MICROBIAL CELL IMAGING USING ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 47

3.2 IMMOBILIZATION OF MICROBIAL CELLS FOR AFM
IMAGING

The problem of cell immobilization has come to be recognized as a signiicant
barrier to the application of AFM to microbiology. As indicated by the number
of papers that continue to be published on the topic, it is also a persistent one.
Immobilization is necessary to prevent displacement of the cell by the scanning
tip. Because the shape, size, rigidity and chemical properties of cells can differ
dramatically, so must strategies for their immobilization. Drying bacteria
onto the substrate before imaging is a popular choice for immobilization. 12-15
Unfortunately, drying the cells can result in cell dehydration and a lattened
or collapsed appearance in the resulting AFM images. 16,17 Moreover, cells
immobilized this way may not be viable and are frequently not stable when
imaged in liquid. Imaging in liquid is a requirement for live-cell imaging but
adds additional challenges because of the tendency of the tip to more easily
disturb hydrated bacteria. Nevertheless, these challenges must be confronted
if imaging dynamic processes are to be realized.
Bacterial surfaces vary because of differences in proteins, saccharides
and appendages (pili, imbriae, lagella) as determined by the genetics
of the strain. Competition between these surface constituents and media
components for binding sites on the substrate can prevent immobilization.
This point is demonstrated by an AFM imaging study of purple membranes
wherein Müller and colleagues enhanced immobilization by optimizing
the ion content and pH of the imaging buffer. 18 Cellular imaging, however,
requires an appreciation of how imaging solutions impact the physiology of
the cells. Depending on the focus of the study, changes in properties such
as osmotic potential or metabolism of the microbe may be undesirable. For
example, force-distance measurements of

carried
out in water and media show signiicant differences in bacterial spring
constants. 19 Also, the pH of the liquid has been shown to have a signiicant
inluence on the nanomechanical properties on

Pseudomonas aeruginosa

Shewaenella putrefaciens. 20
Imaging living microbes in liquid requires careful consideration of the
immobilization technique used to ensure that the physiology of the bacteria is
not compromised by the immobilization and imaging conditions. Therefore,
immobilization strategies must be developed and systematically tested for
individual organisms. Several approaches for mounting and immobilizing
microbes have been described. Successful approaches generally fall into one
of two categories: physical entrapment or chemical attachment ( Fig. 3.1 ).