# Laser Scanning versus Wide-Field--Choosing the Appropriate Microscope in Life Sciences
## 6 Representative Applications  
### Live Cell Experiments (2D)  
Cells are commonly cultivated as two-dimensional (2D) monolayers on glass or plastic slides and are readily assessed by wide-field as well as by laser scanning microscopy. In some cases, they may provide results of limited significance due to nonphysiological conditions that directly affect relevant parameters (e.g., resistance to drugs). Conventional wide-field microscopy can be used for fluorescence or light scattering experiments, if the information originates from thin cell layers of only a few micrometers, e.g., autofluorescence measurements (Figure 6a) or fluorescence measurements of dye-staining organelles, e.g., mitochondria (Figure 6b). However, for a numerical aperture \(\mathrm{A_{N}}=0.75\) (typical for a 40\(\times\) magnifying objective lens), the depth of focus \(\Delta\mathrm{z}=\mathrm{n}\lambda/\mathrm{A_{N}}^{2}\) is less than 1 micrometer, so confocal laser scanning microscopy (CLSM) may be advantageous even for experiments concerning cell monolayers, when high axial resolution is required. Total-Internal-Reflection Microscopy (TIRFM), with an evanescent electromagnetic wave penetrating about 100 nm into a cell, is an appropriate wide-field method for measuring the surface of 2D cell cultures, e.g., for studies of their plasma membrane or membrane proximal sites. An example is given in Figure 6c for Enhanced Yellow Fluorescent Protein (EYFP) linked to Paxiline in focal adhesions, which are relevant for cell-substrate interactions. Figure 5: Principle of axial tomography. Optimization of the point-spread function in view of an isotropic high resolution (**a**,**b**); MCF-7 breast carcinoma cell incubated with 20 nm quantum dots (**c**); fluorescence under 0\({}^{\circ}\) (top view) and 90\({}^{\circ}\) (side view calculated from a z-stack at 0\({}^{\circ}\) or measured after rotation, (**d**); laser scanning microscopy using a 63/0.9 water immersion objective lens; \(\mathrm{A_{ex}}=488\) nm, \(\mathrm{\lambda_{em}}\geq 560\) nm. Previously, TIRFM has been combined with super-resolution or super-localization microscopy, e.g., for selection of thin layers for single-molecule detection. There are also reports in the literature on a combination of TIRFM with SIM, when two interfering laser beams are focused close to the edge of the aperture of high-An objective lenses (A\({}_{\text{N}}\geq\) 1.45), so that they are totally reflected on the sample [23; 24; 25]. In this case, high lateral and high axial resolution are combined on the surface of a specimen, resulting in an isotropic resolution of around 100 nm. An example is given in Figure 7 for the stimulus-dependent translocation of the glucose transporter GLUT4 (fused with green Fluorescent Protein, GFP) from intracellular storage compartments to the plasma membrane. Insulin or the insulin-mimetic phytochemical compounds tannic acid, as well as _Bellis perennis_ extract, were used as stimulating agents [49]. As depicted in Figure 7 (left), we observed fluorescence arising from small vesicles and--in particular, after stimulation--from larger areas within the plasma membrane by SIM. When we measured the plasma membrane almost exclusively by SIM-TIRFM (Figure 7, right), we observed a pronounced increase in fluorescence over the whole cell surface after stimulation. Based on the fact that the penetration depth d(\(\Theta\)) of the evanescent electromagnetic wave depends on the angle of incidence \(\Theta\), we previously calculated the fluorescence  
Figure 6: Wide-field fluorescence microscopy of 2D cell cultures: (**a**) Autofluorescence of U251-MG glioblastoma cells (dominated by the coenzyme NADH); (**b**) BKEz-7 endothelial cells incubated with the fluorescent marker rhodamine 123 (5 uM, 45 min. ); (**c**) HeLa cervical carcinoma cells transfected with enhanced yellow fluorescent protein (EYFP) linked to Paxiline upon Total-Internal-Reflection (TIR) excitation (wide-field microscopy with a 40\(\times\)/0.75 microscope objective lens).