Optical and scanning electron microscopy in the single osteoclast resorption assay

Area of Science:

• Cell Biology
• Biomineralization
• Skeletal Biology

Background:

• Osteoclastic resorption assays are crucial for understanding bone diseases.
• Previous methods using stereophotogrammetry had limitations in statistical analysis due to cell variability.
• Accurate visualization of osteoclasts and resorption lacunae is essential for assay development.

Purpose of the Study:

• To evaluate various optical and scanning electron microscopy methods for visualizing osteoclasts and resorption lacunae in vitro.
• To identify rapid and statistically robust methods for analyzing bone resorption.
• To compare the efficacy of different microscopy techniques for studying osteoclast function.

Main Methods:

• In vitro generation of osteoclasts and resorption lacunae on dentine and bone.
• Utilized scanning electron microscopy (SEM) with stereophotogrammetry in previous studies.
• Evaluated optical microscopy techniques including oblique illumination light microscopy (LM) and tandem scanning reflected light microscopy (TSRLM).
• Assessed darkfield reflected LM for rapid scanning of resorption lacunae.

Main Results:

• Optical microscopy provides complementary data to SEM, enabling vital microscopy of cells.
• Oblique illumination LM and TSRLM are highly valuable for visualizing resorption lacunae.
• Darkfield reflected LM and TSRLM allow rapid scanning of fixed, coated specimens for resorption lacunae.
• Newer optical methods offer faster data acquisition for improved statistical analysis.

Conclusions:

• Optical microscopy techniques, especially TSRLM, are effective for visualizing osteoclasts and resorption lacunae.
• These methods enhance the statistical power of bone resorption assays by enabling faster data collection.
• The evaluated techniques improve the study of osteoclast function and bone resorption mechanisms.