Confocal microscopy

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Confocal Microscopy
MeSHD018613
OPS-301 code3-301
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

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

Confocal point sensor principle from Minsky's patent
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

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 exposures 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.[4]

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

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).[1]

Confocal microscopy provides the capacity for direct, noninvasive, serial

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

This projection of multiple confocal images, taken at the EMBL light microscopy facility, shows a group of diatoms 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 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

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 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

AFM or 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

perfluorocarbons such as perfluorodecalin, which readily infiltrates tissues and has a refractive index almost identical to that of water.[9]

Uses

CLSM is widely used in various

It is also used in quantum optics and nano-crystal imaging and spectroscopy.

Biology and medicine

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

endoscopic procedures (endomicroscopy) is also showing promise.[12] 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.[13] 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.[14]

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

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.[16] Another possible approach is to have part of the optics (especially the microscope objective) in a cryogenic storage dewar.[17]
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

  • β-tubulin in Tetrahymena (a ciliated protozoan).
  • Partial surface profile of a 1-Euro coin, measured with a Nipkow disk confocal microscope.
    Partial surface profile of a 1-Euro coin, measured with a Nipkow disk confocal microscope.
  • Reflection data for 1-Euro coin.
    Reflection data for 1-Euro coin.
  • Colour coded image of actin filaments in a cancer cell.
    Colour coded image of actin filaments in a cancer cell.
  • 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.
    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

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.[18] This system is considered by some later authors as the first confocal optical system.[19][20]

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

Scheme of Petráň's Tandem-Scanning-Microscope. Red bar added to indicate the Nipkow-Disk.

In the 1960s, 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.[20][24]

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

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,[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

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.[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,

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.[34]

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

  • 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

  1. ^ .
  2. ^ a b US 3013467, Minsky, Marvin, "Microscopy apparatus", published 1961-12-19 
  3. ^ Memoir on Inventing the Confocal Scanning Microscope, Scanning 10 (1988), pp128–138.
  4. ^ a b Fellers TJ, Davidson MW (2007). "Introduction to Confocal Microscopy". Olympus Fluoview Resource Center. National High Magnetic Field Laboratory. Retrieved 2007-07-25.
  5. ^ 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 
  6. ^ "Data Sheet of NanoFocus µsurf spinning-disk confocal white light microscope". Archived from the original on 2014-01-20. Retrieved 2013-08-14.
  7. ^ "Data Sheet of Sensofar 'PLu neox' Dual technology sensor head combining confocal and Interferometry techniques, as well as Spectroscopic Reflectometry".
  8. .
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  10. on 14 May 2019. Retrieved 24 December 2017.
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  15. ^ The Digitization Process. Project IRENE, University of California, Berkeley Libraries.
  16. PMID 21133476
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  17. .
  18. . Note: Volume 98 is assigned to the year 1939, however on the first page of the article January 1940 is listed as publication date.
  19. ^ a b c d Colin JR Sheppard (3 November 2009). "Confocal Microscopy. The Development of a Modern Microscopy". Imaging & Microscopy.online
  20. ^ , S. 120–121.
  21. ^ Zyun Koana (1942). Journal of the Illumination Engineering Institute. 26 (8): 371–385. {{cite journal}}: Missing or empty |title= (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.
  22. PMID 14866220
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  23. .
  24. , pages 115–122.
  25. .
  26. .
  27. ^ US 3517980, Petran, Mojmir & Hadravsky, Milan, "Method and arrangement for improving the resolving power and contrast", published 1970-06-30, assigned to Ceskoslovenska akadamie 
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  29. .
  30. , pp. 124–125.
  31. ^ .
  32. .
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  37. ^ Anon (2005). "Dr John White FRS". royalsociety.org. London: Royal Society. Archived from the original on 2015-11-17.
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  40. ^ Brent Johnson (1 February 1999). "Image Is Everything". The Scientist. online
  41. ^ Diana Morgan (23 July 1990). "Confocal Microscopes Widen Cell Biology Career Horizons". The Scientist. online
  42. ^ US 4871251, Preikschat, Fritz K. & Preikschat, Ekhard, "Apparatus and method for particle analysis", published 1989-10-03 
  43. ^ US 5012118, Preikschat, Fritz K. & Preikschat, Ekhard, "Apparatus and method for particle analysis", published 1991-04-30 
  44. ^ reserved, Mettler-Toledo International Inc. all rights. "Particle Size Distribution Analysis". Archived from the original on 2016-10-09. Retrieved 2016-10-06.
  45. ^ Weisshart, Klaus. "The Basic Principle of Airyscanning" (PDF). asset-downloads.zeiss.com. Retrieved 6 September 2023.
  46. PMID 37143962
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External links