# Laser Scanning versus Wide-Field--Choosing the Appropriate Microscope in Life Sciences
## 6 Representative Applications
### Live Cell Experiments (3D)  
Single-cell experiments are not restricted to 2D specimens, i.e., cells growing on microscope slides or in Petri dishes. Cells can also be embedded in gels like agarose, e.g., in micro-capillaries, and possibly grow under more realistic conditions. In this case, Optical  
Figure 8: (**a**) Illumination device for variable-angle TIRFM; (**b**) single TIRFM image of U251-MG glioblastoma cells incubated with the fluorescence marker laurdan (8 μm; 60 min) for \(\theta\) = 66\({}^{\circ}\); (**c**) cell–substrate distances in the range of 0–600 nm calculated from variable-angle TIRFM for the U251-MG wild type, and (**d**) the same for U251-MG with activated TP53 suppressor gene. Excitation wavelength: \(\lambda_{0}\) = 391 nm, detection range: \(\lambda_{\text{D}}\)\(\geq\) 420 nm; image size: 210 μm \(\times\) 210 μm. Reproduced from Ref. [51] with modifications. Sectioning Structured Illumination Microscopy (OS-SIM) or Confocal Laser Scanning Microscopy (CLSM) would be appropriate methods to detect them in the optimal vertical position. With rotatable capillaries, they can even be observed from various sides, and as the lateral resolution in microscopy is always better than the axial resolution, the vertical axis can be rotated into the horizontal position to profit from an enhanced resolution (axial tomography with isotropic resolution), as described above. Three-dimensional (3D) cell assemblies, e.g., multicellular spheroids, closely resemble in vivo tissue in terms of cell physiology, cell metabolism, and development of extracellular matrices. Therefore, they offer more realistic conditions for studies of cell metabolism or tissue diagnostics. Cell spheroids are generally characterized by an external proliferating region and an internal quiescent zone (caused by the gradient of nutrient and oxygen diffusion), which may surround a necrotic core. Especially, multicellular (tumor) spheroids have gained significance in preclinical studies. Several techniques are available for the cultivation of multi-cellular specimens, mostly based on preventing cell attachment to surfaces, e.g., hanging drop methods, liquid overlay methods, or agitation-based approaches [52]. In addition, cell cultivation in a solid matrix, e.g., gel, as reported in the literature for about 30 years [53; 54], may permit 3D cell growth under realistic conditions. For measurements of 3D cell cultures, a variety of specific sample holders, e.g., glass or plastic tubes or even micro-capillaries, is available for wide-field and laser scanning microscopy. Light Sheet Fluorescence Microscopy (LSFM) is an appropriate tool for imaging (larger) cell assemblies, as it reduces light exposure of a single cell layer by illuminating only the plane of interest (focal plane). When using micro-capillaries as sample holders, the benefits of axial tomography (see above) can be applied to 3D cell cultures. LSFM can also be combined with microfluidics in order to apply nutrients, pharmaceutical agents, or fluorescent dyes prior to or during the measurement [4; 8]. The distribution of the cytostatic agent doxorubicin in a cell spheroid 6 h after incubation is depicted in Figure 9 for two different layers, as well as in a 3D reconstruction. While doxorubicin (red fluorescence) accumulates preferentially in the peripheral regions of the spheroid, its degradation product (green-yellow color) is mainly located in its central part, e.g., within cell membranes. A combination of 3D live cell microscopy with spectral imaging and fluorescence lifetime imaging has recently been presented [55]. In a sensor for apoptosis, nonradiative energy transfer (FRET) occurs from Enhanced Cyan Fluorescent Protein (ECFP) anchored in the plasma membrane to Enhanced Yellow Fluorescent Protein (EYFP) coupled to ECF via a specific peptide linker. Upon apoptosis, this linker is cleaved by the enzyme caspase-3, and the energy transfer is disrupted.