Dark field microscopy

Dark-field microscopy is a well known for over 250 years variant of light microscopy. It leads to a dark Backgrounds, before take off, the observed structures bright. This can be generated by transparent objects with very low contrast still well-resolved, high-contrast images without any prior staining of the specimen is required. Also living objects are well observable. Until the development of phase-contrast microscopy in the 1930s, the dark-field microscopy was the only method of contrast enhancement in unstained specimens. In contrast to the dark-field microscopy, the technique of " normal" refers to when light microscopy as bright field microscopy.

The principle of dark field microscopy based on the fact that objects not only absorb light but also always divert a portion of the light beam. If the lighting is adjusted so that the direct rays of light pass to the microscope lens, the viewer sees only the deflected light. One of the deflection causes the called Tyndall effect scattering of light by small particles, which is, for example, also be observed when light is incident in a dark room and the dust within the light beam is clearly visible. Even particles that are smaller than the resolution limit of the microscope, deflect light away and let therefore be detected with a dark field microscope. Some properties such as the mobility of particles can be examined so. This application had the early 20th century, larger than ultramicroscopy importance.

The illumination of the specimen can be seen from the lens after the preparation be carried out by (transmitted light) or the lens side (incident light) or sideways, as is the case with the gap ultramicroscope. Transmitted light and reflected light darkfield are possible both in "normal" microscopes and stereo microscopes.

2.1 Common Ground
2.2 Lighting with central aperture
2.3 darkfield
2.4 Due to the light-dark box in stereo microscopes

3.1 Before 1900
3.2 From about 1900

6.1 Classical reflected light dark -field microscopy
6.2 darkfield reflection in stereo microscopes
6.3 Sidestream Dark Field Imaging

7.1 Optical basics
7.2 Examples
7.3 ultramicroscopy

Comparison of bright field and dark field

Bright and dark field in transmitted light illumination

Transmitted light illumination, an arrangement is referred to in microscopy as viewed from the lens when the illumination is from the back of the specimen, the light passes through the specimen (transmission) and then enters the lens. The normal transmitted light microscopy, more precisely transmitted light bright field microscopy, the variant most commonly used in biology and medicine, are also found in school microscopes application.

In classical transmitted light bright field microscopy, the image contrast is produced mainly by the fact that the specimen absorbs some of the incident light, and so the corresponding region appears darker (see Figure 1). However, many microscopic objects are largely transparent or very small and therefore absorb very little light. They produce only a low contrast in the bright field microscope and are therefore difficult to see against the light background ( see Figure 2 left). Such objects can deflect light, thus changing the direction of some light rays due to scattering, diffraction, refraction and / or reflection. Under bright-field illumination, these distractions can hardly notice because the brightness of the deflected light beams is much weaker than the brightly lit image background. Within certain limits, the contrast can be increased in bright-field illumination by using a smaller aperture in the optical path of the illumination ( condenser diaphragm ) is selected ( see Figure 2, center ). At the same time, this also strengthen aberrations and it created at the edge of the objects interfering diffraction pattern (see small image details in Figure 2).

The transmitted-light darkfield microscopy, the preparation of the back is illuminated so that the light does not pass directly into the lens, but only the diffracted light in the specimen. The background of the image thus appears dark, while objects in the specimen appear light ( see Figure 1, right). This also works, and especially in largely transparent samples. While the differences in brightness of the deflected light are difficult to detect due to the high light intensity of the bright field imaging, these differences appear much stronger in the dark field imaging. The fine fat traces of a fingerprint in Figure 2 (right) are therefore clearly visible. However, the already discernible in brightfield illumination impurities ( particles and scratches ) show the dark field such a strong contrast that they are mapped in the picture just as bright, overexposed spots.

In transmitted light darkfield illumination, it is particularly important that slides, cover glass and the glass surfaces in the microscope are clean, because every dust particle by its deflection of light contributes to the background noise. Also, light-deflecting structures must not occur in different levels to each other, because their signals overlap otherwise. Accordingly dark field illumination is not suitable for thicker preparations as typical tissue sections.

Physically can be transmitted-light darkfield illumination describe as an illumination, in which the main maximum diffraction of light ( see diffraction disk ) is not in the back focal plane of the lens passes. Only leaked light, resulting, for example, by diffraction sidelobes take part in the image formation.

Bright and dark field in reflected light illumination

From Barlight one speaks in light microscopy when the light from above (more precisely, from the lens side) falls to the preparation. The lighting takes place either through the lens itself, or through a stand-alone lighting device that is arranged to the side or around the lens. The angle at which the light is incident on the object, determines the appearance of the image. If a majority of the light directed from the sample reflected captured by the lens, the object appears in the picture bright ( bright-field illumination ). If the illumination is, however, so far from the page that directed the reflected light shines on the lens over, it is called dark field illumination.

For material tests, the bright field illumination is the most commonly used technique for the illumination of rough, low reflectivity. The reflected-light brightfield illumination corresponds to the normal way of seeing the human being: Smooth, highly reflective surfaces appear due to its strong luster bright ( Figure 3, left). The reflection gives the typical gloss metal surfaces. Below arranged by glass or other transparent surfaces structures would only be difficult to see in this kind of lighting by the strong reflection at the surface.

In reflected light dark -field illumination smooth, highly reflective surfaces appear dark. However, edges and surface defects such as scratches or deposits shine bright (Figure 3 right). These are highlighted strong and can more easily detected or are detected with simple image processing techniques. With rough, little reflective surfaces, the lateral arrangement of the reflected light dark -field illumination for local shadowing provides, so that surface structures are a bit plastic. This effect can be significantly enhanced by a one-sided illumination.

Dark field microscopy outside the light microscopy

The terms dark field and bright field can be transferred to microscopic methods using for image generation no light, but other signals. It is distinguished according to whether or not deflected excitation signal is registered by the detector (light box ), or whether only the changing of the sample signal to the imaging contributes ( dark field ). With the dark field method, there is described for example in electron microscopy ( see, for example scanning transmission electron microscope) and in the acoustic microscopy.

Dark field illumination in transmitted light microscopes today

The easiest way with a normal light microscope with light source, condenser and objective to produce a dark field illumination at Köhler shear lighting, is to close the condenser diaphragm closely followed so long to move sideways until no direct light more penetrating into the lens. Illumination is here, therefore, only from one side. However, just newer microscopes have often unable to move relative to the diaphragm condenser.

Common ground

Better image quality is achieved with a centered condenser using an auxiliary device. This auxiliary device limits the illumination of the preparation to a cone (yellow in the diagram at right). The inner part of the cone contains no light (gray in the diagram ). The coming from the condenser cone surface is focused in the specimen plane and widens the simplest case, then again, so not completely deflected light passes to the lens aperture, the image background is dark. Only light that is deflected by the observed object, from entering the lens and forms an image with light structures on a dark background. All of today's transmitted-light darkfield illumination produce a taper, but these do not always occur through the whole preparation through: In some cases it leads to total reflection of the undeflected light at the top edge of the cover glass.

To produce the illumination cone, two different methods are used. A central aperture for the generation of the cone is simple to manufacture and use, affordable, and therefore more popular. This method is particularly suitable for lenses with relatively low magnification, the thickness of the light cone surface can be tuned by simply changing the aperture on the lens used optimally. Special darkfield reach by mirrors techniques, a higher light output and can also be higher magnifying lenses meet the requirements through immersion. The picture quality is better.

When the illumination cone shell as in the diagram passes through the specimen through, he must pass outside the lens. A dark field illumination is only possible when the angle of light emerging from the condenser (opening angle ) is greater than the angle of the collected light from the lens. The larger the opening angle of a lens or the condenser, better is the maximum achievable resolution. Instead of the opening angle of the numerical aperture is provided with lenses and condensers, which can amount up to 0.95 and without immersion oil immersion to about 1.4. For dark-field illumination so the numerical aperture of the condenser must be higher than that of the lens in use. Without immersion condenser of the application is therefore limited to lenses with a numerical aperture of about 0.75 or less. 40x objectives, which are used without immersion, often have a numerical aperture of 0.65.

Lighting with central aperture

Here is an annular aperture is used in an otherwise normal transmitted light bright field microscope. This central panel ( 1 in the upper diagram on the right) has a translucent edge or ring, thus reducing the lighting using a normal condenser (2) on a cone (3). To take full advantage of the opening angle of the condenser, one as far outboard part of the condenser will be used. The larger the aperture angle of the lens used, the greater must be the diameter of the central opaque surface, the illuminance is reduced accordingly. From the stage to the slide (4 ) from the light thus extends past the lens (6). Only structures in the specimen deflected light ( 5) enters the lens. The central aperture can be used as an insert beneath the condenser lens through a normal light microscope.

The more widely used phase-contrast microscopy, though based on a completely different optical phenomenon, but even there diaphragms are used. This ring covers can be sometimes divert as dark field aperture. Phase contrast ring diaphragms are designed so that the cone of light entering at the correct setting in the lens and not because will pass, as is required for dark field. Therefore, can be used for a given lens only those phase-contrast aperture ring as dark-field aperture, which are actually meant for lenses with a significantly larger opening angle (higher numerical aperture ). For example, a phase-contrast annular diaphragm for a 100x oil immersion lens usually is as dark field stop for 10x and 20x dry objectives, as oil immersion lenses have a larger opening angle.

Darkfield

For particularly high demands on the imaging quality special darkfield be used instead of the central aperture. There are Trockendunkelfeldkondensoren and Immersionsdunkelfeldkondensoren, the latter immersion oil or water between the condenser and the slide is introduced. Thus, a higher numerical aperture and thus a higher resolution is possible. A Immersionskondensor provides better contrast because reflections on the slide base and condenser surface is avoided, leading to a brightening of the image background. Its handling is more complex, however, partly because oil makes thorough cleaning is required. The disadvantage of both Dunkelfeldkondensorarten against a central aperture is the more complex change to a bright-field illumination, because the condenser needs to be replaced for this. Trockendunkelfeldkondensoren are suitable for lenses with numerical apertures up to 0.65 or 0.75, while Immersionskondensoren for lenses with numerical apertures up to 1.2 can be used.

Modern darkfield are usually Kardiodkondensoren. This initiates convexly curved central mirror, the incident light to the outside around a current concave mirror, so that the cone-shaped shell is generated ( refer to similar drawing 1910 on the right). The concave mirror has a ideally like a cardioid shaped surface, hence the name. For technical reasons, however, this surface is designed as a spherical surface, without this leading to significant loss of quality. In contrast, a Paraboloidkondensor has the shape of a truncated paraboloid. The light is deflected only once, namely by total internal reflection ( see drawing of Wenham Glasparaboloid below), which in turn an all-around running light cone surface is generated.

To ensure that the numerical aperture of the lens is smaller than that of the condenser, can additionally be used a lens, in which the numerical aperture can be limited by a movable iris diaphragm. The opening angle of the lens can thus be optimally adapted to the diameter of the cone of illumination to the latter to be still able to hide.

Cardioid and Paraboloidkondensoren are also referred to as catoptrical darkfield, since the light deflection takes place in them by mirroring while this is happening in the so-called dioptric condensers through glass lenses.

Transmitted-light darkfield stereo microscopes

Even for stereo microscopes transmitted-light darkfield illuminations are available. The illumination device is housed in the stand. Apart from the actual light source, such as a halogen lamp, a central cover and outer upstanding reflective surfaces are used to allow the illumination of the object with a conical surface. Thus, the principle is similar to the mirror condenser described above. The object is placed on a glass plate that closes off the stand base to the top. The image is made up of light rays that have been deflected in the object by reflection, refraction or diffraction. Typically, the central cover against a ground glass can be replaced, so that in addition to dark-field and bright-field transmitted light illumination. The outside mirrors are derived then still the same amount of light at an angle to the specimen as before, but by the much brighter bright field illumination no longer leads to visible effects.

Previous approaches for transmitted light darkfield observation

Before 1900

In the 17th century dark field microscopy of Antoni van Leeuwenhoek, Robert Hooke and Christiaan Huygens was used to observe blood components or small organisms. However, no special equipment were used. Rather, the light source was about a candle, positioned so that no direct light fell on the lens.

Even with a very oblique illumination mirror dark field microscopy is possible. The first to describe a special apparatus for the dark field illumination was 1837 Joseph Bancroft Reade ( 1801-1870 ), whose method in John Quecketts "Practical treatise on the use of the microscope" in 1852 referred to as background illumination. The light source was placed at the side, a positive lens focused the light onto the specimen so that it is not leaked light was guided past the lens. During the 19th century, more lighting apparatuses have been developed by a number of authors. Because refraction at the glass surfaces chromatic aberration causes, which is particularly troublesome in darkfield microscopy, Spiegelkondensoren also been developed, as does not occur in this mirror errors. The reflection was achieved either by reflective surfaces, or by total internal reflection.

Francis Herbert Wenham (1824-1908) described the 1852-1856 work in several different dark-field illumination principles. In addition to a lateral light ( with effect similar to Reade ) were condensers for a centrally positioned light source to including a hollow, silvered paraboloid and a massive Glasparaboloid, in which the reflection by total reflection came into existence ( see figure). Here, the slide was in direct contact with the condenser. The preparation was embedded in Canada balsam or liquid. Between the cover glass and the lens was air. The principle of diffraction, which is essential for an effective dark field illumination of small objects was not understood at that time. Wenham therefore assumed that the observed effects were due to the fact that the object was illuminated from above, namely, by light that has been reflected from the upper glass edge by total reflection on the preparation.

From about 1900

By the end of the 19th century dark field microscopy, but little used by amateurs in science because they are not with higher resolution lenses ( high numerical aperture ) worked. The work of Ernst Abbe end of the 19th century understood the basics such as optical diffraction. This took W. Gebhardt at Zeiss, by having a central aperture for the Abbe illuminating apparatus suggested for the dark field illumination, the Zeiss in 1898 took into the program. If immersion is used between the condenser and the slide, dry lenses could be used with an aperture up to 0.95. At times these central aperture was included with all appropriate equipment, but she found little favor among customers, this has been discontinued. The company Wiener microscope Reichert offered a similar solution.

The discovery of the syphilis pathogen the dark field microscopy experienced an upswing from 1906, as it allowed a good representation of live spirochetes, including the exciting part. Several large companies developed improved darkfield microscope. The Karl Reichert contained a central aperture of variable size. Henry Siedentopf developed in 1907 for Zeiss a paraboloid condenser. Although the design of the corresponding Glasparaboloid Wenham a darkening in the center of the lower side of the paraboloid, but through improved manufacturing techniques, the optical quality could be increased so that the inner and outer aperture of the illumination cone surface 1.1 and 1.4, respectively. Due to the work Abbes was clear that the diffraction has a decisive role in the image formation and that the total reflection at the cover glass only helps to prevent the entry of non- deflected light into the lens. In a later version, the so-called Helldunkelfeldkondensor, the central darkening could be removed via a lever, so that a quick change between dark and light field was possible.

Approaches have been described based on the fact that the illumination of the specimen with a higher numerical aperture, that is, with a wider angle is carried out, as they can be absorbed by the lens. But, the reverse approach is possible: the preparation is illuminated with a full cone of a low numerical aperture ( for example, 0.2 ). This can also be employed high-resolution lenses, because the numerical aperture and thus the opening angle can be any size, but they must be significantly larger than that of lighting. The non-deflected light in the specimen will then occupy only a central zone in the lens, while the outer region remains free of direct illumination light. The undeflected light is effectively removed later in or behind the lens at a suitable location in the beam path. This was called " konaxiale arrangement " or a " central dark field " and the ultra- microscopic methods ( see below ) were counted. Disadvantage of this approach is that in the preparation much higher light levels are achieved than for example with a central aperture in the condenser, thereby disturbing side diffraction patterns arise in preparations with many objects.

Henry Siedentopf used for such a system he developed an objective in which the otherwise hemispherical rear of the front lens was (the first glass in the lens body ) ground flat and painted black. Carl Metz (1861-1941) in 1905 Leitz developed a system with oil immersion objectives, in which a plunger aperture (also: Hopper panel) was introduced from the rear movable in the lens. This made it possible to use the same lens without the aperture for bright field applications without loss of brightness occurred. But the adjustment was difficult.

Vladimir Sergeyevich Ignatowski developed for a Leitz Darkfield, who owned two reflective surfaces but was easier to handle than previous similar models (see diagram from 1910 above). It was sold from 1907. The cross-sectional drawing of the developed by Felix Jentzsch successor model of 1910 was a template for a Leitz logo, the so-called Leitz coffin.

Even Henry Siedentopf at Zeiss designed a condenser with two reflective surfaces, which is very similar to the enhanced condenser of Ignatowski. For theoretical reasons, the second reflecting surface should be of a section of a cardioid. Cardioid surfaces were only difficult to manufacture. Instead, a spherical surface has been used which produced the same effect within the manufacturing tolerances. Nevertheless, the device of Zeiss was marketed as Kardioidkondensor.

Pros and cons of the transmitted-light darkfield illumination at a glance

Advantages:

Small, also uncolored, objects can be observed with high contrast, especially well in low concentration in thin specimens.
Even objects of the resolution limit cause signals when the lighting is strong enough.
Some forms of dark-field illumination, particularly at low magnification, are to be created easily and without significant cost.
In dark field illumination in contrast to the bright field illumination no entoptic phenomena occur, streaks arising in the eye itself and cast shadows on the retina.

Cons:

Although cause surfaces of objects by the refractive index change signals, but not a homogeneous interior, so that then seen in the picture, only the outline.
For thicker preparations or preparations with many objects, the technique is very useful, since then too many signals about from different focal planes counteract the dark-field effect.
Impurities in the beam path also cause interfering signals, so the demands on the cleanliness of equipment and preparation are very high.
For more demanding special condensers are required because the reflections between the various lenses reduce the dark field effect normal condensers.
Since the opening angle of either the condenser or the lens must be reduced, the resolution is reduced as compared to bright field and other contrast enhancing methods, such as phase contrast and differential interference contrast

Rheinberg illumination

The Rheinberg illumination (also: optical coloration or contrast colors, lighting), is a variant of dark field microscopy with central aperture, which was described in London by Julius Rheinberg first time in 1896. The central panel is replaced by a circular filter with two colors in a concentric arrangement: A color forming an outer ring, it corresponds to the ring in the conventional ring aperture. The light passing through here is thus only fall into the lens when it is deflected in the preparation. In the middle, otherwise opaque region is the second color. It sets the image background. The result is aesthetically sometimes very appealing images without any additional structures are visible.

Under the name Mikropolychromar delivered Zeiss around 1939 until after the Second World War Kondensorzubehör from the Rheinberg illumination was possible. A central bright field and dark field illumination, an exterior could be colored differently with filters. Zeiss recommended this device " to facilitate the examination of unstained objects with low contrast ." Gerlach ( 2009) wrote about this facility she had " certainly of some importance before the introduction of the phase contrast method, " had. The company Reichert drove under the name Optikolor a mirror condenser - based solution that also enabled Rheinberg illumination.

With three colored Rheinberg filters, particularly preparations can effectively represent, which are clearly structured. The outer ring of the filter is divided into four 90 ° angle, the respective opposite quadrants are colored similarly but adjacent different colors. The inner circle is colored with the third color. The two-tone outer ring causes structures that scatter from left to right, are shown in a different color than those which scatter in the specimen plane from front to back. Examples of such preparations are diatoms or fabric.

Dark field illumination in reflected light microscopes

Classic reflected light dark -field microscopy

In light microscopy, the light is irradiated from the same side, it is also observed from which. This method is applied to opaque materials, such as minerals or materials testing. In reflected light bright field illumination, the illumination can be fed through the same lens beam path, is the observed.

In reflected light dark -field illumination illumination and observation beam path are separated, however: special lenses have an additional outer region, which is the illumination beam path reserved (see diagram ). The inner region corresponds to a normal lens, with dark-field illumination, it serves exclusively the observation. The outer region corresponds to the condenser. Here, the light is directed obliquely onto the specimen ( 4) (1 in the drawing) by an annular concave mirror in the outer region (3). If the preparation is a flat mirror, the light reflected there would completely bypasses the inner portion of the lens: the image would remain dark. Surface structures, such as scratches, however, deflected light is received by the lens (5).

In some darkfield epi-illumination lenses it is possible to individual sectors of the illumination ring or hide. As a result, shadowing can be reinforced, so that structures that extend in certain directions can be better recognized. In so-called Ultropak lighting devices, the, condenser ' which is attached to the lens to be moved in height to illuminate different levels in the preparation max. At low magnifications, the required light intensity can also be achieved by a laterally established external light source, such as fiber optics lights.

Surface structures such as scratches stand out in reflected light darkfield clearly from the background, as it is directed at them reflected or scattered light partially in the central area of the lens. Such structures are therefore in the picture light on a dark background. According reflected light dark -field illumination is particularly suitable for the study of surfaces, such as in materials science. Dark field illumination is widely used in reflected light microscopes. In contrast to the transmitted-light darkfield illumination can epi- darkfield illumination can also be used with the strongest lenses. To avoid unwanted reflections, is carried out preferably without a coverslip.

Darkfield reflection in stereo microscopes

For stereo microscopes darkfield epi-illumination can be realized by the lighting is to the surface rather glancing and directed the reflected light, the lens does not reach directly. This is possible, for example by a slight tilt of a flat preparation or a clever arrangement freely positionable light sources (eg gooseneck lighting with a long, bendable bracket). For annular dark-field illumination on all sides, there are special ring lights with a beam angle of 60 ° for example, which are located at a short distance of only 5-15 mm above the sample. The corresponding dark-field adapter ( adjustable tube) allows mounting on the lens and prevents stray light. An example of a captured with an illumination sample is the image of the right 2- euro coin in the section above bright and dark field in reflected light illumination. For stereo microscopes reflected light dark -field illumination is partially seen as Standardbeleuchtungsart.

At low reflective objects created by the dark-field imaging, depending on the angle of incidence, a more or less plastic representation. Extreme dark-field conditions can be realized with a line of light that produces a band of light that graze under an extremely shallow angle of illumination from one side of the surface. Through the shadows created very high-contrast images even from small differences in altitude. Fingerprints can be as simple pose on flat, level surfaces.

Sidestream Dark Field Imaging

As sidestream dark field imaging ( SDF abbreviated to German: sidestream dark-field imaging) a method for investigating the microcirculation is called, so the investigation of small and tiny blood vessels. The process is carried out with a small device with which such vessels in patients, for example, under the tongue, can be examined, where no disturbing skin layers are present. The technique uses a central optical waveguide, in which a lens projects the image of the specimen on a camera chip. The light from the green light-emitting diodes (wavelength 530 nm) is irradiated onto the specimen from a ring around the central light conductor.

Due to the scattering in the specimen there is a uniform distribution of light in the observed range, so that a kind of backlight is created. The hemoglobin in red blood cells absorb green light very strong, so that the blood vessels that are densely filled with red blood cells, stand out as dark structures against a lighted background. The maximum penetration depth into the tissue is 500 micrometers.

Detection of submicroscopic particles

Optical basics

The strength of a signal is not dependent on the dark-field microscopy, the size of a structure, but on how much the light is deflected by it. Therefore, some particles or structures with it, similar to the fluorescence microscopy, can be detected, which are smaller than the resolution limit of the particular microscope. However, can not be distinguished then if the signal of one or more closely spaced structures occurs. Also there is no image but a diffraction phenomenon known as point spread, the size of which in turn depends on the resolution of the microscope.

The shape of the particles (round, oblong, square ... ) plays for the size and shape of the diffraction phenomenon produced no role, so that the shape of the particles can not be determined. For smaller particles, however, decreases the intensity, since less light is deflected by them. Therefore, a strong illumination is required for this. The intensity is also dependent on the difference in the optical density ( index of refraction) between the structure and the surrounding medium, since the larger the refractive index differences, more light is deflected.

Examples

Dark field illumination is used in the context of Millikan experiment in which the dark field technique allows the observation of oil droplets in a capacitor. For the determination of the elementary charge of an electron by this experiment Robert Andrews Millikan was awarded the Nobel Prize for Physics in 1923.

For the detection of metal particles in tissue sections, the dark field microscopy can be used (see figure).

Ultramicroscopy

Around the year 1900 around the term " Ultramicroscopy " came up with the dark- field microscopic examination of so-called " ultramicrons " has been designated, the particles are smaller than the resolution limit of the light, ie less than 0.2 microns. The minimum size of such particles which are already in 1902 some bright sunlight in gold ruby glasses by means of the ultra microscope is less than four nanometers.

Developed by Henry and Richard Siedentopf ZSIGMONDY gap ultra-microscope was used for the study of colloids for biomedical studies, it was not suitable. The illumination was in the form of a plane that is laterally coupled into the specimen, similar to the technique of optical discs modern microscopy ( SPIM), can be used in laser and fluorescence can be excited. To generate the level of a gap before the lighting source was placed at the ultra-microscope, the edges of which were only a few hundredths of a millimeter apart. This gap has been reduced 50-fold by a lens system as shown, and finally to the preparation. The Zeiss offered gap ultra microscopes including accessories 1910 for 474.50 Mark ( for colloids in liquids ) or 744.50 Mark ( colloids in solid materials ) at .. To observe particularly nanoparticles in liquids and to study their behavior developed Richard ZSIGMONDY the gap ultramicroscope in Göttingen together with the company R. Winkel GmbH further and introduced in 1912 before the immersion ultra-microscope.

In 1903 developed, simplified ultramicroscope by Cotton and Mouton was a completely different illumination geometry used. A light beam is laterally fed into a glass prism having a parallelogram - side surfaces. On the underside of the glass body was thereby total internal reflection, the light is directed to the preparation. The slide was with immersion placed directly on the glass body. The light rays hit now so obliquely on the specimen that at the top edge of the cover glass also total reflection was caused and no direct light hit the lens. Only in the preparation diffracted light was recorded. This structure could not be used with immersion objectives, otherwise held on cover glass no total reflection.

Other applications

Due to the limited compared to other contrast enhancement methods such as phase contrast or differential interference contrast resolution dark field microscopy it is today. In biology and medicine only for some special applications of importance (See illustrations at right for examples of research from 2007 and 2008. ) For example, it is still used for the microscopic detection of some pathogens in clinical microbiology, such as spirochetes. The ability to detect submicroscopic structures, can be used to investigate isolated organelles and polymers such as flagella, cilia, microtubules and actin filaments.

In the semiconductor industry, reflected light dark -field microscopy is used for the surface inspection of wafers to locate dirt particles. Such inquiries shall (without immersion) performed with dry objectives, the resolution limit in this case is about 0.35 micrometers. Thanks to dark-field illumination but also particles are visible, which exceed this limit.

In metallography most ground investigations are carried out in bright field. In addition, dark-field can be advantageously used to visualize mechanical surface defects (scratches, cracks, inclusions, pores, voids or outbreaks ) and to examine sections of etched grain boundaries. Colors of inclusions ( sulfides or oxides) appear in dark field more clearly than in the bright field, so that assignments are easier.

Due to the aesthetically pleasing images, the dark field microscopy has a certain spread in Hobbymikroskopikern. With it, for example, transparent water micro-organisms ( plankton) can be observed (see the first pictures of the items and links).

Alternative Medicine

The use of dark-field microscopy in alternative medicine as a diagnostic method for blood tests after Günther Enderlein ( Isopathy ) is supposed to enable early detection of cancer. The method is based on scientifically untenable assumptions about the morphology of microorganisms ( so-called pleomorphism ). A scientific study in 2005 came to the conclusion that the dark-field microscopy for the detection of cancer is inappropriate. Another alternative medical blood test, which is performed by means of dark -field microscopy, the dark field blood diagnostics according to Brehmer. This goes back to the pharmacologists Wilhelm von Brehmer and should also allow for early detection of cancer. However, proof of suitability missing. This blood test looks for Propionibacterium acnes (aka Siphonospora p. ), Which is a typical component of the skin flora, and can easily become contaminated smear in the context of blood donation.

Bright-field microscopy Tissue paper Contrast (vision) Rayleigh scattering Condenser (microscope) Optical aberration Scanning acoustic microscope Phase contrast microscopy Waveguide (optics) Tissue (biology) German gold mark Cover slip Wafer (electronics) Casting defect Homeopathy#Isopathy Pleomorphism (microbiology) Digital Object Identifier

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