Brief Introduction to Coating Technology for Electron Microscopy

August 28, 2013

Coating of samples is required in the field of electron microscopy to enable or improve the imaging of samples. Creating a conductive layer of metal on the sample inhibits charging, reduces thermal damage and improves the secondary electron signal required for topographic examination in the SEM. Fine carbon layers, being transparent to the electron beam but conductive, are needed for x-ray microanalysis, to support films on grids and back up replicas to be imaged in the TEM. The coating technique used depends on the resolution and application.

Coating needed prior to SEM imaging

Limited or non-conductive material samples (ceramic, polymers…etc) require carbon and/or metal coating. Cryogenic  samples are freeze fractured, coated with metal (Leica EM ACE600 freeze fracture and Leica EM VCT500) and imaged in a cryo SEM.

Coating needed prior to TEM imaging

The formvar covered TEM grids need to be coated with carbon to be conductive. Grids are treated with glow discharge otherwise solutions would not stick and distribute onto the grid. Freeze fractured samples are coated in a low angle with metal followed by a carbon backing up film (Leica EM ACE600 freeze fracture and Leica EM VCT500 or Leica EM ACE900) to produce a replica which can be imaged in a TEM.

Sputter coating

Sputter coating for SEM is the process of applying  an ultra-thin coating of electrically-conducting metal – such as gold (Au), gold/palladium (Au/Pd), platinum (Pt), silver (Ag), chromium (Cr) or iridium (Ir) onto a non-conducting or poorly conducting specimen. Sputter coating prevents charging of the specimen, which would otherwise occur because of the accumulation of static electric fields. It also increases the amount of secondary electrons that can be detected from the surface of the specimen in the SEM and therefore increases the signal to noise ratio. Sputtered films for SEM typically have a thickness range of 2–20 nm.

Benefits for SEM samples sputtered with metal:

  • Reduced microscope beam damage
  • Increased thermal conduction
  • Reduced sample charging (increased conduction)
  • Improved secondary electron emission
  • Reduced beam penetration with improved edge resolution
  • Protects beam sensitive specimens

Carbon coating

The thermal evaporation of carbon is widely used for preparing specimens for electron microscopy. A carbon source – either in the form of a thread or rod  is mounted in a vacuum system between two high-current electrical terminals. When the carbon source is heated to its evaporation temperature, a fine stream of carbon is deposited onto specimens. The main applications of carbon coating in EM electron microscopy is X-ray microanalysis and specimen support films on (TEM) grids.

E-Beam coating

Metal and carbon can be evaporated. E-beam coating gives the finest layers, is a very directional process and has a limited surface of coated area. Electrons are focused on the target material which is heated and further evaporated. Charged particles are removed from the beam. Therefore a very low charged beam is hitting the sample. Heat is reduced and the impact of charged particles on the sample is reduced. Only a few runs are possible then the source has to be reloaded and cleaned. Usually, e-beam is used where either directional coating is necessary (shadowing and replicas) or fines layers are required.

Introduction into cryo techniques

Freeze fracture includes a series of techniques that reveal and replicate internal components of organelles and other membrane structures for examination in the electron microscope. Freeze etching removes layers of ice by sublimation and exposes membrane surfaces that were originally hidden.

Freeze drying, also known as lyophilization, removes water from a frozen sample under high vacuum conditions (sublimation). The result is a dry and stable sample which can be imaged in the electron microscope.

Applications

Fig. 1: Mosquito antenna (courtesy of Dr. Daniela Gruber, Vienna, Austria)
Fig. 2: Drosophila eye (courtesy of Kim Rensing, applications specialist, Leica Microsystems)
Fig. 3: Spider mite (courtesy of Kim Rensing, applications specialist, Leica Microsystems)
Fig. 4: Mosquito leg (courtesy of Dr. Daniela Gruber, Core facility of cell imaging and ultrastructure research, University of Vienna, Austria)
Fig. 5: Antenna detail (courtesy of Dr. Daniela Gruber, Core facility of cell imaging and ultrastructure research, University of Vienna, Austria)
Fig. 6: Ommatidien mosquito (courtesy of Dr. Daniela Gruber, Core facility of cell imaging and ultrastructure research, University of Vienna, Austria)

Different coating methods

Sputtering

Carbon thread evaporation

Carbon rod evaporation

E-beam evaporation

  • Rather diffuse (coating from all sides), influenced by the sputter vacuum
  • Minimal heat transfer to the sample
  • Easy to operate and set up
  • During coating vacuum at 10–2 mbar range (can be problematic for cryo applications)
  • Great variation of metals
  • Pulsed evaporation highly controllable
  • Easy to operate
  • Low heat transfer to sample due to short pulses
  • No continuous rotation while coating
  • Fine grains
  • Fast coating
  • Continuous rotation
  • Defined purity of the carbon
  • More heat development than carbon thread
  • Finest grains
  • Directional coating (shadowing!)
  • Focused beam of material
  • Target change/adjustment after several runs
  • Mostly carbon and platinum coating
  • More difficult to set up evaporation source

Typical applications of different coating methods

Biology

  • SEM imaging

High resolution SEM analysis

EDX analysis

Room temperature low angle rotary shadowing for TEM

Material

  • SEM imaging

Heat sensitive samples

Carbon films for TEM grids

Highest resolution SEM freeze fracture:

  • Cryo SEM

Medical research

Basic cryo SEM

  • Replica TEM

Process combinations

Following combination of processes are available for the Leica EM ACE Coaters:


Leica EM ACE200

  • Sputter coater
  • Carbon thread coater
  • Sputter and carbon thread coater

Leica EM ACE600

  • Sputter coater
  • Carbon thread coater
  • Sputter and carbon thread coater
  • Double sputter coater
  • Carbon rod coater
  • e-beam coater
  • Sputter and carbon rod coater
  • Sputter and e-beam coater
  • Double e-beam coater

Leica EM ACE600 Freeze Fracture

  • Every Leica EM ACE600 can be configured or upgraded to a cryo coater
  • Basic cryo set
  • Freeze fracture cryo set


Optional: Glow discharge for all instruments to make carbon coated grids hydrophilic.

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