TAIPEI, TAIWAN, REP. OF CHINA

Academia Sinica, National Taiwan University

KAOHSIUNG, TAIWAN, REP. OF CHINA

National Sun Yat-sen University

BUFFALO, NEW YORK

State University of New York

Multi-photon excited fluorescence and absorption properties of maize tissues --Lin, B-L, Kao, F-J, Sun, C-K, Cheng, P-c In order to interpret the images obtained with confocal and multi-photon fluorescence microscopy, it is important to obtain the basic absorption and autofluorescence properties of maize tissues, in particular, the multi-photon excited fluorescence spectra. It has been demonstrated that Arabidopsis mesophyll protoplasts and leaves exhibit significantly different autofluorescence between single- and two-photon excitation (Cheng et al., Proc. Optics and Photonics Taiwan, 1099-1101, 1999; Cheng et al., SPIE Proceedings 3919, 2000; Cheng et al., Micron, 2000). A 495nm two-photon excited fluorescence emission peak was detected from Arabidopsis protoplasts when illuminated with high intensity 760nm IR pulse. This green emission is very close to the emission peak of GFPs, hence, may interfere with the detection of GFP in plant cells under multi-photon fluorescence microscopy.

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.

Figure 1.

Figure 2.

Figure 3.
 
 


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