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| title | chunk | source | category | tags | date_saved | instance |
|---|---|---|---|---|---|---|
| Confocal microscopy | 3/4 | https://en.wikipedia.org/wiki/Confocal_microscopy | reference | science, encyclopedia | 2026-05-05T10:04:03.194579+00:00 | kb-cron |
== Uses == CLSM is widely used in various biological science disciplines, from cell biology and genetics to microbiology and developmental biology. It is also used in quantum optics and nano-crystal imaging and spectroscopy.
=== Biology and medicine ===
Clinically, CLSM is used in the evaluation of various eye diseases, and is particularly useful for imaging, qualitative analysis, and quantification of endothelial cells of the cornea. It is used for localizing and identifying the presence of filamentary fungal elements in the corneal stroma in cases of keratomycosis, enabling rapid diagnosis and thereby early institution of definitive therapy. Research into CLSM techniques for endoscopic procedures (endomicroscopy) is also showing promise. In the pharmaceutical industry, it was recommended to follow the manufacturing process of thin film pharmaceutical forms, to control the quality and uniformity of the drug distribution. Confocal microscopy is also used to study biofilms — complex porous structures that are the preferred habitat of microorganisms. Some of temporal and spatial function of biofilms can be understood only by studying their structure on micro- and meso-scales. The study of microscale is needed to detect the activity and organization of single microorganisms.
=== Optics and crystallography === CLSM is used as the data retrieval mechanism in some 3D optical data storage systems and has helped determine the age of the Magdalen papyrus.
=== Audio preservation === The IRENE system makes use of confocal microscopy for optical scanning and recovery of damaged historical audio. Wax phonograph cylinders inherently degrade due to contact with the stylus when replayed as intended; microscopy is non-contact, and often gives better sound than stylus playback.
=== Material's surface characterization === Laser scanning confocal microscopes are used in the characterization of the surface of microstructured materials, such as Silicon wafers used in solar cell production. During the first processing steps, wafers are wet-chemically etched with acid or alkaline compounds, rendering a texture to their surface. Laser confocal microscopy is then used to observe the state of the resulting surface at the micrometer lever. Laser confocal microscopy can also be used to analyze the thickness and height of metallization fingers printed on top of solar cells.
== Variants and enhancements ==
=== Improving axial resolution === The point spread function of the pinhole is an ellipsoid, several times as long as it is wide. This limits the axial resolution of the microscope. One technique of overcoming this is 4Pi microscopy where incident and or emitted light are allowed to interfere from both above and below the sample to reduce the volume of the ellipsoid. An alternative technique is confocal theta microscopy. In this technique the cone of illuminating light and detected light are at an angle to each other (best results when they are perpendicular). The intersection of the two point spread functions gives a much smaller effective sample volume. From this evolved the single plane illumination microscope. Additionally deconvolution may be employed using an experimentally derived point spread function to remove the out of focus light, improving contrast in both the axial and lateral planes.
=== Super resolution === There are confocal variants that achieve resolution below the diffraction limit such as stimulated emission depletion microscopy (STED). Besides this technique a broad variety of other (not confocal based) super-resolution techniques are available like PALM, (d)STORM, SIM, and so on. They all have their own advantages such as ease of use, resolution, and the need for special equipment, buffers, or fluorophores.
=== Low-temperature operability === To image samples at low temperatures, two main approaches have been used, both based on the laser scanning confocal microscopy architecture. One approach is to use a continuous flow cryostat: only the sample is at low temperature and it is optically addressed through a transparent window. Another possible approach is to have part of the optics (especially the microscope objective) in a cryogenic storage dewar. This second approach, although more cumbersome, guarantees better mechanical stability and avoids the losses due to the window.
=== Molecular interaction === To study molecular interactions using the CLSM Förster resonance energy transfer (FRET) can be used to confirm that two proteins are within a certain distance to one another.
== Images ==
== History ==
=== The beginnings: 1940–1957 ===
In 1940 Hans Goldmann, ophthalmologist in Bern, Switzerland, developed a slit lamp system to document eye examinations. This system is considered by some later authors as the first confocal optical system. In 1943 Zyun Koana published a confocal system. In 1951 Hiroto Naora, a colleague of Koana, described a confocal microscope in the journal Science for spectrophotometry. The first confocal scanning microscope was built by Marvin Minsky in 1955 and a patent was filed in 1957. The scanning of the illumination point in the focal plane was achieved by moving the stage. No scientific publication was submitted and no images made with it were preserved.
=== The Tandem-Scanning-Microscope ===
In the 1960s, the Czechoslovak Mojmír Petráň from the Medical Faculty of the Charles University in Plzeň developed the Tandem-Scanning-Microscope, the first commercialized confocal microscope. It was sold by a small company in Czechoslovakia and in the United States by Tracor-Northern (later Noran) and used a rotating Nipkow disk to generate multiple excitation and emission pinholes. The Czechoslovak patent was filed 1966 by Petráň and Milan Hadravský, a Czechoslovak coworker. A first scientific publication with data and images generated with this microscope was published in the journal Science in 1967, authored by M. David Egger from Yale University and Petráň. As a footnote to this paper it is mentioned that Petráň designed the microscope and supervised its construction and that he was, in part, a "research associate" at Yale. A second publication from 1968 described the theory and the technical details of the instrument and had Hadravský and Robert Galambos, the head of the group at Yale, as additional authors. In 1970 the US patent was granted. It was filed in 1967.