Confocal microscopy
Encyclopedia
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 three-dimensional structures from the obtained image
s. This technique has gained popularity in the scientific and industrial communities and typical applications are in life sciences
, semiconductor
inspection and materials science
.
and aims to overcome some limitations of traditional wide-field fluorescence microscope
s. In a conventional (i.e., wide-field) fluorescence microscope
, the entire specimen
is flooded evenly in light from a light source. All parts of the specimen in the optical path are 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
s are often required.
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 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.
Each of these classes of confocal microscope have particular advantages and disadvantages, most systems are either optimised for resolution or high 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 resolution. Imaging frame rate
s are typically very slow for laser scanning systems (e.g. less than 3 frames/second). Commercial spinning-disk confocal microscopes achieve frame rates of over 50 per second – a desirable feature for dynamic observations such as live cell imaging.
In practice Nipkow and PAM allow multiple pinholes scanning the same are in parallel as long as the pinholes are sufficiently far apart.
Cutting edge development of confocal laser scanning microscopy now allows better than video rate (60 frames/second) imaging by using multiple microelectromechanical systems
based 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.
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 PSFs gives a much smaller effective sample volume.
From this evolved the single plane illumination microscope.
.
Optical resolution
Optical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged.An imaging system may have many individual components including a lens and recording and display components...
and contrast
Contrast (vision)
Contrast 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...
of a micrograph
Micrograph
A micrograph or photomicrograph is a photograph or digital image taken through a microscope or similar device to show a magnified image of an item.Micrographs are widely used in all fields of microscopy.-Photomicrograph:...
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 three-dimensional structures from the obtained image
Image
An image is an artifact, for example a two-dimensional picture, that has a similar appearance to some subject—usually a physical object or a person.-Characteristics:...
s. This technique has gained popularity in the scientific and industrial communities and typical applications are in life sciences
Life sciences
The life sciences comprise the fields of science that involve the scientific study of living organisms, like plants, animals, and human beings. While biology remains the centerpiece of the life sciences, technological advances in molecular biology and biotechnology have led to a burgeoning of...
, semiconductor
Semiconductor
A semiconductor is a material with electrical conductivity due to electron flow intermediate in magnitude between that of a conductor and an insulator. This means a conductivity roughly in the range of 103 to 10−8 siemens per centimeter...
inspection and materials science
Materials science
Materials science is an interdisciplinary field applying the properties of matter to various areas of science and engineering. This scientific field investigates the relationship between the structure of materials at atomic or molecular scales and their macroscopic properties. It incorporates...
.
Basic concept
The principle of confocal imaging was patented in 1957 by Marvin MinskyMarvin Minsky
Marvin 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:...
and aims to overcome some limitations of traditional wide-field fluorescence microscope
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...
s. In a conventional (i.e., wide-field) fluorescence microscope
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...
, the entire specimen
Specimen
A specimen is a portion/quantity of material for use in testing, examination, or study.BiologyA laboratory specimen is an individual animal, part of an animal, a plant, part of a plant, or a microorganism, used as a representative to study the properties of the whole population of that species or...
is flooded evenly in light from a light source. All parts of the specimen in the optical path are excited at the same time and the resulting fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
is detected by the microscope's photodetector
Photodetector
Photosensors or photodetectors are sensors of light or other electromagnetic energy. There are several varieties:*Active pixel sensors are image sensors consisting of an integrated circuit that contains an array of pixel sensors, each pixel containing a both a light sensor and an active amplifier...
or camera
Camera
A camera is a device that records and stores images. These images may be still photographs or moving images such as videos or movies. The term camera comes from the camera obscura , an early mechanism for projecting images...
including a large unfocused background part. In contrast, a confocal microscope uses point illumination (see Point Spread Function
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...
) 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
Optical resolution
Optical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged.An imaging system may have many individual components including a lens and recording and display components...
, 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
Exposure (photography)
In photography, exposure is the total amount of light allowed to fall on the photographic medium during the process of taking a photograph. Exposure is measured in lux seconds, and can be computed from exposure value and scene luminance over a specified area.In photographic jargon, an exposure...
s are often required.
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 achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the numerical aperture
Numerical aperture
In 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 objective lens, but also by the optical properties of the specimen. The thin optical sectioning
Optical sectioning
Optical 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...
possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples.
Techniques used for horizontal scanning
Three types of confocal microscopes are commercially available:- Confocal laser scanning microscopes use a pair of mirrors (one for the x and the other for the y axis) to scan the laser across the sample and "descan" the image across a fixed pinhole and detector.
- Spinning-disk (Nipkow diskNipkow diskA Nipkow disk , also known as scanning disk, is a mechanical, geometrically operating image scanning device, invented by Paul Gottlieb Nipkow...
) confocal microscopes use a series of moving pinholes on a disc to scan spot of light. - Programmable Array Microscopes (PAM) use an electronically controlled spatial light modulatorSpatial light modulatorA spatial light modulator is an object that imposes some form of spatially-varying modulation on a beam of light. A simple example is an overhead projector transparency. Usually when the phrase SLM is used, it means that the transparency can be controlled by a computer. In the 1980s, large SLMs...
(SLM) that produces a set of moving pinholes. The SLM is a device containing an array of pixels with some property (opacity, reflectivity or optical rotation) of the individual pixels that can be adjusted electronically. The SLM contains microelectromechanical mirrors or liquid crystal components. The image is usually acquired by a CCD camera.
Each of these classes of confocal microscope have particular advantages and disadvantages, most systems are either optimised for resolution or high 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 resolution. Imaging frame rate
Frame rate
Frame rate is the frequency at which an imaging device produces unique consecutive images called frames. The term applies equally well to computer graphics, video cameras, film cameras, and motion capture systems...
s are typically very slow for laser scanning systems (e.g. less than 3 frames/second). Commercial spinning-disk confocal microscopes achieve frame rates of over 50 per second – a desirable feature for dynamic observations such as live cell imaging.
In practice Nipkow and PAM allow multiple pinholes scanning the same are in parallel as long as the pinholes are sufficiently far apart.
Cutting edge development of confocal laser scanning microscopy now allows better than video rate (60 frames/second) imaging by using multiple microelectromechanical systems
Microelectromechanical systems
Microelectromechanical systems is the technology of very small mechanical devices driven by electricity; it merges at the nano-scale into nanoelectromechanical systems and nanotechnology...
based scanning mirrors.
Confocal x-ray fluorescence
X-ray fluorescence
X-ray fluorescence is the emission of characteristic "secondary" X-rays from a material that has been excited by bombarding with high-energy X-rays or gamma rays...
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.
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 4 microscopy4Pi Microscope
A 4Pi Microscope is a laser scanning fluorescence microscope with an improved axial resolution. The typical value of 500-700 nm can be improved to 100-150 nm which corresponds to an almost spherical focal spot with 5-7 times less volume than that of standard confocal microscopy.-Working...
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 PSFs gives a much smaller effective sample volume.
From this evolved the single plane illumination microscope.
Super resolution
There are confocal variants that achieve resolution below the diffraction limit like STED microscopySTED 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...
.
External links
- Molecular Expressions: Laser Scanning Confocal Microscopy
- Nikon's MicroscopyU. Comprehensive introduction to confocal microscopy.
- Emory’s Physics Department. Introduction to confocal microscopy and fluorescence.
- The Science Creative Quarterly's overview of confocal microscopy - high res images also available.
- Programmable Array Microscope - Confocal Microscope Capabilities.
- Introduction to Confocal Microscopy (video) by Kurt Thorn (UCSF)