**1. Introduction**

Confocal laser scanning microscopes (CLSMs) are distinguished by their high spatial and temporal resolution [1, 2]. Modern laser scanning microscopes are unique tools for visualizing cellular structures and analyzing dynamic processes inside single cells. They exceed classical light microscopes especially in their axial resolution, which enables to acquire optical sections (slices) of a specimen. An object can thus be imaged completely in three dimensions and subsequently visualized as a 3D computer image. Apart from simple imaging, confocal laser scanning microscopes are designed for the quantification and analysis of image-coded

information. Among other things, they allow easy determination of fluorescence intensities, distances, areas, and their changes over time. In particular, they are capable of quickly detecting and quantitatively unmixing the spectral signatures of fluorescent objects. Many software functions analyze important parameters such as the degree of colocalization of labeled structures, or the ion concentration in a specimen. New acquisition CLSM tools include the detection of quantitative properties of the emitted light such as spectral signatures and fluorescence lifetimes. The most impressive feature of modern CLSMs is their capability for single-cell microscopic spectroscopy, which allows to obtain spectroscopic information inside single cells and small regions. Another group of applications is the quantitative investigations of dynamic processes in living cells using techniques such as fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET), photoactivation, and photoconversion.

The specific field of CLSM application is the investigation of self-fluorescence of living cells. The conventional biological fluorescent studies are based mostly on using fluorescent dyes and labels (chromophores). But it is well-known that the fluorescent dyes, even most flexible, affect living cells considerably. Thus self-fluorescence should become the most suitable tool for noninvasive investigation of changes in physiological state of living cells. For instance, the photosynthesis research employs the detection of self-fluorescence as a key method to study the metabolic mechanisms in photosynthetic cells and to detect photosynthetic efficiency in vivo. Recent rapid development of confocal microscopes functionality initiates new directions in subcellular biology research. However, the experiments with photosynthetic cells require some additional specific skills and techniques to perform measurements and to carry out data processing [3–7]. The efficiency of photosynthesis and photosynthetic rate are highly dependent on irradiance. This can be seen in the light-dependency of various photosynthetic parameters [8]. Moreover, not only light quantity, but also light "quality" (wavelength) is an important factor. Thus, special spectroscopic methods are required to study the physiology of phototrophic microorganisms [9]. These organisms employ light-dependent photosynthesis as the main energy source for their metabolism and the detected self-fluorescence finally reflects the diversity in morphological and physiological states of their photosynthetic cells [3–7].

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 confocal laser scanning microscopy. 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.

Recent progress in confocal laser scanning microscopy (CLSM) gives an opportunity to investigate different physiological processes in photosynthetic organisms on a single-cell level. Such CLSM applications as spectral unmixing and lambdascanning provide the recording of spectral characteristics from living cells. FRAP and FLIP applications allow to study dynamic processes such as cell membrane fluidity and phycobilisome diffusion along thylakoid membrane. The nondestructive spectroscopic analysis conducting in vivo at a sub-cellular level allows to obtain more complete information about special features of individual cyanobacterial cells and supports the registration of very weak variations in their physiological state. For example, a novel technique for discrimination of cyanobacterial species and physiological states of the cells belonging to one strain was elaborated by the

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**2.1 Model object**

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

authors of the present chapter [3–5]. The technique is based on a strict relation between physiological state and genera affiliation of cyanobacterial cells and the intensity and the shape of corresponding single-cell fluorescence spectra, obtained by means of confocal microscopic spectroscopy. Light-, heat-, ultrasound- and toxin-induced changes can be distinguished by means of confocal microscopic spectroscopy since all these external actions are stress factors affecting photosynthetic process [5]. The application of such techniques for automation of on-line monitoring will give an additional opportunity to rise an effectiveness of biotechnological production and will bring in a valuable contribution to the development

Here we present several experimental approaches to study the metabolic mechanisms in single photosynthetic cells in vivo. They are accompanied by several examples of in vivo investigations. Three main CLSM tools will be discussed in details: spectral imaging, fluorescent microscopic spectroscopy, and FRAP. All presented results were obtained using 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 as a model objects for CLSM studies.

Opposite to the absorption spectra, the in vivo fluorescence spectra are much more informative. Fluorescence detection is a powerful tool owing to the existence of natural fluorescence from phycobilins and chlorophylls. It is a highly sensitive, nearly instantaneous, noninvasive way to study various components and processes in situ and in vivo. Although the fluorescence spectra contain the information only about photosynthetic apparatus of different algal groups, they include the information about the chemical structure of light harvesting complex (LHC) and accessory pigment-proteins, as well as about the character of links between pigment-protein complexes and the efficiency of energy transfer in the light harvesting process. When compared with absorption, fluorescence is affected by the excitation wavelength and energy. Thus, the use of different excitation wavelengths can provide

Today, there is no doubt that the in vivo analysis of fluorescence parameters of light-harvesting complexes is a powerful tool for studying the effect of a wide variety of environmental factors on photosynthetic organisms. The intensity of fluorescence emitted by single photosynthetic cells in vivo depends on the structure and operational effectiveness of photosynthetic apparatus, reflecting the individual characteristic of cyanobacterial strain and in-time physiological state of the cells under consideration. The environmental changes cause the changes in bioenergetic processes occurring in cyanobacterial cells, and so they significantly affect the kinetics parameters and spectral features of the intrinsic fluorescence of photosynthetic apparatus. Thus, the intrinsic fluorescence spectra of a particular type of cyanobacteria, the so-called "fluorescent fingerprints", can be used to identify photosynthetic pigments and to determine the viability of individual cells. These "fluorescent fingerprints" can be easily obtained by the routine lambda-scanning at

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

more detailed information for the study of single-cell composition.

most of confocal laser scanning microscopes.

of innovative approaches in environmental monitoring [4, 5].

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

**2. Natural fluorescence**
