Sample preparation methods for volume electron microscopy in mollusc Berghia stephanieae

Area of Science:

• Neuroscience
• Electron Microscopy
• Comparative Biology

Background:

• High-resolution brain atlases are essential for understanding neural function and development across species.
• Volume electron microscopy (vEM) is a powerful technique for generating detailed neural maps.
• Efficient sample preparation is critical for accelerating vEM imaging and data processing pipelines.

Approach:

• This study introduces replicable methods for processing gastropod mollusc (Berghia stephanieae) brains for vEM.
• Methods include a BROPA-based approach using pyrogallol and rapid processing techniques to reduce bench time.
• Addresses limitations of high-pressure freezing for larger tissue samples by optimizing cryopreservation depth.

Key Points:

• Presents novel, rapid sample preparation protocols for vEM in a gastropod mollusc.
• Demonstrates a BROPA-based method using pyrogallol for enhanced tissue processing.
• Overcomes cryopreservation depth limitations associated with high-pressure freezing for larger samples.

Conclusions:

• This work establishes the first reported sample preparation and imaging pipeline for vEM in a gastropod mollusc.
• Enables detailed connectomic analysis and facilitates comparisons with neural structures across diverse phyla.
• Advances the field of neuroanatomy by providing tools for high-resolution brain mapping in new model organisms.