8 September 2006 Optimization of nonlinear excitation for reducing light-induced changes in photosynthetic systems during imaging with multimodal microscopy
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Proceedings Volume 6343, Photonics North 2006; 634307 (2006) https://doi.org/10.1117/12.706555
Event: Photonics North 2006, 2006, Quebec City, Canada
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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Richard Cisek, Richard Cisek, Arkady Major, Arkady Major, Nicole Prent, Nicole Prent, Catherine Greenhalgh, Catherine Greenhalgh, Virginijus Barzda, Virginijus Barzda, "Optimization of nonlinear excitation for reducing light-induced changes in photosynthetic systems during imaging with multimodal microscopy", Proc. SPIE 6343, Photonics North 2006, 634307 (8 September 2006); doi: 10.1117/12.706555; https://doi.org/10.1117/12.706555

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