Chapter 1 - Histology and its method of study Flashcards
Microanatomy
- structure of individual cells w/n a tissue, and structure of different types of tissues
- function of cells and tissues, roles in different organ systems
Who were the scientists that received a Nobel prize for histology? What did they do?
- Santiago Ramon y Cajal and Camillo Golgi
- determined the structure of neurons in the brain with staining method
What is the ideal goal of specimen prep?
to preserve the tissue’s structure
- sometimes not possible because of where the sample comes from or other circumstances
Fixation
- Step 1 of specimen prep
- strong chemical used to preserve structure and prevent microbial degradation
- irreversibly cross links and denature proteins
What fixatives are used for LM and EM?
- 4% formaldehyde for LM
- Glutaraldehyde and OsO4 for EM
- both methods prevent immunochemistrical methods
Dehydration and clearing
- Step 2 of specimen prep
- water from fixation is removed using dehydrator like alcohol, and then the dehydrator is cleared
What are the chemicals used for dehydration and clearing?
- ethanol - dehydration
- xylene - clearing
Embedding
- Step 3 of specimen prep.
- Sample is put into some kind of wax or solid matrix, which is then trimmed away so the sample is ready for sectioning and staining
What different matrixes are used for embedding?
- Parffin - LM
- Epoxy resin for EM, much more stable then paraffin b/c EM requires much thinner slices
Sectioning, what are the different types for LM and EM?
- Step 4 of specimen prep
- Microtome - glass knife, used for LM, 1-10 micrometers
- Ultramicrotome - diamond knife, used for EM, 50 nanometers
What type of sectioning does not require fixation?
- cryosectioning
- frozen w/ liquid nitrogren, the sectioned in a cooled microtome
- used in biopsies
Staining
- provides contrast so that the sample can be seen easier w/ the microscope
- also enhances certain features
Histochemical staining
general staining, nonspecific
Special staining
selectively stain certain cellular components of particular molecules like an enzyme
- important in diagnosis of some diseases
Basophilic cell component staining
- negatively charged molecules
- methylene blue, toluidine blue, hematoxylin
- stains intense in the nucleus and some cytoplasmic granules
Acidophilic cell component staining
- positively charged molecules
- eosin
- will stain more intensely in the cytoplasm, the plasma membrane, and some cytoplasmic membranes
H&E Stain
- hematoxylin and eosin
- most common stain
- hematoxylin - stains dark blue/purple the nucleus, ribosomes, RER
- eosin - stains pink/red the cytoplasm, collagen
Methylene blue
stains nucleus and ER/golgi apparatus blue
Silver stain
stains certain proteins and delicate structures dark brown/black
Osmium
stains lipids dark brown/black
Periodic acid (Schiff)
stains complex carbs (basement membrane for ex.) red
Light Microscopy
- specimen illuminated by visible light
- Resolving power = 0.2 micrometers
- Magnifies specimen up to 1000X
Fluorescence microscopy
- specimen is stained w/ fluorescent dye, or a fluorescent-tagged antibody is bound to the specimen
- only detects one wavelength/color at a time
- Resolving power = 0.2 mm
- Mag up to 1000X
Electric microscopy
- specimen stained w/ e- dense compound and lighten with e- beam
- magnets are used as lenses
Transmission EM
- beam goes through the specimen
- ultrathin sections required (40-90 nm)
- resolving power 0.1-3 nm
- Mag up to 120,000X
- highest magnification of any type of microscopy, shows detailed structure of cellular organelles
- many organelles were discovered this way
Scanning EM
- scanning the surface of the specimen, gives you a 3D view of the surface of the specimen
- specimen coated in Au or other e- dense ion
- does not require sectioning
Immunocytochemistry
- specific type of histochemistry
- antibodies are used to detect specific proteins, and are tagged w/ fluorescent molecule
- antibodies binds to specific protein and binds to it when incubated w/ sample, making the protein complex visible using the tag on the antibody y
Direct Immunocytochemistry
- antibody is tagged w/ chromophore of fluorescent molecules
- when the antibody binds to its target, it can be directly visualized (no extra steps required)
Indirect immunocytochemistry
- primary antibody binds to target, but it doesn’t have a label, so you have to use a secondary, labeled antibody that detects the primary antibody
How is Immunocytochemistry used in medical diagnosis?
- epithelial tumors
- breast cancer, colon cancer
- ductal cell tumor
Artifact
- not present in normal tissue, interfere with interpretation of specimens
- EX: distortion of tissue during processing, shrinkage during fixation, separation of layers, folding/wrinkling, precipitation of stain, dust on slide
