View source for Confocal microscopy

{{Short description|Optical imaging technique}}
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  Name        = Confocal Microscopy |
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  MeshID      = D018613 |
  OPS301      = {{OPS301|3-301}} |
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[[File:Fluorescent and confocal microscopes.ogg|thumb|upright=1.5|Fluorescence and confocal microscopes operating principle]]

'''Confocal microscopy''', most frequently '''confocal laser scanning microscopy''' ('''CLSM''') or '''laser scanning confocal microscopy''' ('''LSCM'''), is an optical imaging technique for increasing [[optical resolution]] and [[contrast (vision)|contrast]] of a [[micrograph]] by means of using a [[Spatial filter|spatial pinhole]] to block out-of-focus light in image formation.<ref name="Pawley-2006">{{cite book |editor=Pawley JB  |title=Handbook of Biological Confocal Microscopy |publisher=Springer |location=Berlin |year=2006 |edition = 3rd  |isbn=0-387-25921-X}}</ref> Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as [[optical sectioning]]) within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in [[life sciences]], [[semiconductor]] inspection and [[materials science]].

Light travels through the sample under a conventional microscope as far into the specimen as it can penetrate, while a confocal microscope only focuses a smaller beam of light at one narrow depth level at a time. The CLSM achieves a controlled and highly limited depth of field.

== Basic concept ==
{{refimprove section|date = October 2024}}
[[File:Minsky Confocal Reflection Microscope.png|thumb|Confocal point sensor principle from Minsky's patent]]

[[Image:MP-30-GFP.jpg|thumb|[[Green Fluorescent Protein|GFP]] fusion protein being expressed in ''Nicotiana benthamiana''. The fluorescence is visible by confocal microscopy.]]

The principle of confocal imaging was patented in 1957 by [[Marvin Minsky]]<ref name="Minsky-1961">{{cite patent|country=US|number=3013467|pubdate=1961-12-19|title=Microscopy apparatus|inventor1-last=Minsky|inventor1-first=Marvin}}</ref> and aims to overcome some limitations of traditional wide-field [[fluorescence microscope]]s.<ref>[http://web.media.mit.edu/~minsky/papers/ConfocalMemoir.html Memoir on Inventing the Confocal Scanning Microscope], ''Scanning'' '''10''' (1988), pp128–138.</ref> In a conventional (i.e., wide-field) [[fluorescence microscope]], the entire [[Laboratory specimen|specimen]] is flooded evenly in light from a light source. All parts of the sample can be excited at the same time and the resulting [[fluorescence]] is detected by the microscope's [[photodetector]] or [[camera]] including a large unfocused background part. In contrast, a confocal microscope uses point illumination (see [[Point Spread Function]]) and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal – the name "confocal" stems from this configuration. As only light produced by fluorescence very close to the [[focal plane]] can be detected, the image's [[optical resolution]], particularly in the sample depth direction, is much better than that of wide-field microscopes. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity – so long [[exposure (photography)|exposure]]s are often required. To offset this drop in signal after the ''pinhole'', the light intensity is detected by a sensitive detector, usually a [[photomultiplier]] tube (PMT) or [[avalanche photodiode]], transforming the light signal into an electrical one.<ref name="Fellers-2007">{{cite web |vauthors=Fellers TJ, Davidson MW | title = Introduction to Confocal Microscopy | work = Olympus Fluoview Resource Center | publisher = National High Magnetic Field Laboratory | year = 2007 | url = http://www.olympusconfocal.com/theory/confocalintro.html | access-date = 2007-07-25}}</ref>

As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen. The beam is scanned across the sample in the horizontal plane by using one or more ([[servomechanism|servo]] controlled) oscillating mirrors. This scanning method usually has a low reaction [[Latency (engineering)|latency]] and the scan speed can be varied. Slower scans provide a better [[signal-to-noise ratio]], resulting in better [[Contrast (vision)|contrast]].

The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the [[numerical aperture]] of the [[objective lens]], but also by the optical properties of the specimen. The thin [[optical sectioning]] possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples.

Successive slices make up a 'z-stack', which can either be processed to create a 3D image, or it is merged into a 2D stack (predominately the maximum pixel intensity is taken, other common methods include using the standard deviation or summing the pixels).<ref name="Pawley-2006" />

Confocal microscopy provides the capacity for direct, noninvasive, serial [[optical sectioning]] of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution compared to wide-field microscopy.<ref name="Fellers-2007" /> Biological samples are often treated with [[fluorophore|fluorescent dyes]] to make selected objects visible. However, the actual dye concentration can be low to minimize the disturbance of biological systems: some instruments can track single fluorescent molecules. Also, [[transgenic]] techniques can create organisms that produce their own fluorescent chimeric molecules (such as a fusion of GFP, [[green fluorescent protein]] with the protein of interest). Confocal microscopes work on the principle of point excitation in the specimen (diffraction limited spot) and point detection of the resulting fluorescent signal. A pinhole at the detector provides a physical barrier that blocks out-of-focus fluorescence. Only the in-focus, or central spot of the [[Airy disk]], is recorded.

== Techniques used for horizontal scanning ==
{{refimprove section|date = October 2024}}
[[File:Diatom chain.jpg|thumb|upright=2| This projection of multiple confocal images, taken at the [[European Molecular Biology Laboratory|EMBL]] light microscopy facility, shows a group of [[diatom]]s with cyan cell walls, red chloroplasts, blue DNA, and green membranes and organelles]]

Four types of confocal microscopes are commercially available:

'''Confocal laser scanning microscopes''' use multiple mirrors (typically 2 or 3 scanning linearly along the x- and the y- axes) to scan the laser across the sample and "descan" the image across a fixed pinhole and detector. This process is usually slow and does not work for live imaging, but can be useful to create high-resolution representative images of [[Fixation (histology)|fixed]] samples.

'''Spinning-disk''' ([[Nipkow disk]]) confocal microscopes use a series of moving pinholes on a disc to scan spots of light. Since a series of pinholes scans an area in parallel, each pinhole is allowed to hover over a specific area for a longer amount of time thereby reducing the excitation energy needed to illuminate a sample when compared to laser scanning microscopes. Decreased excitation energy reduces [[phototoxicity]] and [[photobleaching]] of a sample often making it the preferred system for imaging live cells or organisms.

'''Microlens enhanced''' or '''dual spinning-disk''' confocal microscopes work under the same principles as spinning-disk confocal microscopes except a second spinning-disk containing micro-lenses is placed before the spinning-disk containing the pinholes. Every pinhole has an associated microlens. The micro-lenses act to capture a broad band of light and focus it into each pinhole significantly increasing the amount of light directed into each pinhole and reducing the amount of light blocked by the spinning-disk. Microlens enhanced confocal microscopes are therefore significantly more sensitive than standard spinning-disk systems. [[Yokogawa Electric]] invented this technology in 1992.<ref>{{cite patent|country=US|number=5162941|pubdate=1992-11-10|title=Confocal microscope|assign1=[[Wayne_State_University|The Board of Governors of Wayne State University]]|inventor1-last=Favro|inventor1-first=Lawrence D.|inventor2-last=Thomas|inventor2-first=Robert L.|inventor3-last=Kuo|inventor3-first=Pao-Kuang|inventor4-last=Chen|inventor4-first=Li}}</ref>

'''Programmable array microscopes (PAM)''' use an electronically controlled [[spatial light modulator]] (SLM) that produces a set of moving pinholes. The SLM is a device containing an array of pixels with some property ([[Opacity (optics)|opacity]], [[reflectivity]] or [[optical rotation]]) of the individual pixels that can be adjusted electronically. The SLM contains [[microelectromechanical systems|microelectromechanical mirror]]s or [[liquid crystal]] components. The image is usually acquired by a [[charge-coupled device]] (CCD) camera.

Each of these classes of confocal microscope have particular advantages and disadvantages. Most systems are either optimized for recording speed (i.e. video capture) or high spatial resolution. Confocal laser scanning microscopes can have a programmable sampling density and very high resolutions while Nipkow and PAM use a fixed sampling density defined by the camera's resolution. Imaging [[frame rate]]s are typically slower for single point laser scanning systems than spinning-disk or PAM systems. Commercial spinning-disk confocal microscopes achieve frame rates of over 50 per second<ref name="Nanofocus">{{cite web | title=Data Sheet of NanoFocus ''µsurf'' spinning-disk confocal white light microscope | url=http://www.nanofocus.com/products/usurf/usurf-explorer/ | access-date=2013-08-14 | archive-url=https://web.archive.org/web/20140120120344/http://www.nanofocus.com/products/usurf/usurf-explorer/ | archive-date=2014-01-20 | url-status=dead }}</ref> – a desirable feature for dynamic observations such as live cell imaging.

In practice, Nipkow and PAM allow multiple pinholes scanning the same area in parallel<ref name="Sensofar">{{cite web | title=Data Sheet of Sensofar 'PLu neox' Dual technology sensor head combining confocal and Interferometry techniques, as well as Spectroscopic Reflectometry| url=http://www.sensofar.com/products/products_neox.html}}</ref> as long as the pinholes are sufficiently far apart.

Cutting-edge development of confocal laser scanning microscopy now allows better than standard video rate (60 frames per second) imaging by using multiple microelectromechanical scanning mirrors.

Confocal [[X-ray fluorescence]] imaging is a newer technique that allows control over depth, in addition to horizontal and vertical aiming, for example, when analyzing buried layers in a painting.<ref>{{cite journal | title=Confocal X-ray Fluorescence Imaging and XRF Tomography for Three Dimensional Trace Element Microanalysis | author=Vincze L | journal = Microscopy and Microanalysis | volume=11 | issue=Supplement 2 | year=2005 | doi=10.1017/S1431927605503167 | doi-access=free }}</ref>

==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>

==Uses==

CLSM is widely used in various [[biological science]] disciplines, from [[cell biology]] and [[genetics]] to [[microbiology]] and [[developmental biology]].<ref>{{cite book|author=Juan Carlos Stockert, Alfonso Blázquez-Castro|chapter=Chapter 6 Fluorescence Instrumental and Techniques|pages=180–184|title=Fluorescence Microscopy in Life Sciences|chapter-url=https://ebooks.benthamscience.com/book/9781681085180/|access-date=24 December 2017|year=2017|publisher=Bentham Science Publishers|isbn=978-1-68108-519-7|archive-date=14 May 2019|archive-url=https://web.archive.org/web/20190514131504/https://ebooks.benthamscience.com/book/9781681085180/|url-status=dead}}</ref> It is also used in quantum optics and nano-crystal imaging and spectroscopy.

===Biology and medicine===

[[File:STD Depth Coded Stack Slices through Cells.png|thumb|Example of a stack of confocal microscope images showing the distribution of actin filaments throughout a cell.]]
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]].<ref>{{cite journal |vauthors=Patel DV, McGhee CN |title=Contemporary in vivo confocal microscopy of the living human cornea using white light and laser scanning techniques: a major review |journal=Clin. Experiment. Ophthalmol. |volume=35 |issue=1 |pages=71–88 |year=2007 |pmid=17300580 |doi=10.1111/j.1442-9071.2007.01423.x|s2cid=23029612 }}</ref> It is used for localizing and identifying the presence of filamentary fungal elements in the [[cornea]]l stroma in cases of [[fungal keratitis|keratomycosis]], enabling rapid diagnosis and thereby early institution of definitive therapy. Research into CLSM techniques for [[endoscopic]] procedures ([[endomicroscopy]]) is also showing promise.<ref>{{cite journal |vauthors=Hoffman A, Goetz M, Vieth M, Galle PR, Neurath MF, Kiesslich R |title=Confocal laser endomicroscopy: technical status and current indications |journal=Endoscopy |volume=38 |issue=12 |pages=1275–83 |year=2006 |pmid=17163333 |doi=10.1055/s-2006-944813|s2cid=260134204 }}</ref> 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.<ref>{{cite journal |doi=10.1016/S0255-2701(98)00032-4|title=Near infrared drying of pharmaceutical thin films: Experimental analysis of internal mass transport|year=1998|last1=Le Person|first1=S.|last2=Puiggali|first2=J.R.|last3=Baron|first3=M.|last4=Roques|first4=M.|journal=Chemical Engineering and Processing: Process Intensification|volume=37|issue=3|pages=257–263|bibcode=1998CEPPI..37..257L |url=https://hal.archives-ouvertes.fr/hal-01632801/file/Near-infrared-drying-of-pharmaceutical-thin-films-experimental-analysis-of-internal-mass-transport.pdf }}</ref> Confocal microscopy is also used to study [[biofilm]]s — 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.<ref>{{Cite book|last1=Gitis|first1=Vitaly|title=Handbook of Porous Materials|last2=Rothenberg|first2=Gadi|publisher=World Scientific|year=2020|isbn=978-981-122-322-8|editor-last=Gitis|editor-first=Vitaly|location=Singapore|pages=63–64|doi=10.1142/11909|editor-last2=Rothenberg|editor-first2=Gadi}}</ref>

===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 (technology)|IRENE]] system makes use of confocal microscopy for optical scanning and recovery of damaged historical audio.<ref>[http://exhibits.lib.berkeley.edu/spotlight/project-irene/feature/the-digitization-process The Digitization Process]. Project IRENE, [[University of California, Berkeley Libraries]].</ref>

=== 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 [[Etching (microfabrication)|wet-chemically etch]] 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 Microscope|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 [[Light sheet fluorescence microscopy|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 microscopy|super-resolution techniques]] are available like [[Photoactivated localization microscopy|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 [[Cryostat#Types|continuous flow cryostat]]: only the sample is at low temperature and it is optically addressed through a transparent window.<ref>{{cite journal | last1 = Hirschfeld | first1 = V. | last2 = Hubner | first2 = C.G. | year = 2010 | title = A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K | journal = [[Review of Scientific Instruments]] | volume = 81 | issue = 11| pages =  113705–113705–7| doi = 10.1063/1.3499260 | pmid = 21133476 | bibcode = 2010RScI...81k3705H }}</ref> Another possible approach is to have part of the optics (especially the microscope objective) in a [[cryogenic storage dewar]].<ref>{{cite journal | last1 = Grazioso | first1 = F. | last2 = Patton | first2 = B. R. | last3 = Smith | first3 = J.M. | year = 2010 | title = A high stability beam-scanning confocal optical microscope for low temperature operation | journal = [[Review of Scientific Instruments]] | volume = 81 | issue = 9| pages = 093705–4 | doi = 10.1063/1.3484140 | pmid = 20886985 | bibcode = 2010RScI...81i3705G }}</ref> 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 ==

<gallery widths="325px" heights="225px">
File:Tetrachimena_Beta_Tubulin.png|[[Tubulin|β-tubulin]] in ''[[Tetrahymena]]'' (a ciliated [[protozoa]]n).
File:Confocal measurement of 1-euro-star 3d and euro.png|Partial surface profile of a 1-Euro coin, measured with a Nipkow disk confocal microscope.
File:Confocal measurement of 1-euro-star 3d Reflection.png|Reflection data for 1-Euro coin.
File:Depth Coded Phalloidin Stained Actin Filaments Cancer Cell.png|Colour coded image of [[actin]] filaments in a [[cancer]] cell.
File:Mitotic spindle in Arabidopsis primary root meristem cells (anaphase).tif| Green signal from anti-[[tubulin]] antibody conjugated with Alexa Fluor 488) and nuclei (blue signal from DNA stained with DAPI) in root meristem cells 4-day-old [[Arabidopsis thaliana]] (Col-0). Scale bar: 5&nbsp;um.
</gallery>

== History ==

=== The beginnings: 1940–1957 ===
[[File:Minski-confocal-patent-figure1.gif|thumbnail|upright=2|Scheme from Minsky's patent application showing the principle of the transmission confocal scanning microscope he built.]]
In 1940 Hans Goldmann, [[ophthalmologist]] in [[Bern]], Switzerland, developed a [[slit lamp]] system to document eye examinations.<ref name="Goldmann-1939">{{cite journal| author= Hans Goldmann| title= Spaltlampenphotographie und –photometrie| journal= Ophthalmologica | volume= 98| issue= 5/6| year= 1939| pages= 257–270| doi =10.1159/000299716}} Note: Volume 98 is assigned to the year 1939, however on the first page of the article January 1940 is listed as publication date.</ref> This system is considered by some later authors as the first confocal optical system.<ref name="Sheppard-2009">{{cite journal| author= Colin JR Sheppard| title= Confocal Microscopy. The Development of a Modern Microscopy| journal= Imaging & Microscopy|date=3 November 2009}}[http://www.imaging-git.com/science/light-microscopy/confocal-microscopy online]</ref><ref name="Masters-2006">Barry R. Masters: Confocal Microscopy And Multiphoton Excitation Microscopy. The Genesis of Live Cell Imaging. SPIE Press, Bellingham, Washington, USA 2006, {{ISBN|978-0-8194-6118-6}}, S. 120–121.</ref>

In 1943 Zyun Koana published a confocal system.<ref>{{cite journal| author= Zyun Koana| journal= Journal of the Illumination Engineering Institute | volume= 26 | issue= 8| year= 1942| pages= 371–385}} The article is available on the [https://www.jstage.jst.go.jp/browse/jieij1917 website of the journal]. The pdf-file labeled "P359 - 402" is 19,020 kilobytes in size and also contains neighboring articles from the same issue. Figure 1b of the article shows the scheme of a confocal transmission beam path.</ref><ref name="Sheppard-2009"/>

In 1951 Hiroto Naora, a colleague of Koana, described a confocal microscope in the journal [[Science (journal)|Science]] for [[spectrophotometry]].<ref>{{cite journal | last1 = Naora | first1 = Hiroto | year = 1951 | title = Microspectrophotometry and cytochemical analysis of nucleic acids. | journal = Science | volume = 114 | issue = 2959| pages = 279–280 | doi = 10.1126/science.114.2959.279 | pmid = 14866220 |bibcode = 1951Sci...114..279N }}</ref>

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.<ref name="Minsky-1961" /><ref name="Minsky-1988">{{cite journal| author= Marvin Minsky| title= Memoir on inventing the confocal scanning microscope| journal= Scanning| volume= 10| issue= 4| year= 1988| pages= 128–138| doi =10.1002/sca.4950100403| author-link= Marvin Minsky}}</ref>

=== The Tandem-Scanning-Microscope ===
[[File:Petran-Patent-Figure2-cutout.png|thumbnail|Scheme of Petráň's Tandem-Scanning-Microscope. Red bar added to indicate the Nipkow-Disk.]]
In the 1960s, the [[Czechoslovakia|Czechoslovak]] [[Mojmír Petráň]] from the Medical Faculty of the [[Charles University in Prague|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.<ref name="Masters-2006"/><ref>Guy Cox: Optical Imaging Techniques in Cell Biology. 1. edition. CRC Press, Taylor & Francis Group, Boca Raton, FL, USA 2006, {{ISBN|0-8493-3919-7}}, pages 115–122.</ref>

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.<ref name="Egger-1967">{{cite journal |vauthors=Egger MD, Petrăn M |title=New reflected-light microscope for viewing unstained brain and ganglion cells |journal=Science |volume=157 |issue=786 |pages=305–7 |date=July 1967  |pmid=6030094 |doi=10.1126/science.157.3786.305 |bibcode = 1967Sci...157..305E |s2cid=180450 }}</ref> 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.<ref name="Petran-1968">{{cite journal|author1=MOJMÍR PETRÁŇ |author2=MILAN HADRAVSKÝ |author3=M. DAVID EGGER |author4=ROBERT GALAMBOS | title= Tandem-Scanning Reflected-Light Microscope| journal= Journal of the Optical Society of America| volume= 58| issue= 5| year= 1968| pages= 661–664| doi =10.1364/JOSA.58.000661|bibcode = 1968JOSA...58..661P }}</ref> In 1970 the US patent was granted. It was filed in 1967.<ref>{{cite patent|country=US|number=3517980|pubdate=1970-06-30|title=Method and arrangement for improving the resolving power and contrast|assign1=[[Czechoslovak_Academy_of_Sciences|Ceskoslovenska akadamie]]|inventor1-last=Petran|inventor1-first=Mojmir|inventor2-last=Hadravsky|inventor2-first=Milan}}</ref>

=== 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.<ref name="Davidovits-1969">{{cite journal | last1 = Davidovits | first1 = P. | last2 = Egger | first2 = M. D. | year = 1969 | title = Scanning laser microscope. | journal = Nature | volume = 223 | issue = 5208| page = 831 | doi = 10.1038/223831a0 | pmid = 5799022 |bibcode = 1969Natur.223..831D | s2cid = 4161644 | doi-access = free }}</ref><ref name="Davidovits-1971">{{cite journal | last1 = Davidovits | first1 = P. | last2 = Egger | first2 = M. D. | year = 1971 | title = Scanning laser microscope for biological investigations. | journal = Applied Optics | volume = 10 | issue = 7| pages = 1615–1619 | doi = 10.1364/AO.10.001615 | pmid = 20111173 |bibcode = 1971ApOpt..10.1615D }}</ref> 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&nbsp;mW Helium-Neon-Laser with 633&nbsp;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&nbsp;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 [[Cathode-ray tube|CRT]] of an oscilloscope, the cathode ray was moved simultaneously with the objective. A special device allowed to make [[Polaroid film|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 College of Medicine|Albert Einstein School of Medicine]] in [[New York City]] where he developed a confocal line scanning microscope,<ref>Barry R. Masters: Confocal Microscopy And Multiphoton Excitation Microscopy. The Genesis of Live Cell Imaging. SPIE Press, Bellingham, Washington, USA 2006, {{ISBN|978-0-8194-6118-6}}, pp. 124–125.</ref> 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<sup>−7</sup> of the illumination light participates in generating the image in the eye piece. Thus, image quality was not sufficient for most biological investigations.<ref name="Sheppard-2009" /><ref name="Inoué-2006">{{cite book| author= Shinya Inoué | editor= James Pawley| title= Handbook of Biological Confocal Microscopy | url= https://archive.org/details/handbookbiologic00pawl | url-access= limited | edition= 3.| publisher= Springer Science and Business Media LLC| year= 2006| chapter= Chapter 1: Foundations of Confocal Scanned Imaging in Light Microscopy| pages= [https://archive.org/details/handbookbiologic00pawl/page/n26 1]–19| isbn= 978-0-387-25921-5}}</ref>

=== 1977–1985: Point scanners with lasers and stage scanning ===
In 1977 [[Colin Sheppard|Colin J. R. Sheppard]] and [[Amarjyoti Choudhury]], [[Oxford]], UK, published a theoretical analysis of confocal and laser-scanning microscopes.<ref name="Sheppard-1977">{{cite journal | last1 = Sheppard | first1 = C.J.R. | last2 = Choudhury | first2 = A. | year = 1977 | title = Image Formation in the Scanning Microscope. | journal = Optica Acta: International Journal of Optics | volume = 24 | issue = 10| pages = 1051–1073 | doi = 10.1080/713819421 | bibcode = 1977AcOpt..24.1051S }}</ref> It is probably the first publication using the term "confocal microscope".<ref name="Sheppard-2009" /><ref name="Inoué-2006" />

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]]".<ref name="Inoué-2006" /><ref>{{cite journal | last1 = Cremer | first1 = C. | last2 = Cremer | first2 = T. | year = 1978 | title = Considerations on a laser-scanning-microscope with high resolution and depth of field. | journal = Microscopica Acta | volume = 81 | issue = 1 | pages = 31–44 | pmid = 713859 }}</ref> This CLSM design combined the laser scanning method with the 3D detection of biological objects labeled with [[fluorescent marker]]s for the first time.

In 1978 and 1980, the Oxford-group around Colin Sheppard and [[Tony Wilson (scientist)|Tony Wilson]] described a confocal microscope with epi-laser-illumination, stage scanning and [[Photomultiplier|photomultiplier tubes]] as detectors. The stage could move along the optical axis (z-axis), allowing optical serial sections.<ref name="Inoué-2006" />

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.<ref name="Amos-2003">{{cite journal|last1=Amos|first1=W.B.|last2=White|first2=J.G.|author-link1=William Bradshaw Amos|author-link2=John Graham White|title=How the Confocal Laser Scanning Microscope entered Biological Research|journal=Biology of the Cell|volume=95|issue=6|year=2003|pages=335–342|pmid=14519550|doi=10.1016/S0248-4900(03)00078-9|s2cid=34919506|doi-access=free}}</ref> 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.<ref name="Masters-2006"/><ref name="Cox-1983">{{cite journal | last1 = Cox | first1 = I. J. | last2 = Sheppard | first2 = C. J. | year = 1983 | title = Scanning optical microscope incorporating a digital framestore and microcomputer. | journal = Applied Optics | volume = 22 | issue = 10| page = 1474 | pmid = 18195988 | doi=10.1364/ao.22.001474|bibcode = 1983ApOpt..22.1474C }}</ref>

=== 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.<ref name="White-1987">{{cite journal|last1=White|first1=J. G.|title=An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy|journal=The Journal of Cell Biology|volume=105|issue=1|year=1987|pages=41–48|issn=0021-9525|doi=10.1083/jcb.105.1.41|pmc=2114888|pmid=3112165}}</ref><ref name="Anon-2005">{{cite web|author=Anon|year=2005|archive-url=https://web.archive.org/web/20151117112042/https://royalsociety.org/people/john-white-12519/|archive-date=2015-11-17|url=https://royalsociety.org/people/john-white-12519/|publisher=[[Royal Society]]|location=London|title=Dr John White FRS|website=royalsociety.org}}</ref> 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&nbsp;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. [[Carl Zeiss AG|Zeiss]], [[Leica Microsystems|Leitz]] and [[Cambridge Instruments]] had no interest in a commercial production.<ref>{{Cite journal |doi = 10.1016/S0248-4900(03)00078-9|pmid = 14519550|title = How the Confocal Laser Scanning Microscope entered Biological Research|journal = Biology of the Cell|volume = 95|issue = 6|pages = 335–342|year = 2003|last1 = Amos|first1 = W.B.|last2 = White|first2 = J.G.|s2cid = 34919506|doi-access = free}}</ref> The [[Medical Research Council (United Kingdom)|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 excitation microscopy|two-photon-fluorescent microscope]] developed 1990 at [[Cornell University]].<ref name="Amos-2003"/>

Developments at the [[KTH Royal Institute of Technology]] in [[Stockholm]] around the same time led to a commercial CLSM distributed by the [[Sweden|Swedish]] company Sarastro.<ref>{{cite journal |last1=Carlsson |first1=K. |last2=Danielsson |first2=P.E. |last3=Lenz |first3=R. |last4=Liljeborg |first4=A. |last5=Majlöf |first5=L. |last6=Åslund |first6=N. |date=1985 |title=Three-dimensional microscopy using a confocal laser scanning microscope |journal=Optics Letters |volume=10 |issue=2 |pages=53–55 |doi=10.1364/OL.10.000053 |pmid=19724343 |bibcode=1985OptL...10...53C }}</ref> The venture was acquired in 1990 by Molecular Dynamics,<ref>{{cite journal| author= Brent Johnson| title= Image Is Everything| journal= The Scientist|date=1 February 1999}} [http://www.the-scientist.com/?articles.view/articleNo/19279/title/Image-Is-Everything/ online]</ref> 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 Microsystems|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,<ref>{{cite journal| author= Diana Morgan| title= Confocal Microscopes Widen Cell Biology Career Horizons| journal= The Scientist|date=23 July 1990 }} [http://www.the-scientist.com/?articles.view/articleNo/11261/title/Confocal-Microscopes-Widen-Cell-Biology-Career-Horizons/ online]</ref> mentioned some manufacturers of confocals: Sarastro, Technical Instrument, Meridian Instruments, Bio-Rad, Leica, Tracor-Northern and Zeiss.<ref name="Amos-2003" />

In 1989, [[Fritz Karl Preikschat]], with his son Ekhard Preikschat, invented the scanning [[laser diode]] microscope for particle-size analysis.<ref>{{cite patent|country=US|number=4871251|pubdate=1989-10-03|title=Apparatus and method for particle analysis|inventor1-last=Preikschat|inventor1-first=Fritz K.|inventorlink1=Fritz Karl Preikschat|inventor2-last=Preikschat|inventor2-first=Ekhard}}</ref><ref>{{cite patent|country=US|number=5012118|pubdate=1991-04-30|title=Apparatus and method for particle analysis|inventor1-last=Preikschat|inventor1-first=Fritz K.|inventorlink1=Fritz Karl Preikschat|inventor2-last=Preikschat|inventor2-first=Ekhard}}</ref> and co-founded Lasentec to commercialize it. In 2001, Lasentec was acquired by [[Mettler Toledo]].<ref>{{cite web|url=http://www.mt.com/us/en/home/products/L1_AutochemProducts/FBRM-PVM-Particle-System-Characterization.html|title=Particle Size Distribution Analysis|first=Mettler-Toledo International Inc. all rights|last=reserved|access-date=2016-10-06|archive-url=https://web.archive.org/web/20161009131635/http://www.mt.com/us/en/home/products/L1_AutochemProducts/FBRM-PVM-Particle-System-Characterization.html|archive-date=2016-10-09|url-status=dead}}</ref> 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.<ref>{{Cite web |last=Weisshart |first=Klaus |title=The Basic Principle of Airyscanning |url=https://asset-downloads.zeiss.com/catalogs/download/mic/104cc06d-f997-4cc0-b679-b2cf93bfc863/EN_wp_LSM-880_Basic-Principle-Airyscan.pdf |access-date=6 September 2023 |website=asset-downloads.zeiss.com}}</ref> 

Other researchers have attempted to digitally refocus the light from a point excitation source using deep convolutional neural networks.<ref>{{cite journal |last1=Xi |first1=Chen |last2=Mikhail |first2=Kandel |last3=Shenghua |first3=He |last4=Chenfei |first4=Hu |last5=Young Jae |first5=Lee |last6=Kathryn |first6=Sullivan |last7=Gregory |first7=Tracy |last8=Hee Jung |first8=Chung |last9=Hyun Joon |first9=Kong |last10=Mark |first10=Anastasio |last11=Gabriel |first11=Popescu |title=Artificial confocal microscopy for deep label-free imaging |journal=Nature Photonics |year=2023 |volume=17 |issue=3 |pages=250–258 |doi=10.1038/s41566-022-01140-6 |pmid=37143962 |pmc=10153546 |arxiv=2110.14823 |bibcode=2023NaPho..17..250C }}</ref>

==See also==
{{div col|colwidth=20em}}
<!-- please keep entries in alphabetical order -->
* [[Charge modulation spectroscopy]]
* [[Deconvolution]]
* [[Fluorescence microscope]]
* [[Focused ion beam]]
* [[Focus stacking]]
* [[Laser scanning confocal microscopy]]
* [[Live cell imaging]]
* [[Microscope objective lens]]
* [[Microscope slide]]
* [[Optical microscope]]
* [[Optical sectioning]]
* [[Photodetector]]
* [[Point spread function]]
* [[Stimulated emission depletion microscope]]
* [[Super-resolution microscopy]]
*[[Total internal reflection fluorescence microscope]] (TIRF)
{{div col end}}
<!-- please keep entries in alphabetical order -->
*[[Two-photon excitation microscopy]]<ref>Although they use a related technology (both are laser scanning microscopes), multiphoton fluorescence microscopes are not strictly confocal microscopes; the term ''confocal'' arises from the presence of a '''diaphragm''' in the '''conjugated focal plane''' (confocal) that is usually absent in multiphoton microscopes due to difficulties descanning the beam.{{says who|date = October 2024}}</ref>

==References==
{{Reflist}}
* {{cite journal|last1=Hoffman|first1=David P.|last2=Shtengel|first2=Gleb|last3=Xu|first3=C. Shan|last4=Campbell|first4=Kirby R.|last5=Freeman|first5=Melanie|last6=Wang|first6=Lei|last7=Milkie|first7=Daniel E.|last8=Pasolli|first8=H. Amalia|last9=Iyer|first9=Nirmala|last10=Bogovic|first10=John A.|last11=Stabley|first11=Daniel R.|last12=Shirinifard|first12=Abbas|last13=Pang|first13=Song|last14=Peale|first14=David|last15=Schaefer|first15=Kathy|last16=Pomp|first16=Wim|last17=Chang|first17=Chi-Lun|last18=Lippincott-Schwartz|first18=Jennifer|last19=Kirchhausen|first19=Tom|last20=Solecki|first20=David J.|last21=Betzig|first21=Eric|last22=Hess|first22=Harald F.|title=Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells|journal=Science|volume=367|issue=6475|year=2020|pages=eaaz5357|issn=0036-8075|doi=10.1126/science.aaz5357|pmid=31949053|pmc=7339343}}

== External links ==
{{Commons}}
{{Library resources box
 |onlinebooks=no
 |by=no
 |lcheading=Confocal microscopy}}
*[https://web.archive.org/web/20160602235702/http://micro.magnet.fsu.edu/primer/virtual/confocal/index.html Virtual CLSM] (Java-based)
*[http://toutestquantique.fr/en/microscopy/ Animations and explanations on various types of microscopes including fluorescent and confocal microscopes] (Université Paris Sud)
*[https://slidingmotion.com/microscope-parts-function-labeled-diagram/Microscope Parts] need to know.

{{Optical microscopy}}
{{Medical imaging}}
{{Lasers}}
{{Authority control}}

[[Category:Microscopy]]
[[Category:American inventions]]
[[Category:Cell imaging]]
[[Category:Scientific techniques]]
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[[Category:Optical microscopy techniques]]
[[Category:Fluorescence techniques]]