U1 p3

Unit Overview

  • Unit 01 – Part 3: Visualizing Cells

  • Course: BIOL 331 – Molecular Cell Biology

  • Source: Molecular Biology of the Cell, 6th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2022.

Topics Covered

  • The Electron Microscope Resolves the Fine Structure of the Cell

  • Special Preparation for Electron Microscopy

  • Heavy Metals for Added Contrast

  • Scanning Electron Microscopy Images

  • Electron Microscope Tomography for 3D Molecular Architecture

  • Cryo-Electron Microscopy for Atomic Resolution

  • Light vs. Electron Microscopy Trade-offs

Comparing Scales

  • Importance of understanding scale in microscopy techniques in cellular biology.

Electron Microscopy Overview

  • Function: Used to resolve ultrastructure of cells.

  • Resolution:

    • Theoretical resolution: 0.002 nm (100,000 X better than light microscopy).

    • Practical resolution: ~1 nm (200X better than light microscopy).

  • Transmission Electron Microscopy (TEM):

    • Involves passing electrons through the specimen to create images.

Transmission Electron Microscope (TEM) Structure

  • Components:

    • Filament or cathode emits electrons from a tall column (approx. 2 m high).

    • Requires vacuum to prevent scattering of electrons.

    • Electrons focused by magnetic coils after passing through a tiny hole.

  • Image Production:

    • Direct passage or scattering of electrons by dense structures generates the image.

    • Images viewed on a phosphorescent screen or high-res digital camera.

TEM: Specimen Preparation Procedures

  • Thin Samples:

    • Essential for electrons to pass through effectively.

  • Fixation:

    • Uses glutaraldehyde (cross-links proteins) and osmium tetroxide (stabilizes lipids/proteins).

  • Embedding:

    • Sequential dehydration with organic solvents followed by resin polymerization creates "blocks" for sectioning.

  • Section Cutting:

    • Slices of 50-100 nm thick are necessary (compared to 1-10 μm for optical imaging).

  • Staining:

    • Sections stained with electron-dense materials (e.g., uranyl acetate) for contrast.

    • Heavy metal salts used to enhance visibility in otherwise low contrast tissues.

Cryo-Electron Microscopy

  • Tissue Preparation: Requires extensive procedures; proposed solution is flash freezing.

    • Rapid freezing (rate > 10,000 degrees C/s) forms vitreous ice to avoid damaging cellular structures.

  • View via TEM: Sectioned samples viewed with cooled holders or through traditional preparation methods.

  • Benefits: Better preservation of the living state and reduced artifact creation.

  • Utilized for immuno-electron microscopy.

Immunoelectron Microscopy: Immunogold EM

  • Gold Particles: Secondary antibodies attached to small colloidal gold particles are viewed as black dots in images.

  • Application: Different sizes of gold can indicate multiple proteins in samples, but only accessible surfaces to antibodies in thin sections.

Visualizing High-Resolution Macromolecules

  • Methodology:

    • Macromolecules shadowed with heavy metal for visibility (e.g., actin).

    • Mixture with heavy metal solution (uranyl acetate) enhances contrast.

    • Target molecules present as light against a dark background.

  • Target Substances: Effective for macromolecular aggregates, such as viruses and ribosomes.

Scanning Electron Microscopy (SEM)

  • Function: Generates 3-D surface images using a fine electron beam.

  • Specimen Preparation: Must be fixed, dried, and often coated with heavy metal.

  • Image Production:

    • Scans surface, detecting scattered/ emitted electrons.

    • Produces images with great depth of field and resolution ~10 nm (20,000 X magnification).

Application Examples of SEM

  • Visualizations:

    • Stereocilia from hair cells in bullfrogs, pollen grains, and bacterial structures.

3-D EM Reconstruction (Tomography)

  • Technique: Computational approach to produce 3-D reconstructions based on multiple angles.

  • Example Application: 3-D visual representation of the Golgi complex with associated vesicles, leading to new insights into organelle structures.

Single Particle Reconstruction

  • Imaging Method: Engages with low-level detection for statistical averaging.

  • Noise Challenge: Variability obscures the image in low-level detection (e.g., cryoEM).

  • Technique Overview:

    • Involves combining thousands of images to produce an average image, clarifying structures beneath noise.

    • Achievable resolution limit is about 0.5 nm, sufficient for observing some protein structures without needing crystalline samples.