Correlative volume-imaging using combined array tomography and FIB-SEM tomography with beam deceleration for 3D...

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biophysics

Background:

• Focused ion beam scanning electron microscopy (FIB-SEM) offers high spatial resolution for volume imaging.
• Analyzing deep biological structures with FIB-SEM is challenging due to limitations in imaging depth and sample preparation.
• Existing methods struggle to provide certainty in selecting analysis areas within resin-embedded samples.

Purpose of the Study:

• To develop a novel volume-imaging method combining array tomography and FIB-SEM tomography.
• To investigate the ultrastructure of chondrocytes, including fine surface details.
• To overcome the limitations of traditional FIB-SEM for analyzing deep biological structures.

Main Methods:

• Integration of array tomography with FIB-SEM tomography for enhanced imaging capabilities.
• Development of a method to precisely determine the analysis area, avoiding defects like cracks or poor staining.
• Application of the combined technique to study chondrocyte ultrastructure.

Main Results:

• The novel method successfully imaged chondrocyte ultrastructure with high fidelity.
• Fine dendritic processes on the chondrocyte surface, thinner than ultrathin sections, were visualized.
• The technique provides certainty in selecting analysis areas, improving reliability.

Conclusions:

• The combined array tomography and FIB-SEM tomography method overcomes previous limitations in analyzing deep biological structures.
• This technique enables detailed visualization of cellular ultrastructure, such as chondrocyte surface morphology.
• The method shows significant promise for mesoscopic architectural analysis, especially when combined with immunostaining.