MICROBIAL CELL IMAGING USING ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 48

(b) (c)

(a)

(f)

(d) (e)

Figure 3.1. Schematics of physical entrapment and chemical attachment of microbial
cells. In (a) and (b), two types of physical entrapment are shown. In (c), a 6 μm 6

μm delection image in liquid of a spherical yeast cell trapped in a ilter pore is shown
(image courtesy of Y. Dufrêne). In (d), immobilization by electrostatic interactions
occurs between the negatively charged cell surface and positively charged imaging
substrate. In (e), covalent attachment occurs by cross linking amino groups on the
cell surface with glutaraldehyde to amino groups covering the substrate surface.
The image in (f ) shows a 1 μm

s

s

1 μm amplitude image taken in liquid of an E. coli
spheroplast covalently linked to an APTES covered surface.

3.2.1 Ph ysical Entrapment

A successfully used method for immobilizing rigid, spherical bacteria, as
well as yeast and fungal spores, is to use polymer membranes with a pore
size comparable to the dimensions of the cell ( Fig. 3.1a ) . The cells become
mechanically trapped in the pores, allowing repeated imaging without cell
detachment or damage. 6,9,21-24 Most commonly, spherical microbial cells have
been examined ( Fig. 3.1c ) using the trapping strategy. The rigidity provided
by the peptidoglycan layer in the cell assists in maintaining the spherical
conformation of the bacteria as well as holding the cells in the pores. Although
the strategy is generally not applicable for immobilizing rod-shaped bacteria