Principle of CLSM

In conventional epi-fluorescence microscopy, one of the major problems is the presence of significant background noise related to the thickness of the object observed. Although focusing is done on a precise focal plane, the recording of the information is marred by a background noise that is superimposed on the image of the observed plane: this noise results from the excitation, by the light source, of all the fluorochromes located in the light path. The confocal microscope acts like an optical microtome, its principle consists in focusing, via an objective, a laser beam that will excite the fluorochromes at a point on the sample, then recovering, on a photomultiplier, the light signal emitted at this point. A pinhole that stops any signal not coming from the focal plane is placed in front of the photomultiplier. The received signal is amplified in the photomultiplier, processed to improve the signal-to-noise ratio, and then digitized. The image is constructed point-to-point by scanning (X,Y) the scanned field using light source deflection mirrors. A motorized stage moves the preparation along the Z axis allowing the capture of different optical planes in the thickness of the object. The images thus formed are stored on the memory of a computer.

Role of the pinhole

This detection diaphragm plays an essential role in spatial resolution: it is an iris diaphragm allowing the adjustment of the elementary volume analyzed by the light probe. The maximum spatial resolution is obtained, in addition to the classical optical conditions (wavelength, numerical aperture of the objective), by adjusting the diameter of the pinhole to the diffraction limit, i.e. equal to the Airy spot.

Benefits

The confocal microscope has many advantages over conventional microscopy. First of all, the microscope's small depth of field (0.5 - 1.5 µm) allows an image of a focal plane (optical slice) to be obtained with a much higher resolution than with a conventional microscope and consequently the continuous fluorescence noise is practically eliminated. This results in a very good detection sensitivity, an increase in contrast and a "clarification" of the images. Another advantage of the confocal microscope is that optical sections can be obtained not only in the XY plane but also in a plane parallel to the optical axis (XZ plane) which can be reconstructed in three dimensions. These optical sections do not affect the integrity of the biological sample in any way, unlike the physical sections required in photonic and electron microscopy. In addition, the digitized acquisition of the images allows, on an image processing station, to increase the possibilities of analysis and quantification.
Confocal microscopy and subcellular cytometry

The sequential acquisition of several fluorescence channels on a set of serial optical slices allows to detect the frequency of spatial coincidences as well as the exclusion zones of the two immunocytochemical markers considered.
The superimposition of two sets of slices as well as the construction of visualization cytofluograms for each slice plane makes it possible to identify and quantitatively estimate the co-localization between two markers.
Spectral analysis

The purpose of spectral analysis is to detect the different wavelengths of light emitted by the sample.
In Leica confocals, the light emitted by the sample is diffracted by a prism. The light then passes through a slit and is collected by a photomultiplier. As the slit moves, it "sweeps" over the desired wavelength range.
In Zeiss confocal lenses, the emitted light is difracted by a grating and is sent to 32 small photomultipliers, each of which detects a different wavelength range.
In Olympus confocal lenses, the light is diffracted by a grating and the wavelengths are selected in front of the photomultiplier by means of a slit.
Spectral analysis enables the separation of fluorochromes with a large overlap of their emission spectra (e.g. CFP/YFP/GFP separation) as well as the determination of the presence of autofluorescence in the sample.

The recording, analysis and spectral separation is carried out in 3 steps :
1.    Acquisition of a lambda stack (series of images representing the intensity of the fluorochrome(s) as a function of the wavelength emitted)
2.    Comparison with a reference spectrum
3.    Spectral deconvolution
Study of dynamics

The sample scanning system using galvanometric mirrors allows the study of molecular dynamics by photoblanching, photoactivation or photoconversion of an area of interest.

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