Backscattered electron imaging. Its application to biological specimens stained with heavy metals

Area of Science:

• Biological imaging
• Scanning Electron Microscopy (SEM)
• Histochemistry

Background:

• Traditional SEM methods primarily focus on surface topography.
• Observing the distribution of specific molecules within biological tissues requires specialized staining techniques.
• Integrating different imaging modes can provide more comprehensive data.

Purpose of the Study:

• To demonstrate the utility of backscattered electron imaging for observing heavy metal-stained biological specimens using SEM.
• To present specimen preparation techniques for backscatter imaging.
• To showcase the application of various staining methods with backscatter imaging.

Main Methods:

• Specimen preparation for backscatter imaging.
• Application of silver staining (argyrophile fibers), Golgi staining (parietal cells), osmium staining (lipid droplets), periodic acid-thiosemicarbazide-osmium staining (mucosubstances/polysaccharides), and enzyme immunohistochemistry (peptides).
• Acquisition and analysis of secondary emission and backscattered electron images, including stereo-pairs and superimposed images.

Main Results:

• Backscattered electron imaging effectively visualizes various heavy metal-stained biological structures.
• Stereo-pairs of backscatter images provide enhanced information on the three-dimensional localization of stained substances.
• Correlations between surface structure (secondary emission images) and stained substances (backscatter images) are clearly visualized.
• Superimposed colored images aid in understanding the orientation of stained substances in 3D.

Conclusions:

• Backscatter imaging is a versatile tool for SEM observation of stained biological specimens.
• The technique facilitates detailed topochemical studies in three dimensions.
• Combining backscatter imaging with other SEM modes offers synergistic advantages for biological research.