**3.3 Single-cell spectroscopy (lambda scanning)**

Fluorescence spectra of living photosynthetic cells can be reliably analyzed by a microspectrofluorometric method (CLSM spectroscopy), which is implemented in many modern CLSMs. This method allows the precise localization of the fluorescence signal, even within a single living cell. Modern fluorescence microscopic spectroscopy or confocal laser scanning microscopic spectroscopy provides a unique opportunity to obtain the intrinsic fluorescence emission spectra from cyanobacterial cells in vivo [40–43, 45–46]. Moreover, using spectral unmixing, the fluorescence of individual spectral components can be resolved, and their relative intensities can be calculated [1–2].

The acquisition of spectral data becomes necessary when the cellular parameter to be measured is coded by changes of the emission spectrum. PMT element registers sequentially a different part of the spectrum, each part having a spectral width of 6 nm with a step 6 nm. The result is a lambda stack of XY images in which each image represents a different spectral window. Lambda stack is a series of images showing the same area, but in different spectral windows. From a lambda stack, the intensity of the signal for each pixel of the image can be extracted as a function of wavelength. These spectral "fingerprints" can easily be obtained for any image area by means of the mean of ROI function.

Although, the pigment structure of different cyanobacterial strains has been intensively investigated, the variations in self-fluorescent spectra of single cells for different cyanobacterial species has not been analyzed yet. We suppose that the best way to investigate the in vivo operation of photosynthetic system is a singlecell fluorescence microscopic spectroscopy. 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 intrinsic self-fluorescence by means of CLSM.

As it was mentioned above, phycobilisomes contains several kinds of biliproteins, and their fluorescence spectra reflect the contribution of each (**Figure 3**). Moreover, depending on the excitation wavelength, the room temperature fluorescence emission spectrum of intact cyanobacterial cells exhibits various extents of contribution of phycobilisome emission to the spectrum. If one exclusively excites Chl a, using a 458-nm line of an Ar laser, the emission spectrum by cyanobacterial cells shows no appreciable emission of PC or APC. This is indicated by PSI emission band at 715 nm and PSII emission band at 682 nm (**Figure 10**). The excitation by intermediate (blue and green) wavelengths (405, 488, and 496 nm) reveals fluorescent maxima of all photosynthetic pigments, as the light in this range is absorbed by all pigment-protein complexes almost in equal portions, and fluorescence emits by all steps of energy transfer chain (**Figure 3**). The direct excitation of cells in the PC absorption region at 514 and 543 nm results in emission spectrum with two main peaks at 580 and 656 nm, which are due to PE, PC, and APC emission. The spectra of the 633 nm excitation directly gives a prominent emission band at 656 nm, that originates from PC, omitting band at 580 nm, which cannot be excited by 633 nm, even for species that have PE. Other small emission bands, corresponding to fine pigment structure of antenna complex, are not resolved at room temperature. Comparative analysis of the series of fluorescence spectra for different cyanobacterial species and strains reveals visible variations in their shape (**Figure 10**). If the fluorescence spectra were taken from alive cells in normal physiological state, which are cultured in the same growth environmental conditions, then the interspecies variations in pigment/Chl a ratios are more pronounced than variations within the individual species. And species/strains differentiation could be carried out on the base of fluorescence analysis [3, 6–7].

**Figure 10** shows the experimental sets of single-cell fluorescence spectra for Microcystis CALU 398, Merismopedia CALU 666, Leptolyngbya CALU 1715,

**53**

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

and Phormidium CALU 624 (cyanobacterial strains are labeled according to CALU collection of the Core Facility Center "Centre for Culture Collection of Microorganisms" of the Science Park of St. Petersburg State University). Each spectrum set was obtained by means of confocal laser scanning microscope (CLSM) Leica TCS-SP5, using different laser-lines for excitation (405, 458, 476, 488, 496, 514, 543, and 633 nm). Corresponding excitation wavelengths are given over each spectrum. All spectra are normalized to the maximum intensity and shifted along

*the individual pigments (PE—580 nm; PC—656 nm; Chl a—682, 715 nm).*

*Single-cell fluorescence spectra of different cyanobacterial strains (unicellular and filamentous): Microcystis 398, Merismopedia 666, Phormidium 624, and Leptolyngbya 1715. The excitation wavelengths (405, 458, 476, 488, 496, 514, 543, and 633 nm) are given over the curves. All spectra are normalized to the maximum intensity and shifted along X-axis for convenience of observation. The dashed lines indicate the fluorescence maxima of* 

Fluorescence of photosynthetic pigments in the intact cells is affected by physicochemical and physiological processes that occur within and across the thylakoid membranes. The structure and function of the thylakoid membrane can alter under stress conditions. The alterations may be both short- and long-term, depending on the nature and duration of the stress. The dynamical changes in fluorescent spectra of living cyanobacterial cells in different stress states can be detected via timeresolved CLSM measurements and can be used for estimation of the physiological state and viability of cyanobacterial cultures at different experimental stages. **Figure 11** illustrates the temporal changes of in vivo fluorescence spectrum taking place in the living cell of cyanobacteria strain Synechocystis CALU 1336 under high-light and heat stress. The excitation wavelength in this experiment was 488 nm. The set of spectra was obtained from a single cell during 45 min with 1 min step under over-excitation conditions (only every fourth spectra is presented in **Figure 11(a)**). At first, several minutes (1–12 min) spectrum changes slightly and mostly in the range of PC-fluorescence (around 656 nm). Since 488 nm light is absorbed primarily by PC and Chl a, photosynthesis and energy transfer chain are quickly saturated and reaction centres closes, thus the fluorescence around 656 nm starts growing. During the next few minutes (12-28 min) a rapid growth of PC-APC fluorescence indicates that over-excitation leads to the uncoupling of phycobilisomes from thylakoid membrane, while the Chl a fluorescence remains almost the same during this time period (see **Figure 11(b)**). Small rise of the Chl a fluorescence intensity at 20–39 min can be explained by strong overlapping of Chl a and PC-APC emission spectra, which leads to a crosstalk between these fluorescent signals. Then, after 30 min of irradiation, the degradation of detached phycobilisomes and PSII

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

X-axis for convenience of observation.

**Figure 10.**

*Confocal Laser Scanning Microscopy for Spectroscopic Studies of Living Photosynthetic Cells DOI: http://dx.doi.org/10.5772/intechopen.84825*

### **Figure 10.**

*Color Detection*

intensities can be calculated [1–2].

by means of the mean of ROI function.

by means of CLSM.

in many modern CLSMs. This method allows the precise localization of the fluorescence signal, even within a single living cell. Modern fluorescence microscopic spectroscopy or confocal laser scanning microscopic spectroscopy provides a unique opportunity to obtain the intrinsic fluorescence emission spectra from cyanobacterial cells in vivo [40–43, 45–46]. Moreover, using spectral unmixing, the fluorescence of individual spectral components can be resolved, and their relative

The acquisition of spectral data becomes necessary when the cellular parameter to be measured is coded by changes of the emission spectrum. PMT element registers sequentially a different part of the spectrum, each part having a spectral width of 6 nm with a step 6 nm. The result is a lambda stack of XY images in which each image represents a different spectral window. Lambda stack is a series of images showing the same area, but in different spectral windows. From a lambda stack, the intensity of the signal for each pixel of the image can be extracted as a function of wavelength. These spectral "fingerprints" can easily be obtained for any image area

Although, the pigment structure of different cyanobacterial strains has been intensively investigated, the variations in self-fluorescent spectra of single cells for different cyanobacterial species has not been analyzed yet. We suppose that the best way to investigate the in vivo operation of photosynthetic system is a singlecell fluorescence microscopic spectroscopy. 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 intrinsic self-fluorescence

As it was mentioned above, phycobilisomes contains several kinds of biliproteins, and their fluorescence spectra reflect the contribution of each (**Figure 3**). Moreover, depending on the excitation wavelength, the room temperature fluorescence emission spectrum of intact cyanobacterial cells exhibits various extents of contribution of phycobilisome emission to the spectrum. If one exclusively excites Chl a, using a 458-nm line of an Ar laser, the emission spectrum by cyanobacterial cells shows no appreciable emission of PC or APC. This is indicated by PSI emission band at 715 nm and PSII emission band at 682 nm (**Figure 10**). The excitation by intermediate (blue and green) wavelengths (405, 488, and 496 nm) reveals fluorescent maxima of all photosynthetic pigments, as the light in this range is absorbed by all pigment-protein complexes almost in equal portions, and fluorescence emits by all steps of energy transfer chain (**Figure 3**). The direct excitation of cells in the PC absorption region at 514 and 543 nm results in emission spectrum with two main peaks at 580 and 656 nm, which are due to PE, PC, and APC emission. The spectra of the 633 nm excitation directly gives a prominent emission band at 656 nm, that originates from PC, omitting band at 580 nm, which cannot be excited by 633 nm, even for species that have PE. Other small emission bands, corresponding to fine pigment structure of antenna complex, are not resolved at room temperature. Comparative analysis of the series of fluorescence spectra for different cyanobacterial species and strains reveals visible variations in their shape (**Figure 10**). If the fluorescence spectra were taken from alive cells in normal physiological state, which are cultured in the same growth environmental conditions, then the interspecies variations in pigment/Chl a ratios are more pronounced than variations within the individual species. And species/strains differentiation could be carried out on the

**Figure 10** shows the experimental sets of single-cell fluorescence spectra for Microcystis CALU 398, Merismopedia CALU 666, Leptolyngbya CALU 1715,

**52**

base of fluorescence analysis [3, 6–7].

*Single-cell fluorescence spectra of different cyanobacterial strains (unicellular and filamentous): Microcystis 398, Merismopedia 666, Phormidium 624, and Leptolyngbya 1715. The excitation wavelengths (405, 458, 476, 488, 496, 514, 543, and 633 nm) are given over the curves. All spectra are normalized to the maximum intensity and shifted along X-axis for convenience of observation. The dashed lines indicate the fluorescence maxima of the individual pigments (PE—580 nm; PC—656 nm; Chl a—682, 715 nm).*

and Phormidium CALU 624 (cyanobacterial strains are labeled according to CALU collection of the Core Facility Center "Centre for Culture Collection of Microorganisms" of the Science Park of St. Petersburg State University). Each spectrum set was obtained by means of confocal laser scanning microscope (CLSM) Leica TCS-SP5, using different laser-lines for excitation (405, 458, 476, 488, 496, 514, 543, and 633 nm). Corresponding excitation wavelengths are given over each spectrum. All spectra are normalized to the maximum intensity and shifted along X-axis for convenience of observation.

Fluorescence of photosynthetic pigments in the intact cells is affected by physicochemical and physiological processes that occur within and across the thylakoid membranes. The structure and function of the thylakoid membrane can alter under stress conditions. The alterations may be both short- and long-term, depending on the nature and duration of the stress. The dynamical changes in fluorescent spectra of living cyanobacterial cells in different stress states can be detected via timeresolved CLSM measurements and can be used for estimation of the physiological state and viability of cyanobacterial cultures at different experimental stages.

**Figure 11** illustrates the temporal changes of in vivo fluorescence spectrum taking place in the living cell of cyanobacteria strain Synechocystis CALU 1336 under high-light and heat stress. The excitation wavelength in this experiment was 488 nm. The set of spectra was obtained from a single cell during 45 min with 1 min step under over-excitation conditions (only every fourth spectra is presented in **Figure 11(a)**). At first, several minutes (1–12 min) spectrum changes slightly and mostly in the range of PC-fluorescence (around 656 nm). Since 488 nm light is absorbed primarily by PC and Chl a, photosynthesis and energy transfer chain are quickly saturated and reaction centres closes, thus the fluorescence around 656 nm starts growing. During the next few minutes (12-28 min) a rapid growth of PC-APC fluorescence indicates that over-excitation leads to the uncoupling of phycobilisomes from thylakoid membrane, while the Chl a fluorescence remains almost the same during this time period (see **Figure 11(b)**). Small rise of the Chl a fluorescence intensity at 20–39 min can be explained by strong overlapping of Chl a and PC-APC emission spectra, which leads to a crosstalk between these fluorescent signals. Then, after 30 min of irradiation, the degradation of detached phycobilisomes and PSII

**Figure 11.**

*Time degradation of living cell of cyanobacterial strain Synechocystis CALU 1336 under high-light and heat stress. (a) Spectra recorded at the excitation wavelength 488 nm with the time-step 4 min. All spectra are shifted along x-axis for convenience of observation. (b) Time dependence of the fluorescence intensity at 656 nm (PC-APC fluorescence) and at 682 nm (Chl a fluorescence).*

pigment-protein complexes begins. The intensity of Chl a and PC-APC fluorescence decreases, and maximum is shifted to shorter wavelengths.

Two presented examples of CLSM lambda-scan application show the unique abilities of CLSM microscopic spectroscopy for investigation of physiological processes in cyanobacterial cells. More examples can be found in [3–5, 47].
