Ch 1 Flashcards
Histology
the study of the microscopic structures of tissues and organs
What is the goal of histology?
to correlate structure with function
What diverse methods does histology utilize?
light, virtual, and electron microscopy
What is the first step to tissue preparation?
Fixation
What is fixation?
the first step in tissue preparation to preserve structure and prevent degradation
What is used in fixation?
formalin
what is formalin
a common fixative
What is the second step in tissue preparation
embed the specimen in paraffin
what does embedding the specimen in paraffin require?
dehydration and clearing, to permit sectioning
What is the third step in tissue preparation?
sectioning
what is sectioning?
slicing the embedded tissue using a microtome
what is the fourth step in tissue preparation
staining
Why is staining necessary
because paraffin sections are colorless
What are two most common staining methods?
Hematoxylin and eosin (H&E)
What does hematoxylin do?
stains nuclei blue
What does eosin do?
stains cytoplasm pink
What is the fifth step in tissue preparation?
mounting the stained section on a glass slide with mounting medium
What are the steps of tissue preparation?
- Fixation
- Embedding in parafin
- Sectioning
- Staining
- mounting
Why are other fixatives sometimes used?
to retain component not preserved by formalin
What are the two the fixatives mentioned?
- heavy metals like osmium tetroxide
- freezing
What are heavy metals such as osmium tetroxide are used for?
to perserve membrane structures
What is freezing sections used for?
to retain neutral lipids
What would other staining procedures be used?
to display specific structural components
What are the two other staining techniques mentioned?
- orcein and resorcin-fuchsin
- Silver impregnation
What is orcein and resorcin-fuchsin are used for?
staining procedure for elastic material
What is silver impregnation is used for?
2 things
Is is a staining method for;
- reticular fibers
- basement membranes
what do all the procedures provide information about?
cell function and extracellular components
What might procedures be based on?
3 things
- specific dying binding
- fluorescent dye labeled antibodies
- inherent enzymatic activity
What is autoradiography?
uses radioactively tagged precursors to localize molecules
What is the chemical basis of staining?
acidic and basic dyes are commonly used
What is an example of an acidic dye?
eosin
What does an acidic dye like eosin do?
carries a negative charge and reacts with cationic components
What is an example of a basic dye?
hematoxylin
What does a basic dye like hematoxylin do?
carries a positive charge and reacts with anionic components
What are other examples of basic dyes?
- methyl green - green
- methylene blue - blue
- pyronin G - red
- toluidine blue - blue
What are other examples of acidic dyes?
- acid fuchsin - red
- aniline blue - blue
- eosin - red
- orange G - orange
what is metachromasia?
a color shift seen with certain basic dyes
what does the periodic acid-Schiff (PAS) reaction do?
stains carbohydrates and carbohydrate-rich molecules
What does feulgen stain do?
sues the Schiff reagent to stain DNA
What can be used to confirm the identity of stained material?
enzyme digestion
what does enzyme histochemistry do?
localizes enzymes in tissue sections
what does immunocytochemistry do?
uses the specificity of antigen-antibody reactions to locate molecules
what techniques does immunocytochemistry use?
direct and indirect
What happens in direct immunofluorescence?
- a fluorochrome-labeled primary antibody reacts with a specific antigen within the tissue sample
- labeled structures are then observed in the fluorescence microscope in which an excitation wavelength (usually UV light) triggers the emission of another wavelength
- the length of the wavelength depends on the nature of the fluorochrome used for antibody labeling
What happens in indirect immunofluorescence?
- specific primary antibodies react with the antigen of interest
- the secondary antibodies, which are fluorochrome labeled, react with the primary antibodies
- the visualization of labeled structures within the tissues is the sam in both methods and requires the fluorescence microscope
What is hybridization used for?
to legalize mRNA or DNA using a complementary nucleotide probe
what is a type of hybridization?
fluorescence in situ hybridization (FISH)
What does FISH do?
uses fluorescent dies with probes
What does autoradiography use?
a photographic emulsion to localize radioactive material
What does expansion microscopy do?
ExM physically expands specimens to improve resolution
- specimens are infiltrated with swellable polymers
What are the steps in preparation of a specimen for expansion microscopy?
- Fixation - cell in formaldehyde
- Anchoring - add anchoring reagents (acryloyl groups) that bind to proteins or other molecules of interest and molecular probes conjugated with fluorescent labels, infuse with superabsorbent (sodium acrylate)
- Genlation - sodium acrylate - triggers development of a sense three-dimensional hydrogel polymer matrix
- mechanical homogenization - allows the cells to break open using ultrasonic homogenizer (creating proteinases),
- Expansion - the specimen is now embedded in a hydrogel matrix, is ready for physical expansion by the addition of water. add water to expand (proportionally in all directions making cell bigger)
how does expansion work - th proteins of interest remain connected to the expanded polymer network which pulls them apart (aprox. 4.5 times bigger)
- examination - fluorescence microscope
What are the types of microscopy?
- light microscopy
- electron microscopy
- atomic force microscopy (AFM)
- virtual microscopy
What is light microscopy?
uses a light source to magnify images
What is resolving power?
the ability to produce separate images of closely positioned objects
that is the most common type of light microscope?
the bright field microscope
What are the other types of optical systems?
- phase contrast
- dark field
- fluorescence
- confocal
- ultraviolet microscopy
What is electron microscopy?
uses electrons to produce high resolution images
What are the types of electron microscopes?
transmission electron microscopy (TEM) and scanning electron microscope (SEM)
- in both of these specimens need to be inserted into a high-vacuum environment
what is atomic force microscopy (AFM)?
uses a probe to scan the specimen’s surface at high resolution
- a tip moves up and down with the surface to produce an image
What does virtual microscopy do?
provides a digital alternative to examining glass slides using a light microscope
