Confocal microscopy
Confocal Microscopy | |
---|---|
MeSH | D018613 |
OPS-301 code | 3-301 |
Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing
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
The principle of confocal imaging was patented in 1957 by
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 (servo controlled) oscillating mirrors. This scanning method usually has a low reaction latency and the scan speed can be varied. Slower scans provide a better signal-to-noise ratio, resulting in better contrast.
The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the
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).[1]
Confocal microscopy provides the capacity for direct, noninvasive, serial
Techniques used for horizontal scanning
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 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.[5]
Programmable array microscopes (PAM) use an electronically controlled
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 rates 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[6] – 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[7] 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.[8]
Resolution enhancement
CLSM is a scanning imaging technique in which the
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
Uses
CLSM is widely used in various
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
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.[15]
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 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
Super resolution
There are confocal variants that achieve resolution below the diffraction limit such as
Low-temperature operability
To image samples at low temperatures, two main approaches have been used, both based on the
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
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Partial surface profile of a 1-Euro coin, measured with a Nipkow disk confocal microscope.
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Reflection data for 1-Euro coin.
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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 um.
History
The beginnings: 1940–1957
In 1940 Hans Goldmann,
In 1943 Zyun Koana published a confocal system.[21][19]
In 1951 Hiroto Naora, a colleague of Koana, described a confocal microscope in the journal Science for spectrophotometry.[22]
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.[2][23]
The Tandem-Scanning-Microscope
In the 1960s, the
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.[25] 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.[26] In 1970 the US patent was granted. It was filed in 1967.[27]
1969: The first confocal laser scanning microscope
In 1969 and 1971, M. David Egger and Paul Davidovits from
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,[30] 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.[19][31]
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.[32] It is probably the first publication using the term "confocal microscope".[19][31]
In 1978, the brothers
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.[31]
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.[34] 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.[20][35]
Starting 1985: Laser point scanners with beam scanning
In the mid-1980s,
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.[39] The venture was acquired in 1990 by Molecular Dynamics,[40] 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,[41] mentioned some manufacturers of confocals: Sarastro, Technical Instrument, Meridian Instruments, Bio-Rad, Leica, Tracor-Northern and Zeiss.[34]
In 1989, Fritz Karl Preikschat, with his son Ekhard Preikschat, invented the scanning laser diode microscope for particle-size analysis.[42][43] and co-founded Lasentec to commercialize it. In 2001, Lasentec was acquired by Mettler Toledo.[44] 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.[45]
Other researchers have attempted to digitally refocus the light from a point excitation source using deep convolutional neural networks.[46]
See also
- 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)
- Two-photon excitation microscopy: 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). This diaphragm is usually absent in multiphoton microscopes due to difficulties descanning the beam.
References
- ^ ISBN 0-387-25921-X.
- ^ a b US 3013467, Minsky, Marvin, "Microscopy apparatus", published 1961-12-19
- ^ Memoir on Inventing the Confocal Scanning Microscope, Scanning 10 (1988), pp128–138.
- ^ a b Fellers TJ, Davidson MW (2007). "Introduction to Confocal Microscopy". Olympus Fluoview Resource Center. National High Magnetic Field Laboratory. Retrieved 2007-07-25.
- ^ US 5162941, Favro, Lawrence D.; Thomas, Robert L. & Kuo, Pao-Kuang et al., "Confocal microscope", published 1992-11-10, assigned to The Board of Governors of Wayne State University
- ^ "Data Sheet of NanoFocus µsurf spinning-disk confocal white light microscope". Archived from the original on 2014-01-20. Retrieved 2013-08-14.
- ^ "Data Sheet of Sensofar 'PLu neox' Dual technology sensor head combining confocal and Interferometry techniques, as well as Spectroscopic Reflectometry".
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- ISBN 978-1-68108-519-7. Archived from the originalon 14 May 2019. Retrieved 24 December 2017.
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- ^ The Digitization Process. Project IRENE, University of California, Berkeley Libraries.
- PMID 21133476.
- PMID 20886985.
- . Note: Volume 98 is assigned to the year 1939, however on the first page of the article January 1940 is listed as publication date.
- ^ a b c d Colin JR Sheppard (3 November 2009). "Confocal Microscopy. The Development of a Modern Microscopy". Imaging & Microscopy.online
- ^ ISBN 978-0-8194-6118-6, S. 120–121.
- ^ Zyun Koana (1942). Journal of the Illumination Engineering Institute. 26 (8): 371–385.
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(help) The article is available on the 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. - PMID 14866220.
- .
- ISBN 0-8493-3919-7, pages 115–122.
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- ^ US 3517980, Petran, Mojmir & Hadravsky, Milan, "Method and arrangement for improving the resolving power and contrast", published 1970-06-30, assigned to Ceskoslovenska akadamie
- S2CID 4161644.
- PMID 20111173.
- ISBN 978-0-8194-6118-6, pp. 124–125.
- ^ ISBN 978-0-387-25921-5.
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- ^ S2CID 34919506.
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- ^ Anon (2005). "Dr John White FRS". royalsociety.org. London: Royal Society. Archived from the original on 2015-11-17.
- S2CID 34919506.
- PMID 19724343.
- ^ Brent Johnson (1 February 1999). "Image Is Everything". The Scientist. online
- ^ Diana Morgan (23 July 1990). "Confocal Microscopes Widen Cell Biology Career Horizons". The Scientist. online
- ^ US 4871251, Preikschat, Fritz K. & Preikschat, Ekhard, "Apparatus and method for particle analysis", published 1989-10-03
- ^ US 5012118, Preikschat, Fritz K. & Preikschat, Ekhard, "Apparatus and method for particle analysis", published 1991-04-30
- ^ reserved, Mettler-Toledo International Inc. all rights. "Particle Size Distribution Analysis". Archived from the original on 2016-10-09. Retrieved 2016-10-06.
- ^ Weisshart, Klaus. "The Basic Principle of Airyscanning" (PDF). asset-downloads.zeiss.com. Retrieved 6 September 2023.
- PMID 37143962.
- 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. 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.