Carbon fixation in cyanobacteria makes a major contribution to the global carbon cycle. The cyanobacterial carboxysome is a proteinaceous microcompartment that protects and concentrates the carbon-fixing enzyme RuBisCO in a paracrystalline lattice, making it possible for these organisms to fix CO 2 from the atmosphere. The protein responsible for the organization of this lattice in beta-type carboxysomes of the freshwater cyanobacterium Synechococcus elongatus , CcmM, occurs in two isoforms

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... ght to localize differentially within the carboxysome matrix. Here, we use widefield timelapse and 3D-structured illumination microscopy (3D-SIM) to study the recruitment and localization of these two isoforms. We demonstrate that this super-resolution technique is capable of successfully resolving the outer protein shell of the carboxysome from its internal cargo. We develop an automated analysis pipeline to analyze and quantify 3D-SIM images and generate a population level description of carboxysome shell protein, RuBisCO, and CcmM isoform localization. We find that both CcmM isoforms colocalize in space and time, prompting a revised model of the internal arrangement of the β-carboxysome.