**2.1 Model object**

Cyanobacteria, used in this study as a model object, are photoautotrophic prokaryotes. They are important microorganisms that contributed to the early oxygenation of the atmosphere and oceans on Earth 3.5 billion years ago. Now, cyanobacteria species are widely spreaded in nature and different morphologies with unicellular and filamentous forms can be found among them (**Figure 1**). Cyanobacteria as oxygen-evolving photosynthetic prokaryotes are good candidates for being a suitable system for numerous biological applications. Cyanobacteria have special metabolic features of autotrophic carbon and nitrogen assimilation and energy supply via photosynthesis, which presents an increased potential for the next generation of sustainable bioproduction.

In the last years, the investigation of taxonomy, physiology, morphology, and genetics of cyanobacteria attracts a considerable attention due to their potential application in biotechnology [10–16] and biosensing [17–19]. Cyanobacterial hydrogen has been considered as a very promising source of alternative energy and has now been made commercially available. Cyanobacteria are also used in aquaculture, wastewater treatment, food, fertilizers, agriculture, and production of secondary metabolites including exopolysaccharides, vitamins, toxins, enzymes, and pharmaceuticals. In addition, the ecological aspect of the harmful bloom monitoring and control makes an important contribution to this rising interest to cyanobacterial problem. A vast amount of different techniques were elaborated to achieve a nowadays insight of the physiological processes that rules cyanobacterial life and their genetic background.

As far as cyanobacteria are ancient photosynthetic microorganisms, they have complex and effective light harvesting apparatus, which exhibit multifarious self-fluorescent properties. These intrinsic natural features are essential for fluorescent nondestructive analysis on a single-cell level and give a unique opportunity for steady-state and time-resolved investigations of various physiological processes in vivo.

In these studies, several cyanobacterial strains from CALU collection of the Core Facility Center "Centre for Culture Collection of Microorganisms" of the Science Park of St. Petersburg State University were utilized.

Before the investigations were carried out, the cyanobacterial cells were cultivated during 7 days in BG 11 medium (at 28°C) under continuous white light irradiation (fluorescent tubes, 40 μmol photons/m2 /s). The cyanobacterial cells for visualization were taken at stationary phase of growth. For CLSM imaging, the

### **Figure 1.**

*Confocal laser scanning photomicrographs illustrating the morphological features and biological diversity of freeliving and laboratory cyanobacterial strains: Synechocystis CALU 1336, Gomphosphaeria (wild type), Anabaena CALU 824, and Geitlerinema CALU 1718. Both fluorescence and transmission photomicrographs are presented. The white bar corresponds to 25 μm.*

**43**

**Figure 2.**

*Confocal Laser Scanning Microscopy for Spectroscopic Studies of Living Photosynthetic Cells*

living cells were placed onto a glass slide and were allowed to settle. A glass coverslip was placed on top and sealed with nail polish. Samples were immediately imaged. All studies were performed with living cells at room temperature. Note here, that any centrifugation and/or resuspending of cyanobacterial cultures before microscopic measurements change considerably the physiological state of cells under

Photosynthetic system of cyanobacteria, in contrast to higher plants, contains the external membrane light-harvesting complexes. Their antenna complex for photosystem II (PS II), and to some extent for photosystem I (PS I), is extrinsic and formed as a large multiprotein organelles, which are located on the stromal side of the thylakoid membranes. These supramolecular pigment-protein complexes are called phycobilisomes (PBSs). The detailed description of the morphology, structure, chemical, and optical properties of light-harvesting complex of cyanobacteria, phycobilisomes, and detached phycobilins can be found in numerous publications [8, 20–34]. Here, we only pointed out several main features that were essential for further discussion.

The main accessory pigments in cyanobacteria are phycobilins. The phycobilins which are bounded to proteins are known as phycobiliproteins. The three classes of phycobiliproteins in antenna complexes are allophycocyanin, phycocyanin, and phycoerythrin. However, in some cyanobacteria, phycoerythrin can be replaced by phycoerythrocyanin or both pigments can be lacking; phycocyanin and allophyco-

PBSs are assembled from 12 to 18 different types of polypeptides which may be grouped into three classes: (1) phycobiliproteins, (2) linker polypeptides, and (3) PBS-associated proteins. Phycobiliproteins, a colored family of water-soluble proteins bearing covalently attached, open-chain tetrapyrroles known as phycobilins.

On the other hand, most of linker polypeptides do not bear chromophores. Phycobilisomes are constructed from two main structural elements: a core substructure and peripheral rods that are arranged in a hemidiscoidal fashion around that core (**Figure 2**). The core of most hemidiscoidal phycobilisomes is composed of three (or two) cylindrical subassemblies, which are arranged side-by-side and form a triangle stack. Each core cylinder is made up of four disc-shaped phycobiliprotein trimmers, allophycocyanin (APC), allophycocyanin B (APC-B), and APC coremembrane linker complex (APC-LCM). By the core-membrane linkers, PBSs are attached on thylakoids and structurally coupled with PSII. The peripheral cylindrical rods (six or eight) radiate from the lateral surfaces of the core substructure and are usually not in contact with the thylakoid membrane. The rods are made up of hexamers, disc-shaped phycobiliproteins, phycoerythrin (PE), phycoerythrocyanin (PEC) and phycocyanin (PC), and corresponding rod linker polypeptides [8, 24–26, 32–35].

*Schematic drawing of a phycobilisome and the photosynthetic energy transfer to the reaction center.*

*DOI: http://dx.doi.org/10.5772/intechopen.84825*

consideration, and thus should be eliminated.

cyanin are constitutively present in all cyanobacteria.

**2.2 Light-harvesting system**
