A2.2.3 Developments in microscopy Notes

Electron Microscopy: Higher Resolution Through Electrons

Advantages of Electron Microscopy

1. Reveals ultrastructure: Can visualize organelles (mitochondria, ribosomes, ER), membranes, and even large molecules like viruses.
2. High magnification: Up to 500,000× or more.
3. Fine detail: Shows internal organization of cells and the arrangement of membrane structures.

Two Main Types Of Electron Micrscopy

1. Transmission Electron Microscopy (TEM)
   1. Electrons pass through thin sections of the specimen.
   2. Reveals internal structures in 2D cross-section.
   3. Best for: Viewing organelles, membranes, and internal cellular organization.
2. Scanning Electron Microscopy (SEM)
   1. Electrons scatter off the surface of the specimen.
   2. Produces 3D surface images.
   3. Best for: Viewing external cell structure and surface topology.

Freeze-Fracture Electron Microscopy: Viewing Membrane Interiors

1. Cells are rapidly frozen (usually in liquid nitrogen).
2. The frozen sample is fractured with a knife.
3. The fracture often splits membranes along their middle (between the two phospholipid layers).
4. The fractured surface is coated with platinum or carbon to create a replica.
5. The replica is viewed under an electron microscope.

Advantages of Freeze-Fracture

1. Exposes membrane interior: The fracture reveals proteins embedded within the lipid bilayer.
2. 3D information: The shadowing effect from metal coating creates depth, showing the distribution and size of membrane proteins.
3. Historical impact: This technique provided key evidence for the fluid mosaic model by showing that proteins are scattered throughout the membrane, not just on the surface.

Cryogenic Electron Microscopy (Cryo-EM): Proteins in Their Native State

1. Samples are flash-frozen in vitreous ice (non-crystalline, glass-like ice).
2. Freezing is so rapid that water doesn't form ice crystals it solidifies instantly.
3. The frozen sample is viewed directly under the electron microscope without staining or chemical fixation.