Specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy.

Area of Science:

• Neuroscience
• Microscopy
• Connectomics

Background:

• High-throughput, high-resolution 3D microscopy generates large-scale neuroanatomical datasets.
• The Knife-Edge Scanning Microscope (KESM) is a pioneering instrument for whole-brain imaging at submicrometer resolution.

Purpose of the Study:

• To present a comprehensive pipeline for utilizing KESM for biological specimen imaging.
• To facilitate broader access and utilization of KESM technology for research.

Main Methods:

• Specimen preparation: fixing, staining (Golgi, India ink, Nissl), and embedding.
• KESM configuration, setup, sectioning, and imaging.
• Image processing, data preparation, visualization, and analysis.

Main Results:

• KESM successfully imaged whole mouse brains, revealing neuronal and vascular networks, and cell body distribution.
• The technique is applicable to various species (octopus, rat) and organs (mouse lung), with ongoing work on zebrafish embryos.
• Acquired data provides ground-truth for connectomics, microcirculation, and stereology research.

Conclusions:

• The detailed KESM protocol supports high-resolution, large-volume biological imaging.
• KESM data significantly contributes to understanding complex biological structures and networks.
• Standardizing the KESM pipeline will advance fields reliant on detailed anatomical data.