Confocal laser scanning microscopy (
CLSM or
LSCM) is a technique for obtaining high-
resolutionImage resolution is an umbrella term that describes the detail an image holds. The term applies to raster digital images, film images, and other types of images. Higher resolution means more image detail....
optical images with depth selectivity. The key feature of
confocal microscopyConfocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of...
is its ability to acquire in-focus images from selected depths, a process known as
optical sectioningOptical sectioning is the process by which a suitably designed microscope can produce clear images of a focal planes deep within a thick sample. This is used to reduce the need for thin sectioning using instruments such as the microtome...
. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of
topologicallyTopology is a major area of mathematics concerned with properties that are preserved under continuous deformations of objects, such as deformations that involve stretching, but no tearing or gluing...
complex objects. For opaque specimens, this is useful for
surface profilingProfilometer is a measuring instrument used to measure a surface's profile, in order to quantify its roughness.While the historical notion of a profilometer was a device similar to a phonograph that measures a surface as the surface is moved relative to the contact profilometer's stylus, this...
, while for non-opaque specimens, interior structures can be imaged. For interior imaging, the quality of the image is greatly enhanced over simple microscopy because image information from multiple depths in the specimen is not superimposed. A conventional microscope "sees" as far into the specimen as the light can penetrate, while a confocal microscope only images one depth level at a time. In effect, the CLSM achieves a controlled and highly limited
depth of focusDepth of focus is a lens optics concept that measures the tolerance of placement of the image plane in relation to the lens...
. The principle of confocal microscopy was originally patented by
Marvin MinskyMarvin Lee Minsky is an American cognitive scientist in the field of artificial intelligence , co-founder of Massachusetts Institute of Technology's AI laboratory, and author of several texts on AI and philosophy.-Biography:...
in 1957, but it took another thirty years and the development of
laserA laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...
s for CLSM to become a standard technique toward the end of the 1980s.
In 1978,
ThomasThomas Cremer , is a German professor of human genetics and anthropology with a main research focus on molecular cytogenetics and 3D/4D analyses of nuclear structure studied by confocal microscopy and live cell imaging...
and
Christoph CremerChristoph Cremer is a German physicist and professor at the Ruprecht-Karls-University Heidelberg, who has successfully overcome the conventional limit of resolution that applies to light based investigations by a range of different methods Christoph Cremer (born in Freiburg im Breisgau, Germany)...
designed a laser scanning process, which scans the three dimensional surface of an object point-by-point by means of a focused laser beam, and creates the over-all picture by electronic means similar to those used in scanning electron microscopes. This CLSM design combined the laser scanning method with the 3D detection of biological objects labeled with fluorescent markers for the first time. During the next decade, confocal fluorescence microscopy was developed into a fully
mature technologyA mature technology is a technology that has been in use for long enough that most of its initial faults and inherent problems have been removed or reduced by further development...
, in particular by groups working at the University of Amsterdam and the
European Molecular Biology LaboratoryThe European Molecular Biology Laboratory is a molecular biology research institution supported by 20 European countries and Australia as associate member state. EMBL was created in 1974 and is an intergovernmental organisation funded by public research money from its member states...
(EMBL) in Heidelberg and their industry partners.
Image formation
In a confocal laser scanning microscope, a laser beam passes through a light source
apertureIn optics, an aperture is a hole or an opening through which light travels. More specifically, the aperture of an optical system is the opening that determines the cone angle of a bundle of rays that come to a focus in the image plane. The aperture determines how collimated the admitted rays are,...
and then is focused by an objective lens into a small (ideally diffraction limited) focal volume within or on the surface of a specimen. In biological applications especially, the specimen may be fluorescent.
ScatteredScattering is a general physical process where some forms of radiation, such as light, sound, or moving particles, are forced to deviate from a straight trajectory by one or more localized non-uniformities in the medium through which they pass. In conventional use, this also includes deviation of...
and reflected laser light as well as any fluorescent light from the illuminated spot is then re-collected by the objective lens. A
beam splitterA beam splitter is an optical device that splits a beam of light in two. It is the crucial part of most interferometers.In its most common form, a rectangle, it is made from two triangular glass prisms which are glued together at their base using Canada balsam...
separates off some portion of the light into the detection apparatus, which in fluorescence confocal microscopy will also have a filter that selectively passes the fluorescent wavelengths while blocking the original excitation wavelength. After passing a
pinhole, the light intensity is detected by a photodetection device (usually a
photomultiplierPhotomultiplier tubes , members of the class of vacuum tubes, and more specifically phototubes, are extremely sensitive detectors of light in the ultraviolet, visible, and near-infrared ranges of the electromagnetic spectrum...
tube (PMT) or
avalanche photodiodeAn avalanche photodiode is a highly sensitive semiconductor electronic device that exploits the photoelectric effect to convert light to electricity. APDs can be thought of as photodetectors that provide a built-in first stage of gain through avalanche multiplication. From a functional standpoint,...
), transforming the light signal into an electrical one that is recorded by a computer.
The detector aperture obstructs the light that is not coming from the
focal pointIn geometrical optics, a focus, also called an image point, is the point where light rays originating from a point on the object converge. Although the focus is conceptually a point, physically the focus has a spatial extent, called the blur circle. This non-ideal focusing may be caused by...
, as shown by the dotted gray line in the image. The out-of-focus light is suppressed: most of the returning light is blocked by the pinhole, which results in sharper images than those from conventional fluorescence microscopy techniques and permits one to obtain images of planes at various depths within the sample (sets of such images are also known as
z stacks).
The detected light originating from an illuminated volume element within the specimen represents one
pixelIn digital imaging, a pixel, or pel, is a single point in a raster image, or the smallest addressable screen element in a display device; it is the smallest unit of picture that can be represented or controlled....
in the resulting image. As the laser scans over the plane of interest, a whole image is obtained pixel-by-pixel and line-by-line, whereas the
brightnessBrightness is an attribute of visual perception in which a source appears to be radiating or reflecting light. In other words, brightness is the perception elicited by the luminance of a visual target...
of a resulting image pixel corresponds to the relative intensity of detected light. The beam is scanned across the sample in the horizontal plane by using one or more (
servothumb|right|200px|Industrial servomotorThe grey/green cylinder is the [[Brush |brush-type]] [[DC motor]]. The black section at the bottom contains the [[Epicyclic gearing|planetary]] [[Reduction drive|reduction gear]], and the black object on top of the motor is the optical [[rotary encoder]] for...
controlled) oscillating mirrors. This scanning method usually has a low reaction
latencyLatency is a measure of time delay experienced in a system, the precise definition of which depends on the system and the time being measured. Latencies may have different meaning in different contexts.-Packet-switched networks:...
and the scan speed can be varied. Slower scans provide a better
signal-to-noise ratioSignal-to-noise ratio is a measure used in science and engineering that compares the level of a desired signal to the level of background noise. It is defined as the ratio of signal power to the noise power. A ratio higher than 1:1 indicates more signal than noise...
, resulting in better
contrastContrast is the difference in visual properties that makes an object distinguishable from other objects and the background. In visual perception of the real world, contrast is determined by the difference in the color and brightness of the object and other objects within the same field of view...
and higher resolution. Information can be collected from different focal planes by raising or lowering the microscope stage or objective lens. The computer can generate a three-dimensional picture of a specimen by assembling a stack of these two-dimensional images from successive focal planes.
Confocal microscopy provides the capacity for direct, noninvasive, serial
optical sectioningOptical sectioning is the process by which a suitably designed microscope can produce clear images of a focal planes deep within a thick sample. This is used to reduce the need for thin sectioning using instruments such as the microtome...
of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution. Biological samples are often treated with
fluorescent dyesA fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
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 proteinThe green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
with the protein of interest).
Resolution enhancement
CLSM is a scanning imaging technique in which the
resolutionAngular resolution, or spatial resolution, describes the ability of any image-forming device such as an optical or radio telescope, a microscope, a camera, or an eye, to distinguish small details of an object...
obtained is best explained by comparing it with another scanning technique like that of the
scanning electron microscopeA scanning electron microscope is a type of electron microscope that images a sample by scanning it with a high-energy beam of electrons in a raster scan pattern...
(SEM). CLSM has the advantage of not requiring a probe to be suspended nanometers from the surface, as in an
AFMAtomic force microscopy or scanning force microscopy is a very high-resolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit...
or
STMA scanning tunneling microscope is an instrument for imaging surfaces at the atomic level. Its development in 1981 earned its inventors, Gerd Binnig and Heinrich Rohrer , the Nobel Prize in Physics in 1986. For an STM, good resolution is considered to be 0.1 nm lateral resolution and...
, 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
diffractionDiffraction refers to various phenomena which occur when a wave encounters an obstacle. Italian scientist Francesco Maria Grimaldi coined the word "diffraction" and was the first to record accurate observations of the phenomenon in 1665...
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 apertureIn optics, the numerical aperture of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. By incorporating index of refraction in its definition, NA has the property that it is constant for a beam as it goes from one...
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. For example, if the pinhole diameter is set to 1 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 which 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 perfluorocarbons such as
perfluorodecalinPerfluorodecalin is a fluorocarbon, a derivative of decalin in which all of the hydrogen atoms are replaced by fluorine atoms. It is chemically and biologically inert, and stable up to 400°C. Several applications make use of its ability to dissolve gases....
, which readily infiltrates tissues and has a refractive index almost identical to that of water
http://www3.interscience.wiley.com/journal/123335070/abstract.
Uses
CLSM is widely-used in numerous biological science disciplines, from
cell biologyCell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...
and
geneticsGenetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
to
microbiologyMicrobiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...
and
developmental biologyDevelopmental biology is the study of the process by which organisms grow and develop. Modern developmental biology studies the genetic control of cell growth, differentiation and "morphogenesis", which is the process that gives rise to tissues, organs and anatomy.- Related fields of study...
.
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
corneaThe cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber. Together with the lens, the cornea refracts light, with the cornea accounting for approximately two-thirds of the eye's total optical power. In humans, the refractive power of the cornea is...
. It is used for localizing and identifying the presence of filamentary fungal elements in the
corneaThe cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber. Together with the lens, the cornea refracts light, with the cornea accounting for approximately two-thirds of the eye's total optical power. In humans, the refractive power of the cornea is...
l stroma in cases of
keratomycosisA fungal keratitis is an 'inflammation of the eye's cornea' that results from infection by a fungal organism. Keratomycosis is the Greek terminology equivalent of fungal keratitis - it is the fungal infection of the cornea, the anterior part of the eye which covers the pupil...
, enabling rapid diagnosis and thereby early institution of definitive therapy. Research into CLSM techniques for endoscopic procedures is also showing promise. In the pharmaceutical industry, it was recommended to follow the manufacturing process of thin film pharmaceutical forms, to control the quality and uniformity of the drug distribution.
CLSM is also used as the data retrieval mechanism in some
3D optical data storage3D optical data storage is the term given to any form of optical data storage in which information can be recorded and/or read with three dimensional resolution ....
systems and has helped determine the age of the
Magdalen papyrusThe "Magdalen" papyrus was purchased in Luxor, Egypt in 1901 by Reverend Charles Bousfield Huleatt , who identified the Greek fragments as portions of the Gospel of Matthew and presented them to Magdalen College, Oxford, where they are cataloged as P. Magdalen Greek 17 and whence they have their...
.
See also
- Confocal microscopy
Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of...
- LSM (Zeiss)
LSM is a line of laser point scanning confocal and two photon microscopes produced by Carl Zeiss AG. The 5th generation, including the LSM 510, LSM 5 Pascal, and LSM 5 Live are now obsoleted by the LSM 700/710/780 series released after 2008....
Laser Scanning Confocal Microscopy platform format form Carl Zeiss
- Two-photon excitation microscopy
Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a very high depth, that is up to about one millimeter. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite...
: 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.
- Total internal reflection fluorescence microscope
A total internal reflection fluorescence microscope is a type of microscope with which a thin region of a specimen, usually less than 200 nm, can be observed.-Background:...
(TIRF)
- Fluorescence microscope
A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption...
- STED microscopy
Stimulated Emission Depletion microscopy, or STED microscopy, is a fluorescence microscopy technique that uses the non-linear de-excitation of fluorescent dyes to overcome the resolution limit imposed by diffraction with standard confocal laser scanning microscopes and conventional far-field...
- Point Spread Function
The point spread function describes the response of an imaging system to a point source or point object. A more general term for the PSF is a system's impulse response, the PSF being the impulse response of a focused optical system. The PSF in many contexts can be thought of as the extended blob...
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