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| title | chunk | source | category | tags | date_saved | instance |
|---|---|---|---|---|---|---|
| Confocal microscopy | 4/4 | https://en.wikipedia.org/wiki/Confocal_microscopy | reference | science, encyclopedia | 2026-05-05T10:04:03.194579+00:00 | kb-cron |
=== 1969: The first confocal laser scanning microscope === In 1969 and 1971, M. David Egger and Paul Davidovits from Yale University, published two papers describing the first confocal laser scanning microscope. It was a point scanner, meaning just one illumination spot was generated. It used epi-Illumination-reflection microscopy for the observation of nerve tissue. A 5 mW Helium-Neon-Laser with 633 nm light was reflected by a semi-transparent mirror towards the objective. The objective was a simple lens with a focal length of 8.5 mm. As opposed to all earlier and most later systems, the sample was scanned by movement of this lens (objective scanning), leading to a movement of the focal point. Reflected light came back to the semitransparent mirror, the transmitted part was focused by another lens on the detection pinhole behind which a photomultiplier tube was placed. The signal was visualized by a CRT of an oscilloscope, the cathode ray was moved simultaneously with the objective. A special device allowed to make Polaroid photos, three of which were shown in the 1971 publication. The authors speculate about fluorescent dyes for in vivo investigations. They cite Minsky's patent, thank Steve Baer, at the time a doctoral student at the Albert Einstein School of Medicine in New York City where he developed a confocal line scanning microscope, for suggesting to use a laser with 'Minsky's microscope' and thank Galambos, Hadravsky and Petráň for discussions leading to the development of their microscope. The motivation for their development was that in the Tandem-Scanning-Microscope only a fraction of 10−7 of the illumination light participates in generating the image in the eye piece. Thus, image quality was not sufficient for most biological investigations.
=== 1977–1985: Point scanners with lasers and stage scanning === In 1977 Colin J. R. Sheppard and Amarjyoti Choudhury, Oxford, UK, published a theoretical analysis of confocal and laser-scanning microscopes. It is probably the first publication using the term "confocal microscope". In 1978, the brothers Christoph Cremer and Thomas Cremer published a design for a confocal laser-scanning-microscope using fluorescent excitation with electronic autofocus. They also suggested a laser point illumination by using a "4π-point-hologramme". This CLSM design combined the laser scanning method with the 3D detection of biological objects labeled with fluorescent markers for the first time. In 1978 and 1980, the Oxford-group around Colin Sheppard and Tony Wilson described a confocal microscope with epi-laser-illumination, stage scanning and photomultiplier tubes as detectors. The stage could move along the optical axis (z-axis), allowing optical serial sections. In 1979 Fred Brakenhoff and coworkers demonstrated that the theoretical advantages of optical sectioning and resolution improvement are indeed achievable in practice. In 1985 this group became the first to publish convincing images taken on a confocal microscope that were able to answer biological questions. Shortly after many more groups started using confocal microscopy to answer scientific questions that until then had remained a mystery due to technological limitations. In 1983 I. J. Cox and C. Sheppard from Oxford published the first work whereby a confocal microscope was controlled by a computer. The first commercial laser scanning microscope, the stage-scanner SOM-25 was offered by Oxford Optoelectronics (after several take-overs acquired by BioRad) starting in 1982. It was based on the design of the Oxford group.
=== Starting 1985: Laser point scanners with beam scanning === In the mid-1980s, William Bradshaw Amos and John Graham White and colleagues working at the Laboratory of Molecular Biology in Cambridge built the first confocal beam scanning microscope. The stage with the sample was not moving, instead the illumination spot was, allowing faster image acquisition: four images per second with 512 lines each. Hugely magnified intermediate images, due to a 1–2 meter long beam path, allowed the use of a conventional iris diaphragm as a 'pinhole', with diameters ~1 mm. First micrographs were taken with long-term exposure on film before a digital camera was added. A further improvement allowed zooming into the preparation for the first time. Zeiss, Leitz and Cambridge Instruments had no interest in a commercial production. The Medical Research Council (MRC) finally sponsored development of a prototype. The design was acquired by Bio-Rad, amended with computer control and commercialized as 'MRC 500'. The successor MRC 600 was later the basis for the development of the first two-photon-fluorescent microscope developed 1990 at Cornell University. Developments at the KTH Royal Institute of Technology in Stockholm around the same time led to a commercial CLSM distributed by the Swedish company Sarastro. The venture was acquired in 1990 by Molecular Dynamics, but the CLSM was eventually discontinued. In Germany, Heidelberg Instruments, founded in 1984, developed a CLSM, which was initially meant for industrial applications rather than biology. This instrument was taken over in 1990 by Leica Lasertechnik. Zeiss already had a non-confocal flying-spot laser scanning microscope on the market which was upgraded to a confocal. A report from 1990, mentioned some manufacturers of confocals: Sarastro, Technical Instrument, Meridian Instruments, Bio-Rad, Leica, Tracor-Northern and Zeiss. In 1989, Fritz Karl Preikschat, with his son Ekhard Preikschat, invented the scanning laser diode microscope for particle-size analysis. and co-founded Lasentec to commercialize it. In 2001, Lasentec was acquired by Mettler Toledo. They are used mostly in the pharmaceutical industry to provide in-situ control of the crystallization process in large purification systems.
=== 2010s: Computational methods for removing the output pinhole === In standard confocal instruments, the second or "output" pinhole is utilized to filter out the emitted or scattered light. Traditionally, this pinhole is a passive component that blocks light to filter the illumination optically. However, newer designs have tried to perform this filtering digitally. Recent approaches have replaced the passive pinhole with a compound detector element. Typically, after digital processing, this approach leads to better resolution and photon budget, as the resolution limit can approach that of an infinitely small pinhole. Other researchers have attempted to digitally refocus the light from a point excitation source using deep convolutional neural networks.
== See also ==
Two-photon excitation microscopy
== References ==
Hoffman, David P.; Shtengel, Gleb; Xu, C. Shan; Campbell, Kirby R.; Freeman, Melanie; Wang, Lei; Milkie, Daniel E.; Pasolli, H. Amalia; Iyer, Nirmala; Bogovic, John A.; Stabley, Daniel R.; Shirinifard, Abbas; Pang, Song; Peale, David; Schaefer, Kathy; Pomp, Wim; Chang, Chi-Lun; Lippincott-Schwartz, Jennifer; Kirchhausen, Tom; Solecki, David J.; Betzig, Eric; Hess, Harald F. (2020). "Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells". Science. 367 (6475) eaaz5357. doi:10.1126/science.aaz5357. ISSN 0036-8075. PMC 7339343. PMID 31949053.
== External links ==
Virtual CLSM (Java-based) Animations and explanations on various types of microscopes including fluorescent and confocal microscopes (Université Paris Sud) Parts need to know.