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Double-layer coating for high-resolution low-temperature scanning electron microscopy

P Walther1, E Wehrli, R Hermann

  • 1Laboratory for Electron Microscopy 1, Institute of Cell Biology, Zürich, Switzerland.

Journal of Microscopy
|September 1, 1995
PubMed
Summary
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Minimize electron beam damage in scanning electron microscopy (SEM) for hydrated samples. A double layer coating technique significantly reduces specimen damage, enabling detailed imaging of frozen-hydrated biological specimens.

Area of Science:

  • Electron microscopy
  • Materials science
  • Biophysics

Background:

  • Electron beam irradiation causes significant mass loss and damage in hydrated specimens during low-temperature scanning electron microscopy (LT-SEM).
  • High magnifications exacerbate specimen damage, limiting the observation of delicate biological structures.

Purpose of the Study:

  • To develop a method to reduce electron beam damage in frozen-hydrated bulk specimens for SEM.
  • To enable routine observation of large frozen-hydrated samples with high structural detail.

Main Methods:

  • Coating frozen-hydrated samples with a 5-10 nm carbon layer to reduce beam damage.
  • Unidirectional shadowing with a 2 nm heavy metal layer (e.g., platinum) before carbon coating to preserve surface details.
  • Utilizing field-emission scanning electron microscopy (FE-SEM) with a backscattered electron detector for imaging.

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Main Results:

  • The double layer coating (heavy metal followed by carbon) significantly reduced specimen damage from electron beam irradiation.
  • The heavy metal layer was visualized using the material-dependent backscattered electron signal in FE-SEM.
  • Structural information comparable to transmission electron microscopy (TEM) freeze-fracture replica techniques was achieved.

Conclusions:

  • The double layer coating method effectively minimizes electron beam damage in LT-SEM of frozen-hydrated samples.
  • This technique allows for routine, high-resolution imaging of large biological specimens in a hydrated state.
  • FE-SEM with backscattered electron detection provides detailed structural insights comparable to established cryo-TEM methods.