IB Biology Topic A2.2.3: Developments in Microscopy Notes

Electron Microscopy: Higher Resolution Through Electrons

Advantages of Electron Microscopy

1. Reveals ultrastructure: it can show organelles, membranes, and even large molecules like viruses.
2. High magnification: it reaches up to 500,000 times or more.
3. Fine detail: it shows the internal organisation of cells and the arrangement of membrane structures.

Two Main Types Of Electron Microscopy

1. Transmission Electron Microscopy (TEM)
   1. Electrons pass through thin sections of the specimen.
   2. It reveals internal structures in a 2D cross-section.
   3. It is best for viewing organelles, membranes, and internal cellular organisation.
2. Scanning Electron Microscopy (SEM)
   1. Electrons scatter off the surface of the specimen.
   2. It produces 3D surface images.
   3. It is 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 from the metal coating creates depth, showing the size and distribution of membrane proteins.
3. Historical impact: this technique gave 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, a non-crystalline, glass-like ice.
2. Freezing is so fast that water solidifies instantly without forming ice crystals.
3. The frozen sample is viewed directly under the electron microscope without staining or chemical fixation.

Advantages of Cryo-EM

1. Preserves native structure: flash-freezing avoids damage from chemical fixatives or drying, keeping biomolecules in their natural state.
2. Dynamic imaging: it can capture proteins in different functional states, such as active and inactive shapes.
3. Atomic resolution: recent advances reach resolutions as fine as 0.12 nm, showing individual atoms within proteins.
4. No crystallisation needed: unlike X-ray crystallography, cryo-EM does not need proteins to form crystals, so it works for flexible or membrane-bound proteins.

Fluorescent Stains and Immunofluorescence: Targeting Specific Molecules

1. Fluorescent stains and immunofluorescence are light microscopy techniques that use fluorescence to highlight specific structures or molecules.
2. Fluorescent molecules absorb light at one wavelength, often UV, and re-emit it at a longer wavelength of visible light.
3. This produces bright, glowing images against a dark background.
4. Fluorescent Stains
   1. These are dyes that bind to specific cellular components, such as DNA, mitochondria, or actin filaments.
   2. They improve contrast and make transparent structures visible.

Immunofluorescence: Pinpointing Specific Proteins

1. Immunofluorescence uses antibodies tagged with fluorescent dyes to bind to specific proteins, called antigens, within cells.
2. Antibodies are designed to recognise a specific target protein.
3. The antibodies are linked to a fluorophore, a fluorescent molecule.
4. When an antibody binds to its target, the fluorophore glows under UV or specific wavelengths of light.
5. This reveals the precise location of the target protein within the cell.

Advantages of Fluorescent Stains and Immunofluorescence

1. High specificity: they can target individual proteins or structures.
2. Multicolor imaging: different fluorophores in different colours let you view several proteins at once, showing their locations and interactions.
3. Living or fixed cells: some fluorescent dyes work in living cells, so cellular processes can be watched in real time.

Comparing Microscopy Techniques