Study sheet: 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

Technique	Detection and principle	Resolution or result
Visible light microscopy	Detects photons using transmitted polychromatic light	200 nanometers
Confocal fluorescence microscopy	Uses one monochromatic wavelength and thin optical slices	Three-dimensional reconstruction with improved resolution
TEM	Detects transmitted electrons through thin sections	0.3 nanometer or less; subcellular structures or protein localization
SEM	Scans a replica and detects surface-emitted signals	0.3 nanometer or less; three-dimensional structures