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.
![](https://s3.amazonaws.com/brainscape-prod/system/cm/416/096/143/a_image_thumb.jpeg?1544892742)
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
![](https://s3.amazonaws.com/brainscape-prod/system/cm/416/096/160/a_image_thumb.png?1544893119)
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
![](https://s3.amazonaws.com/brainscape-prod/system/cm/416/096/171/a_image_thumb.png?1544893371)
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
![](https://s3.amazonaws.com/brainscape-prod/system/cm/416/096/176/a_image_thumb.jpeg?1544893723)
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
![](https://s3.amazonaws.com/brainscape-prod/system/cm/416/096/183/a_image_thumb.png?1544893923)
Why do red blood cells appear white in the middle?
They are biconcave and no nucleus, all haemoglobin on the edges
![](https://s3.amazonaws.com/brainscape-prod/system/cm/416/096/189/a_image_thumb.png?1544893988)
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