Basics of Light Microscopy and Metallography

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1 ENGR45: Introduction to Materials Spring 2012 Laboratory 8 Basics of Light Microscopy and Metallography In this exercise you will: gain familiarity with the proper use of a research-grade light microscope learn how to optimize the resolution and image quality obtained with the microscope use your knowledge and understanding of phase diagrams and transformation diagrams to explain the microstructures of the specimens provided. The following resources provide extensive background material on image formation, and the optimization of resolution and contrast, when using a light microscope. They are well worth downloading and saving for future reference in your studies:

2 ENGR 45: Introduction to Materials UCMerced Metallographic Examination Optical Microscopes Objective To enable the student to acquire a basic understanding of the use and role of optical microscopes in metallographic examination. Introduction Metallographic examination is the process of preparing and viewing the microstructure of metal specimens for several purposes. It is often a means of relating the physical and mechanical properties of an alloy to its observed microstructure. For example, a metal may be observed to verify whether or not it has been processed appropriately by documenting the size and condition of the grain structure. The effects of post-processing steps on microstructure such as work hardening, machining, and heat treating can be observed. It is also an integral part of failure analysis on components that have undergone failure, whether catastrophic or not. Phases can be identified, as well as grain boundaries, slip bands, twins, inclusions, cracks, and other various microdefects. The most basic and fundamental examination of a metal is performed with an optical microscope (or light microscope). Other methods such as electron microscopy and x-ray diffraction are possible and useful, however the use of a light microscope is often the starting point for examination. Since metals and alloys are always opaque and do not allow light to transmit through a sample, it is not possible to use a transmission microscope, the type that is used in observation of biological samples which allow light to transmit through. The basic operation of a transmission microscope involves a light source that passes light through a sample and then through a series of lenses that produce a magnified image. Because of the opaque nature of metals, a reflective or metallographic microscope must be employed. The light source in these tools is directed toward the sample and the reflection of the light is passed through a series of lenses to create a magnified image. Many modern microscopes (including those in the UCM microscopy lab) are capable of both types of observation as they are equipped with dual light sources. It should be noted that many materials, in addition to metals, require the use of a reflective light microscope for examination. Examples include ceramics, ores, some polymers, semiconductors and other materials. It should be noted that this document will concentrate on the reflective mode of operation of a microscope. Figure 1 - Reflective microscope schematic

3 Basic Operation In a reflective microscope, a bundle of rays of light (seen horizontal in Fig. 1) is emitted from the light source or illuminator. After passing through a set of lenses, diaphragms and filters, the light is converged upon a reflective glass (also called a half mirror) and diverted downwards to pass through an objective lens and onto the specimen that has been prepare for examination. The light that is reflected off of the specimen is then passed through the objective again for enlargement. Light then encounters the glass reflector again, but due to special characteristics of the glass, (not described here) light is transmitted through and into the eyepiece lens which provides further magnification. Figure 2 is an illustration of the light pathway (from lamp to sample) superimposed on the parts of a typical reflective microscope. Recall that the pathway in a transmission microscope would be different. The power of magnification is a product of the objective and eyepiece lenses. In modern microscopes the objective lens can easily be changed to values of 5x, 10x, 20x, and 50x or more. The eyepiece usually provides an additional 10x magnification. Total magnifications of up to 1000x are common, however the clarity of the obtained image decreases at higher magnifications. Figure 2 - Light pathway in a reflective microscope (Olympus America Inc.) There are two characteristics in simple lenses that reduce sharp definition in magnified images: chromatic aberrations and spherical aberrations. Chromatic aberration occurs because white light is a mixture of colors that have different wave lengths and therefore are refracted by the optical glass at different angles. Figure 3 shows the varying focal point of three images of different colors. The remedy for this problem is to use either an achromatic or apochromatic objective. In the mid-nineteenth century, achromatic lenses were developed and decreased the amount of chromatic aberration. As can be seen in Fig. 4, achromatic correction brings the green and red images into focus on the same plane, thereby Figure 3 - Chromatic aberration of light Figure 4 - Achromatic lens correction improving the image. In 1886, the apochromatic objective was developed and further improved the image by bringing all three colors into sharp focus (Fig. 5). Research grade microscopes (as used in the UCM lab) use apochromatic lenses to correct the aberration. Figure 5 - Apochromatic lens correction

$Spherical aberration is caused by the fact that light is refracted differently at the outer margins of a lens compared to the rays of light passing near the principal axis. As shown in Fig.$

4 Spherical aberration is caused by the fact that light is refracted differently at the outer margins of a lens compared to the rays of light passing near the principal axis. As shown in Fig. 6, this results in two images, one for the marginal rays and one for the central rays. Fortunately, an apochromatic objective corrects this problem as well. With regards to chromatic aberrations, it becomes evident that the use of monochromatic light would result in the best definition of an image. (Monochromatic light is single-color light with a definite wave length.) Therefore, light filters are often used that allow only a single color of light to pass through. Filters can be relatively simple and composed of colored glass, with blue, green, and yellow being the more commonly used colors. Some filters consist of a dyed gelatin film sandwiched between two thin glass slides. Other filters contain a colored solution captured inside a cell. Figure 6 - Illustration of spherical aberration In microscopy, there are two terms often used that should be familiar to a user of microscopes. They are resolving power and numerical aperture. Resolving power refers to the ability of an optical system to clearly show lines that are very close together without aberration or blurring. High resolving power will distinctly show two lines while a lower resolving power microscope will blur the image such that a single line appears. To understand numerical aperture, another characteristic called angle of aperture must first be explained. The angle of aperture of a magnifying lens (objective) refers to the angle between the rays of light coming from the focal point and outer edges of the lens. The higher the angle of aperture, the higher the resolving power. This is due to the fact that more light is collected if the angle is larger. The numerical aperture value is the product of the sine of the angle of aperture and the index of refraction of the medium between the lens and the specimen (usually air). The numerical aperture value gives the user an indication of the resolving power of the optical system. Figure 7 is a cutaway view of the Olympus BX51 microscope, currently in place in the light microscopy lab at UCM. Note that the BX51 is both a reflective and transmission microscope, capable of either function depending on which light source is powered. Methods of Illumination Examination under any microscope begins with the light source or illuminator. Correct illumination of the sample is Figure 7 - Olympus BX51 transmission/reflective microscope (Olympus America Inc.)

perhaps the single most important aspect of microscopy. Without properly illuminating the specimen, the full detail and color cannot be seen or photographed.

5 perhaps the single most important aspect of microscopy. Without properly illuminating the specimen, the full detail and color cannot be seen or photographed. It is not simply a matter of using a light bulb to direct light down a tube so that a specimen can be seen and therefore focused upon. There are many components involved in illumination and adjustments are necessary in order to optimize illumination of the sample. It is important to note that the procedure for adjustment will also depend on whether the microscope is reflective or transmission. For this lab exercise, all viewing will be done in the reflective mode and adjustments will not be made. The factory adjustment will be sufficient. Today s sources of light for specimen illumination of course vary considerably from the earliest days of microscopy. Therefore, the techniques of focusing the light on the specimen have evolved. The components and geometry necessary for focusing the light of a candle flame will obviously be different than those used with a halogen bulb illuminator. Engineers and scientists have developed various ways to maximize illumination efficiency for a given source and have their names associated with the respective methods. Abbe, Nelson and Kohler are three examples of illumination methods. Early methods were suitable for the uniform brightness of a flame. However, since light bulbs contain a filament of varying intensity and evenness, a new method was necessary to create a focused beam of light of uniform intensity. By far, Kohler illumination is the most common method in modern microscopes. This method provides the highest intensity of even illumination for a nonhomogeneous source such as a light bulb. For reflective microscopy, the components of Kohler illumination include (see Fig. 8) a lamp, a lamp condenser, and two diaphragms. (Diaphragms are variable diameter holes that control the amount of light or angle of light passing through.) After leaving the light source, rays of light pass through the lamp condenser (or collector lens) which gathers the rays into a focused beam. Light then passes through the aperture diaphragm which controls the angle of the cone of light striking the specimen. This angle plays a role in determining the numerical aperture and therefore the resolving power of the microscope. The field diaphragm controls the size of the illuminated field of view in the microscope. The ability to do so allows certain adjustments to be made such as centering of the lamp during Kohler alignment (not to be performed for this lab exercise). For a more detailed description of the functions of the various parts, refer to: Optical Techniques Various optics can be placed in the light path before entering the eyepiece of the microscope. These optics change what is referred to as the contrast mode. While operating a reflective microscope, light that strikes the specimen may either reflect efficiently off of the surface (as with a mirror) or scatter if the sample is not highly polished. Since most samples are highly polished for metallographic examination, the image in the eyepiece is rather uninteresting. It is therefore necessary to use one of two methods (or both) to increase the contrast between grains, grain Figure 8 - Kohler illumination components (Olympus America Inc.)

boundaries, cracks and other microdefects. The first technique is to etch the sample. The second technique to increase contrast is to use one of various contrast modes.

This mode relies on the reflective nature of a highly polished sample.

Figure 9 shows a representative image of two grains shown at the bottom of the figure reflecting light.

6 boundaries, cracks and other microdefects. The first technique is to etch the sample. The second technique to increase contrast is to use one of various contrast modes. There are many different modes, of which three are described below. Bright Field The bright field mode is the most common method for observation and is often the starting point. This mode relies on the reflective nature of a highly polished sample. Polished areas will appear light in the eyepiece of the microscope, while textured areas or grain boundaries will appear dark since light does not reflect efficiently in these areas. Figure 9 shows a representative image of two grains shown at the bottom of the figure reflecting light. The textured grain and the grain boundary scatter the light rather than reflecting at a sharp angle back through the eyepiece. Because of this, the textured grain and the boundary are dark when viewed through the eyepiece as seen in the top portion of the figure. However, the smooth grain reflects light effectively and when seen through the microscope, a bright area appears, as seen in the top portion of the figure. It is important to repeat that features such as grain boundaries are difficult or impossible to see in metals when they samples are polished. The boundaries and surface reflect almost all light due to the uniform surface. It is only when a chemical etchant is used that grain boundaries become visible. This etching process, which makes use of an acid, preferentially removes more material at the grain boundaries because of the higher reactivity at the boundaries. Figure 10 shows the result of a chemically etched steel sample viewed with bright field optics. Dark Field Dark field optics create a contrast mode where only scattered light is used to produce the image and not reflected light. The areas of the sample that scatter light show up as light areas in the eyepiece, the opposite of bright field. Although not entirely correct, a dark field image is similar to a negative of the bright field image. This is not completely true as certain effects are visible in one mode and not the other. Figure 10 is a dark field image of a steel sample. In summary, bright field viewing will most often provide the most detailed image and will be used for the majority of viewing. Certain samples will benefit from dark field viewing as grain boundaries can actually be more vivid and certain defects will only be seen in this mode. Figure 9 - Illustration of reflection of light off of smooth and textured surfaces. Figure 10 - Image of an etched 1018 steel sample (bright field). Figure 11 - Dark field image of a 1018 steel

7 Differential Interference Contrast This method reveals topographic detail present on the sample surface. Chemical etchants will often attack and etch surfaces at different rates depending on the phase or its orientation. This creates height variations that can be seen with differential interference contrast (DIC). Images can be viewed with natural colors as in the bright field, or artificial coloring introduced with a tint plate. Figure 12 is an example of a DIC image taken with artificial coloring introduced. Lab Safety and Cautions Working with microscopes poses very few hazards. The halogen bulb will become extremely hot during operation but is not accessible. However, it will cause the lamp housing to become warm and caution should be taken. Personal protection equipment is not required. Figure 12 - DIC image of gray 20 cast iron. Note the height contrast between the phases. Great care must be taken to protect the microscopes from damage. The Olympus BX51 microscopes are precision instruments and very expensive, both to repair or replace. The following guidelines must always be followed. No food or drink in the lab. Use care when rotating knobs as some have a mechanical stop. Excessive torque may cause damage. When pushing or pulling on levers, do not use excessive force. Note that most filters can be pulled all the way out. Be careful not to remove them completely. Use caution when rotating the nose piece (containing the objectives). Make sure there are no obstructions on the stage. Do not let anything touch the objective lenses (other than appropriate cleaning materials). They must be clean and scratch-free. Keep the eyepiece lens clean and free of debris. The single most comment accident and cause for damage is moving the mechanical stage too far up during focusing. This causes the specimen to collide with the objective. This can damage the objective and other parts. Use caution when focusing. To minimize the possibility of a collision, start by raising the stage until the specimen is close to the objective. Then, move the stage down in order to bring the image into focus. Always place the dust cover on the microscope when finished.

Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 1: THE LIGHT MICROSCOPE

Biology 29 Cell Structure and Function Spring, 2009 Springer LABORATORY 1: THE LIGHT MICROSCOPE Prior to lab: 1) Read these instructions (p 1-6) 2) Go through the online tutorial, the microscopy pre-lab