FACULTY OF ENGINEERING TECHNOLOGY

MATERIAL ENGINEERING TECHNOLOGYBNQ30503

INDIVIDUAL ASSIGNMENT

TITLE: OPTICAL MICROSCOPIC EXAMINATION AND ANALYSIS

NAME :KHAIRUL ANWAR BIN ROSLIMATRIC NUMBER :AN120228LECTURERS NAME :ENGR. DR. NASRUL FIKRY BIN CHE PADATE OF SUBMISSION:5TH MAY 2015

INDEXES

CONTENTSPAGE1.0 Objective 12.0 Literature Review2.1 Background and history of optical microscopic.2.2 Fundamental of image formation of optical microscopy examination.2.3 Specimen illumination2.4 Image magnification13553.0 Discussion 3.1 Techniques of Optical Microscopy3.2 Samples preparation in Optical Microscopy analysis3.3 Examples and Product Safety of Optical Microscopy analysis69104.0 Conclusion13References14

1.0

2.0 Objective of the Study.The aims of this study are:i. To discuss the basic principle and fundamental of optical microscopy techniques.ii. To introduce the available techniques in optical microscopic.iii. To study the uses and application of optical microscopy.iv. To relate the importance of optical microscopy analysis on product safety.

3.0 Literature Review.2.1Background and history of optical microscopic.The history of optical microscopy began a long time ago during 15th century. According to Vignati (2005) the history begins with the invention of simple microscope which it is credited to Zacharias Jansen, in Middleburg, Holland around the year of 1595. The microscope was a single convex lens through which specimen can be focused and magnifies on the observers eye. The development continues in the 1600s whereas Anton von Leeunwenhoek successfully develops compound microscope which was able to observe larger bacteria. The compound microscope consists of an objectives lens placed near to the specimen and an eyepiece close to observers eyes. This development enables a two staged magnification. However, compound microscope with multiple lenses encounter more spherical and chromatic aberration compared to the simple microscope. To counter the problems, more research and development on microscope are done. In 19th century chromatically corrected microscope are invented which is built with different coloured dispersion lenses. By the end of 19th century, there were many competitions among microscope manufacturers to develop mechanically and optically high quality of compound microscope. Modern microscope in the 1990s undergoing advancement such as in glass formulation and lens technology allowing an excellent optical aberration correction. Furthermore, the advancement also enables the resulting image to be detected directly by the eyes or by various types of light detecting devices such as photographic plate, charge-couple device (CCD) cameras, photodiodes and other optical sensors.

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Figure 2.1: Simple and compound microscope in the early development. (a) Anton von Leeunwenhoek's simple microscopes; (b) Hooke compound microscope

Figure 2.2: Past and present microscopes. (a) Powell and Lealand No 1 microscope 1850; (b) Olympus Provis AX 70

2.2 Fundamental of image formation of optical microscopy examination. In the optical microscope, when light from the lamp passes through the condenser and pass through the specimen, some of the light passes around and through the specimen then produce magnified images the specimen on a detector such as human eyes or light detecting devices such as camera (Davidson M. W. and Abramowitz M., 2002). In human eyes, there are 2 types of receptors which are cone cells and rod cells. Cone cells are used for detecting colours while rod cells are used to distinguishing levels of intensity. These cells are located on the retina, iris, and cornea while the crystalline lens is the device for disclosing the light and focusing it on the retina.Optical camera on the other hands, the image was formed on the films. Digital cameras comprises of a dense matrix of photodiodes that accumulate electric charges as light imposes on them. The magnified image must meet these requirements to be seen clearly by the detector:i. It must be spread on the detector at a sufficient angle, unless the light falls on non-adjacent receptor.ii. There must be adequate contrast between adjacent details and background to condense the image visible. Conventional optical microscopy containing one or more lenses that produce enlarged image of an object placed in the focal of the lens. There are two possible configurations for optical microscopy:i. Transmission (Figure 2.3 (a) ) a. The beam of light passes through the sample.b. The specimen is placed between the objective and the condenser lens. The illuminating light formed by the condenser lens falls in the specimen and collected by the objective.c. Example of transmission microscopy is polarizing or petrographic microscope.d. Sample types are usually fine powder or thin transparent slices.ii. Reflection (Figure 2.3 (b))a. The beam of light reflected off the sample surface. b. The objective is used as matching well-corrected condenser and as image forming lens which the light passes the sample twice.c. Example of reflection microscopy is metallurgy microscope.d. The microscopes usually use to analyse the surface of the material especially the opaque material.

Figure 2.3: Type of microscopy light configuration. Khler illumination for transmitted and reflected microscopy (a) Brightfield transmitted light; (b) Brightfield reflected light.2.3 Specimen illuminationIllumination is the important variable in achieving high quality images and it is use for viewing thick specimen. The illumination should be adequately bright, glare free and evenly dispersed in the field of view. Most of microscope's manufacturers have adjusted the arrangement of optical elements in the Khler illumination configuration to optimise the illumination (Vignati E., 2005). This arrangement establishes two sets of conjugates planes. The ones in the path of the illuminating rays are: The lamp filament. The condenser aperture diaphragm. The back focal plane of the objective. The eye-point of the eyepiece.The other set of conjugates planes are related to image formation light path and consist of: The field diaphragm. The focused specimen. The intermediate image plane. The eye's retina or the film plate of the camera.

2.4 Image magnificationMagnification of microscope is dependent on the objective, the tube lens, the eyepieces and additional lenses. Consider an object of size lobj at a distance a from a lens of focal length f. The object's image, of size limg, is formed at a distance b according to the lens law (Vignati E., 2005):

The lateral magnification M is the ratio of the linear size of the image to the linear size of the object:

3.0 DiscussionIn material engineering, the optical microscopy is used to analyse the microstructure, optical illumination system of the elements. For materials that are opaque to visible light such as all metals, some ceramics and polymers, the surface is the subject to be observed and the reflective light microscope must be use. Different types of metals or compounds required the light microscope to be set in different mode. Thus, optical microscopy analysis introduces techniques for analysing different types of materials. 3.1 Techniques of Optical Microscopy3.1.1 Darkfield Microscopy.In Darkfield microscopy, the light illumination involves blocking out of the central light pass through or around the specimen and permitting the only oblique rays to the specimen to be illuminated. This is the most simple and popular for imaging unstained specimens, which will appear as bright illumination on dark background. The right rays came from darkfield condenser strike the specimen from every azimuth, diffracted and reflected into the objectives lens. 3.1.2 Phase Contrast MicroscopyPhase Contrast microscopy is primarily used to detect phase specimens. Phase specimens is the unstained object which do not absorb light and slightly alter the light diffracted by the specimen, usually retarding light to wavelength but remains in amplitude. Phase Contrast involves the separation of the direct zeroth order light from the diffracted light at the rear focal plane of the objective. This is done by placing a ring annulus is in direct position under the lower lens of the condenser at the front focal plane of the condenser, conjugate to the objective rear focal plane.3.1.3: Polarized Light A polarizer place under the sub-stage condenser is focused on the polarized light exiting the polarizer. The plane polarized in a vibration direction with respect to the optic axis of the microscope. The polarized light waves then pass through the specimen and objective before reaching a second polarizer or analyser that is oriented to pass a polarized vibration direction perpendicular to that of the sub-stage polarizer. Therefore, the analyser passes only those components of the light waves that are parallel to the polarization direction of the analyser. The retardation of one ray with respect to another is caused the difference in speed between the ordinary and extraordinary rays refracted by the anisotropic crystal.3.1.4Fluorescence MicroscopeFluorescence microscopy is an excellent technique to study material that can fluoresce, either naturally which is auto-fluorescence or when treated with chemicals capable of fluorescing. The basic fluorescence microscope is to allow light to irradiate the specimen and then separate the much weaker re-radiating fluorescent light from the brighter excitation light, resulting fluorescing areas shine against a dark background with sufficient contrast to allow detection. For example, ultraviolet (UV) light of a specific wavelength or set of wavelengths is produced by passing light from a UV-emitting source through the exciter filter. The filtered UV light illuminates the specimen, which emits fluorescent light of longer wavelengths while illuminated with ultraviolet light. Visible light emitted from the specimen is then filtered through a barrier filter that does not allow reflected UV light to pass.

Table 3.1: The schematic diagram of optical microscopy techniques and description (Davidson M.W and Abramowitz M., 2002)Techniques of Optical MicroscopyDescription

Schematic configuration for darkfield microscopy. The central opaque light stop under the condenser to eliminate zeroth order illumination. The condenser produces a hollow cone of illumination. The reflected, refracted, and diffracted light from the specimen enters the objective front lens.

Schematic configuration for phase contrast microscopy. Light passing through the phase ring is first concentrated onto the specimen by the condenser. The light enters the objective before interference at the rear focal plane of the objective.

Schematic microscope configuration for observing specimens under crossed polarized illumination. White light passing through the polarizer is plane polarized and concentrated onto the specimen by the condenser. Light rays emerging from the specimen interfere when they are recombined in the analyser, subtracting some of the wavelengths of white light, thus producing a myriad of tones and colours

Schematic diagram of reflected light fluorescence microscopy. Light emitted from UV concentrated by the collector lens before passing through aperture and field diaphragms which are reflected down through the objective to illuminate the specimen. Longer wavelength emitted through the objective and dichroic mirror before filtered by the emission filter.

3.2 Sample preparation in Optical Microscopy analysis

Figure 3.1: Basic general steps for specimen preparation microscopy (Adapted from Mukhopadhay S. M., 2003)

Table 3.2: Description of steps in sample preparation for optical microscopy techniquesStepsDescription and function of the steps

CleaningCutting process is carried out in order to get the desired size and dimension of the sample so that it can be easily molded and handle.

MouldingMolding process is carried out to make the sample easier to hold throughout the polishing procedure.

GrindingRough grinding: To remove rough scale and gross imperfections on the surface of sample.

Fine grinding: To improve the specimen's surface so it is shines and reflects light slightly.

PolishingRough polishing: To remove the imperfections that grinding has left.

Fine polishing: To remove scratches and leave a mirror like surface.

Etching Various etchants are used to selectively attack the surfaces of metals to reveal grain boundaries, phase, precipitates, inclusion and composition.

3.3 Examples and Product Safety of Optical Microscopy analysisOptical microscopic are the methods that are extensively applied in material characterization. This is due to the capability of optical microscopy to allow the observation of the internal structure of the material. Microstructure features of the materials such as grains and grain sizes, pores and pore sizes, precipitates, inclusions, alteration products, textures, shapes and morphology of crystals or aggregates, cracking and interfacial reactions, twin boundaries can be recognized and measured. For the determination and recognition of minerals and mineral phases, a polarized optical microscope is usually used and considered to be the basic tool. Two main types of polarizing optical microscopes that are used for the characterization includes transmitted light and reflected light. A transmitted light polarizing microscope is used for the observation of transparent minerals while a reflected or incident light microscope is used for the observation of metallic and opaque minerals. Optical microscopy has the advantages of direct imaging and straightforward information. Certain features are better visualized at low magnification, for example directional deformation and twin boundaries, while porosity and phase fractions can be better quantified. The disadvantage of this method is the low resolution. Due to the light diffraction limit, a common optical microscope can distinguish objects that range with size from 1 to100m, at a magnification of about 1500X. Another drawback can be the sample preparation since the thin sections are required from all samples studied under the polarizing microscope.

3.3.1 Detection of deteriorationMost building material is porous and susceptible to deterioration due to the long and slow effect of the environment conditions on the materials. Microscopic techniques can assist on the study of the whole spectrum of deterioration and find the major mechanism starting from the macroscopic image and even deeper up to the crystal unit means that a researcher can see all the path of the procedure.The cause, the type and the complexity of deterioration is possible to be determined by microstructure investigation. Determining the cause of deterioration is important to understand errors on design or materials use. The information is important to improve the property of the material or the type of material use and also it is essential in order to organize the appropriate restoration procedure. For the heterogeneous materials such as concrete and mortars with features ranging from nano to centimetres, microscopy can give information concerning the size, the distribution and the topology in detail.

Figure 3.2: Mortar from the medieval walls in Rhodes (a) under Stereoscope (x8) where the pore within crystallized salts is observed; (b) under Polarized microscope (x150) the presence of angular crystal are analysed.

3.3.2 Characterization of porosity and crack patternPorosity is a basic characteristic of materials that influence most of the material properties such as mechanical strength, deformation, resistance to weathering and insulting properties. Optical microscopy is among the widely applied techniques for measurement of porosity. The advantage of optical microscopic techniques is the exact size and position of the pore can be identified which the pores and cracks occurring inside the matrix or in the transition zones can be distinguished. Cracks in the microstructure of a material might be pre-exist or are formed during the service life of the materials. Their structure recorded by microscopy of different magnification capacity, allows to determination of the main mechanism responsible for the crack. For example, cracks in the cement matrix of concrete may be related to sulfate attack and ettringite or thaumasite can be easily identified from the characteristic crystals. Cracks in lime based mortars in the other hands are mainly localized in the transition region around aggregates and they are usually filled with re-crystallized calcite which contributes to the increase in density and strength of mortars.

Figure 3.3: Re-crystallization of calcite in the contact zone of binder-aggregate filling the crack3.3.3 Detection of interfacesIn composite materials, the existence of many interface are considered as the weak phases of the material and requires special treatment to reinforce the material. Good compaction, proper additives often use to avoid weak zones and enhance the material strength and other properties. The microscopic analysis of the interfaces provides information about the techniques used for building with these materials. For examples, high quality old mortars taken from monuments, the interfaces between brick-mortars or bedding mortar-renders or even mortar-mortar are quite compact (Figure 3.4). In many cases the microscopic examination shows the chemical reaction products between the bonding materials. In the case of a roller compacted concrete pavement the cold joint due to premature setting and inadequate compaction of concrete implies for a low performance pavement.

Figure 3.4: Interface zones under polarized microscope (x150) (a) brick-mortar; (b) mortar-render; (c) two mortar layers.

4.0 ConclusionOptical microscopy applying the light from the sources that passes through the condenser and specimen either will be reflected or transmitted to produce magnified images of the specimen on the detector such as human eyes or devices such as camera. The main components of optical microscopy are the objective lens, eyepiece, condenser, detection devices and light sources. There are many available techniques in optical microscopy such as Darkfield microscope, Phase Contrast microscopy, Polarized Light microscopy, Reflected Light Fluorescence microscopy and many others. The techniques are different depends on the as the light ray path, the position of the samples, sample types and characteristics and others. The optical microscopy is used in materials analysis. For examples, optical microscope is used in the investigation and analysis of the behaviour of materials such as brick, mortar in the building and construction field. Most common application of optical microscope is in the detection of deterioration in materials, characteristics of porosity and crack pattern on the materials, detection of interface and others. This application is very crucial in building and construction field, as the materials need to be first analysed and determine the strength, compatibility of the material to be use, durability or to be checked any defects so that the quality and the function of the materials can be fully utilized.

References1. Davidson, M. W. and Abramowitz, M. 2002. Optical Microscopy. Encyclopedia of Imaging Science and Technology. .2. Lee T. et al., 2011. Optical Microscopy Of Soft Matter System 3. Mukhopadhyay, S. M., 2003. Sample Preparation for Microscopic and Spectroscopic Characterization Of Solid Surface And Films. Sample Preparation Techniques in Analytical Chemistry. John Wiley & Sons, Inc. (pp. 377412).4. Patzelt W.J, 1985. Polarized Light Microscopy: Principles, Instruments, Application 3rd Edition. Ernst Leitz Wetzlar GmbH, Germany. 5. Stefanidou M. et al., 2014. Applying Microscopic Techniques for the Investigation of the Behaviour of Building Materials. Microscopy: Advances In Scientific Research and Education, FORMATEX 2014, p:1065-10706. Venkannah S., 2004. Metallurgy Laboratory, Material Science. University of Mauritius. 7. Vignati E. 2005. Optical Methods for Investigation of Application-Oriented Complex Fluids. Ph.D Thesis, Radiation Science & Technology, Polytechnic University of Milan.