Optical Microscope

The optical microscope a instrument optical provided with an objective and an Ocular which makes it possible to enlarge the image of a low-size object (Grossissement) and to separate the details from this image ( resolution) so that they are observable with the naked eye. It is used in Biologie, to observe the cells, the fabrics, in Pétrographie to recognize the rocks, in Métallurgie and Métallographie to examine the structure of a metal or an alloy.

History

The first microscope was undoubtedly manufactured by Hans and Zacharias Jansen, two manufacturers of Dutch lenses, in 1590, but this point is disputed. Galileo uses one of its design of them, in 1615. The final model was invented by Antoine van Leeuwenhoek in 1664, then was improved by Christiaan Huygens.

A contrario of other optical systems which are defined by their optical Grossissement (Télescope) or them growth (Camera), the adapted term, for the microscope, is its Puissance , report/ratio of the angle, under which the object through the instrument is seen, with the length of this object.

Not to confuse it with the binocular Magnifying glass which not requiring flat low thickness, or reflective samples, makes it possible to observe natural parts without preparation by enlarging the image of a factor relatively low, but keeping a stereoscopic vision favourable with the revealing macroscopic examination of grains, splits, cracks, etc

Preparation of the samples

The sample observed must meet certain conditions:

of flatness, so that the objective gives a clear whole image of it, or else one can observe only one restricted portion of it
in transmission, it must be low thickness so that the light crosses it and makes visible only some elements (cells) in the case of biology;
in reflection, surface must be in general polished so that the stripes do not mask what one wants to observe;
the parts to be observed must be able to be different:

differentiation of colors by the chemical coloring of standardized solutions, for biology;
attacks chemical by acids to reveal metallurgy defects;
of other differentiations by lighting in polarized light, ultra-violet (fluorescence), or by interferential principle, revealing other aspect, invisible with the naked eye.

In biology, it is necessary, beforehand, to place, the fabric cut or the liquid containing of the living organisms, between a blade and a plate of glass. The objective must approach the blade for the development without, by awkwardness, to destroy the preparation become very fragile.

Because of preparation, optical microscopy requires an important quantity of complementary apparatuses for the only destination of the microscopic observation.

Let us take the case of the Biopsie in medicine and biology (Anatomopathologie): the diagnosis by microscopy, of biological parts taken by Biopsy during an operation, imposes short times. To prepare blade, one uses apparatus called Cryotome, kind of “slicer with ham”, placed in Cryostat (freezer), which makes it possible to cut out very fine sections of the body which will be to observe by cooling it quickly, then by cutting out it using the blade of a special razor, sharpened on another machine with glass plate using set with diamonds pastes. If one wants to work with room temperature, the times are longer and impose dehydrations and replacement of the water removed by paraffin (24 hours) so that the sample keeps its rigidity; then, it is coloured by several substances of alternate actions of duration very long, they too.

Constitution of the microscope

Upwards:

Mirror: is used to reflect the ambient light to light the sample by in lower part, in the case of a transparent sample (for example a thin blade in biology or geology, or a liquid);
artificial Source of light of better temperature of color and stability and by the use of a condenser which makes it possible this light to fill in a homogeneous and regular way the field observed, and especially not to show, by its adequate adjustment, mechanical details of the source of light (whorls of the filament of the bulb). The source of lighting can be more elaborate and comprise a case independent, possibly in polarized light or ultraviolet, to emphasize certain chemical properties of the matter, or to light the sample over (in particular out of metallurgy)
diaphragm: variable opening of diameter allowing to restrict the quantity of light which lights the sample;
platinum carry-sample: where the sample is posed; the grips are used to hold the sample when this one is thin (for example a blade of glass); the turntable can be mobile (left-right-hand side and before-back), which makes it possible to sweep the sample and to select the part observed;
objective S: lens or together of lenses carrying out the enlargement; there are several objectives, corresponding to several enlargements, assembled on a turret; Certain objectives are said to immersion because their power can be reached only by eliminating the blade from air between the sample covered by the plate and the frontal one of the objective. One uses for that of the oil of cedar or oils of synthesis to which the index of refraction is close to that of glass.
developed coarse and fine; so that the image is clear, it is necessary that the object is in the focal plan of the objective; these serrated rollers reduce to assemble and the objective-eyepiece unit with a system from toothed rack, in order to lead the focal plan on the zone of the sample to observe;
Ocular: lens or together of lenses forming the image in a way resting for the eye; the rays arrive parallel, as if they came by far, which allows a relaxation of the muscles controlling the crystalline lens; two eyepieces placed on a head known as binocular makes more comfortable the observation (even if it does not bring stereoscopic vision).

The eyepiece can be replaced by a camera (becoming objective the apparatus is characterized by its growth and either by its power) by, a video camera for the moving objects (alive cells) or a camera CCC to make a numerical acquisition. This makes it possible to make the observation on a monitor (screen of the television type) and to facilitate the use and the image processing (impression, data processing).

Principle of the optical microscope

The optical microscope is based on the lenses to obtain an image increased of the sample to observe.

One can make a microscope simplified with two convergent lenses. The object to be observed is placed in front of the first lens called “objective”. If the object is beyond the focal distance, that forms a real image reversed and of different size; the image is larger than the object if this one is located at a distance lower than the double of the focal distance from the objective.

The second lens is the Oculaire: it is positioned so that the image is in its focal plan. Thus, the eye observes an image “ad infinitum”, therefore by slackening the muscles charged with the Accommodation, which represents a better visual comfort.

It is about a dioptric centered system, partly composed of doublets to correct of them some of the aberration S optics.

Objectives of microscope

In the preceding paragraph, the objective directly provides an image in the focal plan object of the eyepiece: it is said that it is corrected (within the meaning of the correction of the aberrations) for a finished length of tube. In the modern professional photonic microscopes, the objective provides an image ad infinitum (the object being then in the focal plan object) and it known as “is corrected ad infinitum”: a lens of tube or lens of Telan, interdependent of the stand, reforms the image in the focal plan of the eyepiece. The objective is then characterized by its commercial enlargement, and not by its growth.

The various characteristics are engraved by the manufacturer on the mounting of the objective: growth or enlargement, opening numerical, standard from correction of the objective (length of the tube), correction of aberration, thickness of the plate cover-glass, medium of immersion, distance work… For example, a mounting of objective carrying the following inscriptions:

mean that: the enlargement is of 60, the numerical opening of 1,4, that the objective is corrected ad infinitum and that the thickness of the plate cover-glass must be of 0,17 Misters the distance from work (WD for “working distance”) is of 0,21 Misters.

Reversed systems

The sample being above and the objective in lower part (metal or mineralogical samples polished), the image is then reflected by a mirror, the eyepiece is on the side of the microscope, always directed downwards for the comfort of the user.
Pour the massive parts, which would crush the microscope or would damage its mechanical structure, polishing takes place directly on the part, sight then by a right microscope (examination of large, or even rail casting of railroad).

Limits of the optical microscope

The resolution of a microscope depends on its capacity to separate the details (according to the Critère of Rayleigh for example): it is indeed useless to increase the enlargement if the details are not solved. In addition to the correction of the aberrations, its limit is dependant on the phenomena of Diffraction and by the structure of the retina of the eye (optimum to 0,25 mm in pupil of exit).

Independently of the associated sensor (eye, camera CCC…) and of the aberrations, the light diffraction degrades the Impulse response microscope: the image of a point is not a point, but a Tache of Airy. Thus, two close points will have as images 2 spots of Airy, from which covering prevents from distinguishing the 2 points images: the details are not solved any more in space image.

Two solutions are thus offered to push back this limit (see discussion on the formula of the Resolving power), in short:

By using an objective with immersion (with an index of refraction bathing frontal objective close to the maximum of 1,5 - that of glass), the numerical Opening of such an objective being higher.
By decreasing the wavelength (impossible beyond 400 Nm for the optical microscope), dimensions of the spot of Airy decreasing with the wavelength.

The limit of resolution of a traditional photonic microscope is approximately 0,2 μ. For all these reasons, the electron microscope will reach, him, a limit 100 times smaller.

It still should be noted that in the case of the imagery in three dimensions, the transverse resolution (in a plan perpendicular to the optical axis) is higher than the longitudinal resolution (according to the optical axis). Techniques known as tomographic however make it possible to improve the resolution.

See too

External bonds

http://www.microscopies.com/ Microscopy amateur with French Magazine
the microscope, practical works of discovered
To observe with a microscope
Microscopes of the years 1820-1940, French and German with more than 2500 photographs (German text)
a collection of antiquities of the microscopes

Simple: Light microscope

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