Protocol to investigate gene expression heterogeneity in cyanobacteria using mRNA CARD-FISH

Here, we present a protocol for visualizing gene expression in the filamentous cyanobacterium Trichodesmium and the single-celled species Synechocystis and Cyanothece using the catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) technique. We describe steps for fixation, agarose coating, enzymatic permeabilization, and sample handling. This protocol is broadly applicable to cyanobacteria, and the detection of rbcL mRNA in Trichodesmium, used as an example, supports its use to study the heterogeneity of physiological processes at single-cell level.