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Nonlinear microscopy is a very attractive tool for studying photosynthetic organisms on cellular and subcellular levels.
The multimodal microscope can be employed to image photosynthetic structures simultaneously with multiphoton
excitation fluorescence (MPF), second harmonic generation (SHG), and third harmonic generation (THG) contrast
mechanisms. Although the multimodal nonlinear microscope delivers invaluable information about the structure,
spectroscopic properties, and functional dynamics of photosynthetic systems, the prompt light-induced changes of highly
light sensitive pigment-protein complexes complicate the extensive study of photosynthetic organisms. In this work, we
investigated the extent of light-induced changes in chloroplasts from higher plants by imaging with a Ti:Sapphire
femtosecond laser and a Yb-ion doped potassium gadolinium tungstate (Yb:KGW) femtosecond laser. The Ti:Sapphire
laser delivered 800 nm wavelength and ~25 fs duration pulses at a 26.7 MHz repetition rate. In comparison, the
Yb:KGW laser provided a 1042 nm wavelength, ~200 fs pulses at a repetition rate of 14.6 MHz. The 800 nm pulses
predominantly excited chlorophyll pigments via two-photon excitation, while 1042 nm excitation resulted in two-photon
absorption of carotenoids. The induced fluorescence quenching, and decrease in SHG and THG signal was much
stronger when imaged with a Ti:Sapphire laser. Prolonged imaging of up to tenths of minutes with the Yb:KGW laser
did not result in appreciable changes of all three nonlinear signals. The difference in the light-induced changes most
probably appears due to the difference in excited state dynamics following chlorophyll or carotenoid excitation. The
slow component of MPF and THG changes as well as change in SHG reflects the light-induced macroorganization of the
grana, while the fast MPF and THG component is tentatively attributed to the generation of quenchers from chlorophyll
molecules. The success in imaging photosynthetic samples for prolonged periods of time with a Yb:KGW laser opens a
new window of opportunity for thorough in vivo investigations of photosynthetic structures.
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