Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves.
This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of Arabidopsis thaliana and Alocasia wentii under excitation-annihilation free conditions, both for the F (0)- and the F (m)-state. The corresponding average lifetimes are approximately 250 ps and approximately 1.5 ns, respectively, similar to those of isolated chloroplasts. These values appear to be the same for chloroplasts in the top, middle, and bottom layer of the leaves. With the spatial resolution of approximately 500 nm in the focal (xy) plane and 2 microm in the z direction, it appears to be impossible to fully resolve the grana stacks and stroma lamellae, but variations in the fluorescence lifetimes, and thus of the composition on a pixel-to-pixel base can be observed.
Broess
Koen
K
Laboratory of Biophysics, Wageningen University, PO Box 8128, 6700 ET, Wageningen, The Netherlands.
Borst
Jan Willem
JW
van Amerongen
Herbert
H
D031055:Alocasia / Q000502:physiology; D017360:Arabidopsis / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods*; D017785:Photons; D010788:Photosynthesis / Q000502:physiology*; D018515:Plant Leaves / Q000502:physiology*; D013696:Temperature; D013997:Time Factors