# Laser Scanning versus Wide-Field--Choosing the Appropriate Microscope in Life Sciences
## 2 Wide-Field Microscopy  
Samples in life sciences include two-dimensional and three-dimensional cell cultures, biopsies, and living organisms with (at least) a minimum of optical transparency. In conventional wide-field microscopy, an image is generated from the focal plane of a sample with superimposed out-of-focus information that severely diminishes spatial contrast. As this method does not provide any possibilities of sectioning per se, 3D microscopyneeds some special equipment. One method is Optical Sectioning Structured Illumination Microscopy (OS-SIM), where an optical grid is imaged in the plane of the sample in three phase positions \(\Phi_{1}=0\), \(\Phi_{2}=2\pi/3\), and \(\Phi_{3}=4\pi/3\), resulting in the intensities I\({}_{1}\), I\({}_{2}\), and I\({}_{3}\). An algorithm I = [(I\({}_{1}-I\)\({}_{2}\))\({}^{2}+(I\)\({}_{1}-I\)\({}_{3}\))\({}^{2}+(I\)\({}_{2}-I\)\({}_{3}\))\({}^{2}]^{1/2}\) permits calculation of an image of the focal plane, while out-of-focus images add up to zero, as depicted in Figure 1. Upon variation of the sample's vertical position, several focal planes can thus be detected, and a 3D image with high contrast may be calculated [3]. Another method is Light Sheet Microscopy, where a sample is illuminated from the side (at 90\({}^{\circ}\) with respect to the microscope axis). The light sheet and the microscope objective lens used for detection can be shifted simultaneously in the axial direction, so that the illuminated part of the sample is always at the focus of the objective lens (both shifts may be different due to the refractive index of the immersion fluid, but this can be corrected either mechanically [4] or by software). Thus, z-stacks can be recorded, and a 3D image may be calculated with low fluorescence background and high contrast, as depicted in Figure 2. For illumination, Gaussian-shaped laser beams may be either scanned or used in combination with a cylindrical lens. Alternatively, a lattice of so-called Bessel beams can be applied to generate an extremely thin light sheet, as further reported in Section 4. The main advantage of the Light Sheet over OS-SIM is that only those planes are illuminated, which are recorded simultaneously, so that light exposure is considerably lower than for those methods, where the recording of each image requires illumination of the whole specimen. Commercial light-sheet microscopes (e.g., Carl Zeiss, Olympus, Nikon), as well as open-source solutions or add-ons for existing microscopes, are presently available [5; 6; 7]. Resolution is commonly based on the Abbe criterion y\({}_{\text{min}}\)\(\geq\)\(\lambda\)/2A\({}_{\text{N}}\) (for coherent illumination with \(\lambda\) representing the wavelength of light and A\({}_{\text{N}}\) the numerical aperture of the microscope objective lens), or on the [PERSON] criterion y\({}_{\text{min}}\) = 0.61 \(\lambda\)/A\({}_{\text{N}}\) representing the radius of the diffraction maximum of an incoherently luminescent spot (Airy disk). In both cases, values around 200 nm are attained for high numerical apertures (A\({}_{\text{N}}\)\(\geq\) 1.30) and around 400-500 nm for moderate apertures (A\({}_{\text{N}}\)\(\leq\) 0.60), e.g., when imaging larger objects with long-working-distance objective lenses. Only in the last 20 years could these values be reduced by super-resolution or super-localization methods. The latter are based on single-molecule detection within a thin illuminated layer of a sample. Figure 1: Principle of Optical-Sectioning Structured Illumination Microscopy (OS-SIM). Figure 2: 3D imaging of CHO-pAcGFP1-Memcell spheroids with membrane-associated Green Fluorescent Protein (GFP). Light Sheet Fluorescence Microscopy (LSFM) using rectangular micro-capillaries (scheme, two single planes and 3D reconstruction); beam waist: \(\Delta z\) = 5-10 μm, focal depth: \(\Delta y\) = 100–200 μm (reproduced from Ref. [8] with modifications).