**3.2 3D bio-imaging**

In addition to the possibility to observe a single plane (or slice) of a thick specimen in good contrast, optical sectioning allows a great number of slices to be cut and recorded at different planes of the specimen, with the specimen being moved along the optical axis (Z) by controlled increments. The result is a 3D data set, which provides information about the spatial structure of the object. The quality and accuracy of this information depend on the thickness of the slice and on the spacing between successive slices.

Once a 3D stack of images has been recorded, the user has various presentation options. The data may be displayed as a gallery of depth-coded images or as orthogonal projections of the XY, XZ, and YZ planes. To create a 3D impression on a 2D monitor, animations of different viewing angles versus time, 3D reconstruction, shadow projections, and surface rendering techniques are possible.

**51**

should be acquired.

**Figure 8.**

**Figure 9.**

**3.3 Single-cell spectroscopy (lambda scanning)**

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

In **Figure 8**, several two-dimensional optical sections of a filament of Anabaena

CALU 824, obtained by Carl Zeiss LSM 710 with LSM Software ZEN 2009, are shown. A series of XY images (z-stack), acquired in different focus positions represents the Z dimension of the specimen. Here we present images from two channels: green—PE-fluorescence, red—Chl a-fluorescence, and the bottom panel—two channels superimposed. To calculate 3D model from z-stack, much more sections

*Several frames from animation of 3D-model at different viewing angles. Two colors correspond to two fluorescent channels: green channel—PE-fluorescence and red channel—Chl a-fluorescence.*

*Pigment localization through the depth. Two-dimensional sections of Anabaena CALU 824. Green channel:* 

*PE-fluorescence, red channel: Chl a-fluorescence, and bottom panel: two channels superimposed.*

**Figure 9** shows several frames from 3D reconstruction animation computed from a 3D data set in ImageJ 1.46r software (http://imagej.net/). To render 3D-model z-stack containing 80 images were recorded with 0.1 μm increment along z-axis.

Fluorescence spectra of living photosynthetic cells can be reliably analyzed by a microspectrofluorometric method (CLSM spectroscopy), which is implemented

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

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

### **Figure 8.**

*Color Detection*

**Figure 7.**

of pigments from certain organisms may differ crucially from the properties of the dissolved ones, for example, spectra of the components can vary in peak widths and may be shifted in wavelength due to the different pigment-protein and linker connections. In this case, only hyperspectral CLSM measurements [42] can give

*Fluorescence spectra in (e) have the colors corresponding to (a) and (b). Scale bar = 5 μm.*

*Simultaneous observation of self-fluorescent photosynthetic cells of Microcystis CALU 398 and labeled by quantum dots extracellular polysaccharide. (a) Channel 1: quantum dots fluorescence (green), (b) channel 2: natural pigment fluorescence (red), (d) channel 3: corresponding transmission image, (d) all three channels superimposed.* 

If the overlapping of the emission spectra of the fluorescent components is not very much, then the image spectral unmixing can be easily obtained without any calculations. The example of such unmixing is presented in **Figure 7**. In **Figure 7**, the usage of combination of natural pigment fluorescence and fluorescence probes for imaging of cyanobacteria and their extracellular polymeric substances (polysaccharides) is demonstrated. To target extracellular substances, fluorescent labels should be used. Here, nonfluorescent polysaccharides were labeled by quantum dots CdSe/ZnS. Quantum dots have a fluorescence in green (520–580 nm) when excited by ultraviolet light (405 nm) and photosynthetic pigments of Microcystis CALU 398 have a fluorescence in red (620–780 nm). Thus the fluorescent signals will not overlap. The reference spectra for quantum dots and Microcystis CALU 398

In addition to the possibility to observe a single plane (or slice) of a thick specimen in good contrast, optical sectioning allows a great number of slices to be cut and recorded at different planes of the specimen, with the specimen being moved along the optical axis (Z) by controlled increments. The result is a 3D data set, which provides information about the spatial structure of the object. The quality and accuracy of this information depend on the thickness of the slice and on the

Once a 3D stack of images has been recorded, the user has various presentation options. The data may be displayed as a gallery of depth-coded images or as orthogonal projections of the XY, XZ, and YZ planes. To create a 3D impression on a 2D monitor, animations of different viewing angles versus time, 3D reconstruction,

suitable reference spectra for decomposition.

cells were obtained by means of lambda scanning (**Figure 7(e)**).

shadow projections, and surface rendering techniques are possible.

**50**

**3.2 3D bio-imaging**

spacing between successive slices.

*Pigment localization through the depth. Two-dimensional sections of Anabaena CALU 824. Green channel: PE-fluorescence, red channel: Chl a-fluorescence, and bottom panel: two channels superimposed.*

### **Figure 9.**

*Several frames from animation of 3D-model at different viewing angles. Two colors correspond to two fluorescent channels: green channel—PE-fluorescence and red channel—Chl a-fluorescence.*

In **Figure 8**, several two-dimensional optical sections of a filament of Anabaena CALU 824, obtained by Carl Zeiss LSM 710 with LSM Software ZEN 2009, are shown. A series of XY images (z-stack), acquired in different focus positions represents the Z dimension of the specimen. Here we present images from two channels: green—PE-fluorescence, red—Chl a-fluorescence, and the bottom panel—two channels superimposed. To calculate 3D model from z-stack, much more sections should be acquired.

**Figure 9** shows several frames from 3D reconstruction animation computed from a 3D data set in ImageJ 1.46r software (http://imagej.net/). To render 3D-model z-stack containing 80 images were recorded with 0.1 μm increment along z-axis.
