**3.4 Using fluorescence recovery after photobleaching (FRAP)**

A field of growing importance is the investigation of living specimens that show dynamic changes. Dynamic processes in living specimens can be recorded by means of time series implemented in modern CLSMs. Data once acquired can be analyzed "offline," that is, after image acquisition. Time series are defined by a start time and the time interval between two successive images. To analyze a time series, some option allows fluorescence intensity changes to be quantified in defined regions of interest (ROIs).

Within a time series, the CLSM permits selective, point-accurate illumination of ROIs with laser light. This function is useful for generating a photobleaching routine, for example, within a FRAP experiment (fluorescence recovery after photobleaching), for analyzing dynamic processes. Complex time series experiments, with different images to be taken at different sites within a specimen according to a defined time pattern, can be carried out by means of CLSM software.

Fluorescence recovery after photobleaching (FRAP) is a technique widely used in cell biology to observe the dynamics of biological systems. In photosynthetic organisms, it can be directly used, for example, for investigation of the dynamics of thylakoid membranes, including the diffusion of membrane components [39, 48–52].

Diffusion of membrane components is involved in a number of processes, for example, in investigation if the chromatic adaptation of light-harvesting apparatus, in describing of the electron transport in photosynthetic membranes and in studies of the biogenesis, turnover, and repair of the photosystems. Diffusion coefficients for certain thylakoid membrane components can also be estimated by indirect methods [53, 54], but FRAP and related optical techniques may be used for direct observation of the diffusion in biological systems.

The component whose diffusion is to be observed must be bleached by high laser power with the confocal spot scanning briefly over a small area of the sample. After bleaching, the laser power is decreased, and the whole sample is imaged. The bleached area of the sample will be seen as a dark, nonfluorescent patch on the image. Then the sample is repeatedly imaged, and if the bleaching will change

**55**

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

somehow during the time, the diffusion coefficient can be derived from these characteristic changes [48, 49]. Usually the component whose diffusion is to be observed must be tagged with a fluorophore, but, as it was mentioned above, the great advantage of photosynthetic membranes is that their protein complexes are naturally fluorescent, so their diffusion can be observed without any necessity for specific fluorophore binding or GFP gene fusions. The only requirements for quantitative FRAP are that the membrane geometry is known, and the membrane environment is uniform over an area considerably larger than the area of the bleach. Unfortunately, most green plant thylakoid membranes have an intricate, convoluted structure, and extensive lateral heterogeneity on small scales. On the other hand, some cyanobacterial species have the membrane systems located in the periphery of the cell and are uniform enough in small regions. Anyway, by means of FRAP technique, it is always possible to see if particular components are mobile or not, to make a rough estimation of time-scales and to investigate a number of factors that

In contrast to typical green plants, in most cyanobacterial strains, there is no thylakoid membrane stacking and no extensive lateral heterogeneity. The preferred model organism used in previously published papers [39, 48, 49] is the cyanobacterium Synechococcus sp. PCC7942, which has suitable membrane conformation and is also well-characterized and transformable. Its cells can be elongated up to 10–20 μm by means of some growth techniques to achieve lateral heterogeneity in relatively large region. But in this work, we demonstrate that reasonable results can

It is well known that phycobilisomes diffuse rapidly on the surface of the thylakoid membrane, while PS II reaction centers are normally almost immobile. Here we used FRAP to measure the mobility of phycobilisomes in the intact cyanobacterial cells. These experiments indicate that phycobilisomes may frequently decouple from reaction centers and diffuse randomly before attaching to another reaction center. FRAP measurements were carried out with a laser-scanning confocal microscope Leica TCS-SP5. During the measurement, a series of images of the cell is recorded, and it is important to keep the laser power low enough not to cause significant further bleaching of the cell during repeated imaging. The basic procedure for the measurement is to record an image of the sample cell before bleaching (**Figure 12(a)**(1). Then the laser power is increased by a factor of 8–10, and the confocal spot is scanned only over a restricted area of the sample (light-blue vertical rectangle) to bleach out most of the fluorescence in that area. The laser power is then reduced again, and a series of post-bleach images is recorded. The best timescale for the measurement depends on the rate of diffusion and has to be found by trial and error. In thylakoid membranes, the diffusion of both lipids and proteins is generally rather slow and a 1 s bleach, followed by a series of images recorded at

**Figure 12** summarizes the steps involved in recording and analyzing FRAP data. In the presented experiment, a one-dimensional diffusion of phycobilisomes was measured in a cell of the cyanobacterium Microcystis firma CALU 398. Confocal laser scanning microscope Leica TCS-SP5 with immersion objective HCX PL APO 63.0x1.30 GLYC 37°C UV was utilized in this investigation. The He-Ne laser (633 nm, 150 mW) was used for excitation in both bleaching and scanning modes, and the laser power was 25 and 2% correspondingly. The vertical (Z) resolution was 0.2 μm, and the resolution in the x-y plane was about 24.1 nm. Digital zoom was 20. The laser was scanned over the sample with oscillating mirrors. Scan speed was 400 Hz. Fluorescence from the sample was separated from the excitation light with a beamsplitter, passed through a 102.9 μm pinhole and detected with a photomultiplier with PMT voltage 1000 V. The emission band for recorded images was 673–678 nm.

be obtained even for spherical cells of Microcystis firma CALU 398.

1.3 s intervals, was adequate to capture diffusion.

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

affect their diffusion coefficient.
