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    Video 1: Surface Characterization Techniques
    Good morning everyone, today I will be talking about Characterization of Materials.So, until now, you guys might have already studied about what are biomaterials and differenttypes and what are it is application and all.. So, now you have to learn why we need to characterizethe materials.So, materials when we are characterizing, we will make it to understand their properties.So, that it can be whether a material is accepted as a implant or a medical devices and all.So, the biocompatibility of the material is primarily dependent on its property.So, to find out it is properties we have to characterize the materials.So, for developing a new materials also you have to understand the new property.So, that it mimics the natural existing organs and other applications and all.So, to identify those properties also you need to characterize the materials.The material characterization is primarily divided into two categories, one is basedon the engineering properties such as surface properties, mechanical properties, chemicaland physical properties and other is totally dependent on biological relationship withthe material which is in vitro biocompatibility, in vivo biocompatibility.So, surface properties.So, surface property is a property primarily dependent on the material and the host cellinteraction interfaces.So, when a material is implanted into the body, the first point of contact is the materialsurface and the host cells.So, the surface should be biocompatible.So, that it would not be rejected by the host mechanism.Mechanical properties, while you are considering a implant such as hip prosthesis and otherheavy load bearing applications and all, you need to have a good mechanical property whichwill be understood by characterizing the bulk properties of the material, that can be doneusing the mechanical characterization.Physical and chemical properties, so, physical and chemical properties involves understandingthe material composition and what are its thermal properties and what are it is, ifyou are using a scaffold for tissue engineering applications and all, you have to understandthe porosity and permeability and how much the water intake it can actually have.So, those properties and all come under chemical and physical properties.In vitro biocompatibility, so, before going for the clinical application; we have to previouslytest the material with in vitro and in vivo testing.So, in vitro biocompatibility involves whether a material is suitable for the cell adhesion,whether there is a immune, immunogenic response from the cells we are, in vitro studies weare doing, whether there is a protein absorption, whether there is a blood coagulation occurson the material surfaces and all.In vivo biocompatibility involves a testing the material in a animal models like rats,pig and dog models and all, to check whether the material has a same function when youare a introducing into a human body.So, after all these properties has been done for both at the biomaterial level and thebiomedical devices level.It cannot be only for material, if a material, I am preparing a material and if it is a biocompatiblewe cannot say that will be exactly replicated in the devices also, because device will havea different structures and different totally function varies.So, we have to check in the biomaterial level at first then you have to go for the biomedicalapplications also.And all these properties cannot be applied for all the material.So, it depends on what type of material, some are nanoparticle, some are films and someare scaffolds, some are huge bulk materials and all.So, each of this materials will have to be characterized based on the material properties,what type of characterization you have to look and what type of application you aregoing to look into.. So, coming into characterization techniquesthese are the predominantly five different types.Surface characterization, what are the techniques you can, there are lot of techniques availablefor the lot of properties, but these are the widely used techniques used in the biomaterialfield.So, surface characterization techniques such as wettability or contact angle measurement,this is a technique where you will find a material surface is hydrophobic or hydrophilic,hydrophobic is water repelling and hydrophilic is water attracting.X-ray photoelectron spectroscopy, which will calculate the elemental composition of thematerial, what are the elements present on your material surface and what are the chemicalbonds present on the material surface that can be found out using XPS analysis.Secondary ion mass spectroscopy, it is also a surface technique, where you can find outthe surface, particular atomic composition of the material.Whether the, how much electron available on the material surfaces and all, those can becalculated by secondary ion mass spectroscopy.So, microscopy techniques also comes under the surface characterization, but there arelot of microscopic techniques available.So, one of the mainly used technique is SEM, scanning electron microscopy, that is usedin used to observe the morphology of the material surfaces and all.Then transmit, transmission electron microscopy, which is used to see the inside the materialsurfaces and all.So, I will explain both of those techniques later.Atomic force microscopy it will check the surface roughness of the materials.So, you can check how much of the material surface is smooth or whether it is rough,those parameters will greatly affect the cell attachment and other properties.Then optical microscope will have a variety of microscopic technique such as phase contrast,fluorescence microscope, confocal microscope all of those comes under optical microscopy.Physicochemical characterization involves FTIR which will characterize the functiongroups present on the materials.NMR spectroscopy we can find out what are the bondings available on the, what are thebondings and what are the element, elements present on the materials.X-ray diffraction will tell whether the polymer is amorphous, crystalline or semi crystallinethese things.DSC and TGA analysis is a thermal property characterization techniques where you willfind out the melting point, degradation, glass transition temperature.Those kinds of properties can be found out using a DSC and TGA analysis.Mechanical characterization is usually important for the bulk load bearing applications.So, those experiments involve tension and compression how much of a load it can withstandand how much of compression it can go, if I take how long it can be how long it canbe available without any breaking or some breaking all those things.Creep and viscous flow how the polymer actually affects, actually works inside the host system.Then biological characterization, protein absorption study because the first when youimplant a material into a host system, the first interaction between the host systemand the material is the protein absorption.So, if there is a protein absorption occurring on your material surface then that will leadto cell attachment and blood cells attachment also.So, based on the application that can vary.so, if you want your material to not to have any attachment of cells, it should not haveany protein absorption on the material surface.Because when you are going for a blood contacting devices and all, there you do not need a bloodcells to attach onto the material surface because it will lead to blood clot formationinside the devices which will lead to failure of the device.So, for those applications, we have to check for the protein absorption studies.And if you are going for a tissue engineering application like wound healing and these things,so, you need to have cells attached onto the surfaces and all.So, for that application you have to have a protein absorption on the material surfacesand all.So, these are the basic techniques, there are, other than this there are still lot oftechniques are available which is specific for each of the application, we have to lookinto.So, coming into surface characterization techniques; as I told you earlier Contact angle analysis,XPS, Auger electron spectroscopy which involves the auger electrons; then secondary ion massspectroscopy which is known as SIMS.Then FTIR, STM is scanning tunneling microscopy, then scanning electron microscopy.So, each of a technique has a different resolution of how much properties we can identify usingthose techniques and the cost they have given.So, all these atomic related composition finding techniques and all will be very highly costly.So, the basic techniques like contact angle analysis, FTIR and all it is slightly lesser.So, while developing a new material and all you can first optimize those materials usingthe basic techniques, then you can go for a higher end technique to confirm whetherthat material has been a properly based on your application and all ok.So, each of the application I have told you already.So, let us go into contact angle measurement.. So, contact angle measurement is to determinethe wettability of the surface and to estimate the surface energy.So, when a material is introduced, the first liquid solution from the host mechanism willspread on the material surface.So, that primarily affects how the proteins are absorbing?How the cells are absorbing on the material surface and all?So, to find out that wettability property whether the material is hydrophobic or hydrophilicwe have to use the contact angle measurement.So, the angle between the solid-liquid interface and the liquid interface is the contact angle.So, this relation has been given by Young’s equation which is interfacial tension betweenthe solid-liquid interface, cos theta equal to interfacial energy at solid-vapor minusinterfacial energies solid-liquid interfaces.So, based on these contact angle we can modify the surfaces also.So, already a existing material is available and you want to improve the properties ofthe existing materials and all.So, you coat a material surface using a different polymer or some other treatment available,modification techniques available and all.So, if you do that the contact angle can be varied.So, by varying the surface property you can actually have the better cell attachment,better protein adsorption and all those things are.So, if a material is above 90 degree it is hydrophobic, if it is below 90 degree; itis hydrophilic.So, if a material is above 150 and all, it is super hydrophobic which is known as lotuseffect, where you see a water droplet on a lotus leaf where the water will retain itis bead structure on throughout the surface, that is called lotus effect due to it is microstructureon the lotus leaf available.. So, for measuring the contact angle we usethe instrument called goniometer, contact angle goniometer.So, there are different types of measuring contact angle available, one is sessile dropmethod which is the widely used technique.So, the picture where you can see there is a material surface and you put a drop on thematerial surface and you measure the angle between the solid-liquid interface and theliquid interfaces.So, you draw a tangent at the point of contact and measure the angle between the, angle onthat area, interface area.So, that will give you the contact angle that will show whether the material is hydrophobicor hydrophilic.Then cap captive air bubble method which is similar to the sessile drop method, but insteadof using a liquid we use a air bubble to measure the angle, contact angle.So, you have a material surface and it is immersed in a liquid solution and you introducea drop on the bottom of the surface.So the, it will form a bead like structure which will give you the angle between theliquid interface and the solid surface which is the contact angle.So, why we can use, where we can use for the captive air bubble measurement is, when yourmaterial is intaking water and all.So, if you are just doing it in an air atmosphere.So, the material will hydrate and it will absorb all the water into the material itself.So, if you are using a air bubble method.So, it will show the angle because it will be already hydrated with the liquid presenton the surfaces and all.Then the capillary rise method, this is used to measure the angle in small diameter graftsand these things, where the all the surfaces having the uniform coating and uniform surfacemorphology is available, to check that we use the capillary rise method.So there, we will use the meniscus to find the angle between the liquid surface and thesolid surface.Then the Wilhelmy plate method, where we will have a plates, metal plates and all if youcheck the contact angle we introduce the plate.So, both of the sides will have the same angle and by that we can find out the contact angleok.So, the advantages of using contact angle is very cheap and you can use it anywhereimmediately after preparing a material surfaces and all.The major disadvantages is if there is a very rough surface or the surface is not uniformand all, it will have a different angle produced on different angle will be there.. So, next technique is X-ray photoelectronspectroscopy also known as Electron Spectroscopy for Chemical Analysis.It is a technique where you can find out what are the elements present on the material surfaceand all.So, if you have a material and you do not know what are the elements present on thesurfaces and all, you can just do this analysis and it will show if you have a whatever metalions or any other organic compounds present; so it will, show all the elements presentin the material surfaces and all.So, this XPS is only for the surface technique, where it will go up to a depth of ten angstrom.So, up to that depth level only it can measure the elemental analysis.So, below that, it would not have that elemental composition and all.So, this is mainly used when you are checking for contamination of a material surface orif you are modifying a material surface and to check whether the introduced modificationhave a effect and all, you can check using this XPS method it is a confirmation techniquefor the all those applications and all.So, the it works on the principle that, when a X-ray is introduced onto the surface itwill excite the electrons, though the excited electrons will be detected using the detectorand it will check for the, it will show the binding energy based on the electron intensity,it will show the binding energy and analyze, what it, what category it belongs to, whetherit belongs to carbon well, whether it belongs to oxygen or nitrogen sulfur and all.So, it is works on the principle that binding energy equal to energy of the X-rays minuskinetic energy.. So, this is an XPS instrument and this ishow the spectrum will look like.So, in XPS analysis there are two categories, two types of analysis, one is wide spectrumanalysis and another is a core spectrum analysis.So, wide spectrum analysis you can identify the oxidation states and the elements presentthroughout the surfaces and all.So, like have, you can see at the top graph there is a wide spectrum analysis, where youcan actually see the oxygen, carbon, nitrogen, sulfur, so if you have a other elements presentalso it will show that.So, it will be like a elemental percentage you can find out from this spectrum.Whereas, the core level spectrum where you can identify what type of chemical bonds formedin the carbon-based spectrum.So, carbon-based spectrum would be around 285 of binding energy.So, if you look at that carbon-based spectrum itself, so, you can see that peak each ofthe peaks is for the different binding.So, you have a for the normal C-C bond and C-O bond and acid bonds and ester bonds.So, all these bonds you can find out using the core level spectrum analysis.So, by this core level spectrum analysis you can find out the oxidation state, valencyof the valence electrons, valency state of the elements.Then if you are attaching a molecule, so you can find out whether the bonding has beenproperly done on the material surfaces and all.So, all those things can be confirmed using the core level spectrum ok.One of the disadvantages of using XPS is that, the sample would be analyzed under high vacuumcondition.So, if your sample is not stable in high vacuum condition it is difficult to do XPS.

    Video 2: Microscopic Techniques
    So, coming into Microscopic techniques, microscopictechniques also involve comes under the surface characterization techniques only, but it hasa wide applications, like fluorescence microscope can actually see how the organelles and howthe nucleus and all looks inside the cells and all, then transmission electron microscopewill look inside the material surface not only the surface, look inside the materialsand all.So, to find out the morphology, we primarily use the microscopic techniques and each ofthe microscopic techniques has a different depth and different resolution it can go upto.So, it can go from atoms to the cells which cells and normal materials you can go.The size will range from millimeter to nanometer and some microscopic recent techniques andall have go upto angstrom levels also.So, up for light microscopy you can see up to 200 nanometer and if it is a electron microscopyyou can go up to nanometer size and all.. So, this is a schematic representation ofoptical microscope and a fluorescence microscope.In optical microscope, we will have a lens space and light source and you will keep asample inside and based on the light source the sample would be observed through the magnificationpiece and objective piece to see the whatever sample you are observing.So, the optical microscope has a lot of types.phase contrast to check, whether the cells would be in, observed based on the phase differencebetween the cells because the light would be observing, the cells would be absorbingsome of the light.So, that cells and all would be dark surface and lights which are not observed by the cellswould be a brighter side.So, that will have a phase contrast microscopy and confocal microscopy, based on the magnificationparameter and those things it will vary.And fluorescence microscope, where you will have a fluorescence, where you have a fluorescenceexposed onto the sample and if you have a fluorophore inside the sample that will getexcited and that excited wavelength can be observed using the detector.So, the image shows the fluorescence microscopic image, the image is a fluorescence microscopeimage, where you can, the green elements are the actin filaments which are the stainedusing phalloidin and the blue stains are the nucleus which is stained using hoechst stain.So, if you are having a experiment where you are involving nanoparticle encapsulation orif you are introducing a drug into a cells and all.So, whether the drug has been properly introduced into the cells or into the materials and allif you want to find out, you attach that drug with the fluorophore and if you observe underthe fluorescence microscope, you can check whether that actually happened or not ok.. So, both of those techniques has a lower resolutiononly, you can go up to 200 micron, 200 nanometers only you cannot go below that.So, for that purpose we go into scanning electron microscopy.So, this is a schematic representation of the scalling, Scanning electron microscopy,the major difference is that here you have a electron as the, electron beam as the lightsource.So, you have a electron beam which is passed on through the electro magnets which willcontrol the electron beam and it will focus on your sample and based on the sample propertyyou can observe the images ok.So, there are two types of deduct detection available in scanning electron microscopy,one is secondary electron and another is backscattered electron.So, secondary electron is that whenever your electron source is given and that it willgo into the, it will excite a another electron that scattered electron will be absorbed,absorbed under secondary electron detector.So, that will have a secondary electron source.Then for backscattered electrons, the electron which you are giving, so, some of them wouldbe reflected by the sample.So, the same electron when you are deducting, that is a backscattered electron detector.So, why these two all so, when you are having a, when you want to see the morphology veryin detail and all.So, you want the electrons which are repelled by the surfaces and all.So, that will have only the morphology of the surfaces.So, secondary electron will have a depth and all.So, when you have a porous structure and all if you use a secondary electron it will havea better image, when you want to just observe the morphology and all you can go for backscatteredelectrons.Then Auger electrons spectroscopy which is a another totally different type of spectroscopy,where the deduction is based on Auger electron.So, Auger electrons are the electrons which are excited from the core level, will be replacedfrom the outer electrons, that electrons will produce the energy.So, that will be observed under the detector.So, that will show the atomic composition, so, if you having a copper or any other metalsavailable.So, those metals it will show how much of the things will be available, density.So, based on that Auger electrons because based on the copper, one copper if it is releasingsome amount of electron if there is a composition is high it will release certain number ofelectron.So, that will be observed under the Auger electron microscopy.And scanning electron microscopy also you can find out the elemental composition, whereit will show the percentage of elements present on the surface also ok.So, that is called X-ray energy dispersive method.. So, this is how the SEM images looks like.So, you can see the first five images are where the blood platelets are adhering onthe material surface.So, this is, a is control sample and all these four are the different treated sample.So, modification different techniques has been used.So, after the techniques the platelet adhesion has been reduced.So, that can be observed under scanning electron microscopy.Then the bottom one is vascular stent, where they have coated material onto the stent.So, to observe the coating has been properly done on the vascular stent, they have observedusing scanning electron microscopy.So, there are two parameters which are important in microscopy, one is magnification and anotheris resolution.So, magnification determines how much of a magnify you can do a sample and all, resolutiondetermines how detailed your images are.So, magnification depends on the instrument property and resolution it depends on magnification,but how detailed you can go observe, because in scanning electron microscopy if I go aboveten nanometer size and all the image would not be detailed.So, you have to go for a higher end equipment.So, to have that it will have that magnification, but you would not have a clear picture andall.So, resolution is very important when you are looking into microscopy.. So, TEM it also works on the similar principleas SEM, but the idea is that the electron beam will be passed through the sample.So, transmitted through the sample that is why it is called transmission electron microscopy.As we can see in the SEM the sample is at the bottom and if it is reflected based onbackscattered or select secondary electrons the images would be observed, but in transmissionelectron microscopy the sample would be at the center and the electron beam would bepassed through the sample and that will give the what are the what is present inside thematerial and all.If your material is porous structure and all there the electron would not be would nothave any inhibition passing through that porous structure and all, if you have a materialscompact materials and all it will, based on the density of the material the electron willhave a passing transmission will be varying.So, based on that that image would be observed under the transmission electron microscopy.. Then coming into scanning probe microscopy,so, until now we have observed based on the light source, then electron source, then Scanningprobe microscopy is, where you have a probe which will have a feedback on the, from thesurface and it will input into the detector and it will form the images and all.So, scanning microscope has a lot of different techniques based on the application.So, one of the widely used scanning probe microscopy is AFM, atomic force microscopy.In atomic force microscopy itself; it is having a different types such as noncontact, contact,tapping methods, those three techniques are available in atomic force microscopy then.So, these are the techniques available and based the uses based on the techniques whatare you want to.. So, we will see atomic force microscopy.So, atomic force microscopy the cantilever is there, cantilever is a small sharp object,piezoelectric object which will expand and contract based on the voltage applied ontothe cantilever.So, when your material in contact mode, if you are having a material and the cantileverwould be placed on the top of the material.So, if it is in a flat surface, so the material will move along the flat surface and it willhave a straight, without any moment the voltage difference would be constant.So, it will have a flat image and all.So, if you have a rough surface and all it will move up and down.So, that will have the voltage difference, that would be detected using the detectorand it will have that rough image surfaces on them.In Non contact mode, what actually happens is that, the cantilever will move at a frequencywithin the resonant frequency of that cantilever and based on the surface atomic surface ofthe material, the moment would be inhibited by the electrons on the surface of the material.So, that difference would be observed under the voltage difference, that will have a imagingatomic force image, that is a tapping method which will be within the resonance frequency.In non contact mode it is also vibrating at a high frequency above the resonance frequencywhere it would not touch the surface at all, but van der waals force and other long rangeforces available on the surfaces will have a effect on the non contact mode, that effectwill be observed on the into changed into a image.So, the those are the three different things.So, why these three different things are available is some samples would be very sensitive.So, if you are observing under the contact mode, the sample will get damaged.So, to avoid that people will go for tapping mode usually, tapping mode is one which iswidely used, non contact mode when you have a sample which is very sensitive you do notwant to touch the sample at all.So, these are the three different techniques.. So, this is a schematic representation ofAFM and this is how the image looks like.So, you have a plain surface which is a and it has a some ridges and all seen and if youmodify the surfaces, it will have a rougher surface that will be observed as a 3D contour,we can see that images and all.. So, coming into Fourier transform infraredspectroscopy.So, when a molecule is exposed to IR radiation, it will exert vibrational, stretching, contractionand bending and all.So, those molecular properties can be converted into Fourier transform infrared spectroscopyspectrum.So, if you are having a molecule methane CH3 if you are exposing it into IR spectrum, thatmolecule will have a different bending based on the bond between the atoms.So, that can be observed under a FTIR spectroscopy.So, there are different types of stretching, one is symmetric stretching, asymmetric stretching,scissoring and rocking.So, if you are having a highly nucleophile it will have a repulsion between the neighboringnucleophile.So, that it will have a stretching observed, onto the observed, that will be observed underFTIR spectroscopy.. So, the different methods of FTIR, so, FTIRcan be used for surface technique also and for bulk composition also.So, if you are having a material surface, want to check the functional group.So, each functional groups will have a different stretching and bending.So, based on that, we can identify the functional groups.So, it can observed for surfaces also, it can observed for powder samples also, it canobserved for scaffolds and liquid samples also, we can do under the FTIR analysis whereyou.So, I will first explain the method.So, transmission FTIR spectroscopy is the widely used method, where you keep the samplein the path of the IR and it will detect the spectrum and it will show how much of thelight has been transmitted through the sample and based on that it will plot the spectrum.Then ATR, FTIR spectroscopy is the attenuated total reflectance, where you have a reflectivesurface at the bottom, where the light source would be reflected to and fro to the sampleand the reflective surface and the final amplified that reflection would be detected using detector.Then drift spectroscopy, it is diffuse reflection infrared Fourier’s transform where the,it is similar to the transmission.FTIR spectroscopy, but it would be up to a particular depth it would be diffuse the,IR rays would diffuse and then it would be reflected back into the detector.Then reflectance micro FTIR which is a new technique, where which is a new technique,where you have a very micro sample which would be, which has a high sensitivity where theIR source can be reflected and it can be detected for the very small amount of sample with highsensitivity.So, for liquid sample you can form a thin film like that will be a casing where youcan drop the liquid onto that casing.So, it will form a thin liquid film and that can be measured using transmittance FTIR spectroscopy.So, it is an technique where you can optimize the reactions and all, but you cannot fully depend on FTIR.