Dark field microscopy
The darkfield microscopy is a well known for over 250 years variant of light microscopy . It leads to a dark background against which the structures to be observed stand out brightly. As a result, well-resolved, high-contrast images can still be generated from transparent objects with only very low contrast , without the preparation having to be colored beforehand. Living objects can also be easily observed. Until the development of phase contrast microscopy in the 1930s, dark field microscopy was the only method for contrast enhancement in unstained specimens . In contrast to dark field microscopy, the technology of "normal" light microscopy is referred to as bright field microscopy .
The principle of dark field microscopy is based on the fact that objects not only absorb light , but also always deflect part of the light beam. If the lighting is set so that the direct light rays pass the microscope lens , the viewer only sees the deflected light. One of the causes of distraction is the scattering of light on small particles, known as the Tyndall effect , which can also be observed, for example, when light falls into a dark room and the dust within the light beam is clearly visible. Particles that are smaller than the microscope's resolution limit also deflect light and can therefore be detected using a dark field microscope. Some properties, such as the mobility of particles, can be investigated in this way. This application was more important than ultramicroscopy at the beginning of the 20th century .
The specimen can be illuminated from behind the specimen as seen from the objective (transmitted light) or from the objective side (incident light) or also from the side, as is the case with the slit ultrasonic microscope . Transmitted-light and reflected-light darkfield are possible both in “normal” microscopes and in stereo microscopes .
Comparison of brightfield and darkfield
Light and dark field with transmitted light illumination
In microscopy, transmitted light illumination is an arrangement in which the illumination occurs from the rear of the specimen as seen from the objective, the light passes through the specimen ( transmission ) and finally reaches the objective. Normal transmitted light microscopy, more precisely: transmitted light bright field microscopy, is the variant most frequently used in biology and medicine, it is also used in school microscopes.
In classic transmitted light brightfield microscopy, the image contrast is mainly created by the fact that the specimen absorbs part of the incident light and thus the corresponding area appears darker (see Figure 1). However, many microscopic objects are largely transparent or very small and therefore only absorb very little light. They generate 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, that is, change the direction of some light rays through scattering , diffraction , refraction and / or reflection . However, these distractions can hardly be detected under bright field lighting, since the brightness of the deflected light rays is much weaker than the brightly lit background of the image. The contrast can be increased within certain limits with bright field illumination by choosing a smaller aperture in the beam path of the illumination ( condenser aperture ) (see Figure 2, center). At the same time, however, imaging errors increase and disruptive diffraction patterns arise on the edge of the objects (compare small image sections in Figure 2).
In transmitted light dark field microscopy, the specimen is illuminated from the rear in such a way that the illumination does not reach the objective, but only the light deflected in the specimen. The background of the image appears dark, while objects in the specimen appear light (see Figure 1 on the right). This also and especially works with largely transparent samples. While the differences in brightness of the deflected light are difficult to recognize because of the high light intensity of the bright field image, these differences appear much stronger in the dark field image. The fine traces of fat on a fingerprint in Figure 2 (right) are therefore clearly visible. The impurities (particles and scratches) that can already be recognized with bright field lighting show such a strong contrast in the dark field that they are only shown as bright, overexposed spots in the image.
In the case of transmitted-light dark field illumination, it is particularly important that the slide, cover glass and also the glass surfaces in the microscope are clean, since every grain of dust contributes to the background noise by deflecting the light. Also, light-deflecting structures must not occur in different planes on top of one another, as their signals would otherwise overlap. Accordingly, dark field lighting is not suitable for thick specimens such as typical tissue sections.
In physical terms, transmitted-light dark-field lighting can be described as lighting in which the main diffraction maximum of the light (see diffraction disk ) does not reach the rear focal plane of the lens. Only deflected light, for example secondary maxima caused by diffraction, take part in the image structure.
Bright and dark fields with incident light
In light microscopy, incident light illumination is used when the light falls onto the specimen from above (more precisely: from the side of the lens). The lighting takes place either through the lens itself or by an independent lighting device which is arranged to the side or around the lens. The angle at which the light falls on the object determines the appearance of the image. If a large part of the light reflected by the specimen is captured by the objective, the object appears bright in the image (bright field illumination). However, if the lighting is so far from the side that the directionally reflected light shines past the lens, this is referred to as dark field lighting.
When examining materials, bright field lighting is the most frequently used technique for illuminating rough, less reflective objects. The reflected light bright field illumination corresponds to the normal way of seeing humans: Smooth, highly reflective surfaces appear bright due to their strong gloss (Figure 3 left). The reflection gives metal surfaces their typical shine. Structures arranged beneath glass or other transparent surfaces would be difficult to see with this type of lighting due to the strong reflection on the surface.
With reflected light dark field illumination, smooth, highly reflective surfaces appear dark. Edges and surface defects such as scratches or deposits, however, glow brightly (Figure 3 right). These are highlighted and can be recognized more easily or more easily detected with image processing methods. In the case of rough, less reflective surfaces, the lateral arrangement of the reflected-light dark-field illumination ensures local shadow formation, so that surface structures appear more three-dimensional. This effect can be significantly enhanced by one-sided lighting.
Dark field microscopy outside of light microscopy
The terms dark field and bright field can also be applied to microscopic processes that use other signals instead of light to generate images. A corresponding distinction is made as to whether the excitation signal that is not deflected is registered by the detector (bright field) or whether only the signal changed by the sample contributes to the imaging (dark field). Processes called dark fields exist, for example, in electron microscopy (see, for example, scanning transmission electron microscope ) and in acoustic microscopy .
Dark field illumination in today's transmitted light microscopes
The easiest way to create dark field illumination with a normal transmitted light microscope with light source, condenser and lens under Koehler illumination is to close the condenser aperture tightly and then move it sideways until no more direct light penetrates the lens. The lighting is therefore only from one side. However, newer microscopes in particular often do not offer the possibility of moving the diaphragm relative to the condenser.
Common foundations
Better image quality can be achieved with a centered condenser with the help of an additional device. This additional device limits the illumination of the specimen to a cone envelope (yellow in the diagram on the right). The inner part of the cone contains no light (gray in the schematic drawing). The surface of the cone coming from the condenser is focused into the specimen plane and, in the simplest case, then expands again, so that undeflected light completely passes the lens opening, the image background remains dark. Only light that is deflected by the objects to be observed enters the lens and creates an image with light structures on a dark background. All today's transmitted light dark field illumination generate a conical surface, but does not always pass through the entire preparation through: In some cases it comes to the total reflection of the non-deflected light at the cover glass top.
Two different methods are used to create the illumination cone envelope. A central diaphragm for producing the cone jacket is easy to manufacture and use, inexpensive and therefore widespread. This method is particularly suitable for lenses with a relatively low magnification, the thickness of the illumination cone jacket being able to be optimally matched to the lens used by simply changing the diaphragm. Special dark field condensers achieve a higher light yield through mirror technology and can also meet the requirements of higher magnification lenses through immersion . The image quality is getting better.
When the illumination cone jacket passes through the specimen as in the schematic drawing, it must pass outside the objective. Dark field illumination is only possible if the angle of the light emerging from the condenser ( opening angle ) is greater than the angle of the light captured by the lens. The larger the opening angle of an objective or condenser, the better the maximum achievable resolution . Instead of the opening angle, the numerical aperture is specified for objectives and condensers , which can be up to 0.95 without immersion and up to around 1.4 with oil immersion. For dark field illumination, the numerical aperture of the condenser must be higher than that of the objective used. Without immersion of the condenser, the application is therefore limited to objectives with a numerical aperture of about 0.75 or less. 40x objectives that are used without immersion often have a numerical aperture of 0.65.
Lighting with central screen
Here an annular diaphragm is used in an otherwise normal transmitted light bright field microscope. This central diaphragm (1 in the upper schematic drawing on the right) has a translucent edge or ring and thus reduces the lighting with the help of a normal condenser (2) to a cone jacket (3). In order to make optimal use of the opening angle of the condenser, a part of the condenser that is as far out as possible is used. The larger the opening angle of the lens used, the larger the diameter of the central opaque area must be, and the illuminance is reduced accordingly. Starting from the specimen table with the specimen slide (4), the light thus passes the objective (6). Only light (5) deflected by structures in the specimen reaches the objective. The central diaphragm can be inserted under the condenser lens of a normal transmitted light microscope.
The more widespread phase contrast microscopy is based on a completely different optical phenomenon, but apertures are also used there. These ring diaphragms can sometimes be used for other purposes as dark field diaphragms. Phase contrast ring diaphragms are designed in such a way that the light cone enters the lens when it is correctly adjusted and does not go past it, as is necessary for darkfield. Therefore, for a given objective, only those phase contrast ring diaphragms can be used as dark field diaphragms that are actually intended for objectives with a significantly larger opening angle (higher numerical aperture). For example, a phase contrast ring diaphragm for a 100x oil immersion objective is generally suitable as a dark field diaphragm for 10x and 20x dry objectives, since oil immersion objectives have a larger aperture angle.
Dark field condensers
In the case of particularly high demands on the image quality, special dark field condensers are used instead of central apertures. There are dry dark field condensers and immersion dark field condensers , in the latter case immersion oil or water is placed between the condenser and the slide. This enables a higher numerical aperture and thus a higher resolution. An immersion condenser also provides a better contrast, since reflections on the underside of the slide and the surface of the condenser, which lead to a brightening of the image background, are avoided. However, it is more complex to handle, also because oil requires careful cleaning work. The disadvantage of both types of dark field condensers compared to a central diaphragm is the more complex change to bright field lighting, since the condenser has to be replaced for this. Dry dark field condensers are suitable for objectives with numerical apertures up to 0.65 or 0.75, while immersion condensers can be used for objectives with numerical apertures up to 1.2.
Modern dark field condensers are mostly cardioid condensers. Here a convexly curved central mirror directs the incident light outwards onto a concave mirror running around it , so that the cone surface is created (see comparable drawing from 1910 on the right). The concave mirror ideally has a surface shaped like a cardioid , hence the name. For manufacturing reasons, however, this surface is designed as a spherical surface without this leading to any significant loss of quality. A paraboloid condenser, on the other hand, has the shape of a truncated paraboloid . The light is deflected only once here, namely by total reflection (see drawing of Wenham's glass paraboloid below), which in turn creates an all-round illumination cone.
To ensure that the numerical aperture of the objective is smaller than that of the condenser, an objective can also be used in which the numerical aperture can be restricted via a movable iris diaphragm. The opening angle of the lens can thus be optimally matched to the diameter of the lighting cone so that the latter can just be blocked out.
Cardioid and paraboloid condensers are also referred to as catoptric darkfield condensers, because in them the light is deflected by reflection, while in the so-called dioptric condensers this is done by glass lenses.
Transmitted light dark field in stereo microscopes
Transmitted-light dark field illuminations are also available for stereo microscopes. The lighting device is housed in the stand base. Apart from the actual light source, e.g. B. a halogen lamp , a central cover and external, upright reflective surfaces are used to illuminate the object with a cone envelope. The principle corresponds roughly to the mirror condenser described above. The object is placed on a glass plate that closes the stand base at the top. The image is made up of light rays that have been deflected in the object by reflection , light refraction or diffraction . Typically, the central cover can be exchanged for a ground glass, so that in addition to dark field, bright field transmitted light illumination is also possible. The mirrors on the outside then direct just as much light at an angle onto the specimen as before, but due to the much brighter bright-field lighting, this no longer leads to visible effects.
Earlier approaches to transmitted light dark field observation
Before 1900
As early as the 17th century, dark field microscopy was used by Antoni van Leeuwenhoek , Robert Hooke and Christiaan Huygens to observe blood components or small organisms. However, no special equipment was used. Rather, the light source, such as a candle, was positioned so that no direct light fell on the lens.
Dark field microscopy is possible even with a very inclined illumination mirror. The first to describe a special apparatus for dark field illumination was Joseph Bancroft Reade (1801–1870) in 1837 , whose method in John Queckett's "Practical treatise on the use of the microscope" 1852 was referred to as background illumination . The light source was placed on the side, a converging lens focused the light on the specimen so that undeflected light was directed past the objective. In the course of the 19th century a number of authors developed additional lighting equipment. Since refraction on glass surfaces causes chromatic aberration , which is particularly disturbing in dark field microscopy, mirror condensers have also been developed, since this error does not occur with reflection . The reflection was achieved either through reflective surfaces or through total reflection .
Francis Herbert Wenham (1824–1908) described various dark field lighting principles in several works between 1852 and 1856. In addition to side lighting (with an effect similar to that of Reade), it also included condensers for a centrally positioned lighting source, including a hollow, silver-plated paraboloid and a solid glass paraboloid in which the reflection came about through total reflection (see illustration). The slide was in direct contact with the condenser. The preparation was embedded in Canada balsam or liquid. There was air between the cover slip and the lens. The principle of diffraction , which is essential for effective dark field illumination of small objects, was not yet understood at the time. Wenham therefore assumed that the observed effects were due to the fact that the object was illuminated from above, namely by light that was reflected back onto the specimen by total reflection from the upper edge of the cover glass.
From around 1900
Up to the end of the 19th century, dark field microscopy was used by amateurs, but little in the scientific field, as it did not work with higher resolution objectives (with a high numerical aperture ). Thanks to the work of Ernst Abbe at the end of the 19th century, optical principles such as diffraction were understood. W. Gebhardt at Zeiss took advantage of this by proposing a central screen for Abbe's lighting apparatus for dark field lighting, which Zeiss added to its range in 1898. If immersion between the condenser and the slide was used, dry objectives with an aperture of up to 0.95 could be used. At times this central panel was supplied with all corresponding devices, but since it was not well received by customers, it was discontinued. The Viennese microscope company Reichert offered a similar solution.
With the discovery of the syphilis pathogen, dark field microscopy experienced an upswing from 1906, as it enabled a good representation of living spirochetes , to which the pathogen belongs. Several large microscope companies developed improved dark field condensers. That of Karl Reichert contained a central aperture with a variable size. Henry Siedentopf developed a paraboloid condenser for Zeiss in 1907 . Although the design corresponded to the Wenham glass paraboloid with a darkening in the center of the lower side of the paraboloid, the optical quality could be increased through improved manufacturing techniques so that the inner and outer apertures of the illuminating cone jacket were 1.1 and 1.4. Based on Abbe's work, it was clear that diffraction plays a decisive role in the creation of the image and that total reflection on the cover glass only helps to avoid the entry of undeflected light into the lens. In a later version, the so-called light - dark field condenser , the central darkening could be removed using a lever, so that a quick change between dark and light field was possible.
The approaches described so far are based on the fact that the specimen is illuminated with a higher numerical aperture, that is to say with a wider angle, than can be recorded by the objective. But the opposite approach is also possible: The specimen is illuminated with a complete cone with a small numerical aperture (for example 0.2). High-resolution objectives can also be used here, because the numerical aperture and thus the opening angle can be of any size, but they must be significantly larger than that of the lighting. The light not deflected in the specimen will then only occupy a central area in the objective, while the outer area remains free of direct illuminating light. The undeflected light is quasi subsequently removed in or behind the lens at a suitable point in the beam path. This was referred to as “conaxial arrangement” or “central dark field” and counted among the ultramicroscopic methods (see below). The disadvantage of this approach is that much higher light intensities are achieved in the preparation than, for example, with a central diaphragm in the condenser, which results in disruptive secondary diffraction images in preparations with many objects.
Henry Siedentopf used an objective for such a system he had developed in which the otherwise hemispherical back of the front lens (the first glass body in the objective) was ground flat and painted black. Carl Metz (1861–1941) at Leitz developed a system with oil immersion objectives in 1905 in which a stamp diaphragm (also: funnel diaphragm) was movably inserted into the lens from the rear. This made it possible to use the same lens without this diaphragm for brightfield applications without loss of brightness. Adjustment was difficult for that.
Wladimir Sergejewitsch Ignatowski developed a dark field condenser for Leitz, which had two reflective surfaces but was easier to handle than earlier corresponding models (see schematic drawing from 1910 above). It was sold from 1907. The cross-sectional drawing of the successor model developed by Felix Jentzsch from 1910 became the template for a Leitz logo , the so-called Leitz coffin.
Henry Siedentopf at Zeiss also designed a condenser with two reflective surfaces that was very similar to the condenser developed by Ignatowski. For theoretical reasons, the second reflective surface should correspond to a section of a cardioid . Cardioid surfaces were difficult to manufacture. Instead, a spherical surface was used, which produced the same effect within the manufacturing tolerances. Nevertheless, the device was marketed by Zeiss as a cardioid condenser.
An overview of the advantages and disadvantages of transmitted light dark field illumination
Advantages:
- Small, even unstained, objects can be observed with strong contrast, particularly well in low concentrations with thin specimens.
- Objects below the resolution limit also cause signals if the lighting is strong enough.
- Some forms of dark field lighting, especially at low magnification, can be implemented very easily and without significant costs.
- In contrast to bright field lighting, there are no entoptic phenomena in dark field lighting, streaks that arise in the eye itself and cast shadows on the retina.
Disadvantage:
- Surfaces of objects cause signals due to the change in the refractive index, but not a homogeneous interior, so that only the border can then be seen in the image.
- The technique is not very suitable for thick specimens or specimens with many objects, since too many signals, for example from different levels of focus, counteract the dark field effect.
- Impurities in the beam path also lead to disruptive signals, which is why the requirements for the cleanliness of the device and preparation are very high.
- Special condensers are required for higher demands, as the reflections between the various lenses in normal condensers reduce the dark field effect.
- Since the opening angle of either the condenser or the objective has to be reduced, the resolution is reduced compared to brightfield and other contrast-enhancing methods such as phase contrast and differential interference contrast
Rheinberg lighting
The Rheinberg lighting (also: optical coloring or contrasting color lighting) is a modification of the dark field microscopy with a central diaphragm, which was first described by Julius Rheinberg in London in 1896 . The central screen is replaced by a round filter with two colors in a concentric arrangement: One color forms an outer ring, it corresponds to the ring in the conventional ring screen. The light passing through here will only fall into the lens if it is deflected in the specimen. The second color is in the middle, otherwise opaque area. It defines the background of the image. In this way, aesthetically very appealing images are created without additional structures becoming visible.
Under the name Mikropolychromar , Zeiss supplied condenser accessories around 1939 until after the Second World War, with which Rheinberg lighting was possible. A central bright field and an outer dark field lighting could be colored differently with filters. Zeiss recommended this device “to facilitate the examination of uncolored objects with low contrasts”. Gerlach (2009) wrote about this facility that it "certainly had a certain significance before the introduction of the phase contrast method". The Reichert company sold a mirror condenser -based solution under the name Optikolor , which also made Rheinberg lighting possible.
With three-colored Rheinberg filters, preparations that are clearly structured can be displayed particularly effectively. The outer ring of the filter is divided into four 90 ° angles, the opposing quadrants are colored in the same way, but neighboring ones in different colors. The inner circle is colored with the third color. The two-tone outer ring means that structures that scatter from left to right are displayed in a different color than those that scatter from front to back in the preparation plane. Examples of such preparations are diatoms or textile fabrics .
Dark field illumination in reflected light microscopes
Classic reflected light dark field microscopy
With reflected light microscopy , the light is radiated from the same side from which it is observed. This procedure is used for opaque materials, for example minerals or material tests . With incident light bright field illumination, the illumination can be fed in via the same objective beam path that is also used for observation.
In the case of reflected-light dark-field illumination, however, the illumination and observation beam paths are separate: Special lenses have an additional outer area reserved for the illumination beam path (see diagram). The inner area corresponds to a normal lens, with dark field lighting it is used exclusively for observation. The outer area corresponds to the condenser. Here the light (1 in the drawing) is guided obliquely onto the specimen (4) through an annular concave mirror in the outer area (3). If the specimen were a flat mirror, the light reflected there would completely bypass the inner area of the lens: the image would remain dark. Light deflected by surface structures such as scratches, on the other hand, is picked up by the lens (5).
With some reflected light dark field lenses it is possible to show or hide individual sectors of the lighting ring. This can intensify the formation of shadows, so that structures that run in certain directions can be better recognized. With so-called Ultropak lighting devices, the 'condenser', which is attached to the objective, can be adjusted in height in order to illuminate different levels in the specimen as much as possible. At low magnifications, the required light intensity can also be achieved with an external light source set up on the side, for example fiber optic lights.
Surface structures such as scratches stand out clearly from the background in the reflected-light dark field, as light reflected or scattered on them is partially directed into the central area of the lens. Such structures are therefore light in the image on a dark background. Accordingly, reflected-light dark-field lighting is particularly suitable for examining surfaces, for example in materials science . Dark field illumination is widely used in reflected light microscopes. In contrast to transmitted light dark field lighting, reflected light dark field lighting can also be used with the most powerful lenses. To avoid unwanted reflections, work without a cover slip if possible.
Incident light dark field in stereo microscopes
With stereomicroscopes , reflected-light darkfield can be implemented in that the illumination tends to graze the surface and the directionally reflected light does not reach the objective directly. This is possible, for example, by slightly tilting a flat specimen or by cleverly arranging freely positionable light sources (e.g. gooseneck lighting with a long, flexible holder). For ring-shaped, all-round dark field lighting, there are special ring lights with a beam angle of 60 °, for example, which are arranged at a small distance of only 5–15 mm above the sample. The associated dark field adapter (height-adjustable tube) allows mounting on the lens and avoids stray light. An example of a sample recorded with such an illumination is the image of the right 2 euro coin in the above section bright and dark field with incident light illumination . In stereomicroscopes, reflected-light dark-field illumination is sometimes seen as the standard type of illumination.
In the case of less reflective objects, the dark field image creates a more or less three-dimensional representation depending on the angle of incidence. Extreme dark field conditions can be achieved with line light that creates a band of light that sweeps across the surface from one side at an extremely flat angle of illumination. The formation of shadows creates very high-contrast images, even of small differences in height. Fingerprints can be easily displayed on flat, even surfaces.
Sidestream Dark Field Imaging
Sidestream dark field imaging (abbreviated to SDF, in German: side stream dark field imaging) is a method for examining microcirculation , i.e. for examining small and very small blood vessels . The procedure is carried out with a small device with which such vessels can be examined in patients, for example under the tongue, where there are no disturbing layers of skin. The technique uses a central light guide in which a lens projects the image of the specimen onto a camera chip. The light from green light-emitting diodes ( wavelength 530 nm) is radiated onto the specimen from a ring around the central light guide .
The scattering in the specimen results in an even distribution of the light in the observed area, so that a kind of background lighting is created. The hemoglobin in the red blood cells absorbs green light very strongly, so that the blood vessels, which are densely filled with red blood cells, stand out as dark structures against a light background. The maximum depth of penetration into the tissue is 500 micrometers.
Detection of submicron particles
Optical basics
With dark field microscopy, the strength of a signal does not depend on the size of a structure, but on how strongly the light is deflected by it. Therefore, similar to fluorescence microscopy , it can also be used to detect some particles or structures that are smaller than the resolution limit of the respective microscope. In this case, however, it is not possible to distinguish whether the signal comes from just one or several structures that are close together. There is also no image, but rather a diffraction phenomenon called a point spread function , the size of which in turn depends on the resolution of the microscope.
The shape of the particles (round, oblong, angular ...) is irrelevant for the shape and size of the diffraction phenomenon generated, so that the shape of the particles cannot be determined. For smaller particles, however, the intensity decreases because less light is deflected on them. Therefore, strong lighting is required for them. The intensity is also dependent on the difference in the optical density ( refractive index ) between the structure and the surrounding medium, since more light is deflected with larger refractive index differences .
Examples
Dark field lighting is used in the Millikan experiment , in which dark field technology enables the observation of oil droplets in a condenser . Robert Andrews Millikan received the Nobel Prize in Physics in 1923 for determining the elementary charge of an electron using this experiment .
Dark field microscopy can also be used to detect metal particles in tissue sections (see also figure).
Ultramicroscopy
Around 1900 the term “ultramicroscopy” was used to describe the dark-field microscopic examination of so-called “ultramicrons”, particles smaller than the resolution limit of light, i.e. smaller than 0.2 micrometers . The minimum size of such particles, which was determined in 1902 with bright sunlight in gold ruby glasses with the help of an ultramicroscope, is less than four nanometers .
The slit ultrasonic microscope developed by Henry Siedentopf and Richard Zsigmondy was used to examine colloids ; it was not suitable for biomedical examinations. The lighting took place in the form of a plane that was coupled into the specimen at the side, similar to the more modern technology of light disk microscopy (SPIM), in which lasers are used and fluorescence can also be excited. To create the plane, a gap was placed in front of the source of illumination with the ultramicroscope, the edges of which were only a few hundredths of a millimeter apart. This gap was reduced by about 50 times using a lens system and finally imaged in the specimen. The company Zeiss offered slit ultrasonic microscopes including accessories in 1910 for 474.50 marks (for colloids in liquids) or 744.50 marks (colloids in solid materials). In order to be able to observe nanoparticles in liquids in particular and to study their behavior, Richard Zsigmondy further developed the slit ultrasonic microscope in Göttingen together with R. Winkel GmbH and introduced the immersion ultrasonic microscope in 1912.
A completely different illumination geometry was used in the simplified ultramicroscope developed by Cotton and Mouton in 1903. A cone of light was fed into a glass prism with parallelogram side surfaces. Total reflection was created on the underside of the glass body, which led the light to the specimen. The slide was placed directly on the glass body with immersion. The light rays hit the specimen at such an angle that total reflection was also caused on the upper edge of the cover glass and no direct light hit the objective. Only light diffracted in the specimen was recorded. This setup could not be used with immersion objectives, since otherwise there would be no total reflection on the cover glass.
Other uses
Spirochaetes of the species Borrelia burgdorferi , recorded with darkfield illumination. The scale bar corresponds to 8 µm on the left and 25 µm on the right.
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Zebrafish embryos under dark field illumination. Both were heat treated. In the left embryo with a change in a specific gene , growth damage remains in the delimitation of the body segments (above the arrowhead), which can be determined by the absence of the delimitation under dark field illumination, on the right a normal embryo for comparison.
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Due to the limited resolution of dark field microscopy compared to other contrast enhancement methods such as phase contrast or differential interference contrast , it is only of importance for a few special applications in biology and medicine today . (See images on the right for examples from research work 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 study isolated organelles and polymers such as flagella , cilia , microtubules and actin filaments .
In the semiconductor industry , reflected light dark field microscopy is used to inspect the surface of wafers in order to find dirt particles. Such examinations are carried out with dry objectives (i.e. without immersion ), the resolution limit here is around 0.35 micrometers . Thanks to dark field lighting, however, particles are also visible that fall below this limit.
In metallography , most microsection examinations are carried out in brightfield. In addition, darkfield can be used to advantage to visualize mechanical surface defects (scratches, cracks, inclusions, pores , cavities or breakouts) and to examine grain boundaries on etched sections . The colors of inclusions ( sulfides or oxides ) appear more clearly in the dark field than in the bright field, so that assignments are easier.
Because of the aesthetically pleasing images, dark field microscopy has become more widespread among amateur microscopists. With it, for example, transparent water microorganisms ( plankton ) can be observed (see the first pictures of the article and web links ).
Alternative medicine
The use of dark field microscopy in alternative medicine as a diagnostic method for blood tests according to Günther Enderlein ( isopathy ) is intended to enable early cancer detection. The process is based on scientifically untenable assumptions about the morphology of microorganisms (so-called pleomorphism ). A scientific study in 2005 concluded that dark field microscopy was unsuitable for detecting cancer. Another alternative medical blood test that is carried out using dark field microscopy is the von Brehmer dark field blood test . This goes back to the pharmacist Wilhelm von Brehmer and is also intended to enable early detection of cancer. However, there is no proof of suitability. This blood test looks for Propionibacterium acnes (alias Siphonospora p. ), Which is a typical component of the skin flora and can easily contaminate the smear as part of the blood sample.
Web links
- Michael Volger (Ed .: Irene K. Lichtscheidl): Dark field. Retrieved on June 17, 2012 (Theoretical introduction and instructions for practical application at the University of Vienna. Also available as PDF .).
- Christian Linkenheld: Dark field microscopy with the central aperture method and dark field microscopy with special dark field condensers . 2002, accessed May 14, 2012.
- Paraboloid condenser and cardioid condenser from Zeiss in the Museum of Optical Instruments .
- Dark field lighting in materials science, an overview of lighting technologies at the University of Siegen .
- Immersion ultrasonic microscope according to R. Zsigmondy with optics patented in 1912
Individual evidence
- ↑ a b c d e f D. Gerlach: light microscopy . S. 13-85 . In: Horst Robenek (Ed.): Microscopy in research and practice . GIT-Verlag, Darmstadt 1995, ISBN 3-928865-18-8 , p. 48, 65 .
- ↑ a b c d A. Ehringhaus: The microscope its scientific basis and its application . 2nd Edition. Publishing house B. G. Teubner, Leipzig and Berlin 1938, p. 95-109 .
- ↑ Sophie Perrot: Lighting is the alpha and omega! Selection of suitable lighting for applications in industrial image processing . In: Optics & Photonics . tape 3 . Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2010, p. 51-55 ( wiley-vch.de [PDF]).
- ↑ Oleg Kolosov and Kazushi Yamanaka: Adjustable Acoustic Knife Edge for Anisotropic and Dark-Field Acoustic Imaging . In: Jpn. J. Appl. Phys. tape 33 , 1994, pp. 329-333 , doi : 10.1143 / JJAP.33.329 .
- ↑ a b c d e f g h i Randy O. Wayne: Light and Video Microscopy . Academic Press, Elesevier, 2009, ISBN 978-0-12-374234-6 , pp. 95-98 .
- ↑ a b c d e f g h Dieter Gerlach: The light microscope. An introduction to function, handling and special procedures for physicians and biologists . Georg Thieme Verlag, Stuttgart 1976, ISBN 3-13-530301-2 , p. 119-126 .
- ↑ a b Horst Riesenberg: Optical system of the microscope in: Joachim Bergner, Hermann Beyer (Hrsg.): Handbuch der Mikoskopie . 3. Edition. VEB Verlag Technik, Berlin 1988, ISBN 3-341-00283-9 , p. 24-107 . Page 107
- ^ A b Savile Bradbury, Peter Evennett: Contrast techniques in light microscopy . Ed .: Royal Microscopical Society, Microscopy Handbooks. BIOS Scientific Publishers Limited, 1996, ISBN 1-85996-085-5 , pp. 32-33 .
- ^ Heinz Appelt: Introduction to microscopic examination methods . 4th edition. Academic publishing company Geest & Portig KG, Leipzig 1959, p. 106 f .
- ^ Website Nikon Microscopy U, Stereomicroscopy: Darkfield illumination .
- ↑ JB Reade: A New Method of Illuminating Microscopic Objects . In: CR Goring, Andrew Pritchard (Eds.): Micrographia: containing practical essays on reflecting, solar, oxy-hydrogen gas microscopes; micrometers; eye-pieces, & c. & c . 1837, Appendix 2, p. 227–231 ( limited preview in Google Book search).
- ^ John Thomas Queckett: A Practical treatise on the use of the microscope . 2nd Edition. H. Bailliere Publisher, London 1852, p. 194 ( limited preview in Google Book search).
- ↑ a b c d e f g h i j k Dieter Gerlach: History of microscopy . Verlag Harri Deutsch, Frankfurt am Main 2009, ISBN 978-3-8171-1781-9 , pp. 663-676 .
- ↑ a b Henry Siedentopf : The prehistory of the mirror condensers . In: Journal of Scientific Microscopy . tape 24 , 1907, pp. 382-395 ( online ).
- ^ FH Wenham: On a Method of Illuminating Opaque Objects under the Highest Powers of the Microscope . In: The transactions of the Microscopical Society of London . tape 4 , 1856, pp. 55–60 ( limited preview in Google Book Search).
- ↑ Henry Siedentopf : Paraboloid condenser, a new method for dark field lighting for visualization and for instant microphotography of living bacteria etc. (especially for Spriochaete pallida) . In: Journal of Scientific Microscopy . tape 24 , 1907, pp. 104-108 ( online ).
- ↑ Hubert de Martin, Waltraud de Martin: Four centuries of microscope . Weilburg Verlag, Wiener Neustadt 1983, ISBN 3-900100-06-3 , p. 134 .
- ^ Mortimer Abramowitz, Michael W. Davidson: Rheinberg Illumination. In: Molecular Expressions - Optical Primer. Florida State University, accessed May 17, 2012 .
- ↑ a b Rainer Wegerhoff, Olaf Weidlich, Manfred Kässens: Contrast and Microscopy . In: Basics of Light Microscopy & Imaging. Special edition of Imaging & Microscopy in Collaboration with Olympus . 2nd Edition. 2011, p. 22-33 ( download ).
- ^ HG Kapitza: Microscopy from the beginning . 2nd revised edition. Carl Zeiss Jena GmbH, 1997, p. 32 ( download page ).
- ↑ a b Dieter Gerlach: The light microscope. An introduction to function, handling and special procedures for physicians and biologists . Georg Thieme Verlag, Stuttgart 1976, ISBN 3-13-530301-2 , p. 220 f .
- ↑ brochure from the company Zeiss Stemi DR, Stemi DV4, Stemi 2000 stereomicroscopes , accessed July 8, 2012
- ↑ Ehlert & Partner: LED lighting for stereomicroscopy , accessed on July 8, 2012
- ↑ a b Brochure from Zeiss: KL1500 LCD and KL 2500 LCD cold light sources , accessed on July 8, 2012
- ↑ a b R. Gieseler: Stereomicroscopy . In: Horst Robenek (Ed.): Microscopy in research and practice . GIT-Verlag, Darmstadt 1995, ISBN 3-928865-18-8 , p. 87-127 . P. 109
- ↑ a b P. W. Elbers, C. Ince: Mechanisms of critical illness – classifying microcirculatory flow abnormalities in distributive shock. In: Critical care. Volume 10, number 4, 2006, ISSN 1466-609X , p. 221. doi: 10.1186 / cc4969 , PMID 16879732 , PMC 1750971 (free full text), (review).
- ↑ a b C. M. Treu, O. Lupi, DA Bottino, E. Bouskela: Sidestream dark field imaging: the evolution of real-time visualization of cutaneous microcirculation and its potential application in dermatology. In: Archives of dermatological research. Volume 303, number 2, March 2011, ISSN 1432-069X , pp. 69 ff., Doi: 10.1007 / s00403-010-1087-7 , PMID 20972572 (review).
- ^ Robert Andrews Millikan : The Isolation of an Ion, a Precision Measurement of its Charge, and the Correction of Stokes's Law . In: Physical Review (Series I) . tape 32 , no. 4 , 1911, pp. 349-397 , doi : 10.1103 / PhysRevSeriesI.32.349 .
- ↑ Detection of inorganic substances: 8. General detection of metals. In: Benno Romeis , Peter Böck (Hrsg.): Microscopic technology. 17th edition. Urban & Schwarzenberg , Munich / Vienna / Baltimore 1989, ISBN 3-541-11227-1 , p. 429.
- ↑ H. Siedentopf, R. Zsigmondy: About visualization and size determination of ultramicroscopic particles, with special application to gold ruby glasses . In: Annals of Physics . tape 315 , 1902, pp. 1-39 , doi : 10.1002 / andp.19023150102 .
- ↑ W. Gebhardt: For optical and mechanical workshops I . In: Journal of Scientific Microscopy . tape 24 , no. 4 , 1907, pp. 396-421 ( online ).
- ↑ Timo Mappes, Norbert Jahr, Andrea Csáki, Nadine Vogler, Jürgen Popp and Wolfgang Fritzsche: The Invention of the Immersion Ultramicroscope 1912 - The Beginning of Nanotechnology? . In: Angewandte Chemie . 124, No. 45, 2012, pp. 11307-11375. doi : 10.1002 / anie.201204688 .
- ^ Douglas B. Murphy: Fundamentals of Light Microscopy and Electronic Imaging . Wiley-Liss, New York 2001, ISBN 978-0-471-25391-4 , pp. 112-115 .
- ↑ Jan Albers: Contamination in the microstructuring . 1st edition. Hanser Verlag, 2005, ISBN 978-3-446-40291-1 , p. 110-113 (on Google Books ).
- ↑ ETH Zurich, Practical IV: Metallography Light Microscopy Practical IV: Metallography Light Microscopy ( Memento of the original from January 22, 2016 in the Internet Archive ) Info: The archive link has been inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice. (PDF; 5.5 MB), accessed on July 8, 2012
- ↑ Buehler: The sum of our experience: A guide to the preparation of materials and their evaluation ( Memento of the original from January 20, 2013 in the Internet Archive ) Info: The archive link was automatically inserted and not yet checked. Please check the original and archive link according to the instructions and then remove this notice. (PDF; 5.0 MB), accessed on July 8, 2012
- ↑ Samer El-Safadi, Hans-Rudolf Tinneberg, Friedel Brück, Richard von Georgi, Karsten Münstedt: Does Enderlein darkfield microscopy allow the diagnosis of cancer? A prospective study . In: Research in Complementary and Classical Natural Medicine . tape 12 , 2005, p. 148-151 , doi : 10.1159 / 000085212 .
