**1. Introduction**

Cyanobacteria are the most ancient photosynthetic microorganisms on Earth. Nowadays, cyanobacteria are one of the most widespread organisms in nature, and the ecological aspect in their investigation is quite valuable. On the other hand, thousands of strains belonging to different species are cultivated in biolaboratories all over the world for different cyanobacterial biotechnological applications such as biofuel cells, food production, pharmaceuticals, fertilizers, etc. [1–3]. Thus the noninvasive spectroscopic methods are quite requisite for monitoring of physiological state of cyanobacterial cultures and natural communities.

It is well-known that the analysis of self-fluorescence of photosynthetic system is a powerful noninvasive tool for investigation of microalgae in vivo. It reports on the energy transfer and trapping and, thus, reflects the metabolic mechanisms in photosynthetic cells and their photosynthetic efficiency. The detected selffluorescence finally reflects the diversity in morphological and physiological states of photosynthetic cells [4–6].

Self-fluorescence originates from excited states that were lost before photochemistry took place and usually represents a small fraction of the excited state decay in a functional photosynthetic complex. Nevertheless, this small fraction can be easily detected by confocal laser scanning microscopy (CLSM). With the confocal fluorescence microscopy, a very small excitation and detection areas can be investigated, so that single cells under non-damage conditions can be studied in vivo. Single-cell detection can provide the information on small peculiarities that is regularly buried in normal ensemble average experiments. This is thus a good way to study the time evolution process and spectroscopic properties of individual cells. Both steady-state and time-resolved fluorescence measurements can be used for probing the organization and functioning of photosynthetic systems by means of CLSM.

Till now the best taxonomic differentiation is still obtained using classical inverted microscopy. Unfortunately, this method is time-consuming, human based, and requires appropriate technical skills; this eliminates the possibility of its application for continuous online monitoring. Nearly single-cell flow cytometric analysis, based on light scattering by the cells and fluorescence of the chlorophylls and the phycobilins, can be easily automated, but it is appropriate only for unicellular species and is useless for numerous industrially cultured filamentous strains [7, 8]. The main problem of all chemical methods (e.g., high-performance liquid chromatography (HPLC) [9, 10]) is that during the chemical sample preparation, the most of the information about the peculiarities of individual species is lost and the residual part of the information is not enough for species/strain discrimination inside cyanobacterial genera and is suitable only for the rude differentiation of big classes of phytoplankton. Thus, the analysis of the in vivo fluorescence spectra is the only one noninvasive technique for obtaining qualitative information about the phytoplankton abundance and composition, which is continuously demonstrated by various publications [10–17]. The relative phytoplankton abundance can be calculated once initial assumptions about the phytoplankton classes are presented and their pigment compositions have been made [7, 12, 13].

Maybe the first attempt to use phycoerythrins as chemotaxonomic markers was done by Glazer et al. [18] for red algae in 1982, but until now fluorescence spectra of phycobilins do not appear to be useful at familial, ordinal, and class levels in taxonomic studies. Although the investigation in [18] concerns only purified highmolecular-weight phycoerythrin from red algae, this work clearly demonstrates the possibility of the correct taxonomic analysis on the basis of phycobiliproteins structural differences, which can serve as intrinsical fingerprints for taxons and genera in phytoplankton diversity. Later, the correlation between the distribution of the biliproteins and the genera of Cryptophyceae was discussed in [19]. In 1985, Yentsch and Phinney [20] proposed an ataxonomic technique that utilized the spectral fluorescence signatures of major ocean phytoplankton. Seppälä [16] used spectral fluorescence signals to detect changes in the phytoplankton community. In 2002, Beutler et al. reported a reduced model of the fluorescence from the cyanobacterial photosynthetic apparatus designed for the in situ detection of cyanobacteria and presented a commercially available diveable instrument for online monitoring of phytoplankton structure [11].

However, the correct classification of cyanobacterial species on the basis of their bulk fluorescence signature is hampered by alterations in pigment composition within one strain, which depends on the physiological state of the culture (community) and environmental conditions [21]. On the other hand, several researchers show that the nutrient and light limitations do not significantly change the initial fluorescence spectra and cannot impede the species discrimination [17, 22].

**5**

*Self-Fluorescence of Photosynthetic System: A Powerful Tool for Investigation of Microalgal…*

microscopes are distinguished by their high spatial and temporal resolution. Modern laser scanning microscopes are unique tools for visualizing cellular structures and analyzing dynamic processes inside single cells. One of the specific fields of CLSM application is the investigation of self-fluorescence of living cells. CLSM single-cell microscopic spectroscopy is undoubtedly the most powerful tool for in vivo investigation of physiological processes in photosynthetic organisms (cyanobacteria, algae, and higher plants). The investigation of self-fluorescence of single living cells reveals the relation between the physiological state and the operational activity of photosynthetic system. A lot of interesting static and dynamic effects can be studied by means of CLSM. The investigation of self-fluorescence gives the information about single-cell processes as well as about the collaboration in cell communities. Changes in spectral characteristics of living photosynthetic cells indicate changes in their physiological state and can be applied for the studies of the results of stress states and external actions [4–6]. Moreover, the diversity in single-cell self-fluorescence for different species and strains can serve the basis for

In this chapter, a novel ataxonomic approach to differentiation of cyanobacterial cells based on the numerical analysis of in vivo single-cell fluorescence spectra, recorded by means of CLSM, is presented. The differentiation is conducted according to the structure and operation of their photosynthetic apparatus. An optimal set of the parameters is selected, which is sufficient for determination of the taxonomic position of cyanobacteria by means of mathematical statistics. On the basis of the linear discriminant analysis, the obtained spectroscopic data for 23 cyanobacterial strains from CALU collection were analyzed. It was shown that the presented technique allows an accurate differentiation of cyanobacteria up to the species/strain level and enables to distinguish automatically potentially harmful strains. All presented results were obtained using cyanobacterial strains from CALU collection of the Core Facility Center "Centre for Culture Collection of Microorganisms" of the Research Park of St. Petersburg State University as model

All work on preparing cyanobacteria cultures for this research was carried out at the Core Facility Center "Centre for Culture Collection of Microorganisms" of the Research Park of St. Petersburg State University. In the CALU collection [25] at the core facility center, cyanobacterial strains were maintained in semiliquid agar (0.8%) medium no. 6 after Gromov [26] in test tubes of volume 5–6 mL under cotton plugs. The strains were stored at 14°C under a constant illumination of 2000 lux

Cyanobacteria used in this investigation were grown on liquid medium no. 6. A stock culture was preliminarily prepared, for which it was cultivated in 30 mL of medium and incubated for 2 weeks at room temperature under continuous illumination from fluorescent lamps. To maintain a constant volume, 5 mL of medium were added to the stock culture every 2 weeks. All experiments in this study were conducted with cultures presumably in the logarithmic phase of their growth. In this work, 23 cyanobacterial strains from CALU collection were used:

1.*Anabaena variabilis* Kutz. CALU 824, ponds near Old Petergof, Saint

*DOI: http://dx.doi.org/10.5772/intechopen.88785*

ataxonomic discrimination of cyanobacterial genera.

**2.1 Cyanobacterial strains and cultivation conditions**

and were recultivated with a periodicity of 2–3 months.

objects for CLSM studies.

**2. Materials and methods**

Petersburg, Russia.

Recent rapid development of confocal microscope functionality initiates new directions in subcellular biology research [23, 24]. Confocal laser scanning

#### *Self-Fluorescence of Photosynthetic System: A Powerful Tool for Investigation of Microalgal… DOI: http://dx.doi.org/10.5772/intechopen.88785*

microscopes are distinguished by their high spatial and temporal resolution. Modern laser scanning microscopes are unique tools for visualizing cellular structures and analyzing dynamic processes inside single cells. One of the specific fields of CLSM application is the investigation of self-fluorescence of living cells. CLSM single-cell microscopic spectroscopy is undoubtedly the most powerful tool for in vivo investigation of physiological processes in photosynthetic organisms (cyanobacteria, algae, and higher plants). The investigation of self-fluorescence of single living cells reveals the relation between the physiological state and the operational activity of photosynthetic system. A lot of interesting static and dynamic effects can be studied by means of CLSM. The investigation of self-fluorescence gives the information about single-cell processes as well as about the collaboration in cell communities. Changes in spectral characteristics of living photosynthetic cells indicate changes in their physiological state and can be applied for the studies of the results of stress states and external actions [4–6]. Moreover, the diversity in single-cell self-fluorescence for different species and strains can serve the basis for ataxonomic discrimination of cyanobacterial genera.

In this chapter, a novel ataxonomic approach to differentiation of cyanobacterial cells based on the numerical analysis of in vivo single-cell fluorescence spectra, recorded by means of CLSM, is presented. The differentiation is conducted according to the structure and operation of their photosynthetic apparatus. An optimal set of the parameters is selected, which is sufficient for determination of the taxonomic position of cyanobacteria by means of mathematical statistics. On the basis of the linear discriminant analysis, the obtained spectroscopic data for 23 cyanobacterial strains from CALU collection were analyzed. It was shown that the presented technique allows an accurate differentiation of cyanobacteria up to the species/strain level and enables to distinguish automatically potentially harmful strains. All presented results were obtained using cyanobacterial strains from CALU collection of the Core Facility Center "Centre for Culture Collection of Microorganisms" of the Research Park of St. Petersburg State University as model objects for CLSM studies.
