Rotational Diffusion of Membrane Proteins
The preceding chapter has described the use of triplet probes and their time-resolved optical spectroscopic detection in the measurement of rotational diffusion coefficients of membrane proteins. The present chapter is concerned with the detection of triplet probes by light emission measurements, and I shall describe three methods: phosphorescence (Austin et al.,1979; Moore et al., 1979), delayed fluorescence (Greinert et al., 1979), and fluorescence depletion (Garland and Johnson, 1981). The aim of these alternative methods is to improve sensitivity, so that membrane proteins of low abundance (e.g., insulin receptors) can be studied. Because these methods all involve light emission, they can in principle be applied with a modified fluorescence microscope. The ambition then is to make measurements on the surfaces of intact cells, thereby bringing the sensitivity of rotational diffusion measurements to the same level as that obtainable for lateral diffusion using the fluorescence photobleaching recovery (FPR) method (Axelrod et al., 1976; Koppel et al.,1976), which can make measurements on a 1-um2 spot of membrane containing as few as 103 fluorescently labeled target proteins. To achieve this performance for rotational diffusion requires an improvement in sensitivity of triplet detection of some 10 orders of magnitude over the best reported so far (Moore et al., 1979), and this has now been achieved (Garland and Johnson, 1981).
KeywordsRotational Diffusion Transition Dipole Moment Delayed Fluorescence Pockels Cell Preceding Chapter
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