An Unroofing Method to Observe the Cytoskeleton Directly at Molecular Resolution Using Atomic Force Microscopy.

Eiji Usukura,Shuichi Ito,Akira Yagi,Jiro Usukura,Akihiro Narita

doi:10.1038/srep27472

Abstract

An improved unroofing method enabled the cantilever of an atomic force microscope (AFM) to reach directly into a cell to visualize the intracellular cytoskeletal actin filaments, microtubules, clathrin coats, and caveolae in phosphate-buffered saline (PBS) at a higher resolution than conventional electron microscopy. All of the actin filaments clearly exhibited a short periodicity of approximately 5–6 nm, which was derived from globular actins linked to each other to form filaments, as well as a long helical periodicity. The polarity of the actin filaments appeared to be determined by the shape of the periodic striations. Microtubules were identified based on their thickness. Clathrin coats and caveolae were observed on the cytoplasmic surface of cell membranes. The area containing clathrin molecules and their terminal domains was directly visualized. Characteristic ridge structures located at the surface of the caveolae were observed at high resolution, similar to those observed with electron microscopy (EM). Overall, unroofing allowed intracellular AFM imaging in a liquid environment with a level of quality equivalent or superior to that of EM. Thus, AFMs are anticipated to provide cutting-edge findings in cell biology and histology.

Highlights

Binnig et al.[1] first outlined the principle of the atomic force microscope (AFM) as a type of scanning probe microscopy
An ultrasonic generator was improved to precisely regulate the output power in the range of 0 − 1 W. This custom-made low-power sonicator was further combined with a stereomicroscope equipped with a position controller for the sonication probe and an LED light-sheet illumination system to observe the unroofing procedure in detail (Fig. 1B)
In vivo AFM imaging has long been limited to surface observations of living or fixed cells at a slightly higher resolution than that of light microscopes

Summary

Introduction

Binnig et al.[1] first outlined the principle of the atomic force microscope (AFM) as a type of scanning probe microscopy. When the cell membrane is broken, the intracellular region is exposed by removing the cytoplasmic-soluble components, which allows the AFM cantilever to access intracellular structures such as the cytoskeleton and organelles. This preparation technique, which uses sonication, was originally developed to observe membrane undercoats in freeze-etching EM11,12. The improved (custom-made) sonicator is able to generate cavitation at a much lower power (0.3 − 1 W) than that of the commonly used sonicator (50 W or higher) We used this method in an atomic force microscopy analysis of the intracellular cytoskeleton in phosphate-buffered saline (PBS) at a molecular resolution. To improve the signal-to-noise (S/N) ratio and thereby refine the images obtained with the AFM, serial scanned images of a target organelle were averaged using mathematical calculations

Methods

Results

Conclusion