**3.1 Spectral imaging and spectral unmixing**

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 hybrid detector (HyD).

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

*Color Detection*

differentiation of cyanobacterial species.

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

and confocal fluorescence images of the same specimen area.

diameter of 102.9 μm with the lens used (see [1])).

splitter (AOBS) was used to transmit sample fluorescence to detector.

measure quantitatively the efficiency of energy transfer from one set of pigments to another [26]. Moreover, different species of cyanobacteria contain different accessory pigment-proteins and specific linker-proteins between them, therefore a set of fluorescence emission spectra excited by different wavelengths have its own unique shape for the cells of one strain and are quite distinguishable from other species and strains. Such sets of fluorescence emission spectra can be used for automatic

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

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

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

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

**46**

size, in turn, varies with the objective used, the number of pixels per scan field, and the zoom factor. Pixels that are too large degrade resolution, whereas pixels too small require longer scanning times and thus bleach the specimen. The optimum pixel size for a given objective and a given zoom factor should be obtained for each special sample/experiment.

In this section, we examined changes in pigments of live, unfixed cells using spectral imaging. This technique captures an entire emission spectrum from every cell. The images corresponding to the four photosynthetic pigments have been pseudocolored for visualization purposes. The pseudocoloring is based on the colors shown in **Figure 5**: Chl a is red, PC-APC is green, and PE is light blue.

In **Figure 4**, images illustrated differences in fluorescence between diverse physiological states of Microcystis CALU 398 cells are presented. Two channels for fluorescence detection and one transmission channel were used. Channel 1 (a), colored false green, represents averaged fluorescence of PC and APC in the range 650–660 nm, channel 2 (b), colored false red, shows Chl a fluorescence at 678–710 nm, channel 3 (c) is a transmission microphoto, and in (d) all three channels are superimposed. Here small signal in both fluorescent channels corresponds to healthy cells, cells in bad physiological state (dying cells) have a high PC-fluorescence (green arrows), and dead cells have no fluorescence (white arrows). The high intensity in the shorter wavelength interval reflects that fluorescence in dying cells mainly originates from phycobilisomes at ca 650–660 nm. Healthy cells displayed weaker emission intensity over the whole spectral range, located at the periphery of the cells. This response correlates with effective light utilization and energy balance [26].

**Figure 5** represents confocal laser scanning photomicrographs illustrating photosynthetic pigment localization in filamentous cyanobacterium Phormidium CALU 624. False color overlay images were obtained by simultaneous three channel
