English: Paired micrographs and NanoSIMS images of ¹³C and ¹⁵N distribution in Hemiaulus-Richelia symbioses, which were incubated with ¹³C bicarbonate and ¹⁵N₂ and untreated (A) or treated with a eukaryotic inhibitor (B). Images from left to right include: Epi-fluorescent image taken prior to NanoSIMS analyses and correspond to the parallel NanoSIMS imaging of total secondary ion count (0.001 x Esi), enrichment of ¹⁵N (¹⁵N/¹⁴N), and enrichment of ¹³C (¹³C/¹²C). Epi-fluorescent images (A panel, green excitation: 510–60 nm; B panel, blue excitation: 459–90 nm) taken prior to analyses are used to approximate the cellular location of the symbiont and host chloroplast. The emission visible in the epifluorescent micrographs correspond to the filaments of Richelia and chloroplast of the host diatoms. Each host cell has two symbiotic filaments, and the white arrows designate the terminal heterocysts of the Richelia filaments, which emit red and yellow-orange under green (A) and blue excitation (B), respectively. Note that in the control cells (untreated, A or top images), enrichment of ¹⁵N and ¹³C is uniformly high in both host and symbiont, whereas the cell treated with eukaryotic inhibitor has localized ¹⁵N enrichment to the two symbiont filaments and generally low enrichment of ¹³C in whole cell (B, or bottom images). The enclosed markings in the NanoSIMS images define the regions of interest (ROIs), which were used to determine the ¹³C/¹²C and ¹⁵N/¹⁴N ratios. Scale bars are 5 μm.