6b. b (right) Room temperature fluorescence intensity-based image (measured with FLIM). The chloroplast
in Alacosia wentii leaves are excited with TPE at 860 nm and detected with a bandpass filter centered at 700 nm with a bandwidth of 75 nm. The pixel size is 0.26 μm. Fluorescence in each pixel is detected in 4,096 channels with a time resolution of 3 ps per channel. (left) Global fitting with linked lifetimes (τ 1, τ 2, and τ 3) and independent amplitudes for the black trace (1) and blue trace (2) shown in Fig. 6a For learn more Arabidopsis thaliana leaves, it appears to be not at all possible to resolve variations ABT-263 manufacturer in lifetimes between pixels, which is probably due to the fact that for Arabidopsis thaliana, the grana stacks are smaller than for Alocasia wentii. Conclusions In vivo measurements on chloroplasts are possible under low-light conditions with TPE FLIM and the obtained fluorescence kinetics are very similar to those observed in in vitro measurements on isolated chloroplasts. While scanning through Selleck AZD2014 the leaves of Alocasia wentii and Arabidopsis thaliana that were both grown under low-light conditions, no differences could be observed in the fluorescence kinetics, indicating no variation in the chloroplast composition/organization as a function of depth. The spatial resolution of the FLIM measurements
does not allow to observe individual grana stacks in Arabidopsis thaliana chloroplasts, but in the case of chloroplasts of Alocasia wentii variations in the lifetimes Benzatropine are observed, which may be ascribed to variations in the grana density. In the future, the TPE fluorescence lifetime
imaging microscope can be used to study individual chloroplasts in leaves under different stress conditions. Acknowledgments This study is part of the research programme of the “”Stichting voor Fundamenteel Onderzoek der Materie (FOM),”" which is financially supported by the “”Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO).”" Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References Albertsson PÄ, Andreasson E (2004) The constant proportion of grana and stroma lamellae in plant chloroplasts. Physiol Plant 121:334–342. doi:10.1111/j.0031-9317.2004.00315.x Anderson JM (1999) Insights into the consequences of grana stacking of thylakoids membranes in vascular plants: a personal perspective. Aust J Plant Physiol 26:625–639 Barzda V, de Grauw CJ, Vroom J, Kleima FJ, van Grondelle R, van Amerongen H, Gerritsen HC (2001a) Fluorescence lifetime heterogeneity in aggregates of LHCII revealed by time-resolved microscopy. Biophys J 81:538–546. doi:10.