Electron microscopy, electron diffraction, and element analysis of wet biological specimens.

Area of Science:

• Electron Microscopy
• Biophysics
• Materials Science

Background:

• Electron microscopy traditionally requires vacuum conditions, limiting the study of hydrated biological specimens.
• Advancements in temperature-controlled, differentially pumped environmental chambers now permit routine examination of wet samples.
• High-resolution electron diffraction from unfixed protein microcrystals serves as a critical test for environmental chamber efficiency.

Purpose of the Study:

• To evaluate the capability of new environmental chambers for high-resolution electron diffraction of wet protein microcrystals.
• To assess the potential of wet specimen analysis for elemental mapping in cells compared to cryo-methods.
• To facilitate comparative studies on ultrastructural preservation between thin water layers and amorphous ice.

Main Methods:

• Utilizing temperature-controlled, differentially pumped environmental chambers for electron microscopy.
• Preparing wet specimens with minimal water thickness (tens of nanometers) to reduce electron scattering.
• Performing high-resolution electron diffraction on unfixed protein microcrystals.

Main Results:

• Environmental chambers demonstrate the ability to obtain high-resolution electron diffraction patterns from wet, unfixed protein microcrystals.
• Minimizing water layer thickness effectively reduces extraneous electron scattering.
• Commercial availability of necessary equipment now makes comparative studies more feasible.

Conclusions:

• Temperature-controlled environmental chambers represent a significant advancement for routine electron microscopy of wet specimens.
• Further investigations are required to ascertain the benefits of wet specimen microprobe analysis (X-ray or EELS) for cellular element localization versus current freezing techniques.
• Direct comparisons of ultrastructural preservation in thin water layers versus amorphous ice are necessary.