2 - Examining Cells and Tissues Flashcards
`What is the limit of resolution?
The smallest distance by which two objects can be separated and still be distinguished as two separate objects
Compare light and electron microscopes.
How would you produce a specimen to view with electron microscopy?
- Fix with glutaraldehyte
- Embed in epoxy resin
- Stain with osmium tetroxide or heavy metal
- Cut with microtome with diamond knife for TEM
What is the difference between SEM and TEM?
SEM:
- Can see 3D
- Lower resolution
TEM:
- Thin section
- 2D
- Can see internal structures
- Higher resolving power
How do electron microscopes produce images?
- Stain with heavy metal, denser areas will take up more metal
- Metal reflects electrons and these are collected
- Denser areas will reflect more
What is freeze fracture EM?
- Tissue frozen to -160 degrees in glycerol to prevent water crystals forming
- Hit with knife to fracture it
Why do you need to preserve and fix samples, and how do you most commonly do this?
Prevent putrefaction, allow storage and allow for thin cutting
- Formalin (37% formaldehyde, 0.9% NaCl)
- Paraffin wax that sets hard
What are common biopsy techniques and what do you do after you have taken the biopsy?
- Preserve in formalin
- Embed in paraffin
- Cut with microtome
- Stain with H and E
How does formaldehyde preserve tissue?
Isotonic so penetrates the cell. Reacts with amino acids forming methylated bridges, dehydrates and stiffens it
How can tissue processing produce artefacts?
- Dehydration
- Cutting
What do H and E stain?
H - Basic dye so binds to acids like DNA.
E - Acidic dye so binds to base like proteins and cytoplasm
Why are fats not visible with H and E staining?
Xylene and toluene based solvents strip lipophilic molecules
How do you embed tissue?
- Wash specimen and dehydrated in series of alcohol solutions, 0 to 100%.
- Wash with xyulol or tolulol
- Immerse in hot paraffin wax and leave to harden over night
- Repeat hot wax
Why do red blood cells appear white in the middle?
They are biconcave and no nucleus, all haemoglobin on the edges
What is a frozen section and what are the advantages and disadvantages?
- Frozen rapidly to -20 to -30 degrees
- Cryostat (microtome in freezer)
- Stained with H and E
+ Super fast so can be done in surgery
- Poor clarity
- Can’t be stoed indefinitely
Compare a paraffin-embedded section an a frozen section.
How can you use immunology for staining?
- Labelled antibodies with fluorescence or enzyme for coloured precipitate
- Very sensitive
- Only stains the cells the antigen antibody complex is on
What are the advantages of the following microscopes:
- Phase Contrast
- Dark Field
- Fluorescence
- Confocal Light
Why does electron microscopy have a higher resolution?
Lower limit of resolution because of using electrons instead of light
How can you make fats visible under the microscope?
- Use periodic-schiff stain
- Frozen section and dyes soluble in fats
- Use heavy metals to preserve structure of membrane
What is the smallest organelle visible under the light microscope?
- Nucleolus
What are the positives and negatives of viewing cells in culture?
+ Manipulate environment
+ All cells are the same
+ Don’t need to test on animals
- 3d Architecture lost
- Influence of other cells not maintained
- Can only grow small amounts
- Dedifferentiation and instability
What do you do with a section after you have cut it with a microtome to around 1-5 micrometres?
- Pick up section with paintbrush and float on surface of warm water bath
- Surface tension and heat cause cross section to stretch to minimise cutting artefacts
- Glass slide placed under floating section
- Leave tissue to dry and stain
