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.

Advantages of Cryo-EM

1. Preserves native structure: Flash-freezing prevents damage from chemical fixatives or dehydration, keeping biomolecules in their natural state.
2. Dynamic imaging: Can capture proteins in different functional states (e.g., active vs. inactive conformations).
3. Atomic resolution: Recent advances achieve resolutions as fine as 0.12 nm, allowing visualization of individual atoms within proteins.
4. No crystallization needed: Unlike X-ray crystallography, cryo-EM doesn't require proteins to form crystals, making it useful for flexible or membrane-bound proteins.

Fluorescent Stains and Immunofluorescence: Targeting Specific Molecules

Fluorescent stains and immunofluorescence are light microscopy techniques that use fluorescence to highlight specific cellular structures or molecules.

1. Fluorescent molecules absorb light at one wavelength (often UV) and re-emit it at a longer wavelength (visible light).
2. This produces bright, glowing images against a dark background.
3. Fluorescent Stains
   1. Dyes that bind to specific cellular components (e.g., DNA, mitochondria, actin filaments).
   2. Improve contrast and make transparent structures visible.

Immunofluorescence

Immunofluorescence uses antibodies tagged with fluorescent dyes to bind to specific proteins (antigens) within cells.

1. Antibodies are designed to recognize a specific target protein.
2. The antibodies are linked to a fluorophore (fluorescent molecule).
3. When antibodies bind to their target, the fluorophore glows under UV or specific wavelengths of light.
4. This reveals the precise location of the target protein within the cell.

Advantages of Fluorescent Stains and Immunofluorescence

1. High specificity: Can target individual proteins or structures.
2. Multicolor imaging: Using different fluorophores with different colors allows visualization of multiple proteins or structures simultaneously, showing their interactions and locations.
3. Living or fixed cells: Some fluorescent dyes work in living cells, enabling real-time observation of cellular processes.

Comparing Microscopy Techniques