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Cyanobacteria are oxygenic photosynthetic prokaryotes that are the progenitors of the chloroplasts of algae and plants. These organisms harvest light using large membrane-extrinsic phycobilisome antenna in addition to membrane-bound chlorophyllcontaining proteins. Similar to eukaryotic photosynthetic organisms, cyanobacteria possess thylakoid membranes that house photosystem (PS) I and PSII, which drive the oxidation of water and the reduction of NADP+, respectively. While thylakoid morphology has been studied in some strains of cyanobacteria, the global distribution of PSI and PSII within the thylakoid membrane and the corresponding location of the light-harvesting phycobilisomes are not known in detail, and such information is required to understand the functioning of cyanobacterial photosynthesis on a larger scale. Here, we have addressed this question using a combination of electron microscopy and hyperspectral confocal fluorescence microscopy in wild-type Synechocystis species PCC 6803 and a series of mutants in which phycobilisomes are progressively truncated. We show that as the phycobilisome antenna is diminished, large-scale changes in thylakoid morphology are observed, accompanied by increased physical segregation of the two photosystems. Finally, we quantified the emission intensities originating from the two photosystems in vivo on a per cell basis to show that the PSI:PSII ratio is progressively decreased in the mutants. This results from both an increase in the amount of photosystem II and a decrease in the photosystem I concentration. We propose that these changes are an adaptive strategy that allows cells to balance the light absorption capabilities of photosystems I and II under light-limiting conditions. [ABSTRACT FROM AUTHOR]

Cellular autofluorescence, though ubiquitous when imaging cells and tissues, is often assumed to be small in comparison to the signal of interest. Uniform estimates of autofluorescence intensity obtained from separate control specimens are commonly employed to correct for autofluorescence. While these may be sufficient for high signal-to-background applications, improvements in detector and probe technologies and introduction of spectral imaging microscopes have increased the sensitivity of fluorescence imaging methods, exposing the possibility of effectively probing the low signal-to-background regime. With spectral imaging, reliable monitoring of signals near or even below the noise levels of the microscope is possible if compensation for autofluorescence and background signals can be performed accurately. We demonstrate the importance of accurate autofluorescence modeling and the utility of spectral imaging and multivariate analysis methods using a case study focusing on fluorescence confocal spectral imaging of host-pathogen interactions. In this application fluorescent proteins are produced when Francisella novicida invade host macrophage cells. The resulting analyte signal is spectrally overlapped and typically weaker than the cellular autofluorescence. In addition to discussing the advantages of spectral imaging for following pathogen invasion, we present the spectral properties and cellular origin of macrophage autofluorescence. [ABSTRACT FROM AUTHOR]

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010. [ABSTRACT FROM PUBLISHER]