HIGH-SPEED ATOMIC FORCE MICROSCOPY FOR DYNAMIC BIOLOGICAL IMAGING - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 161

of our understanding of dynamic processes played by biological molecules is
enhanced. However, this directness is not suficient. These single-molecule
luorescence techniques observe protein molecules indirectly, and therefore,
we still need to ill the gap between the recorded luorescence images and the
actual behaviour of the labelled biological molecules. To further enhance the
directness, we need techniques that allow us to directly observe biological
molecules with nanometre spatial and millisecond temporal resolution.
The atomic force microscope (AFM) is capable of directly visualizing
unstained biological samples in liquids at nanometre resolution. 2 Since the
invention, biologists have hoped that its unique capability would allow us to
observe the dynamic behaviour of protein molecules at work. However, the
imaging speed was limited to several tens of seconds per frame, and hence, it
could not trace the fast dynamic processes progressing within a sample. Over
the past decade, various efforts have been directed towards increasing the
imaging rate of AFM. 3-8 The most advanced high-speed AFM can now capture
images at 30-60 ms/frame over a scan range of ~250 nm with ~100 scan
lines. 5-7 Importantly, the tip-sample interaction force has been greatly reduced
without sacriicing the imaging rate, so that weak dynamic interactions
between biological macromolecules are not signiicantly disturbed.
In this chapter, irst we briely review the limiting factors of imaging
speed, and key techniques for high-speed imaging. For details of the
instrumentation, readers may refer to a comprehensive review. 8 Then, we
demonstrate some examples of successful imaging of protein, focusing on
dynamics in two-dimensional (2D) protein crystals. In the last section, we
describe the potential of high-speed AFM for cell imaging.

8.2 HIGHSPEED IMAGING TECHNIQUES

High-speed AFM for biological samples in solutions is based on the tapping
mode

in which the AFM tip is vertically oscillated and periodically brought
into contact to a sample surface during scanning. The tip oscillation reduces
the lateral force between tip and sample and thus minimizes damage and/or
deformation of biological molecules. The vertical tip force acting on a sample
is controlled by a PID (proportional-integral-derivative) feedback controller
so that the oscillation amplitude of the cantilever is kept constant. Precise
and fast feedback control is highly required for fast and low-invasive imaging.
In this section, we simply describe the quantitative relationship between the
feedback bandwidth and the various factors involved in AFM devices and
the scanning conditions.

9,10

6

Then, the elemental techniques in the AFM for fast

imaging are described.