Читать книгу Cell Biology - Stephen R. Bolsover - Страница 28

The resolution and precision of fluorescence microscopes have steadily improved with time. In 1979 a team in Amsterdam invented the confocal light microscope that scanned a point of excitation light across the specimen to markedly reduce the contribution of out‐of‐focus light. In 1987 a team in Cambridge developed a prototype of a commercial system and within a couple of years all major microscope manufacturers began offering confocal light microscopes.

A series of advanced microscopy techniques start with confocal microscopy and then use clever optical approaches to dramatically improve the resolution so that objects considerably smaller than the wavelength of light are revealed. Collectively these techniques are known as super‐resolution microscopy. One such technique is Stimulated Emission Depletion Microscopy (STED), in which the excitation spot is surrounded by a doughnut of light of a different wavelength that actually de‐excites the dye. Figure 1.14 shows one use of STED. Over 100 proteins are associated with the nuclear pores that perforate the nuclear envelope (Figure 1.2). Göttfert and coworkers used an antibody that recognizes one of these proteins, called gp210, labeled with a dye that emits red light, and a second antibody, labeled with a green emitter, against the transport machinery inside the pore. Figure 1.14 shows how an uninterpretable fuzz of red and green fluorescence in the standard confocal image is resolved into beautiful images by STED, revealing how eight gp210 molecules surround the pore. Data like these contribute to our present understanding of the eightfold symmetry of the structure that braces the pore to keep it open (Figure 12.10 on page 211). Super‐resolution microscopes can resolve objects down to tens of nanometers in size and can visualize biological structures previously thought to be unresolvable using light.