# Laser Scanning versus Wide-Field--Choosing the Appropriate Microscope in Life Sciences
## 3 Laser Scanning Microscopy  
Laser scanning methods often use the principle of Confocal Laser Scanning Microscopy (CLSM) with apertures rejecting out-of-focus light. This results in an enhanced contrast, reduced noise, and high axial resolution (optical sectioning). The thickness of an optical section is commonly adapted to the depth of focus (wavelength of excitation light divided by the square of the numerical aperture of the objective lens). Information (e.g., fluorescence) from individual planes can be used for 3D image reconstruction. In a point-scanning confocal laser scanning microscope, a focused laser beam is scanned over the sample, and the emitted fluorescence light is imaged on a small pinhole in front of a light detector, e.g., photomultiplier, to eliminate out-of-focus light, as depicted in Figure 4. In this configuration, the confocal image of a specimen is reconstructed point by point, which considerably limits the acquisition speed. By exciting and collecting light from more points simultaneously, a higher acquisition speed is possible with similar optical sectioning and resolution, e.g., with a line-scanning configuration, where the excitation beam is focused to a line instead of a point. A considerably larger improvement was achieved with Spinning Disk Confocal Microscopy [26]. Here, a point-scanning configuration is used, with hundreds of micro-lenses arranged in spirals on a rotating disk, which focus the laser beam to pinholes on a second disk (Nipkow disk). By scanning the pinholes across the sample in rows, spinning disk microscopy vastly improves the speed of image acquisition (up to 1000 or 2000 frames per second) and, therefore, considerably reduces total light exposure and photo-damage. Lateral resolution results from the diffraction-limited laser spot, as defined by the Rayleigh criterion. If the pinhole diameter is larger than the diame  
Figure 4: Principle of a confocal laser scanning microscope (CLSM); only a confocal beam passes the pinhole, while non-confocal beams are blocked (reproduced from Ref. [27] with modifications). disk, it differs only very little from wide-field microscopy (with the relevant wavelength being the excitation rather than the emission wavelength). If the pinhole is smaller, the resolution results from a convolution of the so-called point spread function in the plane of the sample and another point spread function defined by the pinhole. A smaller pinhole allows higher resolution but significantly lowers the collected emission intensity. Image Scanning Microscopy (ISM) overcomes this problem by replacing the pinhole by a fast EM-CCD camera chip to detect the Airy disk with a large number of \"infinitely small pinholes,\" thus increasing the resolution by up to a factor of 2 without losing intensity. ISM can be combined with spinning disk microscopy to reduce acquisition time [28] and may offer prospects for super-resolution imaging in deep tissue sections when combined with two-photon excitation [29]. The principle of using an array of very small pinholes has also been applied in Airy Scan Microscopy, where the Airy disc is detected by 32 single detector elements, thus providing improved resolution (typically by a factor 1.7) and increased sensitivity with reduced acquisition times compared to conventional CLSM. A further approach is Re-scan Confocal Microscopy (RCM) [30] with another laser scanning process over the pinhole. This technique maintains the high lateral and axial resolution of Image Scanning Microscopy, but is very sensitive, as it works with an open pinhole and uses highly sensitive and low-noise detectors. In contrast to CLSM, multiphoton microscopy achieves optical sectioning without using a pinhole [31; 32]. By focusing picosecond or femtosecond laser pulses onto a small spot in the sample, fluorescence is excited by two or more photons exclusively in the small focal volume. This permits scanning of different sample planes and restricts photo-damage to the focus of multiphoton excitation.