Imaging metazoan nuclear pore complexes by field emission scanning electron microscopy

Area of Science:

• Cell Biology
• Microscopy Techniques
• Structural Biology

Background:

• Nuclear pore complexes (NPCs) regulate transport between the nucleus and cytoplasm.
• Advancements in microscopy are crucial for understanding NPC structure and function.
• Field emission scanning electron microscopy (FESEM) offers high-resolution 3D imaging capabilities.

Purpose of the Study:

• To provide a retrospective on microscopy techniques relevant to nuclear pore complex imaging.
• To present detailed protocols for preparing and imaging nuclear pore complexes using FESEM.
• To discuss modern techniques for enhancing NPC visualization.

Main Methods:

• Field emission scanning electron microscopy (FESEM) for high-resolution 3D surface imaging.
• Sample preparation techniques including Xenopus cell-free reconstitution and mammalian nucleus surface exposure.
• Immunogold labeling for specific NPC component identification.
• Delicate coating with iridium for improved imaging.

Main Results:

• Successful acquisition of high-resolution three-dimensional surface images of nuclear pore complexes.
• Demonstration of effective protocols for nuclear surface exposure and anchoring.
• Application of immunogold labeling for metazoan NPC visualization.

Conclusions:

• Field emission scanning electron microscopy is a powerful tool for high-resolution 3D imaging of nuclear pore complexes.
• The presented protocols facilitate detailed structural analysis of NPCs.
• Modern preparation and coating techniques enhance the visualization of these essential cellular structures.