Background & History, Microscopy, Tissue Prep & Staining, Epi. Characteristics 1 Flashcards
What are the characteristics of a virtual image?
-Real -Upright -inside focal point -Can be magnified
What is Histopathology?
The study of diseased tissue
What is refractive power?
A measure of how much a lens bends light waves. Measured in diopters
What are the four basic tissue types?
Epithelial tissue Connective Tissue Muscular Tissue Nervous Tissue
What type of epithelial are kidneys?
Simple cuboidal
What is refraction of light?
The bending of light as it passes through a medium. When traveling through a vacuum, velocity is fixed.
What type of epithelial is the ileum?
Simple columnar
What are 4 examples of connective tissue?
Dense Bone Cancellous bone Adipose Tissue Hyaline Cartilage
What is resolution?
The ability of a microscope to distinguish two lines as separate entities. Diameter of diffraction lines around the points must be reduced.
What are 3 examples of muscular tissue?
Smooth muscle Cardiac muscle Skeletal muscle
What are 2 types of nervous tissue?
Purjinke cells Cerebral Cortex
What is the equation for refractive index?
RI = velocity of light/velocity in medium
What is a focal point?
The point at which all lines converge after passing through a lens.
Indicate which major tissue group these each belong: a) ducts of glands b) skin c) lining of blood and lymphatic vessels d) fat e) red and white blood cells f) kidney tubules g) cartilage h) tendons and ligaments
a) epithelial b) epithelial and CT c) epithelial d) CT e) CT f) epithelial g) CT h) CT
What two histologists shared a Noble Peace Prize in Physiology/Medicine?
John O’Keefe and Edward I. Moser
What is the highest resolution for a TEM?
0.5-10 angstroms
What is the equation for resolution?
d = (0.61)wavelength/n sin alpha (want a small d)
What is the concept of humoral pathology?
That diseases are due to an imbalance of 4 fluid humors: blood, phlegm, yellow bile and black bile
Who found out that it wasn’t due to humoral imbalances but based on organ level in pathology?
Giovanni Battista Morgagni
What role did Bichat play in histology?
Frenchman; described 21 membranes “father of modern histology” via tissue level and didn’t use a microscope; organic life
What role did Virchow place in histology?
German; cellular level; used the cell theory that all life forms contain cells from existing cells
What does the degree of bending depend upon? (2)
The angle at which the light strikes the surface and the refractive index (n)
What are the characteristics of a real image?
Located outside of the focal point Inverted Can be projected into a screen A short focal point indicates greatest magnification
What is the max value for n (refractive index)?
1.4 (oil)
What is the max value for sin alpha?
180
What values can be changed to improve resolution? (2)
Shorter wavelength Larger n (refractive index)
What is the highest resolution of a light microscope?
0.5 micrometers
What are the components of a light/bright field microscope? (4)
Condensor Objective lens Ocular lens Stage Light source
What are the pros and cons of light microscope?
Pros: ability to magnify and resolve structural detail Cons: specimen must be thin, little contrast in unstained specimen
How does Phase Contrast Microscope work, what is examined with them?
Views living cells, unstained cells and tissues. Converts phase shifts (invisible to eye) in light passing through a transparent specimen to brightness changes (visible) in the image. Light passing thru cell regions become deflected and out of phase with main stream light waves which are matched with other induced out of phase wavelengths which cancels their amplitude and creates light waves that can be seen.
How does a fluorescence microscope work and what do they detect?
Detects molecules that emit light of wavelengths within the visible range when exposed to a UV light source. Used to detect induced fluorescence (such as Ag, Ab, injected fluorescent tracers)
How does a confocal scanning microscope work and what is visualized?
A pinhole is added to eliminate out-of-focus light to increase optical resolution and contrast. Used to view 3D images. This type uses a mixture of fluorescent and light microscopes with a scanning system that employs a laser beam that produces a scanning spot that utilizes a mirror system that moves across a specimen and data from each spot on the specimen is recorded and stored into a computer, which then creates a visual image.
What are the advantages of a confocal scanning microscope (3)?
Very thin optical images of specimen can be created (1um thick) Out-of-focus images are subtracted by the computer Computer can make 3D reconstructions of specimens by stacking individual images.
What are the components of TEM microscope?
Cathode Heated tungsten filament (electron source) Anode; drives electrons thru the column Series of electromagnets; condenser lens, objective lens, and projection lens. Specimen holder Viewing screen and photographic film
What does TEM microscope utilize rather than light?
A beam of electrons rather than light?
What does a fluorescence microscope detect and what is its most widespread application?
Detects molecules that emit light of wavelengths in the visible range when exposed to a UV light source Detects naturally occurring fluorescent (autofluorescent) molecules i.e. Vitamin A Widespread use of visualization of induced fluorescence: Ag or Ab in immunochemical staining procedures and fluorescent tracers injectoed into animals or cells
What are the 4 steps in tissue fixation and embedding?
Fixation Dehydration Removal of alcohol Embedding
What are the characteristics of fixation, why is it used?
Prevents further deterioration and hardens tissue prior to embedding and sectioning. Any fixative radically distorts a specimen Ideal fixatives give greater optical contrast (w/ staining) with the least amount of distortion
What fixative agent is most widely used?
Formalin
What are the characteristics of Formalin; what can it be mixed with, what does it react with, and not used for
Can be used alone or mixed with acetic acid which softens and counteracts the shrinkage of alcohol Reacts with amino acids of the tissue proteins and stabilizes tissue structure to prevent further deterioration Not good for detailing fine cytological details
What are the acid fixatives? What do acid fixatives fix?
Chromatin, nucleoli, spindle fibers Carnoy’s fluid Zenker’s fluid Bouin’s fluid
What is the basic fixative and what does it fix?
Mitochondria; dissolves chromatin Zirkle-Erliki
What are the Zirkle-Erliki fixative characteristics?
Requires long fixing time (2 days) and washing under running water.
Uses for Carnoy’s fluid
Useful for preserving glycogen in animal tissues
Uses for Zenker’s fluid
Useful when sharp histological detail is desired, must be washed carefully to prevent precipitation of black crystals
Uses for Bouin’s fluid
widely used that gives good cytological detail requires prolonged and careful washing cycles.
Fixative for TEM?
Glutaraldehyde; which preserves proteins by cross-linking them. And Osmium Tetroxide; which react with lipids (esp. phopholipids) and imparts electron density to cell and tissue structures.
What is tissue dehydration used for during tissue fixation and embedding?
To remove water from the sample because sample will be placed in paraffin which is hydrophobic Placed in ethanol, or N-butyl OH or acetone if don’t want other substances such as neutral fats to get dissolved.
What is used during tissue clearing?
Xylene or cedar oil, to remove the OH so that the paraffin will mix with it
What occurs during tissue embedding?
Sample is placed through many paraffin baths and eventually placed on melted paraffin then thrown into a cold water bath to solidify
What occurs for TEM during tissue embedding?
Tissues are infiltrated with a monomeric resin
What are the techniques used to section tissues? (3)
- Rotary Microtome 2. Sharp razor and tubular holder 3. TEM = diamond knife with copper mesh
What are the steps for staining? (5)
- Specimen is removed from paraffin 2. Xylene is removed via OH 3. Stains are applied then dehydrated with OH 4. OH is removed by xylene 5. Drop of cemet is applied then the cover slip
What are the 3 basic staining dyes?
- Hematoxylin 2. Orcein and Resorcin Fuchsin 3. Sudans
What are the 4 acidic staining dyes?
- Eosin 2. Orange G 3. Acid fuchsin 4. Aniline blue
Characteristics of H&E stain
Stains structural features as opposed to chemical features. H stains nuclear material and RER dark blue to light purple. E stains cytoplasmic and ECM yellow to pinkish
Nuclei - dark purple (hematoxylin)
CT - pink (eosin)
Usage for Orcein and Resorcin Fuchsin stain
Elastic material; reticular fibers and basement membranes black
Usage for Sudans
Fat soluble stains
Characteristics of basic dyes
Basophilic; react with anionic groups (phosphate groups, carboxyl and sulfate groups) Binding depends on pH - high pH binds more readily
Characteristics of acid dyes
Acidophilic; react with cationic group via electrostatic interactions (i.e. amino groups of proteins)
Mallory’s triple stain
Aniline blue - collagen Acid fuchsin - cytoplasm Orange G - RBCs
Metachromasia
When a dye changes color after reacting with a tissue component i.e. toluidine blue used for cartilage ground substance or mast cell granules - stains black
What is used for TEM staining and what do they stain
Electron dense heavy metals Osmium tetroxide Uranyl nitrate Uranyl acetate and lead citrate For SEM gold/platinum
What is histochemistry used for?
Study the chemistry of cells and tissues
What is Pearl’s rxn used for?
To display iron in tissues (such as patients with iron storage diseases; hemochromatosis) Tissus incubated in K ferrocyanide and HCl where results are insoluble blue precipitate of ferric ferrocyanide
What are lipid stains used for
Since lipids are soluble in normal tissue prepping techniques
i.e. oil Red O
Usage for Orcein and Resorcin Fuchsin stain
Elastic material; reticular fibers and basement membranes black
Usage for Sudans
Fat soluble stains
Characteristics of basic dyes
Basophilic; react with anionic groups (phosphate groups, carboxyl and sulfate groups) Binding depends on pH - high pH binds more readily
Characteristics of acid dyes
Acidophilic; react with cationic group via electrostatic interactions (i.e. amino groups of proteins)
Mallory’s triple stain
Aniline blue - collagen Acid fuchsin - cytoplasm Orange G - RBCs
Metachromasia
When a dye changes color after reacting with a tissue component i.e. toluidine blue used for cartilage ground substance or mast cell granules - stains black
What is used for TEM staining and what do they stain
Electron dense heavy metals Osmium tetroxide Uranyl nitrate Uranyl acetate and lead citrate For SEM gold/platinum
What is histochemistry used for?
Study the chemistry of cells and tissues
What is Pearl’s rxn used for?
To display iron in tissues (such as patients with iron storage diseases; hemochromatosis) Tissus incubated in K ferrocyanide and HCl where results are insoluble blue precipitate of ferric ferrocyanide
What are lipid stains used for
Since lipids are soluble in normal tissue prepping techniques
Usage for Orcein and Resorcin Fuchsin stain
Elastic material; reticular fibers and basement membranes black
Usage for Sudans
Fat soluble stains
Characteristics of basic dyes
Basophilic; react with anionic groups (phosphate groups, carboxyl and sulfate groups) Binding depends on pH - high pH binds more readily
Characteristics of acid dyes
Acidophilic; react with cationic group via electrostatic interactions (i.e. amino groups of proteins)
Mallory’s triple stain
Aniline blue - collagen Acid fuchsin - cytoplasm Orange G - RBCs
Metachromasia
When a dye changes color after reacting with a tissue component i.e. toluidine blue used for cartilage ground substance or mast cell granules - stains black
What is used for TEM staining and what do they stain
Electron dense heavy metals Osmium tetroxide Uranyl nitrate Uranyl acetate and lead citrate For SEM gold/platinum
What are Schiff reagent reactions?
Reactions occur due to aldehyde formation from exposure to HCl or periodic acid which forms deep pinkish color
-nuclei (pink)
What are 2 examples of Schiff reagents?
Fuelgen reaction Peirodic acid-Schiff rxn (PAS) - forms aldehyde groups by cleaving bonds adjacent C or carbs (deep pinkish color); Glycogen, Glycosaminoglycans, proteoglycans, glycoproteins, glycolipids
What is best carmine used for?
To demonstrate glycogen deposits (red dots)
What is epitheloid?
An activated Macrophage which lack a free surface
What are 5 epithelium characteristics?
- connected via tight and leaky junctions
- avascularized
- are derived from all three germ layers
- contain little intercellular matrix
- display free surfaces (surface to which no cellular or extracellular formed elements adhere. The free surface may contain specialized modifications, such as microvilli or cilia)
Functions of the basement membrane (3)
- Selective filtration barrier
- Scaffold for embryogenesis and regeneration
- Stabilization for tissue shapes
What is keratin?
Dead protein that is waterproof and provides a protective layer
Found in esophagus and vagina (lightly keratinized) and epidermis (heavily keratinized)
Although not common, stratified cuboidal epithelium may be found in which of the following areas?
Vaginal epithelium
Part of male urethra
Urinary bladder
Kidney tubules
Part of male urethra
Characteristics of the basement membrane (3)
- 50-80nm thick
- Separates an epithelial layer from its CT support
- 2 components; a) basal lamina (produced by the epithelium) b) reticular lamina (produced by CT)
What are the components of the basal lamina? (5)
- Laminin
- Fibronectin
- Type IV collagen (produced by epithelial cells)
- Entactin/Nidogen
- Proteoglycans
What are laminin characteristics? (2)
- Major component of the basal lamina that consists of three chains; alpha, beta, gamma
- contains binding sites for integrins, type IV collagen, entactin and proteoglycans (i.e. heparan sulfate)
What are Fibronectin characterisitics? (3)
- A protein made up of 2 polypeptide chains cross-linked by disulfide bonds
- Contains binding sites for heparin, integrins, collagen and fibrin.
- There are 2 forms: cellular and plasma. Cellular = produced by fibroblasts; part of ECM and Plasma = secreted into bloodstream by hepatocytes
