Electron Microscopy Notes

Transmission Electron Microscopy (TEM): Enables high-resolution imaging of thin samples, revealing internal cell and material structures.
Transmission Electron Microscope (TEM)
- Standard mode: High-resolution imaging of resin-embedded thin sections.
- Electron tomography: Advanced EM for 3D imaging.
- Cryo-TEM: State-of-the-art technique with significant advantages in preserving sample structure.
Scanning Electron Microscopy (SEM)
- Samples are coated (e.g., with gold) to enhance electron scattering.
- Examines scattered electrons to produce detailed surface images.
- Advantage: Provides a large depth of field, allowing more of the sample to be in focus.
Serial Block Face EM: Method for 3D imaging by repeated scanning and slicing of a sample block.
Environmental SEM (ESEM)
- Can tolerate some moisture, unlike traditional SEM, which requires complete dehydration, allowing for imaging of hydrated specimens.

1920s: Discovery that electrons behave like light in a vacuum (wave-particle duality).
1931: First prototype electron microscope developed.
1940s: EM used to study cells, revolutionizing biology by enabling visualization of previously invisible cellular structures.

High resolution: Enables detailed visualization of minute structures.
- TEM: Achieves sub-nanometer resolution, ideal for observing atomic-level details.
- SEM: Offers good resolution for larger objects with an exceptional depth of field.

Electromagnetic lenses: Coiled wires produce magnetic fields to focus electron beams, analogous to glass lenses focusing light.
Electron detection:
- TEM: Uses a phosphorescent plate that emits light when struck by electrons, creating an image.
- SEM: Employs a cathode tube scanning device (similar to confocal microscopy) to detect scattered electrons.

Visualizing internal cell structures (organelles, cytoskeleton):
Studying clinical samples (e.g., respiratory epithelium, kidney biopsies) to diagnose diseases at the cellular level.

Optical column: Contains the electron gun, which generates and accelerates the electron beam.
Vacuum system: Maintains a high vacuum to prevent electron scattering by air molecules.
Electronics:
- High voltage source: Generates high-energy electrons (e.g., 120 kilovolts) to provide high resolution.
- Electromagnetic lenses: Focus the electron beam to create a magnified image.
- Camera: Detects phosphorescence to capture the final image.

Filament (tungsten): Heats up to emit electrons via thermionic emission.
Anode: Positively charged electrode that accelerates the electron beam towards the sample.

Negative staining: Stained areas appear dark, while unstained areas appear white, enhancing contrast.
Electrons interact with the sample and are either:
- Elastically scattered: Interact with nuclei, resulting in wide-angle scattering without energy loss.
- Inelastically scattered: Interact with electrons, resulting in smaller-angle scattering with energy loss and phase change.

Internal structure of cells and organelles: Detailed visualization of cellular components.
Structure of microorganisms (viruses, bacteria):
Protein structure (cryo-EM): Determining protein structures at high resolution.
- Cryo-EM vs. X-ray Crystallography: Cryo-EM is better suited for membrane proteins because it does not require crystallization.
Macro molecular organization (protein complexes): Studying the arrangement of proteins in large complexes.
Energy Dispersive X-ray Detector (EDX): Elemental composition analysis (e.g., detecting copper in diabetic tissue).
Transverse tubules in cardiac myocytes: Imaging structural details in heart muscle cells.

Action potential travels through myocytes via T-tubules: Facilitates rapid and coordinated muscle contraction.
T-tubules facilitate synchronous contraction: Ensures uniform contraction of the heart muscle.
T-tubules are closely associated with the sarcoplasmic reticulum (SR): Forming junctions where calcium is transferred.
Cardiac junctions: Small space between T-tubules and SR where calcium is transferred, crucial for excitation-contraction coupling.
Mitochondria make up about 40% of cardiac muscle cell volume: Providing the necessary energy for cardiac function.

3D reconstruction method: Creates three-dimensional images from a series of two-dimensional projections.
Similar to CT scan: The sample is rotated to capture images from multiple angles.
Requires thicker specimens and high-voltage electron guns for adequate penetration.
Tilt series: A set of images taken at different angles, typically ranging from +70 to -70 degrees.
Reconstruction: Processes the tilt series to create a 3D volume, providing detailed structural information.

Proteins are imaged, and thousands of pictures are averaged to improve the signal-to-noise ratio.
A model of the protein structure is then built based on the averaged images.
Currently achieves a resolution of 1.25 Angstroms (0.125 nm), allowing for near-atomic level detail.

An electron beam scans across the sample's surface in a raster pattern.
Detects electrons reflected back (backscattered electrons) or ejected (secondary electrons) from the sample.
The signal is amplified and converted to a point on the screen, creating an image.
Contrast is based on the number of electrons detected, providing information about the sample's surface properties.

Dehydration is required to remove moisture, which can interfere with imaging.
Coating with a metal (e.g., gold) using a sputter coater enhances electron scattering and prevents charging.

Controlled by the aperture size (f-number) of the electron microscope.
Smaller f-number = larger aperture = shallower depth of field.
Larger f-number = smaller aperture = greater depth of field.
Electron microscopes have a very large depth of field due to the small wavelength of electrons, allowing more of the sample to be in focus.

Visualizing cell surfaces (e.g., primary cilia):
Imaging insects (e.g., brown recluse spider) for detailed morphological studies.

Method for 3D imaging by milling through the sample using an ultramicrotome.
SEM scans the block face, and then an ultramicrotome cuts a thin slice.
The process is repeated to create a z-stack of images, which are then reconstructed into a 3D representation.