. Contact angle analysis Instrumentation Dynamic contact angle measurements are also used to measure contact angle hysteresis. Water is slowly added to an area of the surface with a syringe and the advancing contact angle is measured. The water is then removed via the same mechanism and the receding contact angle is recorded. The difference between these two values represents the contact angle hysteresis for that material and describes how the surface tension of the material changes before and after it has been exposed to an aqueous environment. Hysteresis can occur for a variety of reasons; for example, hydrophilic domains within the material may become reoriented outward from the surface after contact with water, whereas they may be “hidden” within the bulk when exposed to hydrophobic environments such as air. Light Microscopy Basic Principles Visual approach to gain primarily qualitative information about surface topography, or to view thin sections of a sample. In light microscope (e.g., the compound microscope), a white light source is projected through the sample, where the combination of ocular lens (in the eyepiece) and objective lenses reflect the light so as to magnify the sample many times, providing a detailed image of features usually undetectable to the naked eye. For opaque samples, the light source can be located above rather than below the sample. The best resolution of light microscope is around 0.2 μm. Features smaller than or more closely spaced 0.2 μm cannot be distinguished using this method. Light Microscopy The objective forms a magnified image of the object that is larger than the original object. This image is then magnified many times by the eyepiece to form the large (inverted) virtual image. Light path of a compound A compound microscope microscope Light Microscopy Four basic components to a light microscope: 1. Source - produces white light. 2. Lenses - glass lenses focus light beam and/or magnify image of sample. 3. Sample stage - holds sample securely. 4. Detector (camera or human eye) - views and captures resulting image. If a camera is used as the detector, the image can be stored on a computer for further image analysis. The sample is placed on the stage. The condenser lens focuses the light beam before it passes through the sample. After the light exits the specimen, the objective and ocular lenses magnify the sample image. The image can then be either directed to a camera or viewed directly through the eyepiece. Electron Microscopy Electron microscopy (EM) is a technique for obtaining high resolution images of biological and non-biological specimens. Used in biomedical research to investigate the detailed structure of biomaterials, tissues, etc. High resolution of EM images results from the use of electrons (which have very short wavelengths ~ 0.01 nm) as the source of illuminating radiation. Two types Transmission Electron Microscopy (TEM) Scanning Electron Microscopy (SEM) Transmission Electron Microscopy (TEM) A transmission electron microscope is similar to a compound microscope but uses magnetic rather than glass lenses. Wavelength associated with electrons is shorter than that for white light  Resolving power of the TEM is greater, and much more detailed images can be obtained. TEM requires very thin samples (20–200 μm thick) because electron beams are completely absorbed by thicker samples and therefore are unavailable to create an image. The amount of energy lost by the electron beam as it is passing through the sample creates an image. Thin sample sections can be taken from any Fig. (a) Light microscopy (b) portion of the sample; thus, TEM is not strictly a TEM which uses magnetic surface analysis technique. lenses, as opposed to glass. Scanning Electron Microscopy (SEM) In SEM, the surface of a sample is scanned with an electron beam. A beam of electrons is accelerated and travels through an electron column that contains lenses to focus the beam onto the sample's surface. SEM operates in a vacuum to prevent electron interactions with gas molecules, which is necessary for high resolution. The electrons from the beam undergo elastic and inelastic scattering as they collide with atoms in the sample. Elastic scattering results in alteration of the trajectory of the electron, but not its energy. In many cases, after a number of elastic collisions, the electron will exit the sample as a backscattered electron. Scanning Electron Microscopy (SEM) Inelastic scattering occurs when the electron transfers part or all of its energy to a sample atom. The atom then emits secondary electrons, Auger electrons (low-energy electrons that are emitted when an atom relaxes to a lower energy state) or X-rays as a means to release this excess energy. SEM images are produced by recording the production of secondary electrons after an area is bombarded with the primary electron beam. Since the intensity of these electrons is dependent on the surface topography of the sample, SEM is considered a surface imaging technique. Resolution of 0.2 nm. Scanning Electron Microscopy (SEM) Five basic components of a SEM 1. Source - produces accelerated electrons. 2. Lenses - magnetic coils focus electron beam and reduce spot size. 3. Sample holder - holds sample securely. 4. Detector - records the spatial position of secondary electron impact and converts this information into an electrical signal. 5. Computer - translates the signal from the detector to produce an image. Note: For optimal imaging, non-conductive samples, such as polymers, must be pre-coated with a thin layer of conductive material (metal) to reduce charge build-up during scanning. This is accomplished via physical sputtering from a metallic target onto the sample before imaging. Scanning Electron Microscopy (SEM) First, the specimen is placed into the sample chamber and the electron beam is scanned over the surface. The secondary electron detector is positioned so that the locations of the emitted electrons are recorded. The signal from the detector is then processed using appropriate software to produce a 3D image. If emitted X-rays are also examined, a specialized detector system called energy-dispersive X-ray analysis (EDXA) is included to collect and analyze this radiation. Scanning Electron Microscopy (SEM) SEM also provides the possibility of gaining information about the chemical composition of the imaged sample by analyzing the X-rays emitted, rather than the secondary electrons, after bombardment by the primary electron beam. Visualize the surface topography of a biomaterial, or a biomaterial with attached tissue or cells. The combination of SEM and EDXA provides information about chemical composition of the sample, although the ability to distinguish surface chemistry is limited. Scanning Electron Microscopy (SEM) Scanning electron microscope Optical microscope image of nanofibers image at 4000x magnification of the same nanofibers SEM vs. TEM SEM Uses a focused beam of electrons to create a magnified image of an object's surface by detecting electrons that are reflected or knocked off the surface. SEM images show information about the object's composition and physical features. SEM scans the surface of a sample. SEM is generally easier to operate, more affordable and requires less sample preparation than TEM. TEM Uses transmitted electrons to create a projection image of a thin specimen's interior. TEM images provide information about the specimen's inner structure, such as its crystal structure, morphology, and stress state. TEM can achieve higher magnifications and better resolution than SEM.