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

The more distal parts of the antenna system, often a peripheral antenna complex (phycobilisome), maximally absorb photons at shorter wavelengths (higher energies) than do the pigments in the antenna complexes that are proximal to the reaction center. Subsequent energy transfer processes are from these high-energy pigments physically distant from the reaction center to lower energy pigments that are physically closer to the reaction center (**Figure 3**). With each transfer, a small amount of energy is lost as heat, and the excitation is moved closer to the reaction center, where the energy is stored by photochemistry. Note, that the probability of excitation energy escapes from the trap in the form of fluorescence at all transfer steps is nonzero and depends on the intensity and wavelength of the excitation light.

During the energy transfer process, the occasional quenching of the absorbed light by fluorescence can occur and this becomes the essential property for fluorescent spectroscopy. It usually represents a small fraction of the excited states and diminishes in a functioning photosynthetic complex. Nevertheless, the fluorescence is an extremely informative quantity, because it reports on the energy transfer and trapping. Both steadystate and time-resolved fluorescence measurements are widely used methods for probing the organization and functional state of photosynthetic systems.

The unique spectroscopic properties of different cyanobacterial strains may become a promising fingerprints for practical and laboratory application [28]. The polypeptide composition of PBS varies widely among strains of cyanobacteria. However, it should be noted that the degree of PBS compositional variability, which reflects the ability of an organism to adapt to environmental changes, varies from strain to strain. Moreover, for a single strain, it also depends upon the environmental conditions such as nutrient availability, temperature, light quality, and light intensity.

The intact PBSs in cyanobacteria harvest sun light in the visible range from 400 to 750 nm and transfer the energy to the chlorophyll a (Chl a) of the photosystems PSII and PSI [24, 34, 35, 39]. When the single pigment-protein complexes aggregate in PBSs, their absorption bands are broadened due to the additional splitting of energy levels in the cause of interaction with other biliproteins and linker polypeptides. This leads to a more efficient harvesting of the light energy. Blue and red wavelengths of the visible light (around 440 and 675 nm) are mainly absorbed by cyclic tetrapyrroles, chlorophylls, combined in PSI and PSII, while green, yellow, and orange wavelengths (between 550 and 650 nm) are mostly absorbed by openchained tetrapyrrole pigments, the phycobilins, composed in extramembranous antenna structures [24].

The fluorescence of intact living cyanobacterial cells is originated from the efficiency of the energy transfer between all components of the energy transfer chain including the final step, the delivery to PSII or PSI (**Figure 3(a)**). Each transfer step results in the spectrum shape as a peak or shoulder (**Figure 3(b)**). This is due to the fact that when phycobilisomes are bound to the thylakoid membrane, most of the energy from the last component of phycobilisome is channeled to chlorophylls in the thylakoid membrane and thus did not shade the fluorescence of the previous steps in energy transfer chain. In the course of the energy transfer from the initially photoexcited phycobiliprotein to the reaction center of photosystems PSI and PSII, fluorescence is emitted from almost every type of pigment and can be used as a probe to examine the mechanism of energy transfer within the light-harvesting system [8, 25, 26, 28].

A convenient way to monitor this energy transfer process is to irradiate a sample with light that is selectively absorbed by one set of pigments and then monitor fluorescence that originates from a different set of pigments. Obviously, if the energy transfer is taken place between pigments, the light absorbed by one set of pigments is emitted by another set differently depending on the excitation wavelength. This type of fluorescence excitation experiment can also be used to

*Color Detection*

chemistry took place.

**2.3 Absorption, fluorescence, and energy transfer**

this fast process has an efficiency of almost 100%.

As it was mentioned above, the fluorescence is one of the most powerful ways to probe photosynthetic systems, because it reports on the energy transfer and trapping. The intrinsic fluorescence of photosynthetic organisms originates from excited states that were trapped by light-harvesting system and lost before photo-

In the light-harvesting pigment-protein complexes of cyanobacteria, the pigment molecules are excited by photons. In a nonradiating-induced resonance transfer process leading up to the reaction centers, they transfer the excitation energy to other pigment molecules which are excited in turn. In phycobilisomes,

ized excitons, applicable at short distances and strong interactions [26].

tive process, which means that no photon emission or absorption is involved. A critical step in the energy storage process is energy transfer between the antenna and the reaction center, where separation of the electron from a positively charged hole occurs. The molecules that accomplish the last event are organized in the photosynthetic membranes in a highly specific fashion to achieve the high

*Schematic illustration of the energy transfer in light-harvesting system of cyanobacteria (a). Panel (b) represents the normalized in vivo single-cell fluorescence emission spectra of two cyanobacterial species: blue line— Leptolyngbia CALU 1713 and red line—Nostoc CALU 1817. Excitation wavelength 488 nm. Dashed lines and numbers over them indicate emission wavelengths of PE, PC, and Chl a of PCII and PCI, correspondingly.*

efficiency of light energy conversion to photochemistry [30, 38].

There are two mechanisms that serve the efficient excitation transfer in the light harvesting complex of cyanobacteria: the inductive resonance (Förster) transfer, applicable at long distances and weak interactions, and the occurrence of delocal-

The most likely mechanism by which excitation hops from one pigment complex to another across distances greater than several Angstroms is inductive resonance transfer, also known as Förster transfer [36, 37]. The interaction between an electronically excited pigment and its unexcited neighbors results in a downward transition of the initially excited group coupled with an upward transition in the nearby acceptor pigment by a through space Coulombic interaction (**Figure 3**). The energy donor and energy acceptor molecules must have an energy state in common, because when the excitation hops from donor to acceptor, the conservation of energy is required. This can be so only if the two molecules have a common energy state and therefore spectral transitions at the same wavelength. It should be noted, that the requirement for overlap of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor does not lead to the process of emission of a photon by the donor, which is followed by absorption of a photon by the acceptor. The Förster transfer is a nonradia-

**44**

**Figure 3.**
