Editing Confocal microscopy (section)

==Resolution enhancement==
{{refimprove section|date = October 2024}}
CLSM is a scanning imaging technique in which the [[angular resolution|resolution]] obtained is best explained by comparing it with another scanning technique like that of the [[scanning electron microscope]] (SEM). CLSM has the advantage of not requiring a probe to be suspended nanometers from the surface, as in an [[atomic force microscope|AFM]] or [[scanning tunneling microscope|STM]], for example, where the image is obtained by scanning with a fine tip over a surface. The distance from the objective lens to the surface (called the ''working distance'') is typically comparable to that of a conventional optical microscope. It varies with the system optical design, but working distances from hundreds of micrometres to several millimeters are typical.

In CLSM a specimen is illuminated by a point laser source, and each volume element is associated with a discrete scattering or fluorescence intensity. Here, the size of the scanning volume is determined by the spot size (close to [[diffraction]] limit) of the optical system because the image of the scanning laser is not an infinitely small point but a three-dimensional diffraction pattern. The size of this diffraction pattern and the focal volume it defines is controlled by the [[numerical aperture]] of the system's objective lens and the wavelength of the laser used. This can be seen as the classical resolution limit of conventional optical microscopes using wide-field illumination. However, with confocal microscopy it is even possible to improve on the resolution limit of wide-field illumination techniques because the confocal aperture can be closed down to eliminate higher orders of the diffraction pattern{{Citation needed|date=January 2016}}. For example, if the pinhole diameter is set to 1 [[Airy disk|Airy unit]] then only the first order of the diffraction pattern makes it through the aperture to the detector while the higher orders are blocked, thus improving resolution at the cost of a slight decrease in brightness. In fluorescence observations, the resolution limit of confocal microscopy is often limited by the [[signal-to-noise ratio]] caused by the small number of photons typically available in fluorescence microscopy. One can compensate for this effect by using more sensitive photodetectors or by increasing the intensity of the illuminating laser point source. Increasing the intensity of illumination laser risks excessive bleaching or other damage to the specimen of interest, especially for experiments in which comparison of fluorescence brightness is required. When imaging tissues that are differentially refractive, such as the spongy mesophyll of plant leaves or other air-space containing tissues, spherical aberrations that impair confocal image quality are often pronounced. Such aberrations however, can be significantly reduced by mounting samples in optically transparent, non-toxic [[perfluorocarbon]]s such as [[perfluorodecalin]], which readily infiltrates tissues and has a refractive index almost identical to that of water.<ref>{{Cite journal|last1=Littlejohn|first1=George R.|last2=Gouveia|first2=João D.|last3=Edner|first3=Christoph|last4=Smirnoff|first4=Nicholas|last5=Love|first5=John|date=2010|title=Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll|journal=New Phytologist|language=en|volume=186|issue=4|pages=1018–1025|doi=10.1111/j.1469-8137.2010.03244.x|pmid=20374500|bibcode=2010NewPh.186.1018L |issn=1469-8137|hdl=10026.1/9344|hdl-access=free}}</ref> When imaging in a reflection geometry, it is also possible to detect the interference of the reflected and scattered light of an object like an intracellular organelle. The interferometric nature of the signal allows to reduce the pinhole diameter down to 0.2 Airy units and therefore enables an ideal resolution enhancement of √2 without sacrificing the signal-to-noise ratio as in confocal fluorescence microscopy.<ref>{{cite journal |last1=Küppers |first1=Michelle |last2=Albrecht |first2=David |last3=Kashkanova |first3=Anna D. |last4=Lühr |first4=Jennifer |last5=Sandoghdar |first5=Vahid |title=Confocal interferometric scattering microscopy reveals 3D nanoscopic structure and dynamics in live cells |journal=Nature Communications |date=7 April 2023 |volume=14 |issue=1 |pages=1962 |doi=10.1038/s41467-023-37497-7 |pmid=37029107 |language=en |issn=2041-1723|pmc=10081331 |bibcode=2023NatCo..14.1962K }}</ref>