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70 Cards in this Set
- Front
- Back
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What is the etymology of histology? |
Greek. "histos" tissue "logos" study of Sometimes referred to as microscopic anatomy. |
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What are the primary foci of the class? |
1. Structure of normal tissues. 2. Relationship of structure to function. |
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Two types of microscopy |
1. Light microscopy 2. Electron microscopy |
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Light microscopy (illumination and resolution) |
Illumination: visible light (540 nm, green) Resolution: 0.2 micrometers |
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Electron microscopy (illumination and resolution) |
Illumination: electron beam (0.1 nm) Resolution: 0.05 nm |
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What is the primary microscopy tool used to identify and study tissue structures? |
Light microscopy |
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What are the 3 types of light microscopy? |
1. Bright field 2. Phase contrast 3. Fluorescence |
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Bright field microscopy |
- must apply stain to see image - light passes through specimen - most commonly used in histology |
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Amplitude contrast |
The method of producing a contrast between sample and background of white light. |
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Phase contrast microscopy |
- Density of sample causes transmitted light to be differentially out of phase. - modified condenser and objective lens convert phase differences into amplitude differences (can be seen by eye). - used for unstained samples: living cells, very thin fresh tissues or frozen sections. - used primarily for research and not for routine histology. |
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Fluorescence microscopy |
- based on the use of fluorescent dyes to label ("stain") - Fluorescent dyes have the property of emitting light of one wavelength (color) when illuminated with light of a shorter wavelength - provides greater resolution - primarily a research tool, but it is increasingly used in routine pathology to detect markers for specific diseases |
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Commonly Used Fluorescent Dyes |
Dye Exciting Light Emitted Light Fluorescence Blue Green Rhodamine Green Red |
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How is specificity of labeling conferred? |
by attaching the fluorescent dye to a specific probe (label) |
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What do the following probes bind to? antibodies ligands enzymes phalloidin lipids |
antibodies - bind to specific antigens ligands - bind to receptors enzymes - bind to substrates phalloidin - binds to actin filaments lipids - label membranes |
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What are the benefits of electron microscopy? |
- higher resolution - e- has shorter wavelength than photon - greater resolving power (by 1000X) - not used in routine histology, but are used for diagnostic pathology |
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Differences between light and electron microscopes |
1. electrons and specimen must be kept in vacuum 2. e- micro. uses electromagnetic glass lenses to focus e- beams 3. electrons must be converted to photons via striking a phosphorescent screen or e- detector to convert image to TV image 4. both light and electrons can make images on photographic film |
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What are the two types of electron microscopy? |
1. Transmission Electron Microscopy (TEM) 2. Scanning Electron Microscopy (SEM) |
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Transmission Electron Microscopy (TEM) |
- equivalent to bright field light microscopy - e- beam is focused on a sample by a condenser lens - transmitted e-'s that pass through the sample are refocused by objective lens to form image - tissues must be sliced thin <90 nm (versus <5 micrometers for light microscope) |
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Scanning Electron Microscopy (SEM) |
- equivalent to dissecting light microscopy - e- beam is focused on sample by a condenser lens - e-'s that are ejected from surface of sample are picked up by a detector that convers them to image on monitor - to form image, the illuminating e- beam is scanned back and forth in a raster pattern across the sample in synchrony with the raster pattern of the e- beam that illuminates the monitor - result is a pseudo-3D image |
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What 3 factors affect the quality of an optical image? |
1. Magnification 2. Resolution 3. Contrast |
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Definition of magnification |
How much the image of an object is enlarged when viewed with the unaided eye as compared with its actual size. |
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What determines magnification? |
Power of the lenses used to view the sample. |
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What does the final magnification depend on? |
Degree of enlargement during printing. |
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What does the amount of info depend more on: resolution or magnification?
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Depends more on resolution than magnification. |
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How to calculate total magnification
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Product of magnification of different lenses used: 40X * 10X = 400X |
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What is empty magnification? |
Enlargement of an image without a corresponding increase in resolution. |
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Definition of resolution
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The ability of a lens to distinguish between two adjacent objects. A measure of the smallest object that can be seen with the microscope. The minimum distance between two points at which they can still be visualized as two separate points. - It is a fixed property of the lens. - Cannot be increased by subsequent processing. |
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Definition of numerical aperture |
light gathering capacity of a lens
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What does resolving power of a lens depend on? |
1. Depends on ability to gather light. 2. The wavelength of the illuminating radiation. |
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Higher numerical aperture leads to...? |
More light is gathered => Higher resolution |
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Formula for resolution: |
asdfasdfasdf |
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A shorter wavelength leads to...? |
Higher resolution |
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How is numerical aperture determined? |
Determined by size of the cone of light that enters lens from specimen when specimen is in focus (aperture angle). Refractive index of the medium through which the light passes from the sample also matters (air vs vacuum). |
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Oil immersion vs dry lens |
Oil immersion (RI of 1.4) has larger numerical aperture and greater resolving power than dry lens. |
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Definition of contrast |
Ability of an object to be distinguished visually from its background. |
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Biological molecules and tissues have high or low contrast? |
Biological molecules and tissues have low contrast. |
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Types of contrast: |
1. Amplitude contrast 2. Dark field contrast 3. Phase contrast 4. Differential interference contrast 5. Fluorescence contrast |
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What does amplitude contrast use to enhance contrast? |
Colored dyes (stains) to enhance contrast |
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Fluorescence contrast |
- Uses fluorescent dyes to stain different components of cells and tissues selectively. - Uses a modified optical system to allow only the fluorescently labeled structures to be visualized. - Powerful modern tool for research and diagnostics. |
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What are the steps required to prepare tissues for microscopic examination? (All affect appearance of the microscopic image) |
1. Fixation 2. Dehydration 3. Embedding 4. Sectioning 5. Staining |
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What is fixation? |
Treat sample to preserve it for study. |
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What are some fixatives? |
Light microscopy uses formaldehyde. Electron microscopy uses glutaraldehyde and osmium tetroxide. |
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What is dehydration? |
Removing water to permit sample to be embedded in a non-aqueous medium. |
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What are some dehydrating agents? |
alcohol, acetone |
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What is embedding? |
Impregnating and surrounding the sample with a medium that is sufficiently hard to allow it to be cut into thin slices. *This step can be omitted if illuminating radiation does not need to pass through sample. |
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What are some embedding agents? |
For light microscopy, use paraffin. For light and electron microscopy, use plastic. |
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What is sectioning? |
Slicing the sample into slices thin enough for the illuminating radiation to pass through it. *This step can be omitted if illuminating radiation does not need to pass through sample. |
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What is staining? |
Differentially coloring different parts of the tissue to enhance contrast. Staining method used largely determines what one sees under the microscope. |
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Why do stains bind to tissue components? |
Most stains bind to tissue components because of chemical affinity of physical affinity. |
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What are the 3 types of electrostatic binding? (positive/basic binds to negative/acidic) |
Acidophilia Basophilia Mordants |
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Electrostatic binding chart |
Dye Cell Component Stained acid dye proteins (-NH3+) [anionic (- charge)] [basic = cationic = acidophilic] basic dye nucleic acids (-PO3-) acidic sugars (-COO-) [cationic (+ charge)] [acidic = anionic = basophilic] |
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What is acidophilia? |
A substance that stains with an acid dye is described as being acidophilic (acid loving). Most acidophilic molecules = proteins. |
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What is basophilia? |
A substance that stains with a basic dye (base loving). Most basophilic substances are nucleic acids or acidic sugars. |
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What are mordants? |
A substance that binds a neutrally charged molecule to tissue components. e.g., hematoxylin - must be mixed with metal atoms (positively charged) to enable it to bid to negatively charged molecules. |
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What is affects the color of the stain?
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The nature of the mordant affects the color of the stain. |
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What color does potassium-alum hematoxylin stain? |
Purple staining. |
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What color does iron hematoxylin stain? |
Black to brown staining. |
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What color does copper hematoxylin stain? |
Green staining. |
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How is covalent binding achieved? |
Osmium tetroxide (OsO4) is sometimes used as a stain in light microscopy because it binds covalently to lipids, causing them to appear black in the light microscope. |
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What is double staining? |
Nuclear stains and counterstains. Most tissues are stained with two stains. |
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What is a nuclear stain? |
Staining of the DNA of the nucleus (e.g., hematoxylin). |
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What is a counterstain? |
Staining components of the cytoplasm and/or extracellular matrix (e.g., eosin). |
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What are polychrome stains? |
Mixture of different stains (e.g., Wright's stain). Stains different components of tissue different colors. |
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What is orthrochromasia? |
Tissue components stained the color of the dye. |
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What is metachromasia? |
Some tissue components are stained a different color from that dye. Azure dyes (blue) stain DNA blue but can stain certain other structures red or purple. |
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What can histochemical staining accomplish? |
Detects and reveals location of specific substances. This method is referred to as histochemistry. If the object of study is individual cells rather than tissues, the method is referred to as cytochemistry. |
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What is the periodic acid-Schiff (PAS) reaction? |
The most important histochemical method used in routine histology. It allows carbohydrates and carbohydrate-rich macromolecules (mucus, glycogen, etc.) to be stained magenta. Converts carbs to aldehydes. |
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What is the Fuelgen reaction used for? |
Used to differentiate DNA from RNA. |
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What is enzyme histochemistry? |
A method used to detect the presence of specific enzymes by using the enzyme to generate a colored reaction product. |
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What is immunihistochemistry? |
Uses labeled antibodies to detect the presence of specific molecules in tissues and cell. |
