Microanatomy Intro.docx
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Microanatomy general info Microanatomy (AKA: Histology) is the study of cells and tissues, as well as the study of how these integrate to form organs. The study of microanatomy requires a microscope for viewing these samples. This differs from gross anatomy which studies larger parts of the body suc...
Microanatomy general info Microanatomy (AKA: Histology) is the study of cells and tissues, as well as the study of how these integrate to form organs. The study of microanatomy requires a microscope for viewing these samples. This differs from gross anatomy which studies larger parts of the body such as organs, which can be seen with the naked eye. Embryology (AKA: Developmental Biology) is the study of the development of an individual (and involves studying embryo and fetus development) Cytology is the study of cell structure and function. An example of a cytology test is a vaginal smear for estrus detection within a canine. Note that histology shows the viewer the relationship of one cell to another, while cytology does not indicate the relationship between cells. Histopathology is the study of diseased tissue. An example of a histopathology test would be an organ biopsy. Cytopathology is the study of diseased cells via fluids or tissue. An example of a cytopathology test would be a fine needle aspirate (of a lump or bump). A biospecimen (AKA: Tissue Sample) is a sample of tissue or a whole organ that was obtained from a non-living animal. A biopsy is the collection of a tissue sample from a living animal. A liquid biopsy involves a blood sample. The microtome cuts sample into 1-7micrometer thin sections. These slices must then be floated in water to be retrieved and stained. Histochemistry is the staining of enzymes. Immunohistochemistry involves the binding of antibodies labeled with fluorescent dyes or enzymes to antigens, resulting in a color change. Immunohistochemistry is very specific. In the Pancreatic Islets of Langerhans, glucagon and insulin can be stained with antibodies that are labeled with visible stain. Glucagon and insulin are both stained brown within this process. Microscopy Light Microscopy In light microscopy, a light beam is being transmitted through tissue. Examples of light microscopy include: Phase-contrast Fluorescence Brightfield Polarizing Dissecting stereomicroscope Advantages of light microscopy include: Inexpensive Rapid diagnosis Viewing live specimens Resolving power of 2 micrometers (um) Disadvantages of light microscopy include: Requires expertise for diagnosis Requires maintenance 2 dimensional image Limited resolving power based on light wavelength Bright field microscopy Bright field microscopy requires staining for contrast. Bright field microscopy has the same objectives as digital scanners to produce a digital image. Phase Contrast Microscopy Phase contrast microscopy allows for the viewing of living, non-stained structures. These structures can include: Cell culture Tissue culture Spermatozoa Leukocytes Dense structures have a higher refractive index in comparison to less dense structures. Fluorescence Microscopy Fluorescence microscopy involves the staining of specific cellular components and viewing them under ultraviolet light. In a cell culture of kidney cells, the blue fluorescent dye binds to the nuclear DNA, while the green fluorescent dye binds to the actin filaments. Polarized Microscopy In polarized microscopy, a polarizing filter is added to a bright field microscope, thus highlighting birefringent materials such as crystalline materials or collagen fibers. Dissecting Stereomicroscopy Advantages of dissecting stereomicroscopy include: Inexpensive Used in microsurgery 3-dimensional image Practical/versatile Disadvantages of dissecting stereomicroscopy include: Requires maintenance Low resolving power Transmission Electron Microscopy (TEM) TEM utilizes electron beams which have a shorter wavelength in comparison to a light beam, causing its images to have a higher resolution by 1000-fold. In TEM, electrons interact with tissue components. Advantages of the TEM include: Resolving power of 0.16-0.18 nanometers Allows for rapid diagnosis of viruses, storage diseases, and other microorganisms Disadvantages of the TEM include: Expensive Requires extensive preparation of the specimen 2-dimensional images Only black and white images Cannot use living specimens Scanning Electron Microscopy (SEM) The SEM uses electron beams to scan the surface of a specimen, resulting in a 3-dimensional effect. SEM only shows external surfaces within the image. The SEM has a lower resolution in comparison to the TEM. Light Microscopy and Electron Microscopy Similarities In both light microscopy and electron microscopy, the specimen must: Be well preserved Be thin Have enough contrast (AKA: staining) Prepping Tissue for Microscopic Examination Step 1: Collect a tissue sample via biopsy or a biospecimen. Step 2: Trim the tissue until it is 1cm^3 in length. Step 3: place the tissue sample into 10 times the volume of 10% formalin fixative to preserve the sample via process called “fixation”. 10% formalin fixative coagulates proteins into a life-like manner and is hazardous. Step 4: Increase the percent of alcohol within the solution for the “dehydration” process. Step 5: Add xylene to the solution to remove the alcohol for the “clearing” process. Step 6: The “infiltration” process takes place at 58-60 degrees Celsius. Step 7: Add paraffin wax to the solution for the “embedding” process, in which, cooling the paraffin wax will cause the solution to solidify. Step 8: Once solid, the sample can be sliced using a microtome. Staining Types Haematoxylin-Eosin (H&E) Haematoxylin Stains the nucleus, DNA, and RNA blue Basophilic (basic) stain Eosin Stains the cytoplasm, and proteins pink Eosinophilic (acidic) stain H&E is the most common stain used for routine evaluations. H&E stains goblet cells pale. Masson’s Trichrome Masson’s Trichrome stains collagen green and smooth muscle grey. Mallory’s Trichrome Mallory’s Trichrome stains collagen blue and nuclei red. Periodic Acid Schiff (PAS) PAS is a specialized stain that localizes glycogen, glycoproteins, and mucin. PAS stains goblet cells magenta. Gomori’s Method In the brush border of the proximal convoluted tubules of the kidney, Gomori’s Method will stain the alkaline phosphatase black.
