Spatial organization of β-carboxysomes in RbcL:eGFP Synechococcus cells. A, Confocal images of a RbcL:eGFP Synechococcus cell. Green, eGFP-labeled carboxysomes; blue, DAPI-stained DNA; red, autofluorescence of the thylakoid membrane. The merged channel revealed that most of the cytoplasmic volume of the Synechococcus cell is occupied by carboxysomes and chromosomes. This subcellular organization indicated that all carboxysomes in the RbcL:eGFP cell can been visualized using confocal microscopy. The confocal image of the Synechococcus construct that expresses free eGFP illustrates that free eGFP is spread throughout the cytoplasm without specific aggregation. B, Computational programming of image analysis allows automatic identification of carboxysomes in cells in confocal images. Bar = 2 μm. C, Statistical determination of the spatial localization of carboxysomes within the cell revealed the distribution profiles of carboxysomes along both the longitudinal and short axes of the cell (n = 300). The orange squares represent the relative frequency of carboxysome localization in the cell. The developed automated analysis software routines were used in this work for analyzing the carboxysome content and positioning.