**3. Confocal laser scanning microscopy (CLSM)**

CLSM applications in biology and medicine predominantly employ fluorescence. Cellular structures can be specifically labeled with dyes (fluorescent dyes = fluorochromes or fluorophores) in various ways. But the cyanobacterial photosynthetic pigments, chlorophyll a and phycobilins, have an inherent fluorescence at 543 and 633 nm excitation and 590–800 nm emission and need no labelling for visualization. To target other specific elements, many fluorescent dyes and labels can be used. In this study, we use CdSe/ZnS quantum dots for imaging extracellular substances. It is also possible to use the transmission mode with conventional contrasting methods, such as differential interference contrast (DIC), as well as to overlay the transmission and confocal fluorescence images of the same specimen area.

In the present investigation, both Leica TCS-SP5 and Carl Zeiss LSM 750 were used for investigation of living cyanobacterial cells. For Leica TCS-SP5, laser power settings are as follows: 29% of Ar laser power was reflected onto sample with Acousto-Optical Tunable Filter (AOTF) and further power percentage for its laser lines was: 30% of 458 nm laser-line and 10% for all other lines. A 405-nm line of diode UV laser was reflected onto sample with 3%, and HeNe laser lines 543 and 633 nm were reflected with 10% and 2%, respectively. An acousto-optical beam splitter (AOBS) was used to transmit sample fluorescence to detector.

For 2D imaging, Leica TCS-SP5 was utilized. To rise the sensitivity and contrast of 2D images, they were recorded at 405 nm excitation wavelength (diode UV laser) and by Leica HyD hybrid detector, which strongly improves contrast in comparison to PMTs. HyD gain: 100%. The images of 1024 × 1024 and 2048 × 2048 pixels were collected with a 63× glycerol immersion lens (glycerol 80% H2O) with a numeric aperture of 1.3 (objective HCX PL APO 63.0 × 1.30 GLYC 37°C UV) and with additional digital zoom factor 10–35. The fluorescence emission images were accompanied with the transmission images (in the parallel channel), collected by a transmission detector with the photomultiplier voltages ranged from 300 to 500 V. The images were recorded with a pinhole setting of 1 Airy unit (the inner light circle of the diffraction pattern of a point light source, corresponds to a diameter of 102.9 μm with the lens used (see [1])).

Carl Zeiss LSM 750 with LSM Software ZEN 2009 was used for 3D-imaging. The images were collected with EC Plan-Neofluar 100×/1.3 oil M27 and with additional digital zoom factor 2.86. XY-scan of 1024 × 1024 pixel images was performed with resolution 8-bit in two channels. One pixel corresponds to 29.7 × 29.7 μm. A multi-dimensional acquisition tool was used for recording z-stack of 80 sections with z-step 0.10 μm.

Fluorescence emission spectra of the intact cells were measured by Leica TCS-SP5 at 8 excitation wavelengths corresponding to all available laser lines. The excitation wavelengths are: 458, 476, 488, 496, 514 nm—the lines of Ar laser, 405 nm is the line of diode UV laser and 543, 633 nm are the lines of HeNe laser. Chlorophyll fluorescence was excited by the 405, 458, 476, and 488 nm laser lines, and phycobilisome (PBS) fluorescence was induced by the 496, 514, 543, and 633 nm laser lines. Fluorescence emission was detected for PE at 570–600 nm, for

**47**

hybrid detector (HyD).

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

PC and APC at 650–670 nm, and for Chl a of both photosystems at 675–720 nm. The whole emission spectrum between 520 and 785 nm was recorded using the lambda scan function of the "Leica Confocal Software" by sequentially acquiring a series ("stack") of 38–45 images, each with a 6-nm fluorescence detection bandwidth and with 6-nm wavelength step. For obtaining fluorescence-intensity information, images of 512 × 512 pixels were collected with objective HCX PL APO 63.0 × 1.30 GLYC 37°C UV and with additional digital zoom factor 5–9. One pixel corresponds to 53.5 × 53.5 nm. The photomultiplier (PMT) voltages were used in the range from 900 to 1100 V. For better signal yield, lambda scans were performed with "low speed" setting (400 Hz) in bidirectional scan mode and with a pinhole setting of 1 Airy unit. Regions of interest (ROIs) representing single cells or subcel-

In CLSM applications, the laser light density in the focus point is high. Dwell time and the intervals between the illuminations may influence photo-damage and saturation of photosynthesis. Thus, since most chromophores and natural pigments bleach under the high laser excitation energies, a bleach-test should be performed [40]. During the detection, the fluorescence of the main accessory pigments for each cyanobacterial strain should be controlled and the changes in their fluorescence should not exceed 10–20%. The power of individual laser lines should be chosen according to the photodamage they cause. In our experiments, the repeated spectra were obtained under selected excitation power at a fixed point in a cell to check whether the excitation would affect the cells. In each case, it was shown that at the chosen excitation energies, the fluorescence spectra did not vary within the experimental error during 10–15 records. When excitation energy was increased, both the height and the center of the fluorescent peaks varied enormously with time

because of photodamage or structure-breakdown in photosynthetic systems.

In the experiments, where several laser lines were involved for the investigation, the first spectrum was recorded again at the end of each series to control the initial state of the cell. To visualize differences between strains with higher spectral and spatial resolution, lambda scans were performed with 6-nm bandwidth and with 6-nm steps. As far as the fluorescence intensities depend on the excitation energy (which varies for different laser lines), sensitivity setting of the photomultiplier, and the distance from the sample, all spectra were usually normalized to their maxi-

In a conventional light microscope, object-to image transformation takes place simultaneously and parallel for all object points. The specimen in CLSM is irradiated in a pointwise fashion, that is, serially, and the physical interaction between the laser light and the specimen fluorescence is measured point by point. To obtain information about the entire specimen, it is necessary to guide the laser beam across the specimen. Line-by-line scanning of the specimen is carried out with a focused laser beam deflected in the X and Y directions by means of two galvanometric scanners, and pixel-by-pixel detection of the fluorescence emitted by the scanned specimen details is performed by photomultiplier tube (PMT) or by more sensitive

For examining flat specimens such as cell culture monolayers, it is usually sufficient to acquire one XY image to obtain the desired information. The same applies if the specimen is a three-dimensional tissue section of which a single optical section is representative. The thickness of the optical section (slice) and the focal position are selected so that the structures of interest are contained in the slice. The lateral resolution of a 2D image is defined by the pixel size in X and Y. The pixel

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

lular regions were used to calculate fluorescence spectra.

mum, and only qualitative analysis was performed.

**3.1 Spectral imaging and spectral unmixing**

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

PC and APC at 650–670 nm, and for Chl a of both photosystems at 675–720 nm. The whole emission spectrum between 520 and 785 nm was recorded using the lambda scan function of the "Leica Confocal Software" by sequentially acquiring a series ("stack") of 38–45 images, each with a 6-nm fluorescence detection bandwidth and with 6-nm wavelength step. For obtaining fluorescence-intensity information, images of 512 × 512 pixels were collected with objective HCX PL APO 63.0 × 1.30 GLYC 37°C UV and with additional digital zoom factor 5–9. One pixel corresponds to 53.5 × 53.5 nm. The photomultiplier (PMT) voltages were used in the range from 900 to 1100 V. For better signal yield, lambda scans were performed with "low speed" setting (400 Hz) in bidirectional scan mode and with a pinhole setting of 1 Airy unit. Regions of interest (ROIs) representing single cells or subcellular regions were used to calculate fluorescence spectra.

In CLSM applications, the laser light density in the focus point is high. Dwell time and the intervals between the illuminations may influence photo-damage and saturation of photosynthesis. Thus, since most chromophores and natural pigments bleach under the high laser excitation energies, a bleach-test should be performed [40]. During the detection, the fluorescence of the main accessory pigments for each cyanobacterial strain should be controlled and the changes in their fluorescence should not exceed 10–20%. The power of individual laser lines should be chosen according to the photodamage they cause. In our experiments, the repeated spectra were obtained under selected excitation power at a fixed point in a cell to check whether the excitation would affect the cells. In each case, it was shown that at the chosen excitation energies, the fluorescence spectra did not vary within the experimental error during 10–15 records. When excitation energy was increased, both the height and the center of the fluorescent peaks varied enormously with time because of photodamage or structure-breakdown in photosynthetic systems.

In the experiments, where several laser lines were involved for the investigation, the first spectrum was recorded again at the end of each series to control the initial state of the cell. To visualize differences between strains with higher spectral and spatial resolution, lambda scans were performed with 6-nm bandwidth and with 6-nm steps. As far as the fluorescence intensities depend on the excitation energy (which varies for different laser lines), sensitivity setting of the photomultiplier, and the distance from the sample, all spectra were usually normalized to their maximum, and only qualitative analysis was performed.
