Microscope confocal

A microscope confocal is a system for which the illumination and detection are limited to the same volume of reduced size: the focal point.

The image confocale (or crosses optical) is then obtained by the displacement of this point of focusing of the light of excitation in three dimensions of sample XYZ.

Obtaining the focal point

Described for the first time by Minsky (), the technique of imagery confocale of Fluorescence represents an important technological advance since it makes it possible to obtain images of high resolution which are like cuts " optiques" of a thickness of approximately 0,4 µm (sweeping in XY), obtained at various levels in the thickness of a sample (displacement in Z). Based on the elimination of the signals of Fluorescence coming from the areas located apart from the focal plan, this technique thus gives access to the information located inside the cells.

Since the model of study of Minsky which used a light of diffuse excitation generated by a lamp (more one window), two technological changes contributed to the increase in the resolution of the images confocales:

  • the use of a LASER as source of excitation constitutes a source of light easily focalisable. Indeed, the LASER light is coherent, one-way and made up of one or more very pure colors (of the same waves frequency and perfectly in phase).

  • the use of objectives having large a numerical Ouverture (N.A, numerical aperture ) ensures a focusing of the source of light in a point of small size.

For microscopy confocale, the light of excitation is delivered by a LASER and detection is assured either by a camera CCC high sensitivity or by photomultipliers. Sweeping XY is ensured by mirrors assembled on engines galvanometric. Displacement in Z is done by vertical displacement of the objective.

There exist two types of confocaux systems today the simple photon and the multiphoton.

Simple photon

The system confocal simple photon (mono-photonic) uses a light of excitation whose Wavelength excites the fluorophore directly. Therefore, the Fluorescence emitted can come all the thickness of the sample crossed by the beam of excitation. The key component of this microscope confocal is then represented by a " fenêtre" (a pinhole or an iris confocal) placed in front of the detector which eliminates the Fluorescence coming from the nonfocal areas. The observation of signals of fluorescences rests on five elements:

  1. a source of light for the excitation,

  2. a fluorophore,
  3. of the filters to separate the photons from emission of the photons of excitation,
  4. a pinhole,
  5. a detector to transform the light signal of the photons into electrical signal.

Multiphoton

The system confocal multiphoton uses lights of excitation close to the infra-red. In this case, only the point of focusing of the beam Laser is exiting (sufficient density of photon to couple the energy of excitation). This system is regarded as an important technological change for three imperative reasons:

  1. It does not require an iris confocal.

  2. the use of a light of excitation to a high Wavelength (> 900 Nm) ensures a greater penetration inside the sample (up to 500 µm instead of 150 µm) making it possible to work on thicker samples.
  3. the excitation of the fluorophores being limited to the point of focusing of the Laser beam , the risk of photoblanchiement is reduced.

In practice, the output of emission of Fluorescence is less good than a confocal simple photon and the signal-to-noise report/ratio is weaker. Thus, it does not show that little advantage for the observation of cells in culture or fabric cuts (50-70 µm thickness).

See too

References

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