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). The epi-fluorescent images show excitation patterns expected for the symbiont and host chloroplast. The secondary ion content images show the host cellular boundaries and destructive nature of the NanoSIMS analyses.

(A) Symbiotic Hemiaulus hauckii-Richelia intracellularis cell collected from 2 m with 2 Richelia filaments emitting a bright orange-red fluorescence under green excitation (510–60 nm). The strong fluorescence associated with the Richelia filaments corresponds to a high ¹⁵N enrichment (¹⁵N/¹⁴N ratio image), whereas cellular ¹³C enrichment (¹³C/¹²C) is above background but uniformly low.

(B) A larger Hemiaulus membranaceus-Richelia intracellularis cell collected from 25 m with two clearly fluorescent filaments of Richelia. Note the terminal heterocysts on either end of the two filaments, indicating recent or in situ cellular division of the Richelia. NanoSIMS images show uniform high enrichment of both ¹⁵N and ¹³C (¹⁵N/¹⁴N and ¹³C/¹²C ratio images respectively) in areas of both symbiont and host chloroplast with the exception of one heterocyst designated with an arrow.