Light and focused ion beam microscopy workflow for resin-embedded tissues

Area of Science:

• Developmental Biology
• Cell Biology
• Microscopy

Background:

• Focused ion beam scanning electron microscopy (FIB-SEM) offers volume reconstructions but struggles with large sample analysis.
• Understanding ovarian development requires detailed 3D structural information across developmental stages.

Purpose of the Study:

• To present an optimized workflow for efficient 3D structural surveys of developing rabbit ovaries using FIB-SEM.
• To introduce a virtual 4D electron microscopy approach by incorporating the time dimension into volume data.

Main Methods:

• Combined modified transmission electron microscope sample preparation with FIB-SEM volume imaging.
• Developed a precise trimming technique for regions of interest to add a temporal dimension.
• Acquired and analyzed serial volume data from rabbit ovaries at consecutive developmental stages.

Main Results:

• Achieved efficient 3D structural surveys of rabbit ovaries across development.
• Successfully constructed a virtual 4D electron microscopy dataset.
• Observed novel filopodia-like processes extending from oocyte cysts, facilitating intercellular connections between oocytes.

Conclusions:

• The presented workflow enhances the analysis of large biological samples in 3D.
• The virtual 4D electron microscopy approach provides new insights into dynamic developmental processes.
• The discovery of oocyte-to-oocyte filopodia offers a new perspective on ovarian cyst development and oocyte communication.