Using Atomic Force Microscopy To Illuminate the Biophysical Properties of Microbes

Abstract

Since its invention in 1986, atomic force microscopy (AFM) has grown from a system designed for imaging inorganic surfaces to a tool used to probe the biophysical properties of living cells and tissues. AFM is a scanning probe technique and uses a pyramidal tip attached to a flexible cantilever to scan across a surface, producing a highly detailed image. While many research articles include AFM images, fewer include force-distance curves, from which several biophysical properties can be determined. In a single force-distance curve, the cantilever is lowered and raised from the surface, while the forces between the tip and the surface are monitored. Modern AFM has a wide variety of applications, but this review will focus on exploring the mechanobiology of microbes, which we believe is of particular interest to those studying biomaterials. We briefly discuss experimental design as well as different ways of extracting meaningful values related to cell surface elasticity, cell stiffness, and cell adhesion from force-distance curves. We also highlight both classic and recent experiments using AFM to illuminate microbial biophysical properties.

Keywords: adhesion; atomic force microscopy; cell stiffness; force–distance curve; membrane elasticity; microbes.

Figure 1.

(A) Schematic representation of the AFM cantilever from both the side and above as well as (B) basic AFM operating principles. Figures are not drawn to scale.

Figure 2.

Three common experimental designs used to obtain force measurements. Cells may be (A) affxed to a surface and probed directly with the tip or (B) probed with a molecule attached to the tip. (C) Bacterial cells may also be affxed to the cantilever.

Figure 3.

Schematic representation of an approach curve. (A) The tip is lowered toward the cell surface (B) where it makes initial contact. (C) As the tip begins to push into the cell wall, there is a nonlinear change in force, which indicates the elasticity of the cell surface. (D) The tip pushes further on the cell and generates a linear change in force, from which the cell stiffness is calculated.

Figure 4.

Interaction of the tip and the cell is modeled as a compression of two springs. The spring constant of the cantilever (k_cantilever) is measured at the beginning of the experiment,and the k_effective is determined from the slope of the linear compression.k_cell is determined using eq 1.

Figure 5.

Schematic representation of a retraction curve. (A) The tip begins to pull away from the cell, (B) with the data generally overlaying the linear compression of the approach curve. (C) Adhesive forces allow the tip and surface to remain connected, and the tip pulls the surface up (D). Eventually, the attractive forces are overcome and the tip and surface separate, creating a snap-off event in which the force rapidly returns to zero.

Figure 6.

Schematic representation of the Gram-negative cell wall. (A) The Gram-negative cell wall contains two membranes, the plasma membrane and the outer membrane, which sandwich the periplasmic space containing the peptidoglycan. (B) The outer leaflet of the outer membrane contains lipopolysaccharide (LPS), a highly diverse molecule. Different strains of bacteria have different polysaccharide combinations, with some strains containing many O-antigen units, while others contain only the inner core.

Figure 7.

Schematic representation of the Gram-positive cell wall. The Gram-positive cell wall contains a single membrane with a thick peptidoglycan layer. Teichoic acids covalently bind to the peptidoglycan while lipoteichoic acids anchor the peptidoglycan to the plasma membrane.

Figure 8.

Schematic representation of a yeast cell wall. Unlike bacterial cell walls, the main components of yeast cell walls are ß-glucan and chitin.