Figure 4
Spectroscopy on ensembles and single SCP complexes that have been stored for 7 months at − 80 °C in the dark. The samples for spectroscopy have been prepared in a cold room at 5 °C (protocol 3). (A) Room temperature absorption spectrum of an ensemble of SCP dissolved in bulk buffer solution at a concentration of 1.45 × 10–7 M (black line). For comparison the corresponding absorption spectrum from the fresh sample (cf. Fig. 2A) has been underlaid as filled spectrum. Both spectra have been peak normalised at 476 nm. (B) Emission spectrum from SCP complexes embedded in PVA for an excitation intensity of 525 W/cm2. The filled area shows the emission spectrum from a sample of fresh SCP complexes embedded in PVA. Both spectra have been peak normalised for better comparison. The excitation wavelength for recording the emission spectrum was 561 nm as indicated by the green arrow in (A). (C) Fluorescence lifetime of an ensemble of SCP complexes immobilized in PVA in semi-logarithmic representation. The full line corresponds to a biexponential fit (see residuals at the bottom) with decay components (amplitudes) of 4.0 ns (79%) and 1.3 ns (21%). The excitation wavelength is 561 nm, and the excitation intensity amounts to 525 W/cm2. (D) Fluorescence lifetimes and spectral peak positions of a single SCP complex. The fluorescence lifetime (scale on the right) was read out every 500 ms and is given by the open dots. The bin time for recording the spectra was 5.1 s (full line) and the observed spectral peak position of the accumulated spectrum is given by the black dot (scale on the left). The sequence of emission spectra is shown on the right-hand side from top to bottom. For better comparison, the broken vertical line refers to the spectral peak position of the ensemble emission spectrum. (E) Two-dimensional representation of the fluorescence lifetime vs. spectral peak position of individual SCP complexes. Due to fluctuations of both lifetime and/or spectral peak position a single complex can contribute more than one data point to the diagram. For the distinction between the data points in the red and blue shaded areas see text. The number of individual complexes that contribute to the pattern is given in the top left corner. The histograms correspond to the distributions of the spectral peak position (top) and fluorescence lifetime (right). For ease of comparison an emission spectrum from a sample of fresh SCP complexes embedded in PVA has been overlaid in the top histogram. The statistical parameters of the two histograms (mean ± sdev) are (14,537 ± 181) cm−1 for the peak positions, and (2.8 ± 0.9) ns for the lifetimes, respectively.
