Study guide: Cell Biology Microscopy Techniques

📋 Course Outline

1. Microscopy Magnification and Resolution
2. Visible Light Microscopy
3. Confocal Fluorescence Microscopy
4. Photon and Electron Microscopy
5. Transmission Electron Microscopy
6. Scanning Electron Microscopy

📖 1. Microscopy Magnification and Resolution

🔑 Key Concepts & Definitions

• Magnification: The ratio between the observed dimension and the actual size of the object.
• Resolution limit: The minimum distance separating two objects that can still be distinguished by a microscope.

📝 Essential Points

• The resolution limit is imposed by light diffraction and depends on the numerical aperture of the lens and the refractive index of the surrounding medium.

💡 Memory Hook

Magnification enlarges the image, whereas resolution separates two nearby objects.

📖 2. Visible Light Microscopy

📝 Essential Points

📌 Visible transmitted light microscopy uses polychromatic light in air, whereas fluorescence microscopy uses a single monochromatic wavelength.

• In visible light microscopy, the resolution limit is 200 nanometers.

📖 3. Confocal Fluorescence Microscopy

📝 Essential Points

• Confocal fluorescence microscopy decreases the resolution limit and divides a sample into thin slices that can be recombined into a three-dimensional image.

💡 Memory Hook

Imagine thin fluorescent slices stacked into a three-dimensional image.

📖 4. Photon and Electron Microscopy

📝 Essential Points

📌 Photon microscopy detects photons and has a resolution limit of 200 nanometers, whereas electron microscopy detects electrons and reaches 0.3 nanometer or less.

💡 Memory Hook

Photon microscopy detects photons at 200 nm, whereas electron microscopy detects electrons at 0.3 nm or less.

📖 5. Transmission Electron Microscopy

🔑 Key Concepts & Definitions

• Transmission electron microscopy: Or TEM, excites the entire surface of a sample and produces a single image by detecting electrons that pass through non-opacified regions.

★ Must-know

• 🔄 TEM preparation for viewing subcellular structures follows these steps:
  1. Fixation
  2. Dehydration
  3. Resin embedding
  4. Thin sectioning
  5. Opacification

• TEM opacification can use osmium tetroxide or immunogold, and immunogold associated with TEM localizes a protein of interest.

Further detail

• Biological samples in TEM do not resist the vacuum and are relatively transparent to electrons, so resin embedding and opacification with heavy ions are required.

💡 Memory Hook

Fixation → dehydration → inclusion → thin sectioning → heavy-metal contrasting.

📖 6. Scanning Electron Microscopy

🔑 Key Concepts & Definitions

• Scanning electron microscopy: Or SEM, progressively excites and detects the sample with a mobile beam, producing signals detected above the sample.

📝 Essential Points

• SEM preparation uses cryofracture, surface sublimation, and shadowing with heavy ions such as platinum or silver to produce a replica.

• SEM can reveal the three-dimensional structure of cells or macromolecules after freezing and shadowing.

💡 Memory Hook

Cryofracture → surface sublimation → heavy-metal shadowing → three-dimensional replica.

📊 Synthesis Tables

Comparison of microscopy techniques