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Photosynthetic organisms adjust to fluctuating natural light under physiological ambient conditions through flexible light-harvesting ability of light-harvesting complex II (LHCII). A process called state transition is an efficient regulation mechanism to balance the excitations between photosystem II (PSII) and photosystem I (PSI) by shuttling mobile LHCII between them. However, in situ observation of the migration of LHCII in vivo remains limited. In this study, we investigated the in vivo reversible changes in the intracellular distribution of the chlorophyll (Chl) fluorescence during the light-induced state transitions in Chlamydomonas reinhardtii. The newly developed noninvasive excitation-spectral microscope provided powerful spectral information about excitation-energy transfer between Chl- a and Chl- b. The excitation spectra were detected through the fluorescence emission in the 700–750-nm spectral range, where PSII makes the main contribution, though PSI still makes a non-negligible contribution at room temperature. The technique is sensitive to the Chl- b spectral component specifically bound to LHCII. Using a PSI-specific 685-nm component also provided visualization of the local relative concentration of PSI within a chloroplast at room temperature. The decrease in the relative intensity of the Chl- b band in state 2 was more conspicuous in the PSII-rich region than in the PSI-rich region, reflecting the dissociation of LHCII from PSII. We observed intracellular redistributions of the Chl- b -related light-harvesting abilities within a chloroplast during the state transitions. This observation implies the association of the state transitions with the morphological changes in the thylakoid membrane. [ABSTRACT FROM AUTHOR]