Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (Ϸ0.03-m 3 ) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exciting primarily phycobilins and carotenoids. Fluorescence from phycocyanin, allophycocyanin, allophycocyanin-B/terminal emitter, and chlorophyll a was resolved. Moreover, resonance-enhanced Raman signals and very weak fluorescence from carotenoids were observed. Phycobilin emission was most intense along the periphery of the cell whereas chlorophyll fluorescence was distributed more evenly throughout the cell, suggesting that fluorescing phycobilisomes are more prevalent along the outer thylakoids. Carotenoids were prevalent in the cell wall and also were present in thylakoids. Two chlorophyll fluorescence components were resolved: the shortwavelength component originates primarily from photosystem II and is most intense near the periphery of the cell; and the long-wavelength component that is attributed to photosystem I because it disappears in mutants lacking this photosystem is of higher relative intensity toward the inner rings of the thylakoids. Together, the results suggest compositional heterogeneity between thylakoid rings, with the inner thylakoids enriched in photosystem I. In cells depleted in chlorophyll, the amount of both chlorophyll emission components was decreased, confirming the accuracy of the spectral assignments. These results show that hyperspectral fluorescence imaging can provide unique information regarding pigment organization and localization in the cell.cyanobacteria ͉ photosynthetic pigments ͉ multivariate curve resolution C yanobacteria convert light energy to chemical energy by means of photosynthesis, using water as a source of electrons for CO 2 reduction and O 2 production. A key part of the photosynthesis process is light absorption (harvesting) by pigments, followed by excitation transfer to reaction center chlorophyll (Chl) a of photosystems (PS) II and I (1). These processes take place in thylakoid membranes that in cyanobacteria generally form an extensive internal membrane complex of several layers along the periphery of the cytoplasm, with thylakoids found less frequently toward the center of the cell (2).The pigments associated with the photosynthetic apparatus are bound to thylakoid proteins, modifying their spectral properties and providing a spatial distribution that aids in the efficiency of light harvesting and energy transfer to reaction center Chls. Pigments bound to integral membrane proteins in reaction center complexes in thylakoids of cyanobacteria include Chl a [Ϸ40 per PS II (3) and Ϸ100 per PS I (4)] and carotenoids; the latter act in photoprotection and 3 Chl quenchin...