Electron Microscopy

Timeline

1. 1897: JJ Thomson announced the existence of electrons.

2. 1924: de Broglie proposed that a moving electron has wavelike properties.

3. 1926: Busch laid the foundations of electron optics

4. 1931: Max Knoll and Ernst Ruska built first transmission electron microscope.

5. Development of electron microscopy comprised of only 35 years in comparison to light microscopy.

Uses electron beam that is deflected and focused by electromagnetic field.

Electromagnetic spectrum

Shorter wavelength than light. Up to 100000x magnification. Detailed ultrastructural detail. Ribosomes, cell membranes, microtubules, microfilaments and macromolecules.

Scanning electron microscopy

SEM. Uses electrons to scan the surface of the specimen by deflection. 3D image formed.

Lower accelerating voltage, 5-30kV. Images are generated from deflected electrons on the specimens outer surface. Scans the specimens surface with the electron beam.

Structure SEM

Electron gun. Vacuum chamber. Anode. Condenser lens. Electron beam. Condenser lens. Objective lens. Sample chamber. X-ray detector. Backscatter detector. Secondary detector. Sample

Electron pathway SEM

Primary electron beam passes through the anode ro condenser lens. Focus beam into an intense spot that is moved back and forth over the specimen by charged plates (beam deflector). Attract or repel the beam according to the received signal. Beam passes over specimen, molecules within are excited to high levels- emit secondary electrons. Captured by detector. Emits photons when excited by electrons. Electronic signal to a video screen. Point/point or line/line sweep of specimen

Image formation SEM

Responsible for generating a 3D image complete with all grooves and engravings of biological sample surface.

Scanning coils: magnetic field with fluctuating voltage to manipulate electron beam as secondary electrons are dislodge in unique patterns from specimen surface. Electrons collected by its detector and analyzed for different levels of brightness.

Increase image magnification- electron beam scan a smaller area of the sample.

Image development SEM 1

1. Electrons are emitted into vacuum pump by heading cathode filament in the Electron gun.

2. The cathode ray then passes through an Anode, which accelerates and focuses the beam.

3. The condenser aperture prepares the beam for condenser lens by blocking off axis/energy electrons from proceeding.

4. Spray aperture works in conjunction with magnetic condenser lenses which apply a magnetic field to the beam inducing a helical path that focuses the beam onto a spot.

5. Within the objective lens is embedded pairs of deflection coils which deflection the Electron beam to produce a rasterized scan (rectangular pattern of image capture) of the sample

Image development SEM 2

6. Electrons in the beam interact with the sample. As they do so, they will randomly scatter and absorb within the sample.

7. The X ray detector, primary, of the high energy electrons which can map the sample. Secondary Electron detector picks up electrons scattered from the sample surface, generating a topographic image. Backscatter electron detector identifies chemical phase differences in the sample by picking up electrons scattered from the interactions volume of the specimen.

8. Photomultipliers are then used to convert this signal into a voltage signal which is amplified to create the image on a PC screen.

Transmission electron microscopy

TEM. Uses electrons that are transmitted through the specimen. 2D image formed.

Accelerating voltage 100-300kV. Physical orientation reversed to that of LM. Illumination source is at the top and the ocular lens and detector are at the bottom.

Electron pathway TEM

Dependent on wave like and particle like nature of electrons. After electron beam leaves the gun controlled by electromagnets. Enters series of electromagnetic lenses. Various aperture prepare the beam for the different lenses. Lenses focus beam onto specimen. Image develops on viewing screen.

Image formation TEM 1

1. Electrons are emitted into vacuum chamber from heating cathode filament in electron gun

2. Cathode ray then passes through an Anode, which accelerates and focuses beam. Alignment coils further accelerate beam.

3. Condenser aperture prepares beam for condenser lens by blocking off energy electrons from proceeding.

4. Magnetic condenser lens applies a magnetic field inducing a helical path for the electrons and leading the cone shaped electron beam to converge on a spot.

5. Stigmator helps to adjust the beam and prevent astigmatism in the optical system.

6. Electrons pass through the thinly sliced sample inserted onto a grid like stage

Image formation TEM 2

7. Objective lens focuses the image of the sample

8. Diffraction lens is used to apply Braggs scattering (effects of the reflection of electromagnetic waves on periodic structures whose distances are in the range of wavelength) to the electrons

9. Objective aperture, positioned on the back focal plane of the scattering rays, selects the portion of the sample that produced the scattering

10. Projector lenses calibrate the magnification of the image.

11. The image is visualized through ocular or by an image recording system underneath the fluorescent screen.

Image development TEM

Density of electrons. Degree of opacity: Highly opaque= darker appearance. Less opaque= permittance of electrons, light. Contrasting light, dark and intermediate areas create final image. Captured within microscope on photographic paper of fluorescent screen or modern camera (digital imaging)/detector. Generate an electron micrograph.