**1. Introduction**

Cyanobacteria have gained huge attention in recent years because of their potential application in biotechnology [1–5]. For example, cyanobacteria are considered as a rich source of biologically active compounds with antiviral, antibacterial, antifungal and anticancer activities. Several strains of cyanobacteria were found to accumulate polyhydroxyalkanoates, which can be used as a substitute for nonbiodegradable petrochemical-based plastics. Recent studies showed that oil-polluted sites are rich in cyanobacterial consortia capable of degrading

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

oil components. 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, 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 in this rising interest to cyanobacterial problem.

community composition, therefore, requires frequent, high-resolution sampling. Historically, community characterization has been done by chemical preservation of samples and analysis by bright-field or epifluorescence microscopy. Although optical microscopy allows direct measurements of cell size and identification to species level it is laborious and time-consuming, limiting the number of samples that can be analyzed in a day. Minor variations in the composition of phytoplankton are consequently not revealed when using optical microscopy technique. More recently, *in situ* flow cytometric instruments capable of automated characterization of phytoplankton communities have been developed [10, 11]. These instruments have excellent resolution over a wide range of cell sizes but have a great disadvantage of high requirements for sample preparation and no possibility of cell-viability control. Alternative methods that are based on differences in accessory pigments among phytoplankton taxonomic groups [12] such as chemotaxonomic and spectrofluorometric methods have been proposed and included. First requires the high pressure liquid chromatography (HPLC) analysis of pigment contents [13–15]. The introduction of pigment analyses by HPLC facilitated easy and accurate separation, identification, and quantification of phytoplankton pigments. The large number of samples that can be processed by HPLC allows a more thorough examination. However, it does not allow for high-resolution data acquisition and again give no information about physiological state of single cells. And the last, spectrofluorometric method, although enables low-cost, rapid measurements, but till now deals with fluorescence-based chlorophyll *a* (*Chla*) quantification methods, that were proposed by several authors in the early 1960s and were applied either *in vitro* or *in vivo* to continuous measurements of algae and higher plants [16–22]. Unfortunately, these methods cannot be directly applied to cyanobacteria and usually give incorrect results. Recently, attempts to conduct the discrimination among microalga on the base of absorption or fluorescence spectra were reported [19, 21, 23–26]. Most of them use only absorption spectra. Absorption spectrum includes the information only about the chemical structure of photosynthetic cells, so it results in a rough discrimination of big classes of phytoplankton: diatoms, dinoflagellates, prymnesiophytes, euglenophytes, prasinophytes, raphidophytes, cryptophytes, chlorophytes, chrysophytes, and cyanobacteria. However, among the species of one class, for example, cyanobacteria, the chemical characteristics are quite similar, except several cases when phycoerythrin occurs in phycobilisome in addition to phycocyanin as an accessory pigment. In the last case, the only differentiation can be made among two big groups: the species containing phycoerythrin and those who lack it. All other differences are so small that cannot be used for further differentiation of cyanobacterial species and strains, so more precise classification, that is, among cyanobacterial species and strains, is impossible using only the absorption spectra. Opposite to the absorption spectra, the *in-vivo* fluorescence spectra are much more informative. Fluorescence detection is undoubtedly 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 more

Fluorescence Microscopic Spectroscopy for Investigation and Monitoring of Biological Diversity…

http://dx.doi.org/10.5772/intechopen.78044

13

detailed information for the study of single-cell composition.

In the last few years, the investigation of taxonomy, physiology, morphology and genetics of cyanobacteria attracts a considerable attention. A vast amount of different techniques were elaborated to achieve, nowadays, an insight into the physiological processes that rules cyanobacterial life and their genetic background. Large-scale industrial production of the cyanobacterial products requires optimization and more detailed control of incubation conditions in order to increase productivity. Future research will be focus on isolating and study of new cyanobacterial strains and the improvement of different treatments that will support or inhibit their growth.

In this chapter, the novel and most powerful part of the optical spectroscopy, which could make a considerable contribution to the future investigations—a confocal microscopic spectroscopy—will be presented and illustrated by several examples of application. It should be noted, that due to space limitations, only few citations could be incorporated in this chapter. They represent a limited selection from a large amount of works and should be used as a source for further references.

As it was pointed out earlier, there are two opposite aspects in the cyanobacterial problem: first, to prevent ecological hazards, that is, toxic cyanobacterial blooms, and, second, to improve industrial incubation of cyanobacteria, involved in such important applications, as food and fuel production. The former deals with the study and treatment of natural samples and includes the investigation of biological diversity, monitoring of physiological state of natural communities, record and analyze the results of external actions and changings in environmental conditions. In many situations, taxonomic phytoplankton composition is of crucial importance when toxic or other harmful substances might be produced by cyanobacteria. The latter aspect concerns the registration and control of the optimal physiological state and viability of the laboratory or industrial culture in specified conditions. Thus, there are two main problems that are involved in all areas of application mentioned above and meet some obstacles while using conventional methods of investigation. They are: a correct classification and discrimination of present and new cyanobacterial strains, and monitoring of physiological state of cyanobacterial cells in natural communities and in laboratory cultures during industrial incubation.

All these different tasks deal with a big data processing and possibility of process automation are quite desirable. Confocal microscopic spectroscopy gives a unique opportunity for direct automation of all these processes or, otherwise, indirect application of the results of the detailed single-cell spectroscopic investigations for implementation in innovative devices or technique.

The taxonomic composition of cyanobacterial communities is of interest in water-quality field and ecology, where the effect of nutrient pollution on coastal and freshwater resources should be controlled [6, 7], as well as in industrial biomass production, where the additional undesirable strains may appear during the long cultivation process. In nature, the composition of phytoplankton communities can be highly variable in space and time [8, 9]. Characterization of the community composition, therefore, requires frequent, high-resolution sampling. Historically, community characterization has been done by chemical preservation of samples and analysis by bright-field or epifluorescence microscopy. Although optical microscopy allows direct measurements of cell size and identification to species level it is laborious and time-consuming, limiting the number of samples that can be analyzed in a day. Minor variations in the composition of phytoplankton are consequently not revealed when using optical microscopy technique. More recently, *in situ* flow cytometric instruments capable of automated characterization of phytoplankton communities have been developed [10, 11]. These instruments have excellent resolution over a wide range of cell sizes but have a great disadvantage of high requirements for sample preparation and no possibility of cell-viability control. Alternative methods that are based on differences in accessory pigments among phytoplankton taxonomic groups [12] such as chemotaxonomic and spectrofluorometric methods have been proposed and included. First requires the high pressure liquid chromatography (HPLC) analysis of pigment contents [13–15]. The introduction of pigment analyses by HPLC facilitated easy and accurate separation, identification, and quantification of phytoplankton pigments. The large number of samples that can be processed by HPLC allows a more thorough examination. However, it does not allow for high-resolution data acquisition and again give no information about physiological state of single cells. And the last, spectrofluorometric method, although enables low-cost, rapid measurements, but till now deals with fluorescence-based chlorophyll *a* (*Chla*) quantification methods, that were proposed by several authors in the early 1960s and were applied either *in vitro* or *in vivo* to continuous measurements of algae and higher plants [16–22]. Unfortunately, these methods cannot be directly applied to cyanobacteria and usually give incorrect results.

oil components. 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, production of secondary metabolites including exopolysaccharides, vitamins, toxins, enzymes and pharmaceuticals. In addition, the ecological aspect of the harmful bloom monitoring and control

In the last few years, the investigation of taxonomy, physiology, morphology and genetics of cyanobacteria attracts a considerable attention. A vast amount of different techniques were elaborated to achieve, nowadays, an insight into the physiological processes that rules cyanobacterial life and their genetic background. Large-scale industrial production of the cyanobacterial products requires optimization and more detailed control of incubation conditions in order to increase productivity. Future research will be focus on isolating and study of new cyanobacterial strains and the improvement of different treatments that will support or

In this chapter, the novel and most powerful part of the optical spectroscopy, which could make a considerable contribution to the future investigations—a confocal microscopic spectroscopy—will be presented and illustrated by several examples of application. It should be noted, that due to space limitations, only few citations could be incorporated in this chapter. They represent a limited selection from a large amount of works and should be used as a

As it was pointed out earlier, there are two opposite aspects in the cyanobacterial problem: first, to prevent ecological hazards, that is, toxic cyanobacterial blooms, and, second, to improve industrial incubation of cyanobacteria, involved in such important applications, as food and fuel production. The former deals with the study and treatment of natural samples and includes the investigation of biological diversity, monitoring of physiological state of natural communities, record and analyze the results of external actions and changings in environmental conditions. In many situations, taxonomic phytoplankton composition is of crucial importance when toxic or other harmful substances might be produced by cyanobacteria. The latter aspect concerns the registration and control of the optimal physiological state and viability of the laboratory or industrial culture in specified conditions. Thus, there are two main problems that are involved in all areas of application mentioned above and meet some obstacles while using conventional methods of investigation. They are: a correct classification and discrimination of present and new cyanobacterial strains, and monitoring of physiological state of cyanobacterial cells in

All these different tasks deal with a big data processing and possibility of process automation are quite desirable. Confocal microscopic spectroscopy gives a unique opportunity for direct automation of all these processes or, otherwise, indirect application of the results of the detailed single-cell spectroscopic investigations for implementation in innovative devices or technique.

The taxonomic composition of cyanobacterial communities is of interest in water-quality field and ecology, where the effect of nutrient pollution on coastal and freshwater resources should be controlled [6, 7], as well as in industrial biomass production, where the additional undesirable strains may appear during the long cultivation process. In nature, the composition of phytoplankton communities can be highly variable in space and time [8, 9]. Characterization of the

natural communities and in laboratory cultures during industrial incubation.

makes an important contribution in this rising interest to cyanobacterial problem.

inhibit their growth.

12 Cyanobacteria

source for further references.

Recently, attempts to conduct the discrimination among microalga on the base of absorption or fluorescence spectra were reported [19, 21, 23–26]. Most of them use only absorption spectra. Absorption spectrum includes the information only about the chemical structure of photosynthetic cells, so it results in a rough discrimination of big classes of phytoplankton: diatoms, dinoflagellates, prymnesiophytes, euglenophytes, prasinophytes, raphidophytes, cryptophytes, chlorophytes, chrysophytes, and cyanobacteria. However, among the species of one class, for example, cyanobacteria, the chemical characteristics are quite similar, except several cases when phycoerythrin occurs in phycobilisome in addition to phycocyanin as an accessory pigment. In the last case, the only differentiation can be made among two big groups: the species containing phycoerythrin and those who lack it. All other differences are so small that cannot be used for further differentiation of cyanobacterial species and strains, so more precise classification, that is, among cyanobacterial species and strains, is impossible using only the absorption spectra.

Opposite to the absorption spectra, the *in-vivo* fluorescence spectra are much more informative. Fluorescence detection is undoubtedly 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 more detailed information for the study of single-cell composition.

Fluorescence spectra have been used to classify phytoplankton populations since the approximately early 1970s [20]. However, because of the generally low number of available excitation wavelengths in the conventional devices the rate of species discrimination was relatively low. Researches again can separate only algal groups that differ greatly in pigmentation and, therefore, in fluorescence spectra (e.g., cryptophytes, chlorophytes and cyanobacteria), but cannot separate groups that are more similarly pigmented (e.g., among cyanobacterial species) [22, 27]. Discrimination between similarly pigmented taxa or even between species within a taxon requires high-resolution spectra and the use of a set of excitation wavelengths to reveal small peculiarities in configuration and functioning of light harvesting system. The rigorous discrimination is possible if the inter-species differences are greater than those within a species. These requirements can be fulfilled only when for each species a set of single-cell fluorescence spectra, excited by a number of wavelengths, are obtained and analyzed. The possible contribution of environmental adaptation effects to the resulting fluorescence spectra can be minimized by an accurate definition of the corresponding spectral regions in the spectra under consideration.

that are photosynthetically active, that is, alive. Thus, additional signals from dead cell debris do not interfere with the measurements. Long-term DF emission also prevents interference problems with fluorescent backgrounds in natural samples [38]. Furthermore, DF can measure nano- and pico-plankton, which may be lost during filtration or may be unaccounted in direct microscopic analysis. However, by means of DF, only the ensemble spectra of the whole culture can be measured and the physiological state of individual cells is unavailable as before. Thus, a new precise, nondestructive and sensitive method for registration of weak reversible and irreversible changes in the physiological state of cyanobacterial cells should

Fluorescence Microscopic Spectroscopy for Investigation and Monitoring of Biological Diversity…

http://dx.doi.org/10.5772/intechopen.78044

15

Historically, since the 1960s, investigators have noted that changes in the physiological state of cyanobacterial cells occurring when it is damaged are reflected in the corresponding changes in the intrinsic fluorescence spectrum [39]. Several studies have shown that a decrease in the pigment fluorescence is associated with a decrease in the enzymatic activity of the cell and an increase in the permeability of the cell membrane, which can be used as an indicator of aging for cyanobacterial species [31]. However, this fact has not yet been widely used to assess the

To date, 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 only 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; 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, as well as for preliminary taxonomic analysis of full-scale samples [40, 41]. These "fluorescent fingerprints" can be easily obtained by the

We present a novel technique based on a strict relation of the physiological state of cyanobacterial cells and their genera affiliation with the intensity and shape of the intrinsic single-cell fluorescence spectra, obtained by means of confocal microscopic spectroscopy. The nondestructive spectral analysis conducting *in vivo* at a 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. The application of this technique for automation of control processes gives an additional opportunity to rise an effectiveness of production in biotechnology and brings in a valuable contribution to the development of

In this chapter, two main application problems are investigated by means of fluorescent microscopic spectroscopy. Firstly, how the composition of photosynthetic pigments affects the shape of *in-vivo* single-cell fluorescence spectra and, secondly, how the differences in the fluorescence response of cyanobacterial cells may be used for investigation of their physiological state and

viability of individual cells of cyanobacteria and cultures.

routine lambda-scanning at most of confocal laser scanning microscopes.

innovative approaches in environmental monitoring.

biological diversity.

be elaborated.

The problem of registration and control of the physiological state of single cells in natural communities and the viability of cultures during incubation is a primary task in both ecological and biotechnological fields of application of cyanobacteria. This problem is more complex than the species discrimination because it deals with very weak variations in chemical and optical characteristics of single cells and a culture as a whole. Despite the importance of this problem in vast amount of tasks, the methods for studying and monitoring of physiological state of cyanobacteria are still based mainly on traditional optical methods of registration, as well as on the analysis of fixed or dissociated samples [28–31]. These approaches do not allow one to register small changes in the physiological state of cyanobacterial cells, which are extremely important during weak external treatments or environmental changes. Usually, this study is reduced to a manual counting of the total number of cells in the experimental samples and determining the total volume of chlorophyll [32–34].

The last method is very effective for algae and higher plants, where chlorophyll is a main pigment, determining the viability. It quickly disintegrates in dead cells and therefore can really serve as an indicator of the viability of single plant. In cyanobacteria, the main pigments responsible for photosynthetic activity are phycobilins, and chlorophyll does not disintegrate for a long time in dead cells, even after their disruption. Therefore, the methods associated with the analysis of the chlorophyll fraction do not give satisfactory results in the study of cyanobacterial cultures. The analysis of the light fraction of water-soluble phycobilins may give some additional information, but it is also ambiguous, since the pigmentprotein complexes in the disrupted cells degrade rapidly and carry no information about the initial viability of living cells. With such approach, weak changes in the physiological state of cyanobacterial cells cannot be detected, since the results of the experimental treatment are more influenced by the used methods of investigation than the directional external action, which is studied.

The only method that seems to be appropriate for physiological state investigation and can diverse live and dead cells is the delayed fluorescence (DF) technique. Delayed fluorescence is the long-term emission of light from cells triggered by illumination [27, 35, 36]. It has the same emission spectrum as chlorophyll *a* fluorescence, but occurs with a time delay (from milliseconds to minutes) [37]. The major advantage of DF is that it is emitted only from cells that are photosynthetically active, that is, alive. Thus, additional signals from dead cell debris do not interfere with the measurements. Long-term DF emission also prevents interference problems with fluorescent backgrounds in natural samples [38]. Furthermore, DF can measure nano- and pico-plankton, which may be lost during filtration or may be unaccounted in direct microscopic analysis. However, by means of DF, only the ensemble spectra of the whole culture can be measured and the physiological state of individual cells is unavailable as before. Thus, a new precise, nondestructive and sensitive method for registration of weak reversible and irreversible changes in the physiological state of cyanobacterial cells should be elaborated.

Fluorescence spectra have been used to classify phytoplankton populations since the approximately early 1970s [20]. However, because of the generally low number of available excitation wavelengths in the conventional devices the rate of species discrimination was relatively low. Researches again can separate only algal groups that differ greatly in pigmentation and, therefore, in fluorescence spectra (e.g., cryptophytes, chlorophytes and cyanobacteria), but cannot separate groups that are more similarly pigmented (e.g., among cyanobacterial species) [22, 27]. Discrimination between similarly pigmented taxa or even between species within a taxon requires high-resolution spectra and the use of a set of excitation wavelengths to reveal small peculiarities in configuration and functioning of light harvesting system. The rigorous discrimination is possible if the inter-species differences are greater than those within a species. These requirements can be fulfilled only when for each species a set of single-cell fluorescence spectra, excited by a number of wavelengths, are obtained and analyzed. The possible contribution of environmental adaptation effects to the resulting fluorescence spectra can be minimized by an accurate defini-

The problem of registration and control of the physiological state of single cells in natural communities and the viability of cultures during incubation is a primary task in both ecological and biotechnological fields of application of cyanobacteria. This problem is more complex than the species discrimination because it deals with very weak variations in chemical and optical characteristics of single cells and a culture as a whole. Despite the importance of this problem in vast amount of tasks, the methods for studying and monitoring of physiological state of cyanobacteria are still based mainly on traditional optical methods of registration, as well as on the analysis of fixed or dissociated samples [28–31]. These approaches do not allow one to register small changes in the physiological state of cyanobacterial cells, which are extremely important during weak external treatments or environmental changes. Usually, this study is reduced to a manual counting of the total number of cells in the experimental

The last method is very effective for algae and higher plants, where chlorophyll is a main pigment, determining the viability. It quickly disintegrates in dead cells and therefore can really serve as an indicator of the viability of single plant. In cyanobacteria, the main pigments responsible for photosynthetic activity are phycobilins, and chlorophyll does not disintegrate for a long time in dead cells, even after their disruption. Therefore, the methods associated with the analysis of the chlorophyll fraction do not give satisfactory results in the study of cyanobacterial cultures. The analysis of the light fraction of water-soluble phycobilins may give some additional information, but it is also ambiguous, since the pigmentprotein complexes in the disrupted cells degrade rapidly and carry no information about the initial viability of living cells. With such approach, weak changes in the physiological state of cyanobacterial cells cannot be detected, since the results of the experimental treatment are more influenced by the used methods of investigation than the directional external

The only method that seems to be appropriate for physiological state investigation and can diverse live and dead cells is the delayed fluorescence (DF) technique. Delayed fluorescence is the long-term emission of light from cells triggered by illumination [27, 35, 36]. It has the same emission spectrum as chlorophyll *a* fluorescence, but occurs with a time delay (from milliseconds to minutes) [37]. The major advantage of DF is that it is emitted only from cells

tion of the corresponding spectral regions in the spectra under consideration.

samples and determining the total volume of chlorophyll [32–34].

action, which is studied.

14 Cyanobacteria

Historically, since the 1960s, investigators have noted that changes in the physiological state of cyanobacterial cells occurring when it is damaged are reflected in the corresponding changes in the intrinsic fluorescence spectrum [39]. Several studies have shown that a decrease in the pigment fluorescence is associated with a decrease in the enzymatic activity of the cell and an increase in the permeability of the cell membrane, which can be used as an indicator of aging for cyanobacterial species [31]. However, this fact has not yet been widely used to assess the viability of individual cells of cyanobacteria and cultures.

To date, 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 only 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; 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, as well as for preliminary taxonomic analysis of full-scale samples [40, 41]. These "fluorescent fingerprints" can be easily obtained by the routine lambda-scanning at most of confocal laser scanning microscopes.

We present a novel technique based on a strict relation of the physiological state of cyanobacterial cells and their genera affiliation with the intensity and shape of the intrinsic single-cell fluorescence spectra, obtained by means of confocal microscopic spectroscopy. The nondestructive spectral analysis conducting *in vivo* at a 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. The application of this technique for automation of control processes gives an additional opportunity to rise an effectiveness of production in biotechnology and brings in a valuable contribution to the development of innovative approaches in environmental monitoring.

In this chapter, two main application problems are investigated by means of fluorescent microscopic spectroscopy. Firstly, how the composition of photosynthetic pigments affects the shape of *in-vivo* single-cell fluorescence spectra and, secondly, how the differences in the fluorescence response of cyanobacterial cells may be used for investigation of their physiological state and biological diversity.
