A preparation technique for observing cytoskeletons by high resolution scanning electron microscopy

Area of Science:

• Cell Biology
• Microscopy
• Cytoskeletal Dynamics

Background:

• The cytoskeleton provides structural support and is crucial for cell motility and division.
• Understanding the precise arrangement of cytoskeletal elements is key to deciphering cellular functions.
• Previous imaging techniques had limitations in resolving fine cytoskeletal structures in relation to membranes.

Purpose of the Study:

• To investigate the three-dimensional architecture of the cytoskeleton in cultured fibroblasts.
• To examine the spatial relationship between cytoskeletal components and membranous structures.
• To utilize a novel cell-interior revealing technique combined with ultrahigh resolution scanning electron microscopy.

Main Methods:

• Employed a newly devised technique for revealing cell interiors.
• Utilized an ultrahigh resolution scanning electron microscope (UHS-T1).
• Applied immunogold staining for identifying actin filaments, intermediate filaments, and microtubules.

Main Results:

• Successfully preserved both the cytoskeleton and membranous structures (plasma membrane, endoplasmic reticulum).
• Identified and characterized actin filaments (10 nm), intermediate filaments (12 nm), and microtubules (26 nm).
• Observed actin filaments forming stress fibers, sheaths, and meshworks, with a 2D network beneath the plasma membrane; intermediate filaments were mainly in the perikaryon; microtubules and clathrin-coated vesicles were clearly visualized.

Conclusions:

• The study provides high-resolution insights into fibroblast cytoskeleton organization.
• Demonstrates the utility of the novel technique for visualizing cytoskeletal architecture and its membrane interactions.
• Offers a detailed map of cytoskeletal components and their arrangement within the cell.