Why is Kohler Illumination Utilized - Assignment Example

Microscopic Report Student Number: Institution: Date: Question 1 Why is Kohler Illumination utilized? Kohler Illumination is a specimen illumination method used for reflected and transmitted light optical microscopy. The method is used to produce an extremely homogenous illumination of the specimen to ensure that the image of the source of illumination does not become visible in the image formed. The incident light is properly aligned and the image of the illumination source is perfectly defocused, allowing the best imaging possible in a field of light that is evenly dispersed. This enables collection of more imaging data. The contrast between areas of interest is increased, generating a sharper final image with high overall resolution. It is the modern technique for specimen illumination in modern light microscopy. Other reasons why this method is used is because it eliminates glare and reflections in the optical system, and specimen heating is also reduced. Excessive light reflections affect quality of image contrast while heating of specimens is problematic especially when viewing living specimens (Hibbs, 2004). Question 2 Why would you stain specimens prior to examining under a microscope? Cells and most elements that make up a specimen are colorless, except a few natural pigments, e.g. melanin. In order to obtain a detailed structural information using a microscope, staining of the specimen is essential. This is made possible by the fact that staining increases contrast by changing the color of the specimen under observation, allowing for clearer imaging. Different staining techniques are employed in biology and medicine to define particular microorganisms, or highlight structures of interest in biological tissues so as to have a clear view under a microscope. Stains can be used in the examination and defining bulk tissues, for example connective tissue or muscle fibers, or examining cell populations, e.g. for classification of different blood cells, or cell organelles (Karp, 2009). The detailed information provided through specialized staining techniques is important in histopathology to obtain a full differential diagnosis. Staining technique is not limited to biological tissues, but it can also be used to examine morphology of materials such as the lamellar structures in semi-crystalline polymers. Question 3 What structures do haematoxylin and eosin stain and with what colours? Haematoxylin and eosin (H&E) stain is a combination of these two dyes routinely and universally used as a point of start in providing essential structural information. Hematoxylin is a positive/basic violet or dark blue stain that binds to substances that are basophilic. Eosin is a negative/acidic pink or red stain that binds to substances that are acidophilic (Crocker & Burnett, 2005). Using this method, eosin stains the cytoplasm, proteins and other extra-cellular components with shades of pink, orange or red, while haematoxylin stains the cell nucleus, and other objects such as calcified material and keratohyalin with a deep blue-purple color. Generally, oesinophilic structures are composed of extracellular or intracellular protein. Examples of such structures include the Mallory bodies and Lewy bodies, and the red blood cells which are intensely stained with red color. The cytoplasm is largely eosinophilic (Bancroft & Gamble, 2008). In micrscopy, we observe: Cytoplasm as red Nuclei as deep blue/purple Muscles as dark red Basophils as purple/red Collagen as pale pink Erythrocytes as cherry red Mitochondria as pale pink It is possible to diagnose many conditions in histopathology by examining an H&E only. Question 4: Why might you use microscope co-ordinates? Microscope coordinates are important in relocating an organism or artifact on the slide. Each feature on a specimen is located on a unique X-Y coordinate that can be used to identify a particular feature on the slide. This is particularly important to point out for referral or share your findings with people who are interested. The coordinates are normally recorded on the X (from left to right) axis and the Y axis (from top to bottom). It enables the observer to make notes and record positions on the specimen for future references. To move to a particular reference point, the X-Y coordinates are simply entered on the microscope. Coordinate assignment is also important in the case of specimen identification and validation, and taxon types, especially where it will require future researchers to locate a particular specimen as referenced in the protologue, and specimens from materials – specifically, vouchered or type specimens (Paddock, 1999). Question 5: What different epithelial cells might you observe? Epithelium form part of the main tissues in the body. It is usually made up of millions of epithelial cells that are arranged in tubules or sheets attached to the underlying membrane (Krause, 2004). There are four different epithelial cells that can be observed: Squamous epithelial cells – These are flattened cells that are mostly found lining on surfaces that need a thin surface for molecular transfer, such as air sacs found in the lungs, or a smooth fluid flow such as the blood vessels. Cuboidal epithelial cells – These are single layered cube-shaped cells that are typically found in substance secreting or absorbing tissues, such as the glands (e.g. the thyroid gland), ducts and kidney tubules (Krause, 2004). Columnar epithelial cells – These cells are long, thin and columnar-shaped arranged in a single layer. They are found lining in areas that secrete mucus such as the intestine, stomach, and in gall bladder and the uterus. Ciliated columnar cells – These cells usually have their apical surface covered with cilia – tiny little hairs. They are useful in pushing mucus or other particles for smooth flow (Krause, 2004). Question 6: What different types of epithelial cells might you observe from your cheek cells? The human cheek cell is typically thin, flat and irregular in shape and has a large nucleus with the DNA. It is made up of a tissue called basal mucosa that lines the inside walls of the mouth. This tissue is composed of squamous epithelial cells whose function is to continually secrete mucus that play an important role of keeping a moist environment inside the mouth. In association with salivary glands, the cheek epithelial cells provide enough supply of moisture for enzymes to function well. The moisture assists in softening food, swallowing and starts the first steps of food digestion. The epithelial cells are relatively permeable to selectively allow certain particles or molecules in and out of the cell (King, et al., 2001). These cells can be obtained through mouth rinsing or a simple swab. Though simple, these cells contain the whole genetic makeup of an individual. Thus, they are used in investigations involving DNA identification, e.g. in establishing paternity (King, et al., 2001). Question 7: Can you determine from which part of the body the epithelial cells came from, simply by looking at it through the microscope? It is possible to determine the part of the body from which the epithelial cells observed through the microscope. Different epithelial cells have different apical shapes that vary with the part of the body from where they are obtained. There are four main shapes of epithelial cells: squamous, columnar, cuboidal and ciliated columnar. Squamous epithelial cells are thin and flat-shaped and are normally found in blood vessels of air sacs in the lungs. Columnar epithelial cells form long, thin and tiny columns and are normally found in mucus-secreting places such as the stomach, intestines, and on the linings of the gall bladder and in the uterus. Cuboidal epithelial cells have a cubical cross section and are typically obtained in tissues such as glands and the kidney. Ciliated columnar cells are characterized by small hairs known cilia covered on their outer face. Other cell shapes used to identify epithelial cells depends on whether they are simple stratified. Stratified cells are commonly found in areas that are subjected to wear and tear, e.g. the skin (Solomon, 2015). Question 8: Are there any other techniques which might identify from which part of the body the epithelial cell originated? There are other techniques available used to identify the part of the body from which epithelial cells have originated. These are discussed below: The Lugol’s Iodine Staining technique – This technique involves using iodine in the detection of extracellular glycogen. It is believed that some areas have higher concentration of epithelial cells than other areas of the body (Wise, 2002). Dane’s, staining technique – This technique uses histological staining to distinguish between buccal, vaginal and skin epithelial cells. The samples are stained and analyzed quantitatively by examining the patterns or colour, and the morphology of the cells. Under a microscope, epithelial cells from different areas of the body will have different staining patterns and morphology. Immunohistochemical staining techniques – This technique involves staining a sample of epithelial cells with antibodies to identify certain proteins that make up a specific cell. It is widely used to identify vaginal epithelial cells. It has increased the ability to identify other categories of cells under a microscope (Wise, 2002). Question 9: Why might identifying the origin of the epithelial cell be useful in forensic science? There are several forensic cases that may require the identification of the type of epithelial cells from which certain DNA originated. This would provide significant probative evidence. Many types of human body fluids may be retrieved from a crime scene and can be potentially analyzed and become useful in identifying the perpetrator of the criminal act. For example, when investigating exhibits retrieved from a sexual assault scene, forensic science target to obtain evidence that will confirm or refute allegations of occurrence of a sexual intercourse that is non-consensual (James, et al., 2005). Evidence may be unfolded as trace evidence or stains from the body fluid of the suspect, the victim or the scene of crime. Saliva is typically composed of enzymes, water, mucus, various electrolytes, and epithelial cells originating from the cheeks. It contains the same proteins as those found in blood and urine, thus, it is one ideal body fluid for DNA profiling. References Read More