HIGH-SPEED ATOMIC FORCE MICROSCOPY FOR DYNAMIC BIOLOGICAL IMAGING - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 166

weight ratio). Dried lipid ilms were obtained by mixing lipids dissolved in
chloroform followed by evaporating the solvent with argon. The lipid ilms
were further dried in a desiccator by aspirating for more than 30 minutes. To
obtain multilamellar vesicles (MLVs), the dried lipid ilms were resuspended
in a buffer (10 mM HEPES-NaOH, 150 mM NaCl, 2 mM CaCl 2 [pH 7.4]) by
vortexing. Small unilamellar vesicles (SUVs) were produced from the MLV
suspension by sonications with a tip-sonicator for a few seconds.

(b)

(a)

Figure 8.1. (a) Schematic of a streptavidin molecule on a biotinylated lipid bilayer. (b)
Schematic of streptavidin arrays in a C 222 crystal.

SLBs were prepared by depositing 0.1 mg/ml SUVs onto a freshly cleaved
mica surface and incubated for 30 minutes in a chamber with saturated
humidity at room temperature. After that, the excess lipids were washed
out with the buffer. 2D crystallization of streptavidin on biotin-containing
SLBs was performed by injecting streptavidin dissolved in an appropriate
buffer at a inal concentration of 0.1 mg/ml and incubating for 2 hours in
a chamber with saturated humidity at room temperature. The buffer used
for streptavidin crystallization has the same composition as the one used
for the SLB formation. Then, excess streptavidin molecules were washed out
with the buffer. As shown in Fig. 8.1b , in the 222 crystal, the intermolecular

contacts between biotin-bound subunits are contiguously aligned along one
crystal axis (

C

-axis), while the contacts between biotin-unbound subunits are
contiguously aligned along the other axis (

a

-axis).
Monovacancy defects in the streptavidin 2D crystals were produced by
increasing the tapping force onto the sample from the oscillating tip. Then,
diffusion of point defects in the crystals was observed. Figure 8.2a shows
images of the streptavidin 2D crystal and monovacancy defects therein,
which are clipped from successively captured high-speed AFM images. In
Fig. 8.2b , the trajectories of two monovacancy defects are shown. The mobility
of the monovacancy defects was obviously anisotropic with respect to thew

b