Interference reflection microscopy
Interference reflection microscopy ( also: Reflection contrast microscopy ) is a light microscopy method, in which the thickness of very thin structures is determined. It is based on the formation of interferences that occur when light is refracted at the upper and lower boundary surface of the structure, refracted light from the two interfaces to interfere with each other. This results in interference patterns that can be observed, and provide the information about the thickness of the structure.
The resulting interference colors make thickness measurements in the range below 200 nanometers, ie below the normal resolution of the light microscope. Since the interference colors are observed through a light microscope, these thickness measurements can be classified according to microscopically identifiable structures such as individual foothills of cells. Similar to the total internal reflection fluorescence microscopy (TIRF ) of the observation object to the specimen surface, ie in the vicinity of the cover glass is limited.
The process has received different names by different authors. The oldest description ( 1964) is Interference Reflection Microscopy (IRM ), to German interference reflection microscopy. Other names are Reflection Contrast Microscopy ( RCM 1975), on German reflection contrast microscopy, and Reflection Interference Contrast Microscopy ( RICM, 1981). An English review article from 2000 lists also include interference contrast, interference reflection contrast, reflection interference contrast, surface reflection interference contrast microscopy and surface.
History and Applications
The technique was first used in 1958 or 1960 for the study of thin films and introduced in 1964 in the cell biology. It was not until about ten years later, further works were published. In the 1970s, the technique was further developed and commercially offered by Ernst Leitz and Carl Zeiss. The process has not been able to put wide, for specific applications, it came in the 1970s and 1980s to an accumulation of publications.
Interference reflection microscopy was used most frequently for the study of cell adhesion to glass and to determine the thickness of cell processes ( pseudopodia ). Focal adhesion points were first described in 1976 by this technique. Other applications were the investigation of reticulocytes in Blutaustrichen as these in this process of red blood cells (RBCs ) can be distinguished, as well as assays for chromosomal preparations and the cytoskeleton in fixed cells.
Functional principle of the cell adhesion
Interference reflection microscopy based on reflected light illumination, reflection and interference. In order to minimize the contrast -reducing reflections from glass surfaces, oil immersion comes with a low-reflection lenses used. A central aperture in the illumination beam path is also blocked reflections from the central area of the lens, where these are particularly disturbing. For practical reasons, a reverse (inverse ) microscope is used in cell biological applications, so that the cells are on a cover glass and observed from below.
Reflected light at the image of a transparent object is caused by reflection of the light at interfaces at which the refractive index changes. The intensity of the reflected light is the greater the greater the difference in refractive index. Immersion oil and glass have very similar refractive index. In the case of a preparation of live cells grown on a cover glass and immersion oil with incident light illuminated ( in the inverted microscope from below), hence the first refractive index difference occurs at the transition from the coverslip on to the aqueous medium in which the cells are located. This results in a relatively strong reflection. However, there is a cell at the site, then the difference in refractive index, in this case correspondingly weaker between the cover glass and the cell or the cell membrane, significantly less reflection.
Located between the cell and the coverslip or a fluid-filled space, its thickness can be investigated by means of interference reflection microscopy: reflection takes first at the transition coverslip - medium and then at the transition medium - cell instead. If the distance between these two transitions is on the order of the wavelength of the light used, the two reflected beams can interfere with each other. In the use of monochromatic light, which contains only one or a few wavelengths of light and dark areas thus result. In white light, however, arise colorful areas, depending on which wavelengths interfere negatively or positively. From these observations, it is thus possible information on the distance from cell to read the cover glass, in sizes that are below the standard resolution limit of a light microscope ( ≈ 200 nanometers).
However, the described effect can be significantly disturbed by reflected rays originating at other border areas, such as flat cell processes by the transition from the rear cell membrane into the surrounding medium. This problem is reduced if the lighting is done with a high numerical aperture and the cell is at least one micrometer thick, since the upper cell membrane then due to the optical conditions can no longer contribute to the interference pattern. Considering the cell thickness then can be carried out quantitative studies. Frequently, however, qualitative studies have been conducted, for example, the distribution Focal adhesion points.
By the use of polarization filters in the beam path and a 4 wavelength plate in the lens ( Antiflex lens), light reflected at the glass surfaces within the lens itself, be filtered out. At the same microscope can also be accessories for other special procedures to install, such as fluorescence microscopy and differential interference contrast, so that these techniques can be combined.