A Line-Scanning Multiphoton Fluorescence Spectromicroscope Applied to the Study of the Thylakoid Membrane in Chloroplasts

Abstract

We have constructed a laser-line-scanning semiconfocal multiphoton fluorescence microscope with a capability of simultaneous broadband spectral acquisition from a whole linear region. It was applied to the study of the thylakoid membrane of chloroplasts in a plant, Zea mays and Chlorella, all of which were studied at room temperature. In the case of Zea mays, we have determined spectral regions by which photosystem I and II including their associated antenna complexes are almost exclusively probed. The photosystem II distribution showed locally intense spots on a sub micrometer scale, resembling typical images of grana as reported by electron microscopy. The photosystem I fluorescence was rather homogeneously distributed and its image is in good agreement with bright-field images of the same chloroplast. This supports a model in which photosystem I is distributed on both stroma thylakoid and stroma-exposed thylakoid surrounding the granal thylakoid. It is thus shown that simultaneous detection of both photosystem I and photosystem II fluorescence images is possible at room temperature. Spectral images of Chlorella chloroplasts were also compared with those in Zea mays in order to understand different features of the thylakoid membranes.