43 (Chow et al. 1988). This corresponds to 59% Chl of PSII and 41% Chl of PSI. If all the PSIIs are closed, one might expect 59% Chl contribution of slow lifetimes

and 41% of fast lifetime. The amplitudes of the lifetime of 116 ps for both groups of pixels is more than 41%, so the conclusion should be such that not all the PSII reaction centers are closed by the DCMU. The two slow lifetimes of ~1 and ~4 ns must correspond to closed PSII reaction centers because these lifetimes are absent for open RCs. The 6.3% difference in the amplitude of the slow lifetimes for the high- and low-intensity Autophagy activator pixels is probably caused by the fact that the high-intensity pixels comprise more PSII than PSI. This is expected because the grana, where PSII is concentrated, have a higher chlorophyll concentration per pixel than the stroma lamellae. There are two straightforward explanations for the lifetime differences in the pixel groups: (i) The DCMU buffer is not penetrated evenly in every part of the chloroplasts which results in different lifetimes and intensities for each pixel; (ii) In one pixel group, there are more grana than in the other pixel group which will also result in different lifetimes and intensities for each pixel. In Fig. 6b, the

intensity of the different pixels seems to have a random distribution in the chloroplast, which is not expected as a result of varying penetration of the DCMU buffer. The differences in lifetimes for Copanlisib nmr only the two pixel groups can thus better be explained by pixels with more or less grana. It should be kept in mind that the model that is used here (PSI and PSII fluorescence kinetics are both homogeneous) is oversimplified, for instance, because of the action of the PSII repair cycle and the presence of PSII heterogeneity. In conclusion, it appears to

be very difficult to distinguish between regions with more or less grana. Fig. 5 Room temperature fluorescence decay traces (measured with FLIM). The chloroplasts in Arabidopsis thaliana leaves are excited with TPE at 860 nm and are detected with a bandpass filter centered at 700 nm with a bandwidth of 75 nm. Black squares represent a “”normal”" fluorescence decay trace of chloroplasts in an Arabidopsis leaf with an average lifetime of 290 ps. Round open circles represent a fluorescence decay trace of a vacuum infiltrated leaf with a 0.1 mM DCMU buffer with an average lifetime of 1.3 ns Fig. 6 a Room temperature fluorescence decay traces (measured with FLIM) of chloroplasts in Alocasia wentii leaves excited with TPE at 860 nm detected with a bandpass filter centered at 700 nm with a bandwidth of 75 nm. The leaves are vacuum infiltrated with a 0.1 mM DCMU buffer for closing the PSII reaction centers. The black (1) trace with its fit corresponds to the summed fluorescence decay of 10 white (high) pixels from the chloroplast in the intensity-based image in Fig. 6b.