Electron Microscopy Concepts and Principles

Discussion on electron microscopy as a deviation from the typical course.
Justification of the focus on electron microscopy:
- Inclusion of electron microscopes in the laboratory.
- Light microscopy's inherent diffraction limit necessitates exploration of alternative microscopy techniques.

Light microscopy is limited by its diffraction (Abbe) limit.
Definition of Key Term: Diffraction Limit
- The inability to distinguish two points as separate when their diffraction spots overlap.
Introduction of techniques to overcome diffraction limit.

Definition and influence of the aberration disc on resolution and perceived image quality.
Explanation of how two closely spaced points of light appear as one when they overlap.
Importance of recognizing when resolution is insufficient to discern detail between two points (resolution limit).

Factors contributing to resolution in light microscopy:
- Wavelength of light.
- Numerical aperture (NA) of the optical system:
- Importance: Higher NA results in smaller diffraction spots and improved resolution.
Resolution criteria for calculations:
- Formula:
  $Resolution = 0.61 \times \frac{\lambda}{NA}$
- Where:
- λ = wavelength of light
- NA = numerical aperture, defined as $n \times \sin(\alpha)$ where n is the refractive index, and α is the angle.

Theoretical resolution calculations:
- For instance, with a numerical aperture of 1.4 and wavelength of 400 nm, theoretical resolution is 150 nm.
Practical limitations include:
- Average achievable resolution of around 200 nm.
- Z-axis resolution can be approximately four times worse than lateral resolution.

Methods to improve resolution:
- Reducing the wavelength (e.g., using electrons instead of light).
- Improving the numerical aperture (limited to about 1.45 for objectives).

Fundamental differences in working principles:
- Electromagnetic Radiation vs Subatomic Particles:
- Electrons have much shorter wavelengths than visible light (0.001 to 0.01 nm versus 400 to 700 nm).
- Resulting in much higher resolution in electron microscopy.
Comparison on scattering:
- Electrons are more heavily scattered by gas particles leading to the necessity for high vacuum environments.

Importance of using a high vacuum in electron microscopy:
- Prevents scattering of the electron beam.
- Ensures clear imaging of the sample without gas particle contamination.

Principle: The electron beam scans across the sample generating surface images.
Detection methods based on interaction effects:
- Elastic reflection: Electrons reflect without energy loss.
- Secondary electrons: Produced from the sample upon impact, analyzed for surface features.
- Backscattered electrons: Provide compositional information on the sample based on atomic number.

Electron Gun:
- Uses thermionic emission to generate electrons.
- Different types of electron guns may be employed with varying elements of stability and longevity.

Manipulation of electron beams is executed via electromagnetic lenses due to their charge.
Function of lenses includes focusing and scanning to produce sharp images; aberrations need correcting.

Definition of aberration: Deviation of light waves from their ideal path, causing blurriness in images.
Correction using stigmators:
- Adjust beam shape to be circular to ensure clarity in imaging.

Electron beam scans the sample in a systematic raster pattern, yielding surface images similar to confocal microscopy.

Ensuring that samples are conductive to prevent charge buildup on non-conductive samples, which can distort imaging.
Summary of significant considerations before usage in electron microscopy.