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BioScience AFM


Figure 5 : Lateral membrane patch dynamics of bacteriorhodopsin. Addition and dissociation of trimers at the periphery of a membrane patch (relative differences between frames shown with yellow arrows) observed with fast-scanning AFM at time intervals of 17 seconds. The images are crops of the original 512×512 pixel-frames recorded at a line rate of 30 Hz. The z -scale in (a–c) is 1 nm.


Figure 4a shows an example with a soſt lambda phage DNA molecule imaged in buff er solution. T e double helix of DNA is typically revealed by applying a force range between 50–100 pN [ 12 ]. Crystallographic data give the size of the 2 nm thick DNA double helix, with one whole revolution around its axis of 3.4 nm, comprised of a major groove (2.2 nm, see Figure 4a , red arrows) and a minor groove (1.2 nm, see Figure 4a , blue


arrows). T e DNA data obtained by AFM in solution are in very good agreement with the one from crystallographic measure- ments reported in literature. Another example is the high- resolution imaging of bacterio- rhodopsin (BR). Arranged as a 2D protein crystal of trimeric polypeptide molecules consisting of seven transmembrane alpha helices each, BR functions as a light-driven proton pump ( Figure 4b ). T e vast majority of previous high-resolution con- ventional AFM scans of this mol- ecule reported in the literature were almost exclusively done with static mode imaging [ 13 ]. T e application of fast scanning shown allows AFM to get struc- tural information that is compa- rable to the details coming from crystallographic data ( Figure 4c ). T ere was no need to average several data sets.


Figure 6 : Photodynamic cycle changes in bacteriorhodopsin. Frames of (a) non-photolyzed and (b) photolyzed states of BR in a membrane patch as a part of the reversible photoactivation series. The light-illumination part of the cycle is designated with green at the bottom of (b). Upon switching the green light off, the BR molecules revert back to their non-photolyzed-state conformation over a period of seconds. The outlined sector in (a) and (b) shows the associated structural change (displacement of the monomers with respect to the trimeric center of about 0.7 nm). (a) and (b) were recorded at a line rate of 32 Hz. The z -scale is 0.8 nm.


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Dynamic changes in proteins . Fast-scanning AFM allows the imaging of soſt samples in liquid. It is now possible to visualize a range of dynamic processes taking place on the scale of seconds and milliseconds with adequate spatial resolution. Figure 5 shows another example with BR, a light-driven proton pump found in some purple membrane-containing Halobacterium species. By using fast scanning with a line rate of 30 Hz, it is possible to visualize the membrane patch fl uidity. In particular, the images depicted in Figure 5 reveal the addition and removal of BR trimers without introducing scanning defects/ artifacts into the patch. T e newly bound and dissociated BR molecules can be identifi ed in groups of trimers between the individual frames. T is is mostly because the temporal resolution of lateral bond lifetime of such individual events is typically on the timescale of a few hundred milliseconds to a few seconds, which is much less than the time separation of 17 s of the original frames shown.


Conformational change


in molecules . Fast scanning allows study of the photocycle kinetics of the BR molecule. The BR molecule is light-sensitive


www.microscopy-today.com • 2015 November


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