Visualization of cytoskeletal elements by the atomic force microscope

Abstract

We describe a novel application of atomic force microscopy (AFM) to directly visualize cytoskeletal fibers in human foreskin epithelial cells. The nonionic detergent Triton X-100 in a low concentration was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized in either liquid or air-dried ambient conditions. These two types of scanning provide complimentary information. Scanning in liquid visualizes the surface filaments of the cytoskeleton, whereas scanning in air shows both the surface filaments and the total "volume" of the cytoskeletal fibers. The smallest fibers observed were ca. 50 nm in diameter. The lateral resolution of this technique was ca.20 nm, which can be increased to a single nanometer level by choosing sharper AFM tips. Because the AFM is a true three dimensional technique, we are able to quantify the observed cytoskeleton by its density and volume. The types of fibers can be identified by their size, similar to electron microscopy.

Human epithelial cells increase their rigidity with ageingin vitro: direct measurements

“…For scanning in air, the cells were washed with MilliQ ultrapure water and dried under ambient conditions. The details of this technique are described elsewhere (Berdyyeva et al 2004).…”

Molecular imaging of membrane proteins and microfilaments using atomic force microscopy

“…To remove the plasma membrane of cells, two approaches, de-roofing by short ultrasonic burst and detergent extraction, have been demonstrated (Heuser, 2000;Berdyyeva et al, 2005). The ultrastructural insight into the architecture of focal adhesions in de-roofed cells was provided by AFM topographs complemented with optical microscopy (Franz and Muller, 2005).…”

“…Several reports describe investigations of intracellular cytoskeletal structures after detergent extraction using the nonionic detergent Triton X-100 (Berdyyeva et al, 2005;Meller and Theiss, 2006). A method of permeabilization and embedding of lens epithelial cells was used to study the organization and distribution of intracellular proteins with AFM and confocal microscopy (Meller and Theiss, 2006).…”

“…In this study, confocal microscopy was used to identify actin microfilaments by fluorescent labeling with rhodamine phalloidin, whereas AFM enabled the study of three-dimensional structures of microfilaments at the molecular level in fixed and living cells. Berdyyeva et al (2005) described a novel application of AFM to directly visualize cytoskeletal fibers in human foreskin epithelial cells. In this approach, they used Triton X-100 to remove the membrane, soluble proteins, and organelles from the cell.…”

“…De-roofing and detergent extraction to remove the plasma membrane may cause damage to samples during their preparation (Svitkina et al, 1995;Small et al, 1999;Berdyyeva et al, 2005). Recently, we reported a semi-in situ system through photodynamic therapy (PDT) to study the ultrastructure of microfilaments without additional treatment and under near-native conditions (Jung et al, 2009a).…”