# Laser Scanning versus Wide-Field--Choosing the Appropriate Microscope in Life Sciences
## 4 Comparison of Wide-Field and Laser Scanning Microscopy  
As mentioned before, conventional wide-field microscopy has no inherent optical sectioning capability, but there are several techniques available to overcome this limitation. With OS-SIM, axial optical sections can be made as thin as 200 nanometers with a high-numerical-aperture objective lens and a fine grid spacing. While the two-beam configuration of SR-SIM provides super-resolution in the lateral direction, three-beam SR-SIM additionally doubles the resolution in the axial direction. Using a lattice pattern rather than grid lines gives higher contrast and a more robust image reconstruction, which allows deep tissue imaging up to 100 \(\upmu\)m. Excessive illumination may cause light-induced damage that can alter cell physiology and, therefore, lead to incorrect results, especially in long-term experiments. To avoid photo-toxicity during and subsequent to experiments, light exposure should be kept as low as possible. Although structured illumination requires the acquisition of 9-15 individual images, photo-toxicity is still low in comparison with other super-resolution methods. While an irradiance of about 100 mW/cm\({}^{2}\)--corresponding to solar irradiance--is sufficient for SIM, a considerably higher irradiance is necessary for single-molecule methods (where each molecule is excited repeatedly), as well as for stimulated emission in STED microscopy (for an overview, see [27]). Light Sheet Microscopy offers an optical section thickness ranging from about 1 \(\upmu\)m (Lattice Light Sheet: dz = 280 nm [35]) up to about 15 \(\upmu\)m, depending on the setup and the relevant application. With a comparably high penetration depth, most light-sheet systems are suitable for imaging applications over different scales: From single cells over cell assemblies (e.g., spheroids) up to small organisms. The main advantage of light sheet microscopy, in comparison with laser scanning techniques, is that light exposure is restricted to the examined planes of a specimen. Therefore, light sheet microscopy is ideal for low-light exposure, low photo-toxicity, and long-term observation of samples. The primary advantage of laser scanning microscopy is the ability to produce serially thin optical sections of about 0.4 to 1.5 \(\upmu\)m, depending on the numerical aperture, excitation wavelength, and pinhole size. Typical penetration depths for multiphoton LSM are 250-500 \(\upmu\)m, although imaging as deep as 1 mm has been reported in the literature [36], compared to \(\sim\)100 \(\upmu\)m for CLSM. While the detection with CLSM is limited to the focal plane, a large sample volume is exposed to optical excitation, and significant photobleaching or photo-toxicity is likely to occur in living specimens. Simultaneous detection of multiple sample points and improved detection systems may reduce photo-toxicity by reducing the acquisition time and light exposure. With multiphoton excitation, phototoxic damage may be restricted to the laser focus, thus reducing the impact on other regions of the sample. For a comparison of wide-field and laser scanning techniques, see Table 1.  
The acquisition of color images is an essential aspect of many biological applications. Wide-field methods offer a wide range of detection modes, including monochrome cameras for video-rate imaging with high sensitivity or color options for full RGB imaging. Laser scanning methods employ highly sensitive photomultipliers or cameras (spinning disk) to generate intensity-based images. For multicolor/pseudo-color imaging, multi-channel excitation and/or emission devices are available (for further information, see [37]). In summary, conventional wide-field, OS-SIM, or CLSM, including Airy Scan, appear appropriate for 2D specimens or samples of low thickness, when the number of light exposures remains limited. For 3D specimens, particularly those that do not require very high resolution, light sheet microscopy appears to be an appropriate method, e.g., if multiple exposures or long measuring periods are desired.