Pigment Organization Effects on Energy Transfer and Chl a Emission Imaged in the Diatoms C. meneghiniana and P. tricornutum In Vivo: A Confocal Laser Scanning Fluorescence (CLSF) Microscopy and Spectroscopy Study

The.(auto)fluorescence from three diatom strains, Cyclotella meneghiniana (Cm), Phaeodactylum tricornutum la (Pt1a), and Phaeodactylum UTex (PtUTex), has been imaged in vivo to submicrometer resolution using confocal laser scanning fluorescence (CLSF) microscopy. The diatoms are excited at 473 and 532 nm, energy primarily absorbed by the carotenoid fucoxanthin (Fx) found within the fucoxanthin chlorophyll a/c proteins (FCPs). On the basis of the fluorescence spectra measured in each image voxel, we obtain information about the spatial and energetic distribution of the terminal Chl a emitters, localized in the FCPs and the reaction centers of the PSII protein complexes, and the nature and location of the primary absorbers that are linked to these emitters; 532 nm excites the highly efficient Fx(red) light harvesters, and lesser amounts of Fx(green)s, that are enriched in some FCPs and preferentially transfer energy to PSII, compared to 473 nm, which excites almost equal amounts of all three previously identified sets of Fx - Fx(red), Fx(green) and Fx(blue) - as well as Chl c. The heterogeneous Chi a emission observed from the (C)LSF images indicates that the different Fx's serve different final emitters in P. tricornutum and suggest, at least in C. meneghiniana, a localization of FCPs with relatively greater Fx(red) content at the chloroplast edges, but with overall higher FCP concentration in the interior of the plastid. To better understand our results, the concentration-dependent ensemble-averaged diatom solution spectra are compared to the (auto)fluorescence spectra of individual diatoms, which indicate that pigment packing effects at an intracellular level do affect the diatoms' spectral properties, in particular, concerning a 710 nm emission band apparent under stress conditions. A species-specific response of the spectral signature to the incident light is also discussed in terms of the presence of a silica shell in Cm but not in PtIa nor PtUTex.