Electron Microscopy Notes

Electron Microscopy

Types of Electron Microscopy

• Two main types:

  • Transmission (TEM)

    • Standard imaging mode

    • Electron tomography

    • Cryo TEM

  • Scanning (SEM)

    • Standard

    • Serial block face SEM

    • Environmental SEM

History of TEM

• 1920s: Discovery that electrons behave like light under vacuum.

• 1931: Ernst Ruska built the first prototype TEM.

• 1945: Porter et al. study of tissue culture cells using electron microscopy.

Why Use Electron Microscopy?

TEM Resolution

• Achieves resolution less than 1nm.

• Examples:

  • Centrioles in Chondrocytes.

  • Motile cilia from respiratory epithelium.

How TEM Works

• Electrons behave like light under certain conditions.

• An accelerated beam of electrons is created by the electron gun.

• Electrons interact with and are transmitted through the specimen.

• Detected on a fluorescent screen.

• Converted into a digital signal.

Resolution Achievement in TEM

• Accelerated electrons behave like light in a vacuum.

• Travel in a straight line.

• Wavelength is 100,000 times smaller than light.

• Magnetic and electric fields act on electrons as glass lenses act on light.

Components of a TEM

• Electron Optical Column

• Vacuum System

• Electronics

  • High voltage source to electron gun

  • Electromagnetic lenses

TEM Specifications

• Five electromagnetic lenses

• Resolving power of $0.1nm$ (theoretical)

• Magnification of 1 million X

Electron Penetration and Sample Preparation

• The column is under vacuum to prevent electrons from being stopped or deflected.

• Specimens must be very thin for TRANSMISSION (< 200nm thick).

• Sample preparation is key.

TEM Sample Preparation Overview

• Fixation e.g. $2.5\%$ glutaraldehyde

• Dried to remove water as going into a vacuum

• Embedded in resin for thin sectioning <200 nm

• Negative staining Uranyl acetate / immuno gold

• Samples are small!

Electron Interactions and Contrast

• Unscattered electrons

  • Go straight through, resulting in no contrast

• Elastically scattered electrons

  • Interact with nuclei.

  • Scattered at wide angles with no energy loss.

  • High atomic number = more scatter = better contrast (amplitude).

• Inelastically scattered electrons

  • Interact with electrons

  • Low angle scatter/energy loss

  • Change in wavelength / phase contrast

Objective Aperture

• The objective aperture stops electrons that are scattered through a large angle (elastically scattered electrons).

Applications of TEM

• Internal cellular structures - organelles

• Microorganisms

• Viruses, phages, DNA

• Protein structure, membrane interfaces

• Macromolecular organisation

• Energy Dispersive X-ray detector (EDX) - detect x-rays emitted from the specimen, which tells us about elements present.

Electron Tomography

• 3D transmission electron microscopy

• Similar to a medical CT scan

• Thick sections and higher kV to penetrate the specimen

• Collect a tilt-series

• Volume reconstruction and surface rendering

3D Tomography

• Data acquisition over a range of angles (e.g., +70° to -70°).

• Volume reconstruction to create a 3D model.

• Visualization and surface rendering.

Transmission Electron Cryomicroscopy (CryoTEM)

• $1.25 \text{\AA}$ resolution has been reported for apoferritin

• $1 nm = 10 \text{\AA}$ (Amstrong)

Scanning Electron Microscopy

• Electrical Optical Column

• Vacuum System

• Electronics

  • High voltage source to electron gun

  • Electromagnetic lenses

  • Beam scanning with deflection coil

Comparison of Microscopy Types

A table provides a visual comparison between Light Microscopy, Transmission Electron Microscopy, and Scanning Electron Microscopy, highlighting differences in:

• Light/Electron Source

• Condenser Lens

• Specimen Type

• Objective Lens

• Eyepiece/Projection Lens

• Image Viewing Method

How SEM Works

• Image formation is different from TEM.

• Can be compared to reflected light.

• Both have an electron gun, are evacuated, and have electromagnetic lenses.

• Resolution is limited to the size of the beam spot, usually about $5nm$.

SEM Imaging Process

1. Electron source focused on a spot on specimen

2. Electrons are either ‘reflected’ (BSE’s) or knocked out of the specimen’s atoms (SE’s)

3. Electrons are detected by different detectors, amplified and converted electronically to form a spot on a monitor

4. Beam is scanned across specimen

5. The number of electrons detected varies across the specimen (more electrons/ brighter the image)

Applications of SEM

• Surface morphology/topography

• Has a large depth of field – the amount of sample that can be focused

• Magnifications of $10x - 100,000x$

• Easy preparation - no sections – Just drying and coating with electron dense material using gold -”splutter coating”

Depth of Field

• Depth of field explained using photography as an example.

• f number (photography term): $n = \frac{f}{d}$

  • where $f$ = focal length, and $d$ = lens diameter

Serial Block Face Scanning Electron Microscopy (SBF SEM)

• Several hundred micron-imaging depth is possible!

Environmental SEM (ESEM)

• Samples must be dry in a conventional SEM due to the vacuum - this can lead to artifacts.

• ESEM - the sample chamber is kept at a low vacuum, allowing 'wet samples'.

• Uses Gaseous Secondary Electron Detector - and uses cascade amplification to enhance the signal.

Applications of ESEM

• Problematic materials such as wood, foodstuff

• Living preps such as tissues, cells, and insects