**4. Conclusion**

*Color Detection*

**56**

**Figure 12.**

*coefficient is indicated on the plot.*

The first image (**Figure 12(a)**(1) was recorded prior to bleaching, by scanning the confocal spot in the XY mode. Excitation wavelength was at 633 nm, and the detection range was from 650 to 750 nm. The scan was then switched to another mode, the laser power was increased by a factor of 12, and the sample was bleached by scanning the laser repeatedly in one dimension across the cell for 1 s. Light-blue vertical rectangle indicates bleaching area. Then the laser power was reduced again to the initial value, the scan was switched back to the XY mode, and the cell was imaged by scanning over a square of 12.30 × 12.30 μm in the x-y plane (512 × 512 pixels) (**Figure 12(a)**(2–3). There was no detectable photobleaching during the recording of successive image scans. A series of 20 further images were recorded at time intervals 1.3 s and then another 10 images at time intervals 5 s. Images (a3) and (a4) show the state at 20th and 30th point (10.2 and 71.1 s, correspondingly). Within a few minutes, the bleaching profile spreads, becoming broader and shallower. This shows that phycobilisomes are

*Extracting data from a FRAP experiment on a cell of Microcystis CALU 398. (a) Fluorescence intensity images from FRAP-sequence, taken at −5, 0, 10.4, and 71.1 s, correspondingly. ROI 1 (light blue rectangular): bleaching area and ROI 2 (orange rectangular): the area for controlling fluorescence intensity profile across bleaching region. Scale bar = 1 μm. (b) Total fluorescence intensity of bleached area as a function of time. The recovery of the fluorescence is presented as open circles and exponential fitting function—solid line. (c) Pre-bleach and post-bleach fluorescence intensity profiles across bleaching region at t = 0, 1.3, 10.4, 22.1, and 71.1 s. (d) The subtracted postbleach and pre-bleach fluorescence intensity profiles and corresponding Gaussian fitting curves at five time-points indicated at (c). (d) A plot of C-function for one-dimensional diffusion versus time. The obtained diffusion* 

diffusing, with unbleached phycobilisomes diffusing into the bleached area.

Light microscopy has been used for studying cells for many years and has advanced our understanding of key cellular processes. However, fixation involves nonphysiological procedures and only provides a snapshot view of cells at a single point in time. To truly understand cellular function, we need to extend our imaging capabilities in ways that enable us to follow sequential events in real time, monitor the kinetics of dynamic processes, and record sensitive or transient events. And CLSM gives such opportunity. In addition, CLSM technique allows the investigation not only cultivable, but also the noncultivable phototrophic microorganisms.

This study presents simple and practical advices for performing special confocal microscopy applications. Our approach allows to study detailed mechanisms of photoprotection and stress reactions in different cyanobacterial species. Fluorescence spectra of cyanobacterial photosynthetic pigments are easily recorded by spectral CLSM. The fluorescence shares of individual phycobiliproteins can be reliably determined by spectral unmixing, showing that the spectral resolution of CLSM is well suited for this approach.

Finally, with the advent of live cell imaging and the development of high- and superresolution technologies, it is now possible to acquire data on viable cells in a biologically relevant context providing us with a greater insight of cellular function than has previously been possible.

Note here, that there are a lot of additional techniques already implemented in modern CLSMs, which open new perspectives for single-cell investigation, such as: white laser, which provides the ability to obtain not only fluorescence emission spectra, but also single-cell excitation and absorption spectra [55]; hyperspectral CLSM which allows more precise fluorescence spectra through the cell thickness and gives more detailed fluorescent pigments location [42]; STED and multiphotonic techniques, that extend the CLSM abilities to single-molecular studies [56, 57]. They are the subject for further investigations.
