Detailed Notes on Scanning Electron Microscopy (SEM)

Electron Microscopy History

  • Sir Joseph John Thomson (1856-1940)
    • Discovered corpuscles (electrons) in 1897.
    • Nobel Prize in 1906.
    • Proposed the plum pudding model: electrons in a sea of positive charge.
  • Louis de Broglie (1892-1987)
    • Nobel Prize in 1929.
    • Introduced wave-particle duality and "matter waves" in his 1924 Ph.D. thesis.
    • Matter as Waves: λ=hmv\lambda = \frac{h}{mv}
  • Ernst Ruska (1906-1988)
    • Built the first TEM (Transmission Electron Microscope) in 1931.
    • Nobel Prize in 1986.
  • Max Knoll (1887-1969)
    • Contributed to the development of electron microscopy.

Electrons have wavelengths.

Resolving Power and Wavelength

The wavelengths of electrons are significantly shorter (100,000 times) compared to visible light.

  • Resolution is directly dependent on wavelength.
    • Poor resolution is associated with low frequency and long wavelength.
    • Good resolution is associated with high frequency and short wavelength.
  • Formula: d=DInsinθd = \frac{D}{In sin\theta}

Resolving power:

  • Human eye: 0.2 mm
  • Light microscope (LM): 200 nm
  • Electron microscope (EM): 0.2 nm

TEM vs. SEM

  • TEM (Transmission Electron Microscopy): The electron beam goes through a thin sample, allowing visualization of internal structures.
  • SEM (Scanning Electron Microscopy): Electrons do not pass through the sample but are reflected from the surface and collected by a detector. It's good for observing the surface of a sample.

Scanning Electron Microscopy (SEM)

SEM is used for observation of surfaces.

  • SEM images are often colored.
  • SEM has a better depth of focus than a light microscope at the same magnification.
  • SEM images do not retain the true colors of the sample, unlike light microscopy.

Sample Preparation for SEM

  1. Vacuum Requirement: Samples must be prepared for vacuum conditions because the electron beam can only travel effectively in a vacuum; otherwise, it will scatter.
  2. Fixation: Initial fixation of the sample.
  3. Dehydration Series: A dehydration series using increasing concentrations of ethanol (from low to 100%) to remove water.
  4. Critical Point Drying: Samples are dried using a critical point dryer to prevent damage or distortion to their original shape.
  5. Mounting: If the sample is already dry, it can be directly mounted on an aluminum, non-magnetic SEM stub.
  6. Conductivity: Non-conductive samples need to be coated with a conductive material, typically gold (in this lab) or other noble metals, to prevent heat buildup and charging artifacts.
  • Some rigid samples (e.g., insects with rigid exoskeletons) can be air-dried.
  • The critical point (CP) for CO2CO_2 is at 73.8P and 31°C. Different materials have different critical points; water's CP is much higher, which can be harmful to biological samples.
  • A thin layer of gold is applied to make the sample conductive. This is done by removing the atmosphere and introducing a noble gas, creating an argon gas atmosphere. Argon ions bombard the gold target, causing gold atoms to coat the sample.

Issues with Non-Conductive Samples

If a non-coated sample is overexposed, it can exhibit unusual contrast and appear very white due to the inability of electrons to reflect properly.

SEM Components

  • Electron Gun: Electron source.
  • Sample Chamber: Houses the samples.
  • Condenser Lens: Not made of glass but uses conductors.
  • Beam Detector: Positively charged to attract the negatively charged electron beam.
  • Filament: A thin tungsten filament is heated until it emits free electrons, which are accelerated towards the anode. Some electrons pass through the center of the anode, forming an electron beam. This process requires a vacuum.

Different types of filaments:

  • LaB6LaB_6: Allows more electrons, resulting in a brighter beam.
  • FEG (Field Emission Gun): The most expensive type, providing the highest brightness and resolution.

Vacuum System

  • A vacuum is essential for the electron beam to flow. Vacuum pumps or turbine pumps are typically used to create the vacuum.
  • No vacuum = no beam = no images.

Electromagnetic Lenses

  • Condenser and objective lenses are electromagnetic lenses, consisting of copper coils that generate a precise magnetic field. The field is stronger in the center and weaker at the sides, allowing focusing of the electron beam.
  • The focusing ability of electromagnetic lenses is not as good as glass lenses, but they are still required to obtain an image.
  • In magnetic lenses, the image is inverted and rotated.

SEM Operation

Components needed:

  • Samples that can tolerate electrons and vacuum.
  • Electron beam originating from an electron gun.
  • Vacuum system for the electron beam to travel in.
  • Electromagnetic lenses to control the electron beam.

Sample Stage Controls

  • Stair Controller:
    • Trackball (1)
    • XY switch (2)
  • Movements:
    • Horizontal plane (X)
    • Horizontal plane (Y)
    • Vertical (Z)
    • Tilt (T)
    • Rotation (R)
  • Working Distance (WD): Measured in mm. A larger WD is used to see a low magnification image of the whole sample.

Image Adjustments

  • Raster Scan: Used in SEM. Correct the current in the objective lens to fix blurry images caused by too little or too much current.
  • Correct asymmetrical electron probe shape by adjusting the electron beam.

SEM Magnification

  • No change of objectives.
  • Magnification M=DdM = \frac{D}{d}

Since the viewed area remains fixed, a smaller scan area on the sample results in higher magnification.

Parameters affecting image quality and resolution:

  1. Instrument performance
  2. Selection of imaging parameters
  3. Nature of the specimen and its preparation

Poorly prepared samples will not yield good results with SEM.

Electron Detection

  • At 30kV, the electron beam penetrates a little deeper into the sample.
  • Secondary electrons are ejected from the gold coating with low energy.
  • There is one detector for each type of electron being studied (BSD).

Detector and Signal Output

  • The monitor is synchronized with the scanning electron beam.
  • The detector measures intensity point by point in the image.
  • Each point (pixel) is assigned a numeric grey scale value. These points (pixels) are added next to each other to create an image.

Everhart-Thornley Detector

  • A conventional secondary electron detector.
  • A Faraday cage (kept at a low positive bias, e.g., +300V) attracts low-energy secondary electrons.
  • The electrons are accelerated towards a scintillator (kept at a high positive bias, e.g., +10,000V), and the signal is converted to light photons.
  • The photons are then converted back to an electric signal at the PMT's (Photomultiplier Tube) photocathode.

Signal Amplification and Data Collection

  • A Scintillator coupled to a PMT amplifies the electron signal.
  • Each collected data point (pixel) is assigned a specific numeric grey scale value.

Image Formation

  • Areas where secondary electrons are emitted in all directions appear brighter (edge effect).
  • Flat areas emit fewer secondary electrons and appear darker.
  • The detector's position to the side results in a stronger signal from areas pointing towards the detector.
  • These effects create a 3D-like image from a 2D image.

SEM Image Characteristics

  • SEM creates an intensity map from grey scale values pixel by pixel in a raster scan. This results in a 2D SEM-micrograph that has a 3D appearance due to shadow and edge effects of the sample-beam interaction.