Biomedical Engineering Reference
In-Depth Information
using conductive/magnetically coated cantilevers, electrostatic and magnetic forces
can be sensed to image for instance magnetic domains, surface charge distributions,
local surface capacitance, and local conductance.
3
High-Resolution Imaging
3.1
Imaging Ion Channel Structure
AFM gives a unique opportunity for high-resolution studies of biological materials
since they can be examined under physiological conditions. In particular, the tech-
nique is very suitable for imaging membranes in both native and reconstituted
forms. Purifi ed membrane proteins, such as bacteriorhodopsin (Butt et al.
1990
;
Worcester et al.
1990
), as well as the three-dimensional (3D) crystal of OmpC porin
membrane protein (Hwang et al.
2005
) have been imaged. By using force dissec-
tion, both the extracellular and cytoplasmic faces of the gap junctions could be
imaged (Hoh et al.
1991
) ( Fig.
3
).
Hemichannels have more recently been imaged in lipid bilayers after reconstitu-
tion in liposomes (Thimm et al.
2005
). A distinct difference was observed between
two populations of channel-like structures that correspond to the extracellular and
cytoplasmic side of the hemichannels (Fig.
3
). Furthermore, by changing calcium
levels in the imaging buffer, hemichannels could be imaged both in their open (low
calcium) and closed (normal calcium) states.
Using phase imaging, where the phase lag between the tip oscillation in tapping
mode and the driving signal is monitored, Thimm et al. (
2005
) also showed surface
energy measurements using AFM, observing a spike in the phase signal of open
channels most likely due to exposed hydrophobic domains of the hemichannel in
open state (Fig.
3
). The open state of hemichannels has previously been shown to
regulate cell volume and mechanics that infl uences tissue growth (Quist et al.
2000
) .
Also the presence of hemichannels has been linked to induction of oxidative stress-
induced apoptosis, for instance, caused by smoking (Ramachandran et al.
2007
) .
Other proteins that form ion channels have been reconstituted in lipid bilayers
and imaged with AFM (Fig.
4
) at high resolution to resolve single ion channel struc-
tures. For instance, many amyloids, including amyloid beta protein, amylin, alpha-
synuclein, ADan, ABri, and Serum Amyloid A (Lin et al.
2001
; Quist et al.
2005a
) ,
when reconstituted in membrane form ion channel-like structures. Resolution in
such studies is suffi cient to resolve a central channel and to distinguish different
subunit arrangements. Electrical recording from these preparations shows that these
amyloid channels are active and conduct ionic currents.
This illustrates the application of AFM in resolving the pathogenic mechanism of
channelopathies. For instance, the toxicity observed upon amyloid deposition seems
at least partly to be created by ion channel-induced fl ow of ions that disturbs the cells'
homeostasis. The ability to directly image ion channels gives opportunities in testing
prospective drugs and therapies on a single channel level for channelopathies.
