Confocal Laser Scanning Microscopy for Spectroscopic Studies of Living Photosynthetic Cells

Abstract

Self-fluorescence of light-harvesting complex is a powerful tool for investigation of living photosynthetic microorganisms. As the physiological state of single cells of such microorganisms is closely related to the operation and activity of photosynthetic system, any variations in spectroscopic properties of their self-fluorescence indicate the changes in their physiological state. In this chapter, we present several applications of confocal laser scanning microscopy (CLSM) for investigation of living photosynthetic cells. A set of ordinary CLSM techniques will be applied for studying of cyanobacteria (or blue-green algae) such as 3D imaging, spectral imaging, microscopic spectroscopy, and fluorescence recovery after photobleaching (FRAP). Cyanobacteria were chosen as a model microorganism due to their great importance for different scientific and biotechnological applications. Cyanobacteria are the most ancient photosynthetic microorganisms on Earth. Nowadays, cyanobacteria are one of the most wide-spreaded organisms in nature, and the ecological aspect in their investigation is quite valuable. On the other hand, thousand strains belonging to different species are cultivated in biolaboratories all over the world for different biotechnological applications such as biofuel cells, food production, pharmaceuticals, fertilizers, etc. Thus, the noninvasive spectroscopic methods are quite important for monitoring of physiological state of cyanobacterial cultures and other photosynthetic microorganisms.

Studying Cyanobacteria by Means of Fluorescence Methods: A Review

“…Many useful parameters as the degree of colocalization of labeled structures or the ion concentration in a specimen can be analyzed [128]. The most interesting feature of CLSM is the capability for single-cell microscopic spectroscopy, which allows obtaining spectroscopic information inside small regions and single cells [35]. Another group of facilities deals with the quantitative analysis of dynamic processes in living cells using techniques such as fluorescence recovery after photobleaching (FRAP) [129,130], fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM) [131], photoactivation, and photoconversion.…”

“…However, while working with living cells, the caution must be taken. The studies of the photosynthetic cells require some additional specific skills and techniques to perform measurements and to carry out data processing [35,41,42,44]. The efficiency of photosynthesis and photosynthetic rate are highly dependent on irradiance.…”

“…The most naturally suitable and appropriate methods of investigation of photosynthetic organisms are to use the self-fluorescence of photosynthetic apparatus. In this sense, following in vivo and in situ methods can be suggested: fluorescence spectroscopy (steady-state fluorescence) [24,25], chlorophyll fluorescence induction analysis (fast and slow kinetics) [26,27], pulse amplitude-modulated fluorometry (chlorophyll fluorescence quenching) [28,29], delayed fluorescence [30,31], flow cytometry [32,33], confocal microscopic spectroscopy [34,35], and remote sensing (passive and active, e.g., fluorescence LIDAR technique) [36,37].…”

Self-Fluorescence of Photosynthetic System: A Powerful Tool for Investigation of Microalgal Biological Diversity

“…It reports on the energy transfer and trapping and, thus, reflects the metabolic mechanisms in photosynthetic cells and their photosynthetic efficiency. The detected selffluorescence finally reflects the diversity in morphological and physiological states of photosynthetic cells [4][5][6].…”

“…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 [4][5][6]. Moreover, the diversity in single-cell self-fluorescence for different species and strains can serve the basis for ataxonomic discrimination of cyanobacterial genera.…”