Figure 9–32 TIRF microscopy allows the detection of single fluorescent molecules. (a) tirF microscopy uses excitatory laser light to illuminate the cover-slip surface at the critical angle at which all the light is reflected by the glass–water interface. some electromagnetic energy extends a short distance across the interface as an evanescent wave that excites just those molecules that are attached to the cover slip or are very close to its surface. (B) tirF microscopy is used here to image individual myosin–gFp molecules (green dots) attached to nonfluorescent actin filaments (C), which are invisible but stuck to the surface of the cover slip. (Courtesy of Dmitry Cherny and Clive r. Bagshaw.) particular molecule of interest. This problem can be solved by the use of a special optical technique called total internal reflection fluorescence (TIRF) microscopy. In a TIRF microscope, laser light shines onto the coverslip surface at the precise critical angle at which total internal reflection occurs (Figure 9–32A). Because of total internal reflection, the light does not enter the sample, and the majority of fluorescent molecules are not, therefore, illuminated. However, electromagnetic energy does extend, as an evanescent field, for a very short distance beyond the surface of the cover slip and into the specimen, allowing just those molecules in the layer closest to the surface to become excited. When these molecules fluoresce, their emitted light is no longer competing with outoffocus light from the overlying molecules, and can now be detected. TIRF has allowed several dramatic experiments, for instance imaging of single motor proteins moving along microtubules or single actin filaments forming and branching. At present, the technique is restricted to a thin layer within only 100–200 nm of the cell surface (Figure 9–32B and C).
individual Molecules Can Be touched, imaged, and Moved Using atomic Force Microscopy
While TIRF allows single molecules to be visualized under certain conditions, it is strictly a passive observation method. In order to probe molecular function, it is ultimately useful to be able to manipulate individual molecules themselves, and atomic force microscopy (AFM) provides a method to do just that. In an AFM device, an extremely small and sharply pointed tip, often of silicon or silicon nitride, is made using nanofabrication methods similar to those used in the semiconductor industry. The tip of the AFM probe is attached to a springy cantilever arm mounted on a highly precise positioning system that allows it to be moved over very small distances. In addition to this precise movement capability, the AFM device is able to collect information about a variety of forces that it encounters—including electrostatic, van der Waals, and mechanical forces—which are felt by its tip as it moves close to or touches the surface (Figure 9–33A). When AFM was first developed, it was intended as an imaging technology to measure molecularscale (B) (C) 1000 800 600 400 200 0 0 100 200 300 extension (nm) laser photodiode (detector) detector and feedback electronics springy cantilever arm AFM tip stretching molecule attached to substrate e.g., mica (A) 28 nm force (pN)