Fluorescence and photobleaching dynamics of single light-harvesting complexes

Abstract

Single light-harvesting complexes LH-2 from Rhodopseudomonas acidophila were immobilized on various charged surfaces under physiological conditions. Polarized light experiments showed that the complexes were situated on the surface as nearly upright cylinders. Their fluorescence lifetimes and photobleaching properties were obtained by using a confocal fluorescence microscope with picosecond time resolution. Initially all molecules fluoresced with a lifetime of 1 +/- 0.2 ns, similar to the bulk value. The photobleaching of one bacteriochlorophyll molecule from the 18-member assembly caused the fluorescence to switch off completely, because of trapping of the mobile excitations by energy transfer. This process was linear in light intensity. On continued irradiation the fluorescence often reappeared, but all molecules did not show the same behavior. Some LH-2 complexes displayed a variation of their quantum yields that was attributed to photoinduced confinement of the excited states and thereby a diminution of the superradiance. Others showed much shorter lifetimes caused by excitation energy traps that are only approximately 3% efficient. On repeated excitation some molecules entered a noisy state where the fluorescence switched on and off with a correlation time of approximately 0.1 s. About 490 molecules were examined.

Figure 1

Examples from a series of fluorescence images of single LH-2 assemblies. The image size is 102 × 128 pixels and the integration time per pixel was 4 ms. The excitation power was 290 nW at λ = 800 nm. The molecule labeled 1 is an A state in a. It goes to a B′ state in b and it recovers to an A′ state in c. During the scanning of this image, a B state was formed for ≈0.5 s before recovering to the A′ state. In d a P state is reached. The circle labeled 2 is one of the rare cases where a molecule suddenly appears. Probably it was bleached to a B state during the sample preparation.

Figure 2

Examples of bleaching curves of single LH-2 assemblies with a binning time of 0.25 s. The excitation power at λ = 800 nm was 0.5 μW, 3.7 μW, 0.3 μW, and 0.7 μW for a, b, c, and d, respectively. The state labels are discussed in the text. The corresponding lifetime measurements of the different states in d are given in Fig. 3.

Figure 4

Laser power dependence of bleaching. (a) The mean of the inverse of the survival time in state A (〈1/τ_A〉; •) and the mean of the inverse of the time between state A and the first reemitting state (〈1/τ_B〉; □) are plotted against the excitation power at λ = 800 nm. The increase in 〈1/τ_A〉 is almost linear with the excitation power, whereas 〈1/τ_B〉 is almost independent of it. These results are based on the analysis of 201 and 136 single-molecule measurements for 〈1/τ_A〉 and 〈1/τ_B〉, respectively. (b) “Bulk” measurements on ≈100 molecules were fitted by a double exponential. Only the fast time constant (90%) is plotted against the excitation power. The error bars correspond to a confidence level of 95%.

Figure 3

Lifetime measurements on single LH-2 assemblies in the states corresponding to those in Fig. 2d.