Raman and fluorescence microscopy sensing energy-transducing and energy-storing structures in microalgae

Chlorophylls and carotenoids are core components of photosynthetic energy transduction in algal cells, whereas starch, lipids, and polyphosphates represent energy reserves. All these biomolecules exhibit characteristic molecular vibrations that were sensed and localized in individual cells of Desmodesmus quadricauda and Chlorella vulgaris by confocal Raman microscopy. In the same cells, fluorescence of chlorophylls and of lipid bodies stained with Nile Red was mapped using the same instrument. In the first of the three consecutive scans, a low-power and short-exposure excitation by a green 532 nm laser was used to map chlorophyll fluorescence. In the second scan, the full power of a 785 nm laser was used for Raman mapping of carotenoids. The third scan was performed again with the 532 nm laser to map non-fluorescent biomolecules such as lipids, proteins, starch, and polyphosphates. Before the third scan, chlorophyll fluorescence was suppressed by photobleaching a wide area using a strongly defocused 532 nm laser beam. Raman microscopy was used for the first time to localize polyphosphate granules within a single algal cell in the context of other relevant bio-energetic structures. The new information represents an opportunity for algal cell phenotyping.