Microscopy Flashcards
What are the steps involved in tissue processing?
- fixation
- dehydration
- clearing
- embedding
- sectioning
- section mounting
- section staining
What is the process of fixation?
avoid autolysis/degradation by bacteria and to preserve the structure and molecular composition
What are the two chemical methods of fixation?
- LM-formaldehyde: reacts with amine groups
- TEM-glutaraldehyde: cross-links proteins; addition of osmium tetroxide
What is the process of dehydration?
water is extracted from fixed tissue fragments by bathing them successfully in series of ethanol (70% -> 80% -> 90% ->abs. ethanol)
What is the process of clearing?
ethanol tissue replaced with xylene which makes tissue transparent
What is included in the process of embedding?
- paraffin: tissue placed in melted paraffin; heat causes xylene to evaporate and spaces in tissue get filled with paraffin that hardens
- epoxy resins: tissues cleared using plastic solvents
What is the process of sectioning?
cool/hard paraffin tissue is sectioned on slicing machine (mirotome for LM- 6 thick ; ultramicrotome for TEM- .06 thick)
What is the process of section mounting?
tissue sections are mounted on glass slides (light microscope) or copper wired grid (TEM)
What does the process of section staining include?
- paraffin must be dissolved with xylene and sections must be rehydrated with alcohols stained with hematoxylin in water
- eosin staining after dehydrating sections because counterstain is more soluble in alcohol
What does hematoxylin stain? What color?
cell nucleus, nucleic acid, RNA rich organelles; blue/purple
What does eosin stain? What color?
mitochondria, secretory granules, extracellular material; pink
Which chemical stain is acidic dye, carries “-“ charge, stains “+”, and reacts with cationic groups?
eosin
What are the characteristics of hematoxylin?
basic dye, carries “+” charge, stains “-“, and reacts with anionic groups
What are some neurotransmitters stained with?
heavy metal
What is the ONLY organelle visible at light level?
nucleus; the rest leave an impression
What are the artifacts of preparation?
shrinkage and artificial spaces
What is the problem with interpretations of tissue sections?
distortion (shrinkage)
During interpretation of tissue sections, artificial spaces are caused by ….
shrinkage and loss of molecules
What can cause shrinkage?
fixation, dehydration in ethanol, heat of melted paraffin
What are the artifacts of sectioning?
- a sectioned ball-shaped structure will appear as a circle
- a sectioned tube looks like a ring
- sections taken through a solid ball-like structure/ taken through a solid cylinder are similar
What is resolution (resolving power)?
smallest distance between 2 particles
What does resolving power depend on?
the quality of the objective lens
The quality of an image depends on ….
resolving power
What is a light microscope composed of ?
condenser, ocular, and objective lens
What is the resolving power and magnification of a TEM?
max resolving power: 3nm
max magnification: 400,000x
magnification: 120,000x
What is the resolving power and magnification of a light microscope?
resolving power: .2 microns
magnification: 1000 - 1500x
What are confocal microscopes used for?
immunoflurescence
What are the types of microscopes used?
- polarizing microscopy
- confocal
- light
- electron
- ultra high voltage TEM
What is the exception for an electron microscope?
if .1nm or less, then NOT able to resolve
