Electronic Microscopy in transmission
The electronic microscopy in transmission (English MET or TEM for Transmission Electron Microscopy ) is a technique of Microscopie based on the principle of Diffraction of the electron S and being able to reach a Grossissement X: 5000000. The principle of the electron microscope in transmission was developed in 1931 by max Knoll and Ernst Ruska, this last received besides the Nobel Prize of physics in 1986 for this invention.
It consists in placing a sufficiently thin sample under an electron beam used in coherent beam, and to visualize is the Hologramme obtained that is the figure of diffraction in the focal plan of the objective, that is to say to use another lens to obtain the figure Transformée of Fourier of the figure of observable diffraction by the impact of the electrons on a fluorescent screen or to record it on a photo plate.
The limit of resolution depends on the Wavelength on Broglie on the electrons, therefore of their tension of acceleration, it would be thus of about size of the picometer in an ideal case. But because of the strong aberrations it is actually only of some Ångstroms.
A current error consists in saying “electron microscope to transmission” (by analogy with the “electron microscope with sweeping”). This error is frequent in the scientific circle, and even in books! It is indeed a microscope in transmission (one observes the sample in transparency, in transmission).
Basic principleThere exists a certain analogy between the electron microscope in transmission and the optical microscope with direct light.
It is the radiation used which differs mainly in both cases:
The optical microscope uses as radiation of the Photon S (external light). A system of optical lenses makes it possible to deviate or to focus the luminous ray which crosses a sample " relatively fin". The image obtained is formed directly on the Rétine of the observateur.
The electron microscope in transmission uses, him, as radiation of the electron S. a system of magnetic lenses makes it possible to deviate or to focus the ray of electrons on a sample " extremely fin". The image (or stereotype of Diffraction) obtained can be seen on a fluorescent screen, recorded on a film photographic or detected by a sensor CCC.
The electron microscope in transmission has two principal modes of function according to whether an image or a stereotype of diffraction is obtained:
; image mode: The electron beam crosses the sample. According to the thickness, the density or the chemical nature of this one, the electrons are more or less absorbed. While placing the detector in the Plane image, one can, by transparency, to observe an image of the irradiated zone. It is this principle which is used, in other, in biology, to observe cells or mean cuts of bodies.
; diffraction mode: This mode uses the undulatory behavior of the electrons (wave De Broglie) (this is modelled by the Quantum physics). When they meet organized matter (of the crystals), they thus will be diffracted, i.e. deviated in certain directions depending on the organization of the atoms. The beam is diffracted in several small beams, and those recombine to form the image, thanks to magnetic lenses (electromagnet S which deviate the electrons).
DescriptionAn electron microscope in transmission is composed:
- of a system of pumps with vacuum;
- of a cooling system with nitrogen;
- of a Electron gun composed of a source of electron S, of a system of focusing and an accelerator of electrons;
- of an electronic optical column containing the magnetic lenses and the diaphragm S;
- of a door sample;
- of a Detecting of electrons;
- possibly of another electronic elements of measurement.
Preparation of the samplesThe preparation of the samples for an observation under the electron microscope in transmission is a very important phase. It is it which will partly determine the quality of the results obtained. The electron beam having to cross the sample, its thickness must be ideally about some Nanomètre S. Following the use which one makes of the MET (Biologie or Science of the materials), the technique of preparation of the samples differs.
BiologyThe samples are in the shape of thin blades and are placed under ultra-high vacuum. In biology, the thin blade is obtained by making a cut (ultra microtome). A technique of microcleavage made it possible to obtain profiles of Multicouche S: Microcleavage transmission interfacial electron microscopy applied to the structure off multilayers and microstructure off small particles one has substrate
Negative coloringThe thin samples (of some nanometers to a few tens of nanometers thickness) are absorbed on a metal grid covered with a fine carbon film (some nanometers). They are typically proteinic complexes or viruses. The water excess is absorbed using a blotting paper. A solution containing a contrasting agent, such of the osmium tetroxide or the acetate of uranyl, is added on the grid during a few seconds then absorbed. This one will be fixed preferentially at the edge of the absorptive particles. From its strong atomic mass, contrasting it deviates the electrons in the objective diaphragm. Thus the biological sample appears clearer than what surrounds it, from where the name of negative coloring. The sample appears white on a dark bottom on the photographs.
This technique also called " shade réplique" is a technique of MET which studies the relief of the structures. It consists of the vaporization of a very fine layer of platinum, with a precise angle, on the sample maintained in rotation. This layer of platinum, consolidated with a very fine layer of carbon also, is then separated of the sample then observed directly by deposit on the grids of observations.
Out of metallurgy, it is obtained by a meticulous cutting (for example with a Scie with set with diamonds wire), then a thinning. The most current technique consists in of final stage making a crater with a beam of Ion S (ionic Amincisseur) or by Fraisage, a hole crosses the blade right through, and one looks at the thin edges of the hole.
Systems of illumination
In electronic microscopy, the illumination of the samples is made by means of electrons. The source of electron is called gun or Cathode. There are several types. According to the systems, the electron beam will be more or less coherent, i.e. the electrons will be more or less in phase. A coherent beam increases the resolution of the images.
A metal point in the shape of V is heated at high temperature. Thus the electrons present in metal move very quickly. A small number of electrons arrive so much at the angle of V that they are ejected metal. At the same time an electric field of about size of 100 Kv is applied. The electrons which left metal are accelerated by the electric field in direction of the sample. Generally the gun with heating does not give a very coherent beam. That is due to the fact that speed, and thus the kinetic energy of the emitted electrons, follow a Gaussian distribution. It results from this a chromatic aberration.
There exist filaments out of tungsten and hexaborate of lanthanum. The latter are much more expensive but provide a better coherence.
Canon with emission of field (in English Field Emission Gun or FEG )
This gun also consists of an extremely sharp-edged point of Tungstène crystalline. It is not heated but, on the other hand, an important electric field is applied (2 to 7 Kv). So the emitted electrons have a very low energy variability. The beam is thus very coherent.
The vacuum present in this kind of microscope must be extremely thorough. If it is not the case, the point of the gun very strongly heats and breaks. This particular requirement makes FEG of the very expensive and delicate machines.
Modes of imagery
Mode in clear field
The screen is placed in the image plan. One observes an increased image of the object. This mode is especially used to observe mainly irregular objects, biological cells for example.
Weak mode proportions
This mode is a mode in field clearly optimized for the observation of samples sensitive to the electrons. It is essential to the study of biological samples observed in a vitreous hydrated state. It makes it possible to at least irradiate the zone of the sample which one wants micrographier. The principle of this mode is the following. With weak enlargement (approximately 5 000 X), one selects a zone of interest in the sample. With this enlargement, one irradiates only very slightly the object (the electronic amount is proportional to the square of the enlargement). Starting from this positioning, the zone of exposure and the zone of settling are defined. They are distant of some micrometers one of the other. The development requires to irradiate the sample throughout one several second to the final enlargement (typically 40 000 X). That deteriorates the sample and this is why one does it at a certain distance from the zone of exposure. This last zone is irradiated with the final enlargement only time to record a micrography (approximately 1 second).
Instead of being interested in the formed image, one can be interested in diffraction of the electrons. While placing oneself in the focal plan of the beam and either in the image plan (simply by changing the tension in the electromagnetic lenses), one obtains the figure of diffraction, similar to the stereotypes of Laue obtained in Diffraction of x-rays. One can thus visualize the directions in which the electrons go and thus characterize the crystals (organization of the atoms, orientation,…).
See also: Theory of diffraction.
Mode in field sinks
By selecting a particular beam diffracted to form the image, one obtains a contrast called “in dark field” ( dark field ). According to the local orientation of a crystal, either this one lets pass the electrons in straight line, in which case there is a clear contrast, or it deviates the electrons and one obtains a dark contrast.
Let us suppose that, for a crystal, the beam is almost in conditions of diffraction: the beam is not deviated; it thus crosses crystallite without encumbers and this one appears clear. So now the mesh is locally distorted by a defect (for example a Dislocation), then the beam is locally in condition of diffraction and is deviated. This zone of defect appears dark.
Microscopy with high-resolution (HRMET)
Certain electrons are deviated (diffracted), others are transmitted in hot line. If one makes interfere a beam transmitted in hot line with a diffracted beam, one obtains a figure of interference. Contrasts on the image obtained thus are directly correlated with the projected potential of the sample. According to the defocusing and the size sample, this correlation changes. A simulation of the figure of interference is then necessary to interpret the image obtained and to say if the atomic columns are located on the white points, black or between the two. It should not be believed that an image HRMET is a simple photography where the white points (or blacks) are atoms. These images, after treatments, enable us all the same to draw from information on the crystalline organization as well as the defects which are there (grain boundary, dislocations…).
Microscopy in transmission with sweeping (METB)
This technique, so called STEM ( scanning transmission electron microscopy ), consists in giving a movement of sweeping to the beam. The main advantage is to be able to make an ultimate analysis of x-rays emitted by the atoms under the effect of the electrons (see the article on the Microsonde of Castaing for more details) and thus to draw up a chemical cartography of the analyzed part.
If the electron microscope in transmission were perfect, its resolution would be of about size wavelength of the electrons. For electrons accelerated to approximately 100 Kv, it would be about the Picomètre (10-12 m). However electronic optics is much less effective than the photonic optical . The practical resolution is of some Angström.
When an electron crosses a lens, it undergoes a change of management. The extent of the deviation depends on the wavelength of this last (i.e. of its energy, of its color). However the electrons composing the beam do not have all the same wavelength. It is said that the beam is not monochromatic. So the image of a point will not be a point but a disc.
The beam is not monochromatic for two reasons. Firstly, on the level of the gun, the emitted electrons have an energy which varies around a certain value (chromatic variation). Secondly, at the time when it crosses the sample, an electron of the beam can undergo inelastic shocks with electrons composing the sample. In this case, the electron of the beam loses energy.
The angle of deviation of the electrons is also proportional to the distance between the center of the lens and the place where the electrons cross this lens. But the relation is not linear and the electrons which pass far from the center of the lens are deviated too much and consequently focused ahead.
The lens does not deviate the electrons in the same way according to their initial direction. If the lens objective is astigmatic, a point on the object will have as an image a line.
electronic microscopy by Christian Colliex, research director with CNRS on the site of the physics laboratory of the solids on the site of the University of Paris XI
- Examples of micrographies in transmission
- In the eye of the electron microscope in transmission
- Definition and principle of the electron microscope in transmission
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