ИСТИНА

Autotrophic microalgae use sunlight, carbon dioxide, and inorganic nutrients to biosynthesize complex organic compounds. Algae represent a large group of phylogenetically diverse organisms adapted to live at different, often extreme habitats, and capable of producing various substances that may be used as renewable raw materials. To better understand the peculiarities of their metabolism, as well as for efficient search and phenotyping of new strains suitable for industrial use, methods allowing rapid detection, simultaneously with imaging and quantification of various biomolecules directly within the intact cells are more than desirable. Confocal Raman microscopy, which combines the molecular specificity of vibrational spectroscopy together with the spatial resolution of the confocal optical microscopy, may be a method of choice for chemical mapping of various microorganisms. Nevertheless, its routine applicability to photosynthetic microorganisms, especially microalgae, has long been hindered by a strong autofluorescence of photosynthetic pigments interfering with Raman spectra. Recently, we have developed a simple methodology [1] for fast and efficient suppression of the chlorophyll fluorescence, which opens the door wide into this exciting but little-explored field. Besides the simultaneous detection, visualization, and quantification of already known compounds [1, 2], conventional Raman microscopy based on commercially available Raman microscopes can be of great help for identifying the real chemical nature of various intracellular structures [3, 4], frequently visualized by electron microscopy but still of unknown or questionable molecular composition. In such a way, inclusions found in some microalga were identified as crystalline guanine [3]. Similarly, enigmatic particles embedded into the plastids of extremophilic Arctic microalga Cylindrocystis were for the first time identified as polyphosphate granules [4]. As Raman spectra are sensitive also to isotopic labeling, Raman microscopy can be useful for metabolic studies at a single-cell level, representing thus less laborious alternative to nanoscale mass spectrometry [5]. Recent progress in the application of confocal Raman microscopy in algal research will be presented and demonstrated using our original research results. Advantages and perspectives, but also limitations and pitfalls of the method will be pointed out and discussed.