Abstract:
In the procedure, the light beams are incident parallel to the transparent medium and are deflected toward a focal point inside the transparent medium. This avoids the use of refractive lens systems. The focusing lens system according to the invention enables the bundling of light beams of different wavelength to a point inside the sample. 
     This clearly simplifies the design and adjustment of a multicolor fluorescence correlation spectroscopy device.

Description:
FIELD OF THE INVENTION 
     The invention relates to a procedure and a device for fluorescence correlation spectroscopy, in particular for multicolor fluorescence correlation spectroscopy, during which light beams are focused in a transparent medium. 
     BACKGROUND OF THE INVENTION 
     In multicolor fluorescence correlation spectroscopy (FCS), as described by P. Schwille et al., Biophysical Journal, Vol. 72 (1997), 1878 to 1886 or M. Völcker et al. tm—Technisches Messen, 36 (1196, 128 to 135), molecular interactions are studied by marking two reactants with different fluorescent dyes and allowing them to freely diffuse in a liquid, transparent medium. The reactants generate fluctuations in fluorescent intensity while diffusing through the focal point that can be detected with a confocal lens system. If predominantly correlated intensity fluctuates are detected between the emission wavelengths of the two fluorogens, this points to a complexing between the two partners. 
     Given the varying excitation wavelengths of the fluorogens, use must be made of two laser wavelengths, which are focused on the smallest possible, identical volume in the sample. As a rule, microscope lenses with a high numerical aperture, i.e., large aperture ratio, are used to this end to achieve as small a focal point as possible on the one hand, and gather as much of the emitted light 
     Another disadvantage to the available lens is that an optimal, diffraction-limited focal point is only ensured if the refractive index of the immersion medium and sample solution reflects the value for which the lens was optimized. Since fluorescence correlation spectroscopy is very sensitive to changes in focus volume, a change in the refractive index can have a highly adverse effect on the results. Among other things, changes in the refractive index can be caused by a change in the temperature of the sample or salts dissolved in the buffer, and hence in the parameters that are commonly varied in biochemical experiments. This problem is already encountered in single-color fluorescence correlation spectroscopy. 
     SUMMARY OF THE INVENTION 
     In terms of the procedure, the object of the invention is to develop a procedure and device that do not exhibit the described disadvantages. 
     This object is achieved by means of a generic procedure in which the light beams are incident on the transparent medium in an approximately perpendicular manner, and are only deflected toward the focal point inside the transparent medium. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention is based on the knowledge that the known errors stem from the use of refractive lens systems. The angle of incidence and angle of reflection on a surface between two media are linked by the refractive index according to Snell&#39;s Law. If the refractive indices change owing to dispersion, i.e., a change in the refractive index with wavelength, or resulting from the use of other buffers, a change takes place in the beam path, and hence in the focus volume. This set of problems affects every beam path except for the light beams perpendicularly incident on the boundary surface. Only a light beam perpendicularly incident on the boundary surface passes the boundary surface without being deflected, independently of the refractive index. As a result of this knowledge, only reflective lens systems came to be used in the focusing arrangement to solve the task, and all light beams penetrate the boundary surfaces between different optical media only perpendicularly. 
     In this regard, the term “approximately perpendicular” is defined by the desired measuring accuracy. Deviations are possible within the framework of a desired measuring accuracy. 
     Hence, the described procedure enables a simple optimization of the used lens, and permits the focusing of laser beams with different wavelengths on an identical, as diffraction-limited as possible volume inside the sample. 
     One relatively easy way to realize the invention is to reflect the light beams inside the transparent medium toward the focal point. 
     In terms of the device, the object is achieved with a general device in which the sample vessel exhibits a focusing, metal-coated floor, wherein the focal point lies inside the sample vessel. In this case, the floor can be designed in such a way as to take the light beams penetrating into the sample vessel essentially parallel to each other and focus them essentially on the focal point. 
     This device makes it possible to use the sample vessel as a focusing element. The parallel light beams incident in the sample vessel are focused by its floor on a point. Since this point lies inside the sample vessel, there is no further deflection of the parallel incident light beams on the boundary surfaces between two media. The light beams only need to get into the sample once, and since parallel and perpendicular incident light beams are used here, they are not deflected when penetrating through the cover slip or making the transition between the cover slip and sample liquid. No boundary surfaces between different media need be overcome in the process of deflection inside the transparent sample. 
     It is advantageous if the sample vessel is saucer-shaped, and if the focal point lies inside the saucer. An optimal shape for the floor is achieved by giving the floor a parabolic or slightly elliptical shape. 
     To achieve a good measurement result, it is proposed that the floor be precisely fabricated to a fraction of the used wavelength. The high level of precision achievable with the device also requires that the metal-coated floor surface be accurately fabricated. 
     To ensure the long-term durability of the reflective metal coating, it is additionally proposed that the floor be metal-coated with a layer resistant to conventional buffer solutions. 
     Any material in which a fluorescence correlation spectroscopy can be performed may be used as the transparent medium. In particular, this can be a transparent fluid or transparent liquid or gel. 
    
    
     An embodiment of the device according to the invention is shown in a drawing in a comparison with a known device, and will be described in greater detail below. 
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a diagrammatic view of a known device for multicolor fluorescence correlation spectroscopy, and 
     FIG. 2 is a diagrammatic view of device for multicolor fluorescence correlation spectroscopy according to the invention. 
    
    
     The device  1  shown on FIG. 1 reflecting prior art consists of a cuvette  2 , which stands on a cover slip  3  and contains the sample  4  in a buffer solution. Located under the cover slip  3  is the last lens  5  of an objective, while immersion liquid  6  is located between this last lens  5  and the cover slip  3 . 
     Parallel light beams  7 ,  8 ,  9 ,  10  pass through the lens  5  into the immersion liquid  6  and travel on through the cover slip  3  into the buffer solution  4  located in the cuvette  2 , where they converge in a focal point  11  or  12 . 
     As a result of the different wavelengths of light beams  7 ,  8  or  9 ,  10 , the beams are deflected differently at each boundary surface between two media, and hence converge at different focal points  11  or  12 . The greater the distance  13  between focal point  11  and focal point  12 , the more impaired the measurement. 
     The described device shows that light beams  7 ,  8 ,  9 ,  10  from lasers with different laser wavelengths have to pass through several boundary layers until the converge in a focal point  11  or  12  that corresponds to the wavelength of the respective light beam. These transitions greatly impair the measurement. 
     The device  20  shown on FIG. 2 consists of a block  21  with a flat surface  22  that incorporates a recess  23  as the sample vessel. This sample vessel is shaped like a parabola in cross section and has a metal-coated floor area  24  that acts as a parabolic mirror. This parabolic mirror is arranged in such a way that the focal point  25  of parallel incident light beams is located inside the recess  23 . 
     Placed over the recess  23  on the flat surface  22  of the block  21  is a cover slip  26 , which covers the recess  23  filled with the sample in buffer solution. 
     To optimize the focusing at point  25 , the floor  24  is precisely fabricated to a fraction of the wavelengths of the used light beams, and the reflective material is adjusted to the buffer solutions in such a way as to prevent the buffer solutions or sample from changing the material. 
     When using the device  20  according to the invention, the sample is first poured into the recess of block  21  with a buffer solution. The completely filled recess  23  is then covered with the cover slip  26 . As a result, parallel incident beams  27 ,  28 ,  29 ,  30 ,  31 ,  32  perpendicularly pass through the surface of the cover slip, penetrate into the sample arranged in the recess  23 , going through the sample and arriving at the floor area  24 . Since the light beam intersects each boundary surface between two media (air/cover slip) and (cover slip/sample) perpendicularly up to this point, it is not deflected. Only after hitting the metal-coated floor area  24  inside the sample liquid are the light beams  27  to  32  deflected toward the focal point  25 . Since this reflection on the floor area  24  is independent of the wavelength of the light used, all light beams  27  to  32  converge in focal point  25 , even if the light beams  27  to  32  exhibit different wavelengths. 
     Therefore, the advantage to the device is that the mirror ground to a parabolic or slightly elliptical shape serves as the focusing element and simultaneously as a holder for the sample, thereby essentially eliminating all imaging errors.