# Laser Scanning versus Wide-Field--Choosing the Appropriate Microscope in Life Sciences
## 2 Wide-Field Microscopy
If a single molecule is detected n times, its localization can be determined with a precision \(\Delta\)x = \(\Delta\)x\({}_{0}\)/\(\surd\)n with \(\Delta\)x\({}_{0}\)\(\approx\) 200 nm resulting from the Rayleigh criterion. Therefore, localization occurs with a precision \(\Delta\)x = 20 nm for n = 100 and a precision \(\Delta\)x = 10 nm for n = 400. Methods based on super-localization microscopy include Stochastic Optical Reconstruction Microscopy (STORM), Photoactivation Localization Microscopy (PALM), and related techniques [9; 10; 11]. However, in comparison with conventional fluorescence microscopy, the irradiance in single-molecule experiments is typically 500 to 1000 times higher, which results in a high risk of phototoxic damage. Super-resolution Microscopy by (coherent) Structured Illumination (SR-SIM) [12; 13] avoids this high light exposure and appears to be a promising alternative for live cell microscopy, particularly in the cases of longer exposure times or repeated measurements. A periodical illumination pattern permits transmission of spatial frequencies, which are up to a factor of 2 higher than for conventional microscopy, resulting in a two-fold enhancement of resolution in comparison with the Abbe criterion. While illumination by three laser beams creates an interference pattern with high spatial resolution in all three dimensions [14], illumination by two laser beams, e.g., the first diffraction orders of an optical grating or a spatial light modulator (SLM) [15; 16], creates an interference pattern in two dimensions, if these laser beams can be rotated to (at least) three different positions. Calculation of a super-resolved image commonly needs 9-15 single images with structured illumination of different phase and rotation angles [14; 15; 16]. This can be achieved by the fast scanning of a diffraction element with galvano mirrors, as described in [17] and further realized in a commercial system (Elyra 7, Carl Zeiss Microscopy Deutschland GmbH, Oberkochen, Germany). Use of a (switchable) SLM, however, obviates any movable component and seems to be a promising alternative. As an example of live cell microscopy, the intracellular distribution of the glucose transporter GLUT4 [18; 19] transfected with green Fluorescent Protein (GFP) and stimulated by insulin is depicted in Figure 3, and a region of interest is compared for wide-field microscopy and SIM. While GLUT4 accumulates in intracellular vesicles, as well as in the plasma membrane, Figure 3 demonstrates an enhancement of resolution by a factor of 2 due to SIM in the plasma membrane. It should be emphasized that calculation of SIM images requires original images of sufficiently high contrast, using, e.g., fluorescent dyes or fluorescent proteins. Total Internal Reflection Fluorescence Microscopy (TIRFM) permits the selective detection of surfaces, e.g., cell membranes, in the 100 nm range, and by variation of the angle of incidence, an axial resolution of only a few nanometers becomes possible. This permits the study of cell-substrate topology, e.g., upon application of cytotoxic or phototoxic agents  
Figure 3: Fluorescence image of CHO-K1-hIR-myc-GLUT4-GFP cells upon stimulation by insulin (1 \(\upmu\)M; 60 min): (**a**) Wide-field microscopy with Wiener filter; (**b**) comparison of filtered wide-field (upper part) and super-resolution microscopy by (coherent) structured illumination (SR-SIM) (lower part) for a selected field; (**c**) line scan with Gaussian fit over a marked range of 0.8 \(\upmu\)m across the plasma membrane for filtered wide-field and SIM; excitation wavelength: \(\lambda_{0}\) = 488 nm; detection range: \(\lambda_{0}\)\(\geq\) 515 nm; Plan Neofluar 40\(\times\)/1.30 oil immersion objective lens. or in the case of diseases. \"Prism-type TIRF\" with light incidence via a glass or quartz prism whose refractive index exceeds that of the specimen has been used for live cell measurements for almost 40 years [20,21], while \"Objective-type TIRF\" only became available 20 years later.