Microscopy Confocale - SpeedyLook encyclopedia

The microscopy confocale is a Technique derived from the Microscopie to fluorescence; it is thus a photonic form of microscopy.

Microscopy confocale inspired various techniques of Microscopie, certainly less powerful but having the advantage of working in direct light.

Advantages of this technology

One of the problems of standard optical microscopy is the Depth of field: the higher the enlargement is, plus this depth is weak, which prevents from having a clear image on the totality of a a little wide object. This is particularly tedious for the objects lengthened like the Nerf S; they are thus fuzzy on part of their way some is the ability of the preparer. In fact, microscopy with traditional fluorescence poses a problem for all the objects having a certain thickness. Indeed, the fluorescence emitted by the focal plan, therefore clear, is lost in the fluorescence emitted by the plan adjacent in the focal plan, which by definition are vague.

Microscopy confocale, brings a solution to this problem by the means of an opening called " pine-hole" (" hole of épingle" in English), located in manner confocale at the focal plan, so that only fluorescence coming from the focal plan is able to cross it. The light coming from the adjacent plans (fuzzy) is stopped by the edges of the hole. It is thus possible to obtain a perfectly clear optical cut corresponding only to the focal plan. While varying this plan one obtains a succession of cuts giving of clear and precise information in three dimensions of the object observed. In addition, to induce the fluorescence of the fluorochromes used, the source of excitation used is not any more one arc lamp, but one (or several) beam (X) laser. The advantage of the laser is to provide a monochromatic light more specific and easy to control (in term of filtering). Moreover the smoothness of the laser brush allows to improve considerably the resolution in XY (in the plan) because of a less diffusion. N the other hand, the use of laser requires: 1) the use of mirrors galvanometric which allow a sweeping of the whole of the field observed: a very fast mirror (between 200Hz and 2KHz according to the models) carrying out sweeping in X, follow-up of a mirror allowing sweeping in Y; 2) the use of photomultipliers (PMT) like sensors, in the place of the eye or the camera, because of the very weak signal emitted for each position of the beam. For each one of these positions, the PMT will produce an electrical signal propostionel on the level of light collected. This electrical signal is then digitalized to constitute a matrix of pixels then. While working in 8 bits one thus obtains an image in 256 levels of gray, which are then " colorés" artificially in order to differentiate in a superposition fluorescence emitted by several fluorochromes. The images obtained have a resolution varying of 64x64 with 2048x2048. The higher the resolution is, the longer time of acquisiton is (becomes a limit for the fast acquisition of phenomena biological on alive material), and the more the generated file is " lourd" (problem of report storage). Microscopy confocale allows studies on fixed material, but also makes it possible to study dynamic phenomena, on cells or living tissue, in particular thanks to the molecules of family GFP (Green Fluorescent Protein). In addition the permanent technological developments make it possible today to consider processes of interaction molecular (Technical of FREIGHT), or of molecular dynamics (FRAP, FLIP).

Treatments

The result of an observation in microscopy confocale can be regarded as a Tomographie on a cellular scale. Once the tomography obtained, it can be treated various ways:

by joining together the various images, one reconstitutes a sight in 2D of the unit of the perfectly clear preparation in any point;
by creating a stereoscopic image giving a sight in three dimensions of the preparation;
as all the elements of the same image are with the same depth of the preparation, one can determine connections between the various observable elements.

These uses are not however restrictive and depend on the needs and the imagination of the scientists.

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