Interference reflection microscopy

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Interference reflection microscopy (also: reflection contrast microscopy ) is a light microscopic method with which very thin structures can be examined. It is based on the formation of interferences that arise when light is reflected at the top and bottom interfaces of the structure and reflected light from both interfaces interferes with one another. This creates interference patterns that can be observed and provide information about the thickness of the structure.

 The resulting interference colors or interference lines allow thickness measurements in the range below 200 nanometers, i.e. below the normal resolution of the light microscope. The resulting interference lines correspond in their course to the contour lines of a map, provided that the refractive index remains constant within the object. Since the interference patterns are observed through a light microscope, these thickness measurements can be assigned to microscopically recognizable structures, such as individual extensions of cells . For precise measurements of the regional layer thickness or changes in layer thickness, monochromatic light should be used because the wavelength of the illuminating light is included in the calculation. The shorter the wavelength, the finer the vertical resolution. If, for example, monochromatic green light is used (wavelength: 546 nm), thickness measurements can be carried out in red blood cells (erythrocytes) with a resolution of 113 nm. Similar to internal total reflection fluorescence microscopy (TIRF), observation is limited to objects on the specimen surface, i.e. near the cover slip .

The process has been given different names by different authors. The oldest name (1964) is Interference Reflection Microscopy (IRM), in German interference reflection microscopy . Other names are Reflection Contrast Microscopy (RCM; 1975), in German Reflection Contrast Microscopy , and Reflection Interference Contrast Microscopy (RICM, 1981). An English review article from 2000 also lists: interference contrast, interference reflection contrast, reflection interference contrast, surface reflection interference and surface contrast microscopy .

History and areas of application

The technique was first used in 1958 and 1960 for the study of thin layers and introduced into cell biology in 1964 . More work was not published until about ten years later. In the 1970s, the technology was further developed and offered commercially by Ernst Leitz and Carl Zeiss . Leitz has marketed the process under the name "Reflection Contrast", Zeiss under the name "Immersion Contrast". The main difference between the two variants was the angle of incidence of the illuminating light. At Leitz the object was illuminated in a concentrically inclined oblique light (angle of incidence: 45 °), at Zeiss orthogonally (angle of incidence 0 °). The angle of incidence has optical-physical effects on the formation of the image, which is why the two methods are not to be regarded as equivalent. The method did not become widely accepted; there was an accumulation of publications for special applications in the 1970s and 1980s.

By using polarization filters in the beam path (preferably in a cross position) and a λ / 4 plate in the lens (antiflex lens), light that is reflected on glass surfaces within the lens itself is filtered out. Only the light reflected from the object contributes to the creation of the image. If the polarization filters are arranged in a cross position, the objects light up brightly on a black background. Accessories for other special processes, such as fluorescence microscopy or differential interference contrast, can also be attached to the same microscope so that these techniques can be combined. The Leitz reflection contrast lenses were equipped with phase rings as standard, so that a seamless change between reflection contrast and phase contrast could be made and a phase contrast image could also be added if required.

Interference reflection microscopy has been used particularly frequently for the investigation of cell adhesion to glass and the determination of the thickness of cell extensions ( pseudopodia ). Focal points of adhesion were first described using this technique in 1976. Other applications were the reconstruction of the surface relief of erythrocytes and the examination of reticulocytes in blood smears, since these can be distinguished from mature red blood cells ( erythrocytes ) in this procedure , as well as examinations of chromosome preparations and the cytoskeleton in fixed cells. Since 2004, the IRM has experienced a kind of renaissance with the development of the iSCAT microscope .

Functional principle using the example of cell adhesion

Schematic representation of interference reflection microscopy. Two rays of light (thicker green wavy lines) come from below through a cover glass (gray) and hit either a cell (brown) or first on aqueous cell culture medium (ocher yellow) and then on the cell. Where the light beam from the cover slip passes directly into the cell (left side) there is reflection (thin green beam). If, on the other hand, there is a small space between the cell and the cover slip (right side), a reflection occurs at the cover slip-medium boundary and another at the medium-cell boundary. The two resulting beams can interfere with each other. The refraction of light at interfaces was ignored here.

Interference reflection microscopy is based on incident light illumination , reflection and interference. To the contrast -reducing reflections to minimize on glass surfaces, is oil immersion with anechoic lenses used. A central diaphragm in the illumination beam path also blocks reflections from the central area of ​​the lens, where these are particularly disturbing. For practical reasons, an inverted (inverted) microscope is used in cell biology applications so that the cells are on a cover glass and can be observed from below.

In incident light, the image of a transparent object is caused by the reflection of the light at interfaces where the refractive index changes. The intensity of the reflected light is the greater the greater the difference in the refractive index. Immersion oil and glass have a very similar index of refraction. In the case of a preparation with living cells that grow on a cover slip and are illuminated with incident light (in the inverted microscope from below) during oil immersion, the first difference in refractive index occurs at the transition from the cover slip to the aqueous medium in which the cells are located. This creates a comparatively strong reflection. However, if there is a cell at that point, the difference in refractive index, here between the cover glass and the cell or cell membrane , is significantly smaller, and the reflection is correspondingly weaker.

If there is still a medium-filled space between the cell and the cover slip, its thickness can be examined with the help of interference reflection microscopy: Reflection takes place first at the cover slip-medium transition and then at the medium-cell transition. If the distance between these two transitions is on the order of the wavelength of the light used, the two reflected rays can interfere with each other. When using monochromatic light that contains only one or a few wavelengths, light and dark areas are created. In contrast, white light creates colored areas, depending on which wavelengths interfere negatively or positively. Information about the distance between the cell and the cover glass can therefore be read from these observations, namely in orders of magnitude that are below the usual resolution limit of a light microscope (≈ 200 nanometers ).

The effect described can, however, be considerably disturbed by reflected rays that arise at other interfaces, for example in the case of flat cell processes from the transition of the rear cell membrane into the surrounding medium. This problem is reduced if the illumination is carried out with a high numerical aperture and the cell is at least one micrometer thick, since the upper cell membrane can then no longer contribute to the interference pattern due to the optical conditions. Quantitative investigations can then be carried out taking into account the cell thickness . More often, however, qualitative studies were carried out, for example on the distribution of focal points of adhesion .

Web links

Individual evidence

  1. ↑ a b W. J. Patzelt: Reflection contrast, a new microscopic method . In: Natural Sciences . 63, No. 11, November 1976, p. 535. doi : 10.1007 / BF00596860 . PMID 1004619 .
  2. ↑ a b c d H. Verschueren: Interference reflection microscopy in cell biology: methodology and applications . In: J. Cell. Sci. . 75, April 1985, pp. 279-301. PMID 3900106 .
  3. ^ R. Parthasarathy, JT Groves: Optical techniques for imaging membrane topography . In: Cell Biochem. Biophys. . 41, No. 3, 2004, pp. 391-414. doi : 10.1385 / CBB: 41: 3: 391 . PMID 15509889 .
  4. ^ TJ Filler, ET Peuker: Reflection contrast microscopy (RCM): a forgotten technique? . In: J Pathol . 190, No. 5, April 2000, pp. 635-638. doi : 10.1002 / (SICI) 1096-9896 (200004) 190: 5 <635 :: AID-PATH571> 3.0.CO; 2-E . PMID 10727991 .