HIGH-RESOLUTION ATOMIC FORCE MICROSCOPY OF NATIVE MEMBRANES - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 37

biomolecules. The reasons for this are probably the concerns about causing
sample damage due to the higher stiffness of the short high-speed probes
compared with the probes used in contact mode in conventional AFMs, the
instinctive association of high speed with high friction, and the fact that
samples mainly studied with the HS-AFM are isolated biomolecules lying
on lat substrates, thus creating elevated local corrugations. Only recently,

contact mode has been used for high-speed high-resolution AFM imaging.
The sample selected was the purple membrane, a well-structured lattice of
the bacteriorhodopsin (bR) protein that shows a low corrugation ( Fig. 2.9a ).

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The results show that a membrane sample is stable under high-speed contact
mode AFM imaging and, furthermore, that the high-speed contact mode
imaging results in comparable resolution to imaging of the purple membrane
using conventional AFM or HS-AFM in oscillatory mode. 93 Interestingly, the
high-speed imaging rate obtained for the contact and oscillatory modes
was the same in both cases—10 frames per second. This contrasts with
conventional AFM, where the contact mode allows for an imaging rate on the
biological membrane that is between 5 to 10 times faster than the oscillatory
mode. Probably factors not related to the probe, such as piezoelectric stage
or the control electronics, could have limited the speed in contact mode
HS-AFM, and future works could show slightly higher imaging speeds in
contact mode HS-AFM with respect to the oscillatory mode for imaging low-
corrugation samples the same way as in conventional AFM. At lower speed,
the imaging contrast was higher. Individual bR trimers associating and
dissociating to the edges of the bR array could be monitored ( Fig. 2.9b ). 94
Similar results have been acquired using oscillating mode HS-AFM. 93
Finally, it is important to highlight the role of the apex of the AFM probe
on the quality of AFM imaging. An AFM probe comes in contact of the sample
only through the apex of the microfabricated pyramid that is located at
the end of the cantilever. The shape of the tip of the apex determines the
imaging resolution and the pyramid the distribution of the applied force over
the sample. In biological samples with low corrugation, such as the purple
membrane, it has been shown by using conventional slow AFM that a blunt
apex presenting some nanometric protrusion can provide the optimal imaging
conditions for contact mode imaging. The reason is that the force applied by
the probe is distributed over a larger area, but that the protrusion is sharp
enough to provide a local sensitivity and obtain high-resolution images.

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For the high-speed contact mode AFM studies 92 the blunt probes were also
used and provided optimal imaging conditions. In contrast, probes with a
sharper apex tended to easily damage the samples in contact mode HS-AFM.
On the other hand, previous high-speed studies in oscillatory mode typically
used a sharpened apex. The high corrugation of the isolated samples studied
required sharp, high-aspect ratio probes to minimize the convolution and
enhance the resolution. 91

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