This technique is usually carried out with a confocal microscope and, like photoactivation, can deliver valuable quantitative data about a protein’s kinetic parameters, such as diffusion coefficients, active transport rates, or binding and dissociation rates from other proteins (Figure 9–29).
One way to study the chemistry of a single living cell is to insert the tip of a fine, glass, ionsensitive microelectrode directly into the cell interior through the plasma membrane. This technique is used to measure the intracellular concentrations of common inorganic ions, such as H+, Na+, K+, Cl–, and Ca2+. However, ionsensitive microelectrodes reveal the ion concentration only at one point in a cell, and for an ion present at a very low concentration, such as Ca2+, their responses are slow and somewhat erratic. Thus, these microelectrodes are not ideally suited to record the rapid and transient changes in the concentration of cytosolic Ca2+ that have an important role in allowing cells to respond to extracellular signals. Such changes can be analyzed with ion-sensitive indicators, whose light emission reflects the local concentration of the ion. Some of these indicators are luminescent (emitting light spontaneously), while others are fluorescent (emitting light on exposure to light).
Figure 9–28 Photoactivation.
photoactivation is the light-induced activation of an inert molecule to an active state. in this experiment, shown schematically in (a), a photoactivatable variant of gFp is expressed in a cultured animal cell. (B) Before activation (time 0 sec), little or no gFp fluorescence is detected in the selected region (red circle) when excited by blue light at 488 nm. after activation of the gFp with an ultraviolet laser pulse at 413 nm, it rapidly fluoresces brightly in the selected region (green). the movement of gFp, as it diffuses out of this region, can be measured. since only the photoactivated proteins are fluorescent within the cell, the trafficking, turnover, and degradative pathways of proteins can be monitored. (B, from J. Lippincott-schwartz and g.h. patterson, Science 300:87–91, 2003.)
Figure 9–29 Fluorescence recovery after photobleaching (FRAP). a strong focused pulse of laser light will extinguish, or bleach, the fluorescence of gFp. By selectively photobleaching a set of fluorescently tagged protein molecules within a defined region of a cell, the microscopist can monitor recovery over time, as the remaining fluorescent molecules move into the bleached region (see Movie 10.6). (a) the experiment shown uses monkey cells in culture that express galactosyltransferase, an enzyme that constantly recycles between the golgi apparatus and the endoplasmic reticulum (er). the golgi apparatus in one of the two cells is selectively photobleached, while the production of new fluorescent protein is blocked by treating the cells with cycloheximide. the recovery, resulting from fluorescent enzyme molecules moving from the er to the golgi, can then be followed over a period of time. (B) schematic diagram of the experiment shown in (a).  (a, from J. Lippincott-schwartz, Histochem. Cell Biol. 116:97–107, 2001. With permission from springer-Verlag.) only molecules in the evanescent feld fuoresce