Biology Reference
In-Depth Information
imaging of individual molecule at work,
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and this setup is already convenient
to image biological membranes. Rupture of liposomes on mica and formation
of SLB from a ternary mixture of lipids were observed at one image per
second,
meaning that this setup should be very useful to elucidate membrane
phenomena such as microdomain nucleation, diffusion of nanoscale domains
and diffusion of membrane components. Recently, high-resolution movies of
individual bacteriorhodopsin trimers were acquired at a 100 ms frame and
highlighted temporal luctuation at the crystal edges.
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The second main drawback of AFM for imaging complex systems such
as biological membranes or model membranes, including several proteins,
is the identiication of membrane components. One of the solutions is to
combine AFM with luorescence microscopy, even if the lateral resolution
in classical luorescence microscopy (~ 200-300 nm due to the diffraction
limit) is very weak compared with that of AFM. In a seminal paper,
7
membrane microdomains were localized on a wide scale by luorescence in
a DOPC/DPPC supported bilayer, whereas AFM provided topography in the
mesoscopic scale. This combination is now proposed by most manufacturers.
Interestingly, the development of super-resolution luorescence microscopy
should reduce the gap between AFM and luorescence approaches. Using far-
ield optical microscopy or nanoscopy such as stimulated emission depletion
(STED), photoactivated localization microscopy (PALM) or stochastic optical
reconstruction microscopy (STORM), the lateral resolution can reach a few
tens of nanometers.
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Membrane dynamics can also be explored with optical
nanoscopy.
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Combined with AFM, optical nanoscopy might open a new ield
of exploration of biological membranes that sounds very exciting. However,
according to the scanning rate of commercially available microscopes, it is
necessary to immobilize membrane components for superimposing optical
and AFM images. Combining high-speed AFM and optical nanoscopy could
eventually solve this drawback. Another strategy to identify membrane
components by AFM is to perform single-molecule recognition imaging.
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Single-molecule imaging requires tip functionalization with relevant
molecules to recognize a speciic molecule associated with the membrane.
Detection of molecular recognition events can be performed using adhesion
force mapping or dynamic recognition force mapping (see
Chapter 7
). Finally,
we can speculate that combining AFM with nanoSIMS (secondary ion mass
spectroscopy), a new technique that has been used to identify molecules on
top of membranes, should be very interesting. NanoSIMS uses the secondary
emitted ions from a bombarded surface to get a mass spectrum and to
identify the species present on the surface. The beam scans the surface,
and a mass spectrum is made at each pixel. The lateral resolution, which is
given by the ion beam diameter, can presently reach 50 nm. The nanoSIMS
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