Atomic force microscopy applied to study macromolecular content of embedded biological material

Area of Science:

• Biophysics
• Microscopy techniques
• Cell biology

Background:

• Assessing structural preservation in biological samples is crucial for accurate ultrastructural analysis.
• Traditional methods like transmission electron microscopy (TEM) can be influenced by sample preparation artifacts.

Purpose of the Study:

• To evaluate atomic force microscopy (AFM) as a tool for estimating the structural preservation of biological samples in epoxy resin.
• To compare AFM and TEM imaging of Caenorhabditis elegans prepared with different freeze-substitution protocols.
• To propose a novel correlative imaging procedure for AFM and TEM.

Main Methods:

• Atomic force microscopy (AFM) for surface topography and macromolecular distribution.
• Transmission electron microscopy (TEM) for ultrastructural imaging.
• Freeze-substitution protocols with conventional OsO4 fixation and epoxy fixation for sample preparation.
• Correlative light and electron microscopy (CLEM) principles adapted for AFM-TEM integration.

Main Results:

• AFM provides a reliable estimation of macromolecular distribution and structural integrity in epoxy-embedded samples.
• High TEM stainability correlated with low macromolecular density in the cellular matrix.
• The novel procedure enabled correlative AFM-TEM imaging of specific organelles, facilitating interpretation.
• New insights into protein arrangement and ultrastructure were obtained through combined AFM-TEM analysis.

Conclusions:

• AFM is a powerful complementary technique to TEM for evaluating sample quality and structural preservation.
• Understanding macromolecular density is key to interpreting TEM images.
• Correlative AFM-TEM imaging enhances ultrastructural studies by providing multi-modal data on biological samples.