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High resolution biological scanning electron microscopy: a comparative study of low temperature metal coating
1Laboratory for Electron Microscopy I, Institute for Cell Biology, ETH Zentrum, Zürich, Switzerland.
Journal of Electron Microscopy Technique
|August 1, 1991
Summary
Optimizing chromium coating thickness and low-temperature deposition using double-axis rotary shadowing (DARS) enhances the visualization of molecular surface structures on biological specimens like T4 phages. This cryo-method improves structural definition for scanning electron microscopy.
Area of Science:
- Cryo-electron microscopy
- Surface science
- Structural biology
Background:
- Scanning electron microscopy (SEM) requires metal coating for visualizing biological specimen surfaces at molecular resolution.
- The quality of surface structure visualization is highly dependent on metal coating parameters, including thickness and deposition temperature.
- Cryo-methods are crucial for preserving the native state of specimens during SEM analysis.
Purpose of the Study:
- To evaluate the impact of chromium coating parameters on the visual definition of surface structures in T4 polyheads and T4D phages using SEM.
- To compare double-axis rotary shadowing (DARS) with planar-magnetron sputtering (PMS) and unidirectional shadowing techniques.
- To determine optimal conditions for metal coating to achieve high-resolution imaging of biological macromolecules.
Main Methods:
- Specimens (T4 polyheads and T4D phages) were coated using chromium double-axis rotary shadowing (DARS) at various temperatures (-250°C to room temperature).
- Coating thickness was optimized for each temperature condition.
- Micrographs were compared with those obtained using chromium planar-magnetron sputtering (PMS) and platinum/carbon unidirectional shadowing.
Main Results:
- Optimal visual definition of T4 polyhead and T4D phage surface details (e.g., 8 nm capsomeres, 3 nm tail fibers) was achieved with DARS when chromium film thickness exceeded the minimum for metal film coalescence.
- DARS coating at room temperature yielded poor structural definition.
- Good visual detail was obtained with DARS at specimen temperatures of -85°C and -150°C, with slightly reduced definition at -250°C.
Conclusions:
- Chromium DARS coating, particularly at low temperatures (-85°C and -150°C) and with optimized thickness, significantly enhances the visualization of fine surface structures on biological specimens in SEM.
- The study demonstrates the critical role of coating parameters in achieving molecular-level resolution.
- All tested coating techniques (DARS, PMS, unidirectional shadowing) provided adequate structural visibility, but DARS offered superior definition under optimized conditions.