Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/55—Specular reflectivity
  • G01N21/552—Attenuated total reflection
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N2021/6463—Optics
  • G01N2021/6478—Special lenses
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B6/00—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
  • G02B6/24—Coupling light guides
  • G02B6/42—Coupling light guides with opto-electronic elements
  • G02B6/4201—Packages, e.g. shape, construction, internal or external details
  • G02B6/4204—Packages, e.g. shape, construction, internal or external details the coupling comprising intermediate optical elements, e.g. lenses, holograms
  • G02B6/4206—Optical features
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B6/00—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
  • G02B6/24—Coupling light guides
  • G02B6/42—Coupling light guides with opto-electronic elements
  • G02B6/4298—Coupling light guides with opto-electronic elements coupling with non-coherent light sources and/or radiation detectors, e.g. lamps, incandescent bulbs, scintillation chambers

Definitions

• the invention relates to an optical arrangement for detecting light that is emitted by a sample, according to the preamble of patent claim 1.
• Molecules are usually detected using optical biosensors by detecting a specific bond between the molecules and so-called receptor molecules that are immobilized on a surface. The detection of molecules can also be carried out very selectively from a mixture via such a specific binding.
• Typical receptor molecules are antibodies or DNA molecules. Typical surfaces can be found on optical fibers or transparent slides or microscope cover plates.
• a fluorescence signal specific for the bond between the sought-after molecule and the receptor molecule is frequently detected. This can be generated by the searched molecules themselves, provided that they are fluorescent.
• the beam optics it is not possible to radiate a light beam from the optically thinner into the optically denser medium if it spreads in the optically denser medium at an angle to the interface which is greater than the critical angle of total reflection ⁇ .
• the drop in the field strength is of the order of the wavelength of the incident light. Despite total reflection, a certain proportion of the light enters the optically thinner medium. It also applies that light from a location in the optically thinner medium that is close to the interface can also enter the optically denser medium at an angle that is greater than the critical angle of total reflection ⁇ .
• This effect is used in optical fiber sensors. With these sensors, receptor molecules are immobilized on the fiber surface. Excitation light, usually laser light, is injected into the fiber. The laser light is transmitted through the fiber by total reflection. It also passes through the areas of the fiber to the surface of which the fluorescent molecules are bound. Due to the evanescent field of the excitation light on the surface of the fiber, fluorescent molecules can be excited.
• the distance of the molecules from the fiber surface is only a few nanometers in the bound state, so that the exponential drop in the field strength of the excitation light is hardly noticeable.
• the molecules bound to the surface are therefore effectively excited by the excitation light.
• the light emitted by the molecules can also couple back into the fiber, in some cases at angles at which the once-coupled fluorescent light is transmitted through the fiber due to total reflection.
• the transmitted fluorescent light component of the bound molecules can then be detected at one of the fiber ends.
• a disadvantage of optical fiber sensors that detect evanescent radiation is that the efficiency with which they can collect (capture and transmit) emitted fluorescence photons is very limited. In particular, the sensitivity of such an arrangement is not sufficient to detect individual molecules capable of fluorescence.
• the object of the invention is to improve the light collection efficiency for light coupled evanescently into an optically dense medium.
• optical arrangement with the features of patent claim 1.
• the optical arrangement according to the invention can detect both light that was generated in front of an interface, for example due to chemiluminescence, and light scattered in front of the interface, for example fluorescent light as a result of excitation by a light source.
• the light generated in this way is collected by a light collecting device.
• this can also have further lenses, mirrors, filters and other customary optical components.
• the light collected by the light collecting device is detected by the detection device.
• this in turn can also have further lenses or filters for directing the light onto the detector.
• the first end face can be completely flat or even only in a region in which the sample is arranged.
• the interface in front of which the sample is arranged can be formed directly by the first end face itself.
• the first end face can be optically coupled to an interface between the sample medium and the optically dense medium.
• the optically dense medium can be the medium of a slide which is coupled to the first end face with the aid of an optically dense immersion oil.
• the diameter of the sample carrier and the dimensions of the first end face should be selected such that light emitted by the sample and emitted into the half space facing the optically denser medium is not prevented from entering the first end face by purely geometric restrictions.
• the arrangement and design of the outer surface ensures that the evanescent radiation on the outer surface is completely reflected back into the light guide body. This can be done on the one hand by means of total reflection, but on the other hand by appropriate mirroring of the outer surface.
• the evanescent radiation can then be directed to a detection device in a known manner and detected by it.
• the evanescent radiation component can be detected essentially completely. Since classical radiation, i.e. Light that propagates at an angle (based on the surface normal mentioned above) in the light guide body, which is smaller than the critical angle of the total reflection corresponding to the refractive index ratio of the optically denser and the sample medium, can thus detect almost the entire radiation of a half space become .
• the first end face is flat.
• the lateral surface adjoining this is inclined relative to the first end surface such that the light emitted by the sample is totally reflected on the lateral surface. This enables lossless reflection to be achieved. Furthermore, a special mirroring of the outer surface is not necessary.
• the arrangement and design of the lateral surface are chosen such that essentially only the evanescent portion hits the lateral surface or is totally reflected on it.
• the classic radiation leaves the Lichtleitkorper unreflected. This allows an extremely efficient separation of evanescent and classic radiation.
• Evanescent rays The proportion of radiation can only come from radiation sources close to the interface. As a result, the light emitted close to the surface of a sample can be detected very selectively, as is essential for optical biosensors.
• the light guide body is designed to be rotationally symmetrical about an axis, and the sample is arranged on or in the immediate vicinity of the axis.
• the lateral surface can then be a section of a paraboloid of revolution, the sample being arranged near the focal point. If the emitted light comes from the sample and thus the focal point of the paraboloid of revolution, it is parallelized by reflection on the lateral surface. The light parallelized in this way can be collected and detected in a simple manner. If the second end face is flat, the parallelized light also emerges in parallel from the light guide body. It can then be forwarded particularly easily. In addition, the radial distance of the light beam emerging from the second end face depends in a well-defined manner on the angle of entry into the first end face.
• the lateral surface is a section of an ellipsoid of revolution, the sample being arranged near a focal point of the ellipsoid of revolution on or in front of the first end face.
• the outer surface can also be designed as an outer truncated cone surface. This allows the light guide body to be manufactured easily. In addition, it has been shown that a truncated cone-shaped outer surface leads to an improved imaging quality in comparison to a rotationally paraboloidal outer surface, since deviations in the location of the Radiation source from the axis have less impact on the image sharpness.
• the second end face is convex and serves as a converging lens for the totally reflected light. It can be focused directly on one point.
• the detection device can be located in this focal point, for example.
• a light-absorbing diaphragm is arranged between the second end face and the detection device in such a way that it selectively absorbs the classic radiation component of the light emerging from the light guide body. In most designs of the lateral surface, evanescent and classic radiation components emerge from the second end surface at different locations and / or at different angles.
• a diaphragm is then able to selectively absorb the classic radiation from a mixture of classic and evanescent radiation and only let the evanescent radiation pass through.
• a selective detection of only the evanescent radiation in turn allows the selective detection of light near the surface, which is important for optical biosensors.
• the detection device can be designed such that it detects classic and evanescent radiation separately. This can be done, for example, with the aid of a locally resolving detector.
• the separate detection of evanescent and classical radiation allows a further differentiation between near-surface and far-away light. It can also be used to determine the distance of the sample from the first interface.
• the optical arrangement has a light source for irradiating the sample.
• the light can be directed through the light guide body to the sample by is irradiated, for example, on the second end face or is irradiated in such a way that it excites the sample evanescently. As a rule, however, it is irradiated on the sample side and thus reaches the light guide body through the first end face.
• an absorption device is arranged between the light guide body and the detection device, which absorbs the light from the light source so that it does not reach the detector. In this way, a disruptive influence of the light from the light source on the detection is avoided. This is essential for achieving the desired extremely high sensitivity, which also allows detection of individual fluorescent molecules.
• a thin cutting disk made of an optically denser medium is arranged between the first end face and the interface.
• This can typically be a slide or a microscope cover plate.
• the thickness of the cutting disc is significantly less than the minimum dimension of the first end face, so that the major part of the evanescent radiation can enter the light guide body. This is particularly convenient for routine analysis.
• receptor molecules are immobilized on a sample carrier surface.
• the sample carrier is optically coupled to the first end face by an immersion oil. In this way, direct contact between the light-guiding body and the sample and thus disruptive contamination of the first end face and background signals caused thereby are avoided.
• the sample carrier is slidably mounted parallel to the first end face relative to the light guide body.
• the optical coupling between the Lichtleitkorper and the sample holder is retained by the immersion oil even when the sample holder is moved. This makes it possible to the surface of a sample carrier to find out whether a single or a few individual searched molecules have bound to the receptor molecules immobilized on it. This is essential if extreme sensitivities are to be achieved, since the concentrations of the molecules sought are so low that no molecule searched for is bound to a receptor molecule in a given small observation area.
• the sample carrier is part of a flow cell. This allows automation of measurements and the analysis of continuous sample streams.
• FIG. 1 shows a sectional view of a light-guiding body according to the invention with a rotationally paraboloid-shaped outer surface
• FIG. 2 shows the light guide body according to FIG. 1 with a convex second end surface acting as a converging lens
• FIG. 3 shows a sectional view of a light-guiding body according to the invention with a rotating lipoid-shaped lateral surface
• FIG. 5 schematically shows an exemplary embodiment of the optical arrangement according to the invention, which is formed from an array of individual optical arrangements.
• 1 shows a light guide body 1 with a flat first end face 2, a rotationally paraboloid-shaped jacket surface 3 and a flat second end face 4.
• the 10
• Lichtleitkorper is rotationally symmetrical with respect to an axis 5.
• a typical material for the light guide body is glass with a refractive index of 1.5.
• the sample is usually in an aqueous solution with a refractive index of approximately 1.3.
• the Lichtleitkorper could, for example, be surrounded by a liquid medium or another solid (plastic / glass interface).
• Light that enters the light guide body 1 from a sample 6 initially comprises the so-called “classic” radiation 7.
• the classic radiation enters the light guide body 1 at an angle (with respect to the axis 5) that is smaller than that of the refractive index ratio of the Optical fiber material and the sample medium corresponding limit angle of total reflection ⁇ is.
• light rays can enter at an angle that is greater than the critical angle of total reflection in the light guide body 1. These rays are called evanescent rays 8.
• the distance R between the light beams 7 or 8 reflected on the lateral surface and the axis 5 depends only on the angle at which the respective light beam enters the light guide body 1 starting from the sample 6. It is therefore possible to reconstruct the angular emission characteristic of the sample from the radiation emerging through the second end face 4 with the aid of a spatially resolving detector, for example a CCD camera.
• the evanescent and classic radiation can also be detected and selectively analyzed at the same time. This enables additional information to be obtained, e.g. about the distance of the sample from an interface.
• the diameter B of the first end face can be freely selected.
• the exact course of the parabola belonging to the paraboloid of revolution can be used in the event that sample and focal point in the 11
• B 1 Y - 4 + B ⁇ 2 ' (2)
• x is the distance of a point (x, y) on the parabola from axis 5
• y is the distance of the point from the plane of the first face.
• Typical values for B are in the range of 1 to 5 cm.
• miniaturized shapes of the light guide body 1 are also conceivable with very small values for B, for example less than 100 ⁇ .
• the thickness D of the light-guiding body is chosen such that both evanescent radiation 8 and classic radiation 7 are totally reflected on the rotationally paraboloid-shaped outer surface 3.
• the dimensions of the light guide body 1 can be selected in such a way that no conventional radiation strikes the outer surface and is totally reflected. This can be achieved in that the thickness D of the light guide body 1 is chosen to be correspondingly small.
• the minimum angle at which evanescent radiation with respect to the rotational symmetry axis propagates in the light guide body 1 results from equation (1), taking into account that ⁇ .2 the refractive index of the sample medium (typically water with a refractive index of 1.3) and n ] _ the refractive index of the light guide body (typically that of glass: 1.5). This results in a of approx.
• FIG. 2 shows the light guide body according to FIG. 1, but not with a flat second end face 4, but with a spherically convex second end face 4.
• Light entering the light guide body 1 from the sample 6 is parallelized in the event of a reflection on the rotationally paraboloid-shaped outer surface 3, if the sample 6 is in the focal point of the paraboloid of revolution.
• the resulting parallel light beams 7 and 8 are focused on a point 9 by the spherically convex second end face 4.
• FIG. 3 shows a light guide body 1 with a flat first end face 2, a flat second end face 4, and a rotationally ellipsoid-shaped outer surface 3.
• the sample 6 is located in one of the two focal points of the associated rotational ellipsoid. Most of the light emitted by the sample 6, which enters the light guide body 1, is totally reflected on the rotating ellipsoidal surface 3 and focused in the second ellipse focal point. Since the second focal point in the exemplary embodiment shown here lies outside the light-guiding body 1, the light is refracted in the direction of the axis 5 when it emerges from the flat second end face 4. The focus 9 is therefore somewhat closer to the Lichtleitkorper 1 than the second focal point of the ellipsoid of revolution. The second end face could equally well be spherically convex. This would have the consequence that the focus 9 is even closer to the Lichtleitkorper 1.
• a light beam 21 emerges, which can be a diode laser, for example. This is first spectrally cleaned by an optical filter 22, since the radiation from diode lasers generally contains spectral components that do not match the desired wavelength of the laser radiation. The laser beam is then focused onto the sample 6 through a converging lens 23.
• a microscope objective can also serve as a converging lens.
• the sample 6 is located on the surface of a slide 24.
• the opposite surface of the slide is optically coupled to the light guide body 1 by an immersion oil.
• the slide 24 forms a wall of a flow cell 25.
• the wall 26 of the flow cell 25 opposite the slide 24 has a window which is transparent to the excitation wavelength.
• Antibodies can be immobilized on the slide 24, for example. A solution containing the molecules or antigens sought is then introduced into the flow cell. Instead of a liquid, the medium in the sample cell can also be a gas with which the molecules sought are mixed.
• the solution also contains second antibodies, so-called probe molecules, which selectively bind to the complex of the first antibody and the antigen bound to it.
• the first antibody is selective for the antigen, ie it binds very specifically almost exclusively to the antigen.
• the probe molecule is labeled with fluorescence.
• the fluorescent dye must be selected such that its absorption wavelength matches the emission wavelength of the light source 20. If the light source is a diode laser with an emission wavelength of, for example, approximately 630 nm, then a rhodamine or cyanine dye, for example Cy5, is suitable as the dye. Typical antigens to be detected are poorer in tumors. LO LO t to ⁇ - ⁇ **
• a general paraboloid is also possible, for example an elliptical in cross section, which maps the light of the sample onto a line. It is only important that evanescent radiation is reflected as completely as possible on the outer surface 3.
• the first and the second end face can be arranged both parallel and at an angle to one another.
• the light guide body 1 can have further small-area regions in the transition regions between the surfaces.
• All light sources commonly used in spectroscopy can be used as light sources 20, i.e. especially lamps and lasers of all kinds.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Immunology (AREA)
• Chemical & Material Sciences (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Urology & Nephrology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Molecular Biology (AREA)
• Hematology (AREA)
• Microbiology (AREA)
• Cell Biology (AREA)
• Biotechnology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)
• Optical Measuring Cells (AREA)
• Microscoopes, Condenser (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (1)

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Cited By (4)

Families Citing this family (19)

Citations (3)

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• 1998
  • 1998-03-12 DE DE19810615A patent/DE19810615A1/de not_active Withdrawn
• 1999
  • 1999-03-10 US US09/646,031 patent/US6714297B1/en not_active Expired - Fee Related
  • 1999-03-10 WO PCT/EP1999/001548 patent/WO1999046596A1/de active IP Right Grant
  • 1999-03-10 EP EP99907618A patent/EP1076823B1/de not_active Expired - Lifetime
  • 1999-03-10 JP JP2000535927A patent/JP4259762B2/ja not_active Expired - Fee Related
  • 1999-03-10 AU AU27288/99A patent/AU2728899A/en not_active Abandoned
  • 1999-03-10 DE DE59903655T patent/DE59903655D1/de not_active Expired - Lifetime

Patent Citations (3)

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