Academia Sinica, National Taiwan University
KAOHSIUNG, TAIWAN, REP. OF CHINA
National Sun Yat-sen University
BUFFALO, NEW YORK
State University of New York
Leaves from Ohio43 inbred were used in this study. Figure 1 shows the absorption properties of normal leaf and waterlogged leaf. Note the normal leaf shows a significantly higher optical density as a result of light scattering from air chambers within the leaf. The lower optical density in the longer wavelength region favors the use of multi-photon fluorescence microscopy for the study of thicker tissues.
Figures 2 and 3 show two-photon excited fluorescence spectra of both whole leaf and the acetone extract of the leaf. When 780nm excitation was used, two major fluorescence peaks (678nm and 512nm) were observed in whole leaf. In contrast, fluorescence emission at 512nm and 668nm was observed in leaf acetone extract. Two-photon fluorescence emission at 686nm was detected from leaf blade under 1240nm illumination. A second red fluorescence shoulder was observed around 740nm. Leaf acetone extract shows similar fluorescence peaked at 671nm and a longer shoulder around 730nm. The green fluorescence (512nm) observed in the 800nm excited spectra was absent due to the longer wavelength of the illumination (1240nm) which is not capable of producing 2-photon excitation at this wavelength. Although three-photon excitation is possible, no green fluorescence was observed in our current set-up.
Two-photon fluorescence spectra were obtained by using a Coherent Verdi pumped Spectra-Physics Tsunami mode-locked Ti-sapphire laser operated at 780nm with 100fs IR pulse. The 1240nm IR excitation was obtained from a Spectra-Physics Millennium IR (1064nm) pumped Chromium-doped Forsterite (home-built) laser operated at 1240nm with a pulse width of 130fs.
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