The optical microscope or light microscope is an optical instrument with an objective and an eyepiece that magnifies the image of a small object (which characterizes its optical power) and separates the details of this image (and its power of resolution) so that it is observable by the human eye. It is used in biology, to observe cells, tissues, in petrography to recognize rocks, in metallurgy and metallography to examine the structure of a metal or alloy.
It should not be confused with the binocular loupe which does not require flat samples of thin, or reflective, and allows to observe natural parts without preparation by magnifying the image of a low factor, but keeping a stereoscopic vision conducive to the macroscopic examination revealing grains, cracks, fissures, etc.
- 3 Uses and Development
It's hard to say who invented the compound microscope. It is often said that a Dutch optician Hans Janssen and his son Zacharias Janssen fabricated the first microscope in 1590, but this comes from a statement by Zacharias Janssen himself in the middle of the seventeenth century. The date announced is quite unlikely since it was shown that Zacharias Janssen was born around 1590.
Another favorite as the inventor of the microscope is Galileo. He developed an occhiolino , a microscope composed of a convex lens and another concave in 1609. Athanasius Kircher describes his microscope in 1646 1 which he uses for the observation of blood.
A drawing by Francesco Stelluti of three bees appears on the seal of Pope Urban VIII (1623-1644) and passes for the first image of microscopy published 2 . Christian Huygens, another Dutchman, developed in the late seventeenth century a simple dual eyepiece corrected chromatic aberrations, which was a great step forward in the development of the microscope. The Huygens eyepiece is still manufactured today, but suffers from a fairly small field and other minor problems. Antoni van Leeuwenhoek (1632-1723) is generally credited for having drawn the attention of biologists on the uses of the microscope, even if ordinary magnifying glasses were already manufactured and used in the sixteenth century. Van Leeuwenhoek's hand-made microscopes were simple, small-sized instruments with a single but strong lens. In comparison, multi-lens systems remained difficult to develop and it took no less than 150 years of optical development before the compound microscope could deliver image quality equivalent to that of Van Leeuwenhoek's simple microscopes. Nevertheless, and despite many claims, Antoni Van Leeuwenhoek can not be considered as the inventor of the compound microscope. Robert Hooke is also one of the first to design it.
The optical microscope is an optical lens system whose purpose is to obtain an enlarged image of the observed object.
The object to be observed is placed in front of the first optical group called "objective". If the object is beyond the focal length, it forms a reversed real image of different size; the image is larger than the object if the object is located at a distance less than twice the focal length of the lens.
The second optical group on the observer's side is the eyepiece: it is positioned so that the image is in its focal plane. Thus, the eye observes an "infinite" image (for a standard observer), thus releasing the muscles responsible for accommodation, offering a better visual comfort.
It is a dioptric centered system, composed in part of doublets to correct some of the optical aberrations.
In contrast to other optical systems that are defined by their optical magnification (telescope) or magnification (camera), the appropriate term for the microscope is its power , the ratio of the angle, under which the object is viewed through the instrument, to the length of this object.
The most widely used illumination technique in conventional wide-field microscopy is Köhler illumination, which guarantees the best image quality.
From bottom to top :
- mirror: used to reflect ambient light to illuminate the sample from underneath, in the case of a transparent sample (eg a thin slide in biology or geology, or a liquid);
- artificial light source of better color temperature and stability and by the use of a condenser which allows this light to fill in a homogeneous and regular way the observed field, and especially not to show, by its setting adequate, the mechanical details of the light source (turns of the filament of the bulb). The light source may be more elaborate and include an independent housing, possibly in polarized or ultraviolet light, to highlight certain chemical properties of the material, or illuminate the sample above (especially in metallurgy)
- diaphragm: opening of variable diameter to restrict the amount of light that illuminates the sample. As for a camera, the diaphragm mainly allows to vary the depth of field (fully open for histological sections and more closed for digestive parasite egg searches);
- platinum sample holder: where the sample is placed; "valets" are used to hold the sample when it is thin (eg a blade). The stage can be mobile (left-right and front-back), which allows to sweep the sample and select the observed part;
- objectives: lens or set of lenses achieving magnification. There are usually several objectives, corresponding to several magnifications, mounted on a barrel. Some objectives are called immersion because their power can only be achieved by eliminating the air gap between the sample covered by the coverslip and the front of the lens. For this purpose, cedar oil or synthetic oils whose refractive index is close to that of glass are used.
- rapid and micrometric focus; for the image to be clear, the object must be in the focal plane of the lens; these wheels raise and lower the objective-ocular assembly with a rack system, in order to bring the focal plane to the area of the sample to be observed;
- eyepiece: lens or set of lenses forming the image in a restful manner for the eye; the rays arrive parallel, as if they came from very far, which allows a relaxation of the muscles controlling the crystalline lens; two eyepieces placed on a so-called binocular head makes observation more comfortable (even if it does not bring stereoscopic vision).
The eyepiece can be replaced by a camera, or - in the case of video microscopy - by a video camera or a CCD camera to make a digital acquisition. This makes it possible to observe on a video monitor (television type screen) and to facilitate the use and processing of images (printing, computer processing, telemedicine, etc.).
The resolution of a microscope refers to its ability to separate very close details. Regardless of the sensor used and the aberrations or imperfections of the lenses, the resolution of the optical microscope is fundamentally limited by the diffraction of light. Indeed, because of the diffraction, the image of a point is not a point, but a spot (the spot of Airy or more generally the function of spreading point - PSF). Thus, two distinct but neighboring points will have for images two spots whose recovery can prevent to distinguish the two image points: the details are then no longer solved.
According to Abbe's theory, the resolution limit (transverse) of a microscope, that is to say the smallest distance below which two neighboring points will no longer be distinguished, can be expressed simply by means of the illumination wavelength, the refractive index at the lens output, and the half angle of the maximum accessible light cone.
where NA denotes the product or numerical aperture of the lens. We can increase the resolution in two ways:
- by increasing the refractive index. This can be achieved by using an immersion lens: the lens is immersed in a liquid whose refractive index is close to the maximum of 1.5 - that of glass;
- by decreasing the wavelength. However, if we stay in visible light, it is not possible to go below 400 nm.
The resolution limit of a conventional light microscope is about 0.2 μm. The transmission electron microscope will reach a limit 100 times smaller.
Many photonic microscopy techniques can increase the resolution. When they exceed Abbe's limit, they are called "super-resolution" or nanoscopy. Among others:
- linear structured illumination techniques (eg, the SIM microscope) and tomographic techniques that seek to recover high spatial frequencies cut in a conventional microscope. These techniques allow to increase the resolution, without exceeding the Abbe limit.
- techniques using evanescent waves (SNOM);
- techniques using optical impulse response (PSF) shaping: confocal microscopy, STED microscopy (super-resolved);
- techniques using the successive localization of individually photoactivated molecules, "photoactivation localization microscopy" (PALM, Betzig et al., 2006) and "stochastic optical reconstruction microscopy" (STORM, Rust et al., 2006). These two microscopies are identical in principle, but do not use the same type of fluorophore.
When using a conventional microscope, it is used in transmission, that is to say that the light passes through the sample observed. It is also possible to work "in reflection". In this case, the sample is illuminated on the same side as the observer, either from above for a right microscope and from below in the case of inverted microscopes used in metallography or crystallography. The light produced by the source passes a first time through the lens, arrives on the sample, is reflected and goes back through the lens for observation which requires several sets of mirrors or prisms.
Reflective microscopy makes it possible to examine opaque objects, or too thick for transmission. In return of course, it can only give information on the surface of the sample in the case of observation in white light; in polarized light, it reveals the grain orientations of the constituents of minerals or metals.
A classic case is metallography where observations of metal pieces called micrographs are made in this way. As said above the microscope is often reversed, the piece to be observed placed on the support plate (usually pierced with a circular hole).
In contrast to diascopic lighting ( dia - through), episcopic lighting ( epi - on) makes it possible to observe opaque objects in color and giving them a more "natural" rendering.
The idea of such a lighting is old, since in 1740, Descartes inspired Lieberkühn who created for his observations under the microscope a silver mirror surrounding the lens, the focus of this mirror targeting the preparation.
Lightfield (or "brightfield") light microscopy is the simplest and oldest microscopy technique. The wavelengths used (visible spectrum) limit the separating power of this microscope to 0.2 microns for those of them that have the best optics.
The illumination is by transmission of white light, that is to say that the sample is illuminated from below and observed from above. The limitations of this technique are mainly a low contrast of most biological samples and a low resolution due to blur created by the material off the focal plane. In return, the technique is simple and the sample requires only minimal preparation.
If the sample is illuminated from above, the microscope is called "inverted microscope". The objective is then located below the preparation, and the eye tube straightens the beams of light so that the eyepieces are "normally" positioned for the user.
The dark-field microscope, which uses the principle of "dark field microscopy", improves the contrast of transparent but untinted samples 3 .
Dark field illumination uses a carefully aligned light source to minimize the amount of light directly transmitted and to collect only the light scattered by the sample. It dramatically increases contrast, especially for clear samples, while requiring little equipment and simple sample preparation. However, this technique suffers from a low light intensity collected and is still affected by the resolution limit.
Rheinberg illumination is a variant of dark field illumination in which transparent color filters are inserted just before the condenser, so that more or less oblique light rays are colored differently (the background of the image may be blue while the sample appears bright yellow). The resolution limit is the same as in the dark field. Other combinations of colors are possible, but their effectiveness is quite variable 4 .
Dark field microscopy is particularly suitable for fresh samples and allows microcinematography (for example, bacteria on the move). She has no interest in colored objects (smears or colored cuts). It is particularly useful for:
- to observe beings or flat objects with regular and transparent structure such as diatoms, radiolarians …
- observe very fine punctiform or linear objects, the size of which would be limited for the separation of the light-field microscope. These objects will give an image of points or very luminous features (example: Treponema pallidum , agent of the syphilis) and with sharp outlines if the object is sufficiently thick, or for the largest bacteria (example: Borrelia, agent of the disease of Lyme).
The use of oblique illumination (by the side) gives a three-dimensional appearance image and can highlight aspects otherwise invisible. This is the main advantage. The limitations are the same as those of light field microscopy.
In polarized light microscopy, the sample is placed between a polarizer and an analyzer in order to detect the polarization variations of the light after passing through the sample. This technique is very useful for the observation of birefringent media, especially in mineralogy.
When certain compounds are illuminated by a high-energy light source, they emit light at a lower energy. This is the phenomenon of fluorescence. Fluorescence (or epifluorescence ) microscopy is a technique using an optical microscope equipped with the laser emitter of a photon radiation having a precise wavelength. This radiation will excite a target molecule with fluorescent properties. It makes it possible to take advantage of the phenomenon of fluorescence and phosphorescence, instead of or in addition to the classical observation by Reflection (physics) or absorption of natural or artificial visible light 5 , 6 .
This method is now of prime importance in the life sciences thanks, in particular, to the marking of cellular or tissue structures by fluorescent molecules, such as rhodamine or fluorescein. It can be very sensitive, even allowing the detection of isolated molecules. Different structures or chemical compounds can also be detected simultaneously using different compounds that will be differentiated by their fluorescence color.
The total internal reflection fluorescence microscope (TIRF, total internal reflection fluorescence microscopy), or microscopic evanescent wave, is a particular type of optical fluorescence microscope to examine a very thin slice of a sample (less than 200 nm thick), thanks to a particular mode of illumination: the total internal reflection.
Phase contrast is a widely used technique that makes it possible to highlight the differences in refractive indices as difference in contrast. It was developed by the Dutch physicist Frederik Zernike in the 1930s (he was awarded the Nobel Prize in 1953). The nucleus of a cell, for example, will appear dark in the surrounding cytoplasm. The contrast is excellent, however this technique can not be used with thick objects. Often, a halo is formed around small objects that can drown details.
The system consists of a circular ring in the condenser that produces a cone of light. This cone is superimposed on a ring of similar size in the lens. Each lens has a different size ring, so it is necessary to adapt the condenser to each lens change. The ring in the lens has special optical properties: it reduces the intensity of direct light and, more importantly, it creates a quarter-wavelength artificial phase difference that creates interference with diffused light, which creates the contrast of the image.
Interferential contrast (IC, IC for English speakers) is a technique that allows you to visualize transparent objects by increasing their contrast. It is currently the CI according to Nomarski, invented in the 1950s is the most widespread. This technique brings a more important compared to the phase contrast by suppressing the halo phenomenon specific to the latter. It has established itself in microscopy in many areas today.
The confocal microscope generates an image in a totally different way from normal light field microscopy. The resolution is slightly better, but the most important point is that it allows to form an image of cross sections without being disturbed by the light off the focal plane. It gives a clear picture of objects in three dimensions. The confocal microscope is often used in conjunction with fluorescence microscopy.
The lensless microscope records the diffraction pattern of a laser by the sample (principle of holography), then processes this figure by computer to generate the image.
The interference contrast microscope is a microscope that exploits the interferences of two beams of a light wave passing through a sample.
The observed sample must meet certain conditions:
- of flatness, so that the objective gives a clear whole image, failing which one can observe only a restricted portion
- in transmission, it must be thin so that light passes through it and makes visible only a few elements (cells) in the case of biology;
- in reflection, the surface must be generally polished so that the stripes do not mask what we want to observe;
- the parties to be observed must be able to differentiate themselves:
- color differentiation by chemical staining of standardized solutions, for biology;
- chemical attack by acids to reveal defects in metallurgy;
- other differentiations by lighting in polarized light, ultraviolet (fluorescence), or by interferential principle, revealing other aspects, invisible to the naked eye.
In biology, it is necessary, beforehand, to place the tissue section (or the liquid containing living organisms) between a slide and a slide. The objective must approach the blade for the development without, by clumsiness, destroy the preparation become very fragile.
Because of the preparation, optical microscopy requires a large amount of complementary devices for the sole purpose of microscopic observation.
Take the case of the biopsy in medicine and biology (pathology): the microscopic diagnosis of biological parts taken by biopsy during an operation, imposes short delays. To prepare the blade, we use a device called cryotome, a kind of "ham slicer", placed in a cryostat (freezer), which allows to cut very thin slices of the body that will be observed by cooling quickly, then cutting it with the blade of a special razor, sharpened on another glass plate machine using diamond paste. If you want to work at room temperature, the delays are longer and require dehydration and replacement of water removed by paraffin (24 hours) for the sample to retain its rigidity; then it is colored by several substances of alternating actions of very long duration, too.