Biology Reference
In-Depth Information
Figure 8.9 shows successive AFM images captured during the crystal
growth of annexin V. In this measurement, the buffer solution at the initial
condition does not contain CaCl
2
. During scanning, CaCl
2
was injected into
the sample chamber to give a inal concentration of 3 mM. At 0 second, the
lipid surface is primarily observed because of the absence of Ca
+
ions. This
image shows large noise which is caused by annexin V molecules rapidly
diffusing on the surface. A CaCl
2
solution was injected at 7 seconds. Soon
after the injection, small particles appear (see the image at 31 seconds). This
is probably the irst stage of the assembly in which three molecules cluster
together in an almost irreversible manner to form a trimer
.
Surface-diffusing
annexin molecules come into contact with the surface-bound trimer and
gradually increase the cluster size with time. However, in the captured images,
crystallization progresses more predominantly from the top left in the images.
The precursor protein clusters observed at the irst stage are incorporated
into large crystals during progression of the crystallization. Eventually, the
lipid surface is completely covered by the crystal in a few minutes.
8.4 FUTURE PROSPECTS: TOWARDS DYNAMIC IMAGING OF
LIVE CELLS
Current high-speed AFMs can ilm dynamic processes played by puriied
protein molecules. The video images of molecular processes provide insight
into their functional mechanisms in a much more straightforward manner
than other techniques. However, at present, high-speed AFM cannot be
applied to observe dynamics on cell membranes. To change this situation,
we have to overcome some technical dificulties. In this section, we discuss
whether and how we can achieve such a new generation of high-speed AFM.
8.4.1 Lower Interacon Force and Non-Contact Imaging
Since cell membranes are suspended and hence extremely soft, achieving
small tip-sample interaction force is essential for their stable and high-
resolution imaging. Also, membrane proteins that are not anchored to
cytoskeletons or not clustered diffuse very fast within the membranes.
High-speed AFM requires much higher imaging speed. Generally, to reduce
cantilever stiffness, one must compromise the resonant frequency and
vice
versa
. The most advanced small cantilevers deem to have almost achieved
the ultimate goal of balancing these two mechanical quantities. Therefore,
reduction of the interaction force by using softer and smaller cantilevers
seems impossible. The ultimate minimization of the tip-sample interaction
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