Figure 9–33 Single molecules can be imaged and manipulated by atomic force microscopy. (a) schematic diagram of the key components of an atomic force microscope (aFM), showing the force-sensing tip attached to one end of a single protein molecule, as in the experiment described in (D). (B) and (C) an aFM in imaging mode created these images of a single heteroduplex Dna molecule with a Muts protein dimer (larger white regions) bound near its center, at the point of a mismatched base pair. Muts is the first protein that binds to Dna when the mismatch repair process is initiated (see Figure 5–19). the smaller white dots are single streptavidin molecules, used to label the two ends of each Dna molecule. (D) titin is an enormous protein molecule that provides muscle with its passive elasticity (see Figure 16–34). the extensibility of this protein can be directly tested using a short, artificially produced protein that contains eight repeated immunoglobulin (ig) domains from one region of the titin protein. in this experiment, the tip of the aFM is used to pick up, and progressively stretch, a single molecule until it eventually ruptures. as force is applied, each ig domain suddenly begins to unfold, and the force needed in each case (about 200 pn) can be recorded. the region of the force–extension curve shaded green records the sequential unfolding event for each of the eight protein domains. (B and C, from Y. Jiang and p.e. Marszalek, EMBO J. 30:2881–2893, 2011. reprinted with permission of John Wiley & sons; D, adapted from W.a. Linke et al., J. Struct. Biol. 137:194–205, 2002. With permission from elsevier.) features on a surface. When used in this mode, the probe is scanned over the surface, moving up and down as necessary to maintain a constant interaction force with the surface, thus revealing any objects such as proteins or other molecules that might be present on the otherwise flat surface (Figure 9–33B and C). AFM is not limited to simply imaging surfaces, however, and can also be used to pick up and move single molecules that adsorb strongly to the tip. Using this technology, the mechanical properties of individual protein molecules can be measured in detail. For example, AFM has been used to unfold a single protein molecule in order to measure the energetics of domain folding (Figure 9–33 D).
The variations on light microscopy we have described so far are all constrained by the classic diffraction limit to resolution described earlier; that is, to about 200 nm (see Figure 9–6). Yet many cellular structures—from nuclear pores to nucleosomes and clathrincoated pits—are much smaller than this and so are unresolvable by conventional light microscopy. Several approaches, however, are now available that bypass the limit imposed by the diffraction of light, and successfully allow objects as small as 20 nm to be imaged and clearly resolved: a remarkable, orderofmagnitude improvement.