Decay Associated Spectra (DAS)
Figure 9b, published in (Theiss et al., 2011), shows typical fluorescence decay curves of whole cells of A.marina collected after excitation with 632 nm at room temperature performed with the technique of time and wavelength correlated single photon counting (TWCSPC) (Schmitt, 2011). The number of the registered photons at each wavelength and
Figure 9. a) Color Intensity Plot (CIP) of a measurement on A.marina after excitation at 632 nm at 298 K. b) Fluorescence decay curves at 660 nm and 725 nm. c) Time resolved Fluorescence spectra at 0 ps and 1 ns (at 1 ns multiplied with a factor 5). d) Decay associated spectra (DAS) of a global fit in the range 640 nm - 690 nm (multiplied with a factor 0.3) and a global fit in the range 700 nm - 760 nm (see text). The figure is published in (Theiss et al., 2011 and Schmitt, 2011). Image reproduced with permission.
each time channel was stored in a 2-dim, 256 x 1024 data matrix. In Figure 9a, this data matrix is shown as a color intensity plot (CIP). A CIP is a plot of the fluorescence intensity (pictured by color) as a function of wavelength (y-axis) and time (x-axis). Therefore the CIP contains the time- and wavelength-resolved fluorescence emission data and provides information on the steady state fluorescence spectra and the lifetimes and dynamics of different emitter states. A vertical plot at a constant time t0 results in the time-resolved emission spectrum F(t0, X) (Figure 9c) while a horizontal intersection delivers the fluorescence decay at a constant emission wavelength X0 (Figure 9b). Time-resolved spectra provide information on the fluorescence of certain fluorophores at distinct times when the fluorescence emission of other pigments or scattered light have already decayed or after energy transfer when the main emission shifted in time from the donor pigment to the acceptor pigment.
The spectral resolution of the spectrometer system used to acquire the data shown in Figure 9 which is described in (Schmitt, 2011) is limited to about 2 nm (spectrometer entrance slit < 0.5 mm) due to the distance of the delay-line meanders and electrical crosstalk between the meander lines.
Figure 9b shows the decay curves of A.marina at 660 nm and 725 nm. It is seen that the fluorescence at 660 nm decays much faster than the emission at 725 nm. A closer look at the emission maximum on top of Figure 9b reveals a very small temporal shift between the 660 nm and the 725 nm decay curves. The 725 nm decay is slightly shifted to later times in comparison to the 660 nm decay. A data fit shows that the small temporal shift, in the following called “fluorescence rise kinetics”, has a similar time constant as the fluorescence decay at 660 nm. Both curves are convoluted with the instrumental response function (IRF) which leads to the small visible difference.
In Figure 9c the main emission is observed at 645-660 nm (PBP emission) immediately after excitation (0 ps) while after one nanosecond (1 ns) the strongest fluorescence band occurs at 725 nm (Chl d emission). For better illustration the spectrum after 1 ns is multiplied by a factor of 5. At longer times the Chl d emission exceeds the PBP emission.
Detailed analyses of the fluorescence decay curves were performed by iterative reconvolution of a polyexponential decay model with the IRF using a global lifetime analysis minimizing the quadratic error sum X employing a Levenberg-Marquardt algorithm (for details see Theiss et al., 2011; Schmitt, 2011). The IRF was measured using distilled water as scattering medium. The multiexponential fits of all decay curves measured in one time- and wavelength resolved fluorescence spectrum were performed as global fits with common values of lifetimes r. (linked parameters) for all decay curves and wavelength-dependent preexponential factors A (A) (non-linked parameters, see Figure 9d). The result of such a fit analysis is usually plotted as a graph of A (A) for all wavelength independent lifetimes . This plot represents the so called “decay associated spectra” (DAS) thus revealing the energetic position of individual decay components (for details also see Schmitt et al., 2008).