Spectral microscopic imaging of heterocysts and vegetative cells in two filamentous cyanobacteria based on spontaneous Raman scattering and photoluminescence by 976 nm excitation

Photosynthetic pigment-protein complexes are highly concentrated in thylakoid membranes of chloroplasts and cyanobacteria that emit strong autofluorescence (mainly 600–800 nm). In Raman scattering microscopy that enables imaging of pigment concentrations of thylakoid membranes, near infrared laser excitation at 1064 nm or visible laser excitation at 488–532 nm has been often employed in order to avoid the autofluorescence. Here we explored a new approach to Raman imaging of thylakoid membranes by using excitation wavelength of 976 nm. Two types of differentiated cells, heterocysts and vegetative cells, in two diazotrophic filamentous cyanobacteria, Anabaena variabilis, and Rivularia M-261, were characterized. Relative Raman scattering intensities of phycobilisomes of the heterocyst in comparison with the nearest vegetative cells of Rivularia remained at a significantly higher level than those of A. variabilis. It was also found that the 976 nm excitation induces photoluminescence around 1017–1175 nm from the two cyanobacteria, green alga (Parachlorella kessleri) and plant (Arabidopsis thaliana). We propose that this photoluminescence can be used as an index of concentration of chlorophyll a that has relatively small Raman scattering cross-sections. The Rivularia heterocysts that we analyzed were clearly classified into at least two subgroups based on the Chla-associated photoluminescence and carotenoid Raman bands, indicating two physiologically distinct states in the development or aging of the terminal heterocyst.