A further development in photoactivation is the discovery that the genes encoding GFP and related fluorescent proteins can be engineered to produce protein variants, usually with one or more amino acid changes, that fluoresce only weakly under normal excitation conditions, but can be induced to fluoresce either more strongly or with a color shift (for example, from green to red) by activating them with a strong pulse of light at a different wavelength. In principle, the microscopist can then follow the local in vivo behavior of any protein that can be expressed as a fusion with one of these GFP variants. These genetically encoded, photoactivatable fluorescent proteins allow the lifetime and behavior of any protein to be studied independently of other newly synthesized proteins (Figure 9–28).
A third way to exploit GFP fused to a protein of interest is known as fluorescence recovery after photobleaching (FRAP). Here, one uses a strong focused beam of light from a laser to extinguish the GFP fluorescence in a specified region of the cell, after which one can analyze the way in which remaining unbleached fluorescent protein molecules move into the bleached area as a function of time.
Figure 9–26 Fluorescence resonance energy transfer (FRET). to determine whether (and when) two proteins interact inside a cell, the proteins are first produced as fusion proteins attached to different color variants of green fluorescent protein (gFp). (a) in this example, protein X is coupled to a blue fluorescent protein, which is excited by violet light (370–440 nm) and emits blue light (440–480 nm); protein Y is coupled to a green fluorescent protein, which is excited by blue light (440–480 nm) and emits green light (510 nm). (B) if protein X and Y do not interact, illuminating the sample with violet light yields fluorescence from the blue fluorescent protein only. (C) When protein X and protein Y interact, the resonance transfer of energy, Fret, can now occur. illuminating the sample with violet light excites the blue fluorescent protein, which transfers its energy to the green fluorescent protein, resulting in an emission of green light. the fluorochromes must be quite close together—within about 1–5 nm of one another—for Fret to occur. Because not every molecule of protein X and protein Y is bound at all times, some blue light may still be detected. But as the two proteins begin to interact, emission from the donor blue fluorescent protein falls as the emission from the acceptor gFp rises.
Figure 9–27 Determining microtubule flux in the mitotic spindle with caged fluorescein linked to tubulin.
a metaphase spindle formed in vitro from an extract of Xenopus eggs has incorporated three fluorescent markers: rhodamine-labeled tubulin (red) to mark all the microtubules, a blue Dna-binding dye that labels the chromosomes, and caged-fluorescein-labeled tubulin, which is also incorporated into all the microtubules but is invisible because it is nonfluorescent until activated by ultraviolet (UV) light.
a beam of UV light activates, or “uncages,” the caged-fluorescein-labeled tubulin locally, mainly just to the left side of the metaphase plate. over the next few minutes—after 1.5 minutes in (C) and after 2.5 minutes in (D)—the uncagedfluorescein–tubulin signal moves toward the left spindle pole, indicating that tubulin is continuously moving poleward even though the spindle (visualized by the red rhodamine-labeled tubulin fluorescence) remains largely unchanged. (From k.e. sawin and t.J. Mitchison, J. Cell Biol. 112:941–954, 1991. With permission  from the rockefeller University press.) 546 Chapter 9:  Visualizing Cells