Figure 2
Spectroscopy on ensembles and single SCP complexes. The samples for spectroscopy have been prepared under ambient conditions (protocol 1). (A) Room temperature absorption spectrum of an ensemble of SCP complexes dissolved in bulk buffer solution at a concentration of 1.45 × 10–7 M. (B) Peak normalised emission spectra from the bulk buffer solution for an excitation intensity of 30 μW/cm2 (full line) and from an ensemble of SCP complexes embedded in a thin film of PVA (dashed line) for an excitation intensity of 525 W/cm2. The excitation wavelength for recording the emission spectra 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 (85%) and 1.2 ns (15%). 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 drop out after 4 s presumably results from unresolved blinking. 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. 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 ensemble emission spectrum has been overlaid in the top histogram. The statistical parameters of the two histograms (mean ± sdev) are (14,723 ± 109) cm−1 for the peak positions, and (3.4 ± 0.8) ns for the lifetimes, respectively.
