Comparative study of thylakoid membranes in terminal heterocysts and vegetative cells from two cyanobacteria, Rivularia M-261 and Anabaena variabilis, by fluorescence and absorption spectral microscopy.

Heterocyst is a nitrogen-fixing cell differentiated from a cell for oxygen-evolving photosynthesis (vegetative cell) in some filamentous cyanobacteria when fixed nitrogen (e.g., ammonia and nitrate) is limited. Heterocysts appear at multiple separated positions in a single filament with an interval of 10-20 cells in some genera (including Anabaena variabilis). In other genera, a single heterocyst appears only at the basal terminal in a filament (including Rivularia M-261). Such morphological diversity may necessitate different properties of heterocysts. However, possible differences in heterocysts have largely remained unexplored due to the minority of heterocysts among major vegetative cells. Here, we have applied spectroscopic microscopy to Rivularia and A. variabilis to analyze their thylakoid membranes in individual cells. Absorption and fluorescence spectral imaging enabled us to estimate concentrations and interconnections of key photosynthetic components like photosystem I (PSI), photosystem II (PSII) and subunits of light-harvesting phycobilisome including phycocyanin (PC). The concentration of PC in heterocysts of Rivularia is far higher than that of A. variabilis. Fluorescence quantum yield of PC in Rivularia heterocysts was found to be virtually the same as those in its vegetative cells, while fluorescence quantum yield of PC in A. variabilis heterocysts was enhanced in comparison with its vegetative cells. PSI concentration in the thylakoid membranes of heterocysts seems to remain nearly the same as those of the vegetative cells in both the species. The average stoichiometric ratio between PSI monomer and PC hexamer in Rivularia heterocysts is estimated to be about 1:1.

[Coherent phase microscopy: a new method of identification of intracellular structures based on their optical and morphometric parameters].

A new method for the identification of intracellular structures of a living cell and obtaining the quantitative parameters characterizing these structures by means of coherent phase microscopy is proposed. The method is based on the analysis of the histogram of a cell phase image and its decomposition by phase height levels. In the spherical and cylindrical approximation of the cell, the method makes it possible to separate the contributions of phase-contrast intracellular structures to the integral refractive index of the whole cell. The calculation of refractive indices of intracellular structures is illustrated on a two-component model of a spherical cell. The possibility of determining the refractive indices of cellular organelles was shown by an example of cyanobacterium Anabaena variabilis ATCC 29413 cells and Cancer mammae breast cancer cells. Three most contrast cellular structures in the phase images of the cells were identified, and their refractive indices were determined. For Anabaena variabilis cells, these structures were the cell wall ( = 1.39), tylakoids ( = 1.47), and the nucleoid ( = 1.58); for breast cancer cells, these were the cytoplasm ( = 1.34), the nucleus ( = 1.36), and the nucleolus ( = 1.44). Using this method is possible to identify phase-contrast intracellular structures and calculate their refractive indices. This enables one to follow morphofunctional changes of not only the whole cell but also its individual organelles.