Microscope confocal with laser scanning
The microscope confocal with laser scanning - MCBL (in English CLSM for confocal laser scanning microscope ) is a optical Microscope which with the property to carry out images of very low depth of field (approximately 600 Nm) called “optical sections”. By positioning the focal plan the objective at various levels of depth in the sample, it is possible to carry out series of images from which one can obtain a three-dimensional representation of the object.
The object is not directly observed by the user; this one sees an image recomposed by Ordinateur.
The microscope confocal functions in reflected light or fluorescence. The principle of the microscope confocal was described by Marvin Minsky in 1953, but it is only in the end of the Années 1980 that commercial models appeared, making this technique accessible to many laboratories. Microscopy confocale is very much used in biology like in sciences of materials.
Principle and characteristics
In traditional optical microscopy, so that an image is clear, it is necessary that the object is in the focal plan of the optical system. When an object is thick, presents an important relief, or when it is tilted compared to the objective, only part of the object is clear in the image (see the article on the Depth of field).
To solve this problem, one clarifies surface either by a beam of white light, but by a ray Laser, concentrated by a lens, which sweeps surface by positioning one sténopée (English pinhole) in front of the detector, in a focal plan combined in the focal plan of the objective (confocaux plans). In this manner, only the photons coming from the focal plan pass sténopée and take part in the formation of the image, from where the name “confocal” (synonymous with monofocal).
Sweeping by the laser is done using two orthogonal mirrors. The detectors used are tubes photomultipliers (PMT), the luminous intensity is measured and digitized according to the position of the laser in the sample: digital images directly are obtained.
The use of a coherent source of light (laser) as well as the reduced size of the enlightened field make it possible to obtain a slightly better side resolution (180-160 Nm) with that awaited for a conventional optical microscope (200 Nm). The resolution in Z (depth) is about 600 Nm in microscopy confocale.
The lasers used most frequently are the following:
The positioning of the image in the depth of the sample is generally obtained by moving in Z the objective using a piezoelectric quartz by successive steps of 200-300nm.
Other related techniques
Microscopy of fluorescence per multiphotonic excitation
Technique very similar to microscopy confocale with laser scanning it employs the same material. The low depth of field is obtained by exciting fluorescence only in one volume restricted by multiphotonic excitation using pulsated lasers. This excitation consists of nearly simultaneous absorption several photons of excitation a wavelength close to a multiple of the optimal excitation to a photon. The totality of fluorescence arrives at the level of the detector, it did not sténopée more there. One cannot thus speak any more about microscopy confocale, although one employs sometimes the term of multiphotonic microscopy confocale in an abusive way. Very often the applications are limited to biphtonic microscopy (excitation of the fluorophore by two photons). In addition to the aspect of excitation naturally confocale, this technique is used for the simultaneous excitation of several fluorophores with different emission spectra.
Fluorescence microscope by multiphotonic excitation multifocale
The principle is identical, but the laser beam is divided into several beams what makes it possible to sweep several points simultaneously. This makes it possible to decrease the time of acquisition of the images.
Microscope of fluorescence by total reflection interns
allows to obtain a better depth of field (200 Nm) that microscopy confocale (600 Nm), but only at the base of the sample (more precisely on the level of the interface sample/transparent support.
Other techniques
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Microscope confocal in Rotary Disque (or disc of Nipkow - spinning disk).
- spectral Microscope confocal.
- Spectroscopy by correlation of fluorescence.
- Microscope for the imagery of lifespan of fluorescence.
External bond
- virtual microscope confocal
- Microscopy of Fluorescence per Excitation Multiphotonique
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