**3.1. Monitoring of physiological state of single cyanobacterial cell and a whole culture**

investigation are shown. Analyzing the shape of the ensemble average fluorescence spectrum and counting the relative number of alive and semi-dead cells the conclusion about the viabil-

Special attention should be paid to the sample preparation, as well as we work with alive objects. Coverslip should be pressed very carefully to prevent any glass slide, which can cause cell damage. On the other hand, one should keep in mind that cyanobacteria can move and glide; so to fix the object, the coverslip should be pressed hard enough to prevent any motility of the investigated object, which sometimes have a diameter near 1 μm (e.g., microcystis and

There is another very powerful tool implemented in CLSM—spectral unmixing. Unfortunately, in living cyanobacterial cells, it meets some difficulties. The authors of [43] pointed out that a lot of problems arise during the spectral unmixing procedure, which is based on the spectra of isolated phycobiliproteins. These problems are caused by the fact that the light absorption and emission properties of isolated phycobiliproteins are rather different from those of the intact phycobilisomes in the living cyanobacterial cells. In living cells, the spectral properties of pigments from certain organisms may differ crucially from the properties of the dissolved ones, for example, spectra of the components can vary in peak widths and may be shifted in wavelength due to different pigment-protein and linker connections. Thus, the analysis based on the initial fluorescence spectrum without any decomposition is preferable for living cells.

**3. Examples of fluorescent microscopic spectroscopy application**

The detailed description of the morphology, structure, chemical and optical properties of light-harvesting complex of cyanobacteria, phycobilisomes and phycobilins can be found in numerous publications [55–72]. On the other hand, the fluorescence properties of the intact living cyanobacterial cells differ drastically from the properties of the detached phycobilisomes and its components and originate from the efficiency of the energy transfer between all components of the energy transfer chain included the final step, the delivery to PSII or PSI. Each transfer step result in the spectrum shape as a peak or shoulder. Moreover, fluorescence of photosynthetic pigments in the intact cells is affected by physicochemical and physiological processes that occur within and across the thylakoid membranes. Here we demonstrate on several examples how these peculiarities can be used for investigation of physiological state

The correct identification of cyanobacterial cultures and estimation of their physiological state are quite important in the environmental monitoring and industrial applications. The ability to detect small variations in the physiological state of cyanobacterial culture under weak external treatments is quite desirable in both field and laboratory experiments. The fluorescent CLSM technique is a very powerful tool that can support any on-line field, laboratory

ity of the whole culture at given developmental stage could be made.

**2.5. Sample preparation**

20 Cyanobacteria

synechocystis cells).

**2.6. Spectral unmixing**

and biological diversity of cyanobacteria.

Since the first broad-scale spectroscopic investigations, the authors of many articles note the dependence of the intrinsic fluorescence spectra of cyanobacteria on the developmental stage of the culture and physiological state of single cells. It is well-known that the light-harvesting and energy-transfer capacities of phycobilisomes can react to the environmental changes, as well as to the changes in physiological state of the living cells induced by stress conditions [39, 64, 73–75]. However, this effect has not yet been widely used to assess the viability of the culture. Several authors pointed out that, although a single-cell fluorescence spectra for the diverse physiological states differ significantly, the physiological state of the given cell cannot be estimated correctly because of the absence of a full set of reference spectra [40, 76, 77]. Moreover, the authors of [40, 76, 77] pointed out that while comparing spectra of individual cells and the results of the ensemble average experiments at a culture as a whole (so-called integral spectra), a significant difference was observed. Obviously, this difference is owing to a wide diversity of single-cell physiological states in the bulk growing culture, which in sum gives different integral fluorescence spectra for a specified strain at different developmental stages because of the variations in cell's proportions. This, of course, should be taken into account. On the other hand, the whole culture in addition to a set of single living cyanobacterial cells consists of metabolites, dissolved pigments, other organic substances and cellular debris. All these substances form undesirable and unpredictable fluorescent background in volume samples.

Actually, the intensity of fluorescence emitted by single photosynthetic cell *in vivo* depends only on the structure and operational effectiveness of photosynthetic apparatus, tracing intime physiological state of the cyanobacterial cell. Thus, the fluorescence emission can be used effectively to monitor various physiological processes. **Figure 3** illustrates the temporal changes of in-vivo fluorescence spectrum taking place in one living cell of cyanobacteria strain *Synechocystis CALU 1336* under light and heat stress. On the other hand, this timeline set of fluorescence emission spectra illustrates all stages of cyanobacterial cell degradation, that is, all possible physiological states. It is obvious that during the evolution of the culture and aging of each cell all this stages will be presented in the natural samples simultaneously.

Several newer publications [76–80] clearly demonstrate that the variations in the fluorescence shape and intensity of living cells, presented in **Figure 3**, indicate the consequent degradation in the light harvesting chain (antenna complex—reaction center) and following dissociation of the detached antenna complex. It can be seen that in the alive cell, the chlorophyll *a* fluorescence prevails over the fluorescence of the pigment-protein complexes of phycobilisome. While the single-cell physiological state changes for the worse, the photosynthetic apparatus shows instability in operation, that is, the most part of the absorbed energy emits as fluorescence at the early stages of light harvesting. At the last stages, the changes in fluorescent spectrum, shown in **Figure 3**, are the same as it was demonstrated in the works [80–82], where

this given strain. For accuracy of further estimation of the viability of the whole culture, these spectra should be recorded at several excitation wavelengths for each cell (in our investigations it was set of eight laser lines) and then each one should be averaged through a number of cells. The fluorescence spectra of a single cell excited by one laser line is not enough to have a complete information about deviations in photosynthetic process and pigment composition. **Step 3.** Direct study of the tested sample in order to determine the viability of investigated culture or colony. A representative random sampling is used for the cells from a given strain and single-cell fluorescence spectra excited by chosen excitation wavelengths are recorded. At

Fluorescence Microscopic Spectroscopy for Investigation and Monitoring of Biological Diversity…

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

23

If the aim is to estimate the viability of a specified culture or colony, one can directly count the rate of normal and depressed cells by their fluorescence emission characteristics and make a decision about the viability of the whole culture. In different periods of culture development, the percentage of living and depressed cells changes significantly. If the percentage of alive cell spectra prevails in the sample, then the culture is supposed enough viable. If the sample has more spectra of sick and dead cells, then the culture is weak and incapable of active reproduction. Alternatively, one can make a linear combination from several spectra, belonging to different physiological states, and compare this result with the integral spectra of the whole culture, obtained via conventional fluorimeter. The fitting coefficients in this linear combination will show the rate of the viability of the considered culture. The second way is less accurate, due to the undesirable fluorescent background in the whole culture mentioned above, and can be

If the main purpose of the investigation is to reveal the influence of weak external actions or environmental changes on the physiological state of single cells from the tested sample, the comparative analysis of single-cell fluorescence spectra from the control and treated culture should be carried out. In this case the investigation of the cells in normal (good) physiological state is enough. If any changes in the shape or intensity of single-cell fluorescence spectra are registered compared to the reference sampling, thus the influence takes place. Independently to the form and the sign of this changes, one can fix the result of the external action; however, it is impossible to consider the origin of this result. For the latter, the additional precise inves-

Let us illustrate the effectiveness of this technique on a concrete example of the estimation of the viability of the culture of cyanobacterial strain *Synechocystis CALU* 1336 (from CALU collection), provided by of the Core facility Center for Culture Collection of Microorganisms

The experiment was carried out in the following way. After passing through steps 1 and 2, for the considered culture the spectra of the individual cells in the normal and depressed physiological states were recorded by means of CLSM at eight excitation wavelengths and averaged for each state (**Figure 4a–c**). Usually, for the culture in exponential growing phase, the range of physiological states includes almost all developmental stages presented in **Figure 3**, but only two main single-cell physiological states—"healthy cells" and "cells in strongly depressed state"—can be selected for further calculations, as it was previously shown in **Figure 2** (states I and II, respectively). These two basic sets of spectra for the main physiological states are shown in **Figure 4a–c**. Blue lines show healthy cell in good physiological state, red lines correspond to the strongly depressed cells. For the clarity of further narration, only three of the

this stage, two variants of investigation are possible according to the final purpose.

applied only for the fresh culture at the early growth phase.

of Saint-Petersburg State University.

tigation by means of other physical or chemical methods has to be done.

**Figure 3.** Time degradation of living cell of cyanobacterial strain *Synechocystis CALU 1336* under light and heat stress. Spectra were recorded at the excitation wavelength 488 nm and with the time step 2 min. Spectra are shifted along *x*-axis relative to each other for convenience of observation.

the dissociation of phycobilisomes was investigated. Thus, the estimation of the viability of single living cell and the whole culture is possible via investigation of the changes in fluorescence emission spectra.

Here we present a novel technique, recently elaborated by authors of this chapter, which is based on a strict relation between the shape and intensity of a single-cell fluorescence spectra of cyanobacteria and the physiological state of this cell. This technique is a direct extension of the previously elaborated visual methods of the estimation of the physiological state of cyanobacterial cells by the color of their fluorescence conducted by means of conventional fluorescent microscopy [39]. In contrast to the latter, which is very subjective, a new technique provides a detailed spectroscopic information and variations in "color" of the fluorescence can be measured in nanometers of light wavelength. A general character of the presented technique makes it possible to use it for investigation of any species of cyanobacteria, regardless of their habitat or cultivation conditions. Also the influence of scattering particles and preillumination effects, which are very important in the ordinary fluorescent methods, are absent in single-cell microscopic spectroscopy technique. Moreover, the use of the novel methods of microscopic spectroscopy allows to estimate the viability of colonies of noncultivated cyanobacterial species in natural samples according to the physiological state of individual cells. This fact can considerably facilitate the work with small concentrations of objects under consideration in environmental probes.

According to the proposed technique, the research process can be divided into three steps:

**Step 1.** Obtaining of the complete set of single-cell fluorescence spectra for given cyanobacterial strain in different physiological states by recording a series of spectra during singlecell degradation by means of CLSM at a certain excitation frequencies (e.g., at 458, 488 and 514 nm), as it was previously shown for 488 nm laser line in **Figure 3**. These sets will serve as reference spectra while determining the rate of cell degradation. At the same time, the most informative spectra for further investigation should be selected, which reflects the physiological state of the cells of a given strain.

**Step 2.** Recording of several sets of fluorescence spectra for single cells from the tested sample, which belongs to different physiological states, according to obtained reference spectra for this given strain. For accuracy of further estimation of the viability of the whole culture, these spectra should be recorded at several excitation wavelengths for each cell (in our investigations it was set of eight laser lines) and then each one should be averaged through a number of cells. The fluorescence spectra of a single cell excited by one laser line is not enough to have a complete information about deviations in photosynthetic process and pigment composition.

**Step 3.** Direct study of the tested sample in order to determine the viability of investigated culture or colony. A representative random sampling is used for the cells from a given strain and single-cell fluorescence spectra excited by chosen excitation wavelengths are recorded. At this stage, two variants of investigation are possible according to the final purpose.

If the aim is to estimate the viability of a specified culture or colony, one can directly count the rate of normal and depressed cells by their fluorescence emission characteristics and make a decision about the viability of the whole culture. In different periods of culture development, the percentage of living and depressed cells changes significantly. If the percentage of alive cell spectra prevails in the sample, then the culture is supposed enough viable. If the sample has more spectra of sick and dead cells, then the culture is weak and incapable of active reproduction. Alternatively, one can make a linear combination from several spectra, belonging to different physiological states, and compare this result with the integral spectra of the whole culture, obtained via conventional fluorimeter. The fitting coefficients in this linear combination will show the rate of the viability of the considered culture. The second way is less accurate, due to the undesirable fluorescent background in the whole culture mentioned above, and can be applied only for the fresh culture at the early growth phase.

the dissociation of phycobilisomes was investigated. Thus, the estimation of the viability of single living cell and the whole culture is possible via investigation of the changes in fluores-

**Figure 3.** Time degradation of living cell of cyanobacterial strain *Synechocystis CALU 1336* under light and heat stress. Spectra were recorded at the excitation wavelength 488 nm and with the time step 2 min. Spectra are shifted along *x*-axis

Here we present a novel technique, recently elaborated by authors of this chapter, which is based on a strict relation between the shape and intensity of a single-cell fluorescence spectra of cyanobacteria and the physiological state of this cell. This technique is a direct extension of the previously elaborated visual methods of the estimation of the physiological state of cyanobacterial cells by the color of their fluorescence conducted by means of conventional fluorescent microscopy [39]. In contrast to the latter, which is very subjective, a new technique provides a detailed spectroscopic information and variations in "color" of the fluorescence can be measured in nanometers of light wavelength. A general character of the presented technique makes it possible to use it for investigation of any species of cyanobacteria, regardless of their habitat or cultivation conditions. Also the influence of scattering particles and preillumination effects, which are very important in the ordinary fluorescent methods, are absent in single-cell microscopic spectroscopy technique. Moreover, the use of the novel methods of microscopic spectroscopy allows to estimate the viability of colonies of noncultivated cyanobacterial species in natural samples according to the physiological state of individual cells. This fact can considerably facilitate the work with small concentrations of objects under

According to the proposed technique, the research process can be divided into three steps:

**Step 1.** Obtaining of the complete set of single-cell fluorescence spectra for given cyanobacterial strain in different physiological states by recording a series of spectra during singlecell degradation by means of CLSM at a certain excitation frequencies (e.g., at 458, 488 and 514 nm), as it was previously shown for 488 nm laser line in **Figure 3**. These sets will serve as reference spectra while determining the rate of cell degradation. At the same time, the most informative spectra for further investigation should be selected, which reflects the physiologi-

**Step 2.** Recording of several sets of fluorescence spectra for single cells from the tested sample, which belongs to different physiological states, according to obtained reference spectra for

cence emission spectra.

22 Cyanobacteria

relative to each other for convenience of observation.

consideration in environmental probes.

cal state of the cells of a given strain.

If the main purpose of the investigation is to reveal the influence of weak external actions or environmental changes on the physiological state of single cells from the tested sample, the comparative analysis of single-cell fluorescence spectra from the control and treated culture should be carried out. In this case the investigation of the cells in normal (good) physiological state is enough. If any changes in the shape or intensity of single-cell fluorescence spectra are registered compared to the reference sampling, thus the influence takes place. Independently to the form and the sign of this changes, one can fix the result of the external action; however, it is impossible to consider the origin of this result. For the latter, the additional precise investigation by means of other physical or chemical methods has to be done.

Let us illustrate the effectiveness of this technique on a concrete example of the estimation of the viability of the culture of cyanobacterial strain *Synechocystis CALU* 1336 (from CALU collection), provided by of the Core facility Center for Culture Collection of Microorganisms of Saint-Petersburg State University.

The experiment was carried out in the following way. After passing through steps 1 and 2, for the considered culture the spectra of the individual cells in the normal and depressed physiological states were recorded by means of CLSM at eight excitation wavelengths and averaged for each state (**Figure 4a–c**). Usually, for the culture in exponential growing phase, the range of physiological states includes almost all developmental stages presented in **Figure 3**, but only two main single-cell physiological states—"healthy cells" and "cells in strongly depressed state"—can be selected for further calculations, as it was previously shown in **Figure 2** (states I and II, respectively). These two basic sets of spectra for the main physiological states are shown in **Figure 4a–c**. Blue lines show healthy cell in good physiological state, red lines correspond to the strongly depressed cells. For the clarity of further narration, only three of the

Despite the near qualitative character of the presented analysis, the accuracy and stability of this method are ensured by the simultaneous calculations over a series of eight spectra. If several variants are possible while fitting one spectrum, then simultaneous fitting of eight spectra will provide a sufficient accuracy. This small example demonstrates that the shape of single-cell fluorescence spectra reflect the physiological state of cyanobacteria and the sum of single-cell contributions represents viability of the whole culture because these phenomena are strictly related with the correct or incorrect functioning of the photosyn-

Fluorescence Microscopic Spectroscopy for Investigation and Monitoring of Biological Diversity…

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

25

Finally, it should be noted that the presented technique can be modified for obtaining any developmental stage of the selected cyanobacterial culture, by considering the reference set of fluorescence spectra to be fitted. For instance, in the technological process of the industrial incubation of cyanobacteria, involved in food production, it is quite desirable to estimate the optimal stage of the culture development when the accumulation of biologically active compounds attains its maximum. This process can be controlled via online recording of several fluorescence spectra and comparing them with reference ones. Moreover, in the environmental monitoring, the online estimation of the viability of the cyanobacterial colonies in the field samples via fast and effective fluorescence technique can assist the prediction and prevention

More detailed description of the presented technique and its application can be found in [83].

In recent years, several environmentally friendly methods for preventing of toxic cyanobacterial "blooms" of water bodies have appeared. One of them is a weak ultrasound treatment. Unfortunately, due to the low intensity of the applied sonication and its constant but weak influence on the biophysical parameters of the cyanobacteria most of the results of the previ-

Despite the significance of the problem of cyanobacterial blooms and a variety of applied methods to control them, the sufficient principles of investigation and monitoring the results of various external actions on cyanobacteria are not developed. Currently, the most of all studies are based mainly on traditional visual methods of obtaining results or on the analysis of fixed or dissociated environmental samples. However, standard methods can only record the presence of the bloom in reservoir, but cannot determine at what evolution stage it is situated, or, all the more, to predict the possibility of further cyanobacterial bloom. This owes to the use of rather crude methods of monitoring of the physiological state of the culture during the experiment. All previously elaborated monitoring methods either destroy cells or change significantly their physiological state just before the measurements, so it is impossible

For example, in the paper [87], the ultrasonic inhibition of *Microcystis aeruginosa* cell growth and extracellular microcystins release was examined. The authors reported the decrease of antenna complexes like cyanobacterial chlorophyll a and phycocyanins (PC), and the oxygen evolution rate. The conclusion about slowing down of the photoactivity and damaging of the antenna complexes was made according to the measuring of the growth rate of the whole culture. This is not the case because there is no any confirmation that the single-cell physiological state really

thetic apparatus.

of the hazardous cyanobacterial blooms.

ous investigations are quite ambiguous [32–34, 84–89].

to determine the initial physiological state of the treated culture.

**3.2. Ultrasonic treatments**

**Figure 4.** The illustration to the process of the culture viability estimation. (a–c) Averaged SFS for cyanobacterial strain Synechocystis CALU 1336, excited by three laser lines 458, 488, and 514 nm. *Blue* lines show spectra of alive cells, *red* lines are for the cells in the depressed physiological state. (d–f) Fitted and integral normalized spectra for the whole culture under consideration. *Black* lines show a linear combination of CLSM spectra of alive and depressed cells summed in ratio 2:1. *Green* lines show the integral spectra of the whole culture obtained using Cary Eclipse fluorimeter (Varian Cary) at corresponding excitation wavelengths. At all plots dashed lines indicate the fluorescence maximum of chlorophyll a (680 nm) and phycocyanin (656 nm), respectively. For SFS averaging was carried out over 10–15 cells.

eight obtained spectra are presented in **Figure 4**. Then, the integral spectra of the whole culture were obtained at corresponding excitation wavelengths using Cary Eclipse fluorimeter (Varian Cary) (**Figure 4d–f**; green lines). Two sets of eight fluorescence spectra for cells in normal and depressed physiological state were taken to obtain a linear combination for fitting procedure a set of integral spectra. Each spectrum in two sets was averaged over 10–15 cells. It should be noted that to raise accuracy of calculations, the curve fitting process was done over the whole set of the eight spectra simultaneously, so that to exclude any ambiguity. The fitting coefficients for alive and depressed cells were about 2 and 1, correspondingly. Thus, the whole culture can be considered as a healthy and being in the exponential or logarithmic growth phase.

As it is following from the plots of **Figure 4a–c**, the degree of cell damage is determined mostly by the relative fluorescence intensity of phycobilins at 656 nm and chlorophyll-binding proteins at 680 nm. It is especially clear from the excitation wavelength 488 nm (**Figure 4b**), where the fluorescence spectra for healthy and depressed cells have the mirror shape. For the 458 nm excitation, the shape and the intensity of fluorescence spectra for this two states differ not so much because this wavelength excites better the peak of chlorophyll-binding proteins in PS II (680 nm), which is not affected. On the other hand, the 514 nm wavelength strongly excites the fluorescence of the antenna pigments and the fluorescence spectra intensity differs significantly for the healthy and depressed cells.

Despite the near qualitative character of the presented analysis, the accuracy and stability of this method are ensured by the simultaneous calculations over a series of eight spectra. If several variants are possible while fitting one spectrum, then simultaneous fitting of eight spectra will provide a sufficient accuracy. This small example demonstrates that the shape of single-cell fluorescence spectra reflect the physiological state of cyanobacteria and the sum of single-cell contributions represents viability of the whole culture because these phenomena are strictly related with the correct or incorrect functioning of the photosynthetic apparatus.

Finally, it should be noted that the presented technique can be modified for obtaining any developmental stage of the selected cyanobacterial culture, by considering the reference set of fluorescence spectra to be fitted. For instance, in the technological process of the industrial incubation of cyanobacteria, involved in food production, it is quite desirable to estimate the optimal stage of the culture development when the accumulation of biologically active compounds attains its maximum. This process can be controlled via online recording of several fluorescence spectra and comparing them with reference ones. Moreover, in the environmental monitoring, the online estimation of the viability of the cyanobacterial colonies in the field samples via fast and effective fluorescence technique can assist the prediction and prevention of the hazardous cyanobacterial blooms.

More detailed description of the presented technique and its application can be found in [83].
