DEVICE FOR DETERMINING OPTICAL PROPERTIES OF SAMPLES

Description

FIELD OF APPLICATION AND STATE OF THE ART

[0001] The invention relates to a device for determining optical properties of samples arranged in a matrix arrangement in wells of a microtiter plate.

[0002] Optical methods are among the most important examination techniques in biochemical and pharmacological research. Devices for determining the optical properties of samples are used to acquire measured values. These can be specialized for a specific measurement method, such as fluorescence measurements, or they can be designed as multi-technology devices, such as multi-label readers (MLR) or multi-mode readers, which can be used for two or more different measurement methods, such as fluorescence measurements, luminescence measurements, absorption measurements, etc.

[0003] Sample multiplexing techniques are often used to conduct these studies. These techniques involve arranging the samples to be analyzed in a matrix array in microwell plates and analyzing them either sequentially or in parallel. The types of samples analyzed are extremely diverse. For biological samples, for example, they can range from homogeneous solutions to immobilized cells.

[0004] The application concerns such devices, which are suitable, among other things, for the nephelometric measurement of samples. For a nephelometric measurement, a sample is placed in a well with a transparent base, i.e., permeable to the measuring light used.

[0005] Nephelometry is an optical measurement technique for quantitatively determining the particle fraction in suspensions or aerosols based on the intensity of scattered light generated by suspended particles due to the Tyndall effect. The term "scattered light" refers to light that has been deflected from its original propagation direction due to scattering. A relationship between turbidity and the content of opacifying substances is given by the Rayleigh equation, according to which the light intensity scattered by a turbid solution is proportional to the intensity of the incident radiation, the number of particles, and the sixth power of the particle radius, as well as inversely proportional to the square of the distance from the illuminated volume element and the fourth power of the wavelength of the incident light.Under the same conditions and with the same size of the scattering particles, the intensity of the scattered light is proportional to the number of scattering particles, which can be used for quantitative analysis.

[0006] The intensity of scattered radiation can be measured either directly (scattered light measurement) or indirectly determined from the attenuation of the transmitted light. Scattered light measurement (also called tyndallometry) is usually performed at right angles to the primary radiation. In turbidity measurement (turbidimetry), the scattered portion of the primary radiation is determined from the difference between the incident light and the transmitted light.

[0007] DE 35 35 652 A1 describes a system suitable for nephelometry that can work with microtiter plates. The sample is irradiated vertically from bottom to top. The system can be equipped with a mechanical movement device for horizontal positioning of the samples.

[0008] Patent EP 0 864 083 B1 discloses a nephelometer. The aim of the invention is to be able to measure matrix-shaped samples in open-topped vessels nephelometrically with high sensitivity and free from interfering background signals. The nephelometer comprises an illumination device for exposing a sample to light. The illumination device has a light source, an exit lens arrangement, and an exit aperture diaphragm, which are arranged in this order on a common optical axis emanating from the light source.Furthermore, the nephelometer has a detector device for detecting the scattered light emanating from the sample, wherein the detector device has a central shading element for absorbing light from the light source passing directly through the sample and incident on the detector device, a detector entrance lens arrangement, a second shading element for absorbing light from the light source directly incident on the detector device from the exit aperture stop of the illumination device, and a detector, which are arranged in this order on the optical axis, wherein the exit aperture stop of the illumination device and the entrance lens arrangement as well as the two shading elements of the detector device are arranged such that the central shading element is located in a plane conjugate to the light source and the second shading element is located in a plane conjugate to the exit aperture stop.

[0009] BMG LABTECH GmbH (Offenburg, Germany) offers a laser-based nephelometer called NEPHELOstar®, which can be used for nephelometric measurements of samples held in microtiter plates. It uses a high-intensity light source in the form of a laser diode that emits a highly collimated laser beam at 635 nm, with adjustable intensity and beam width. The laser beam penetrates the sample vertically from top to bottom and, after passing through the transparent base, enters a scattered light detector containing an integrating sphere. An integrating sphere is a hollow sphere with diffuse internal reflection. The associated photosensitive detector is attached to a side light exit opening of the integrating sphere. If the light is not deflected by particles in the sample but passes directly through the integrating sphere, no signal is generated at the detector.If particles are present in the sample, the light is scattered and reflected several times inside the integrating sphere before reaching the detector, which generates a corresponding signal.

[0010] CH 711376 A2 discloses an example of a device for determining the optical properties of samples arranged in a matrix arrangement in wells of a microtiter plate. The device comprises a light source arrangement with at least one light source and an excitation path for transmitting light from the light source as excitation light from an upper light entrance side to a measuring position located in a sample plane, in which a sample is arranged or can be arranged, which is accommodated in a well of a microtiter plate. An angle-selective emission path leads from the measuring position to a first detector on the light entrance side. A concave mirror with a mirror surface rotationally symmetrical to an optical axis of the emission path is arranged in the emission path for collecting emission light emerging divergently upward from the sample.The concave mirror has a light entrance opening on its light entrance side facing the sample plane, which widens into a light exit opening optically facing the first detector. In one embodiment, the wells are equipped with a transparent bottom to enable absorption measurements.

[0011] EP 3 499 200 A1 describes a tunable filter monochromator for spectrally isolating a narrow spectral pass range of a predefined bandwidth around a predefined central wavelength from an incident light intensity with a larger spectral bandwidth. The bandwidth and the central wavelength of the spectral pass range are continuously adjustable independently of each other. A parabolic mirror is arranged in the emission path.

[0012] DE 10 2013 224 463 A1 describes a device for determining the fluorescence properties of samples.

[0013] The documents DE 10 2018 200646 A1, WO 2018 / 033600 A1, EP 2 284 521 B1 and JP S62 66141 A disclose further examples of devices for determining optical properties of samples. TASK AND SOLUTION

[0014] It is an object of the invention to provide a device for determining optical properties of samples arranged in a matrix arrangement in wells of a microtiter plate, which device is suitable, among other things, for nephelometric measurements, has a higher sensitivity than the prior art and offers the possibility of using other measurement techniques on the same sample simultaneously or in quick succession, as an alternative or in addition to nephelometric measurements, without having to reposition the sample.

[0015] To achieve this object, the invention provides a device having the features of claim 1. Advantageous further developments are specified in the dependent claims. The wording of all claims is incorporated by reference into the content of the description.

[0016] The device is suitable, among other things, for the nephelometric measurement of samples arranged in a matrix array in microtiter plate wells. The device can be used as a component of a microplate reader. The device can also be used for other measurement methods. In some embodiments, the device can be used for nephelometry, turbidimetry, fluorescence measurements, absorption measurements, and chemiluminescence measurements in microplates.

[0017] The device has a light source arrangement with at least one light source. This can be a single light source or several, one of which can be selected for a measurement.

[0018] An optically downstream excitation path serves to transmit light from the light source as excitation light to a measuring position. The excitation path can contain one or more optical elements for influencing the light coming from the light source. The excitation path can contain, for example, at least one optical element for adapting the light source to the beam shape for the sample container, at least one optical element for influencing the frequency or wavelength of the excitation light effective on the sample (e.g. filter or monochromator) and / or at least one direction-changing element. It is also possible for there to be no optical element between the light source and the measuring position in the excitation path, so that the light coming from the light source reaches the sample immediately or directly.

[0019] The measurement position is located in a sample plane. The measurement position is the position in which a sample is positioned (during a measurement) or in which a sample can be positioned for a measurement. During measurement, the sample is held in a microtiter plate well with a transparent bottom.

[0020] The excitation light enters the sample or sample plane from one light entrance side. Light exits from the opposite light exit side after interacting with the sample. The device has a first detector, which is a light-sensitive or photosensitive detector.

[0021] The excitation light usually passes through the sample as a parallel beam or convergently with a very small beam angle to the optical axis of the excitation path, whereby the cross-section of the beam should be as small as possible, in any case smaller than the sample in the well.

[0022] The excitation light interacts with the sample as it passes through it. The area in which the interaction between the excitation light and the sample material takes place is referred to here as the measurement volume. The measurement volume is thus the intersection volume between the excitation light and the sample. The light resulting from the interaction of the excitation light with the sample is referred to in this application as "emission light" or "emitted light." The emission light thus originates from the measurement volume irradiated with the excitation light.

[0023] Depending on the type of interaction, different types of emission light can be distinguished.

[0024] If no interaction occurs, or if the interaction consists solely in the absorption of part of the excitation light, the resulting emission light propagates in the same direction or at the same beam angles as the excitation light. Thus, no angular change of the light rays occurs as they pass through the sample.

[0025] There are also interactions that cause the resulting emission light to propagate in a different direction than the excitation light that caused it. Such angle-changing interactions include scattering and the generation of fluorescent light.

[0026] In this application, "scattering" of light generally refers to the deflection of light through interaction with another local object (scattering center). "Scattering" is the angular change of light in a (theoretically infinite) variety of directions with an intensity characteristic that can range, for example, from "lobe-shaped" to "isotropic." Thus, the emission light resulting from the "scattering" interaction has a completely different propagation characteristic than the excitation light.

[0027] Fluorescence is known as the spontaneous emission of light shortly after a material is excited by excitation light. The emitted fluorescent light is usually shifted to the longer wavelength range of the light spectrum compared to the excitation light (Stokes shift). Fluorescence results in a different variant of the emitted light. The fluorescent light propagates more or less isotropically. Thus, a predominant portion of the fluorescent light propagates in a direction different from that of the excitation light.

[0028] The emission light leaving the sample towards the light exit side can be divided into two types depending on its propagation direction.

[0029] First-type emission light, also called first emission light, is emission light whose propagation direction is angularly altered compared to the propagation direction of the excitation light, i.e., it propagates at different angles or directions than excitation light that has passed through the sample without any angle-changing interaction. First emission light can also be referred to as "angle-altered emission light" and can include, for example, scattered light resulting from scattering and / or fluorescent light generated due to fluorescence.

[0030] Second-type emission light, also called second emission light, is emission light whose propagation direction is unchanged or essentially unchanged from the propagation direction of the excitation light. Second emission light thus propagates essentially the same way the excitation light would propagate without interaction with the sample and can therefore also be referred to as "non-angle-altered emission light."

[0031] The device is designed to distinguish between first and second emission light. For this purpose, the device has an angle-selective emission path that leads from the light exit side of the measuring position, opposite the light entrance side, to the first detector. The angle-selective emission path is configured such that at least a portion of the first emission light (angle-changed emission light) falls on a sensitive surface of the first detector, while second emission light does not reach the first detector. The first detector can receive first emission light, e.g., scattered light and / or fluorescent light, and generate detector signals therefrom. Detector signals from the first detector are also referred to below as first signals.

[0032] The term angle-selective emission path refers to an emission path that selectively guides the emitted light (emission light) either to the first detector or in another direction, depending on its propagation direction. The propagation direction is therefore the selection criterion. The emission path is "angle-selective" in the sense that the light passing through without changing the angle (second emission light) is geometrically separated from the light that changes its propagation direction upon interaction with the sample, particularly through scattering, but possibly also through other effects, e.g., through excitation of fluorescence.

[0033] A concave mirror with a mirror surface rotationally symmetrical to the optical axis of the emission path (and the concave mirror) is arranged in the emission path for collecting first emission light (e.g., scattered light and / or fluorescent light). The concave mirror has a light entrance opening on a light entrance side facing the sample plane and widens to a light exit opening optically facing the detector. The light exit opening is thus larger than the light entrance opening. The mirror surface is preferably continuously concavely curved, but can optionally also be approximated by successive conical sections of different cone angles or by a combination of conical surfaces and flat slices of spherical surfaces.

[0034] When using a concave mirror, light enters the reflection chamber defined by the mirror surface of the concave mirror via its light entrance opening, which widens from the light entrance side to the opposite light exit side. This arrangement ensures that divergent first emission light, such as scattered light, which enters the reflection chamber at relatively large angles with respect to the optical axis of the concave mirror, strikes the mirror surface of the concave mirror and is collected and emitted through the light exit opening by simple directed (non-scattering) reflection at the mirror surface. Light components that travel at smaller angles to the optical axis can, if necessary, exit directly through the light exit opening, i.e. without reflection at the mirror surface.

[0035] One advantage of using a concave mirror, particularly a parabolic mirror, to collect the light emerging from the sample at the light exit side is that the collection effect can be achieved with a relatively compact size of the optical element used for collection (the concave mirror). In other words, a favorable ratio can be achieved between the largest possible detection angles and the size of the optical element perpendicular to the optical axis.

[0036] The device can be configured such that the emission path is designed such that first emission light, e.g. scattered light and / or fluorescent light, reaches the first detector directly and / or via a single reflection at the concave mirror.

[0037] In this application, first emission light that reaches the first detector directly is understood to mean first emission light (e.g. scattered light) that reaches the sensitive surface of the detector from the sample without interaction with a curved optical surface, i.e. without refraction at a lens surface or reflection at a concave mirror surface of a concave mirror.

[0038] The emission path is designed so that, in addition to the first emission light reaching the first detector directly, the first emission light can also reach the first detector via a single reflection from the concave mirror. With the help of the concave mirror, the first emission light emerging divergently from the sample and striking the mirror surface of the concave mirror can be collected toward downstream components. The concave mirror thus serves as a reflective, collecting optical element downstream of the measurement position. A portion of the divergent radiation emerging forward from the measurement position can be collected by the concave mirror.

[0039] It is possible that a portion of the generated first emission light reaches the first detector directly, while another portion reaches the first detector indirectly via reflection from the concavely curved mirror surface of the concave mirror. It is also possible that all deflected light entering the emission path reaches the first detector only via reflection from the concave mirror.

[0040] In any case, it is ensured that the first emission light in the emission path, in particular, for example, scattered light and / or fluorescent light, can reach the first detector with at most a minimal loss of intensity from the cause of the change in direction, i.e., from the interaction in the measurement volume in the sample. Since reflection layers for mirrors in the wavelength range of interest of the device can be designed so that the reflectance is largely independent of the wavelength of the light, it can be ensured that, if necessary, the measured values ​​for the intensity of the first emission light (e.g., the scattered light intensity or the intensity of fluorescent light) do not unintentionally vary with the wavelength of the excitation light.

[0041] In preferred embodiments, the concave mirror is designed as a parabolic mirror. A parabolic mirror is a concave mirror whose mirror surface can be described as a section of a paraboloid of revolution (see, for example, EP 2 072 998 B1). A reflection chamber is formed between the light entrance opening and the light exit opening.

[0042] The concave mirror, especially the parabolic mirror, is preferably the only imaging reflective optical element in the emission path. Additionally, a flat deflecting mirror can be connected downstream, which can be used, for example, to fold the beam path or a partial beam path for space reasons.

[0043] If the concave mirror is designed as a parabolic mirror or an approximately parabolic mirror, the concave mirror can be designed and positioned so that one focal point of the parabolic mirror is within the sample volume, i.e., above the transparent base of the well. The deflected light reflected by the concave mirror's surface, such as scattered light, then exits the light exit opening more or less perfectly parallelized, which, among other things, allows for more precise subsequent spectral filtering. Divergent light emerging from the sample at the light exit side can thus be collected, and in particular, largely parallelized.

[0044] By using a concave mirror, in particular a parabolic mirror, in the emission path optically behind the light exit side of the sample plane, refractive optical elements for collecting the divergently deflected light emerging from the sample can be dispensed with.

[0045] Accordingly, in some embodiments there is no refractive optical element, in particular no converging lens, in the emission path between the measuring position and the first detector.

[0046] However, in other embodiments, a converging lens may be provided in the emission path behind the concave mirror, for example if it is desired to use a first detector with a relatively small sensitive area and to concentrate light in the emission path onto this relatively small area.

[0047] The arrangement can be used to detect emission light emerging from the sample in a divergent forward direction, i.e. on the light exit side opposite the light entrance side. The detection angle is usually less than 100°. This maximum value can be achieved, for example, with light that is deflected within the sample immediately close to the bottom of the well. If one refers to sample volumes in the region of the focal point of the parabolic mirror, detection angles in the range of 50° to 60° can be achieved. Assuming that the first emission light is essentially concentrated within a cone with a cone tip on the optical axis, the detection angle corresponds to the interior angle measured at the cone tip or twice the aperture angle of the cone.In preferred embodiments, average detection angles of up to 60° or up to 70° for forward radiation can be realized.

[0048] According to the claimed invention, the concave mirror is arranged below the sample plane such that the light entrance opening is located above the light exit opening of the concave mirror. This allows measurements to be performed in which the excitation light is irradiated from above, i.e., from the open side of a well, and exits downward through the transparent bottom. This arrangement has the advantage, among other things, that no excitation light reflected from the surface of the sample can enter the emission path and thus impair the measurement.

[0049] In variants not covered by the claimed invention, it is possible to irradiate the samples from bottom to top. In this case, the concave mirror, in particular the parabolic mirror, would be positioned above the sample plane, with the smaller light entrance opening located below the larger light exit opening, and the excitation light would be irradiated from below, so that it first passes through the transparent bottom before entering the sample and entering the emission path through the upper opening of the well.

[0050] A variant not belonging to the claimed invention also provides that the concave mirror, in particular the parabolic mirror, is arranged above the sample plane such that the smaller light entrance opening is located below the larger light exit opening above the well. The excitation light is irradiated from above, so that it first passes through the sample and then the transparent base, entering the emission path below the sample plane. In addition to the concave mirror, a plane mirror is provided that can be inserted into the emission path below the measuring position and has a passage opening for the passage of second emission light.If this plane mirror, also known as a pinhole mirror, is inserted into the emission path in an operating position directly below the sample plane such that the mirror surface is orthogonal to the optical axis of the emission path, the second emission light, which does not change its angle, will pass through the aperture, and the first emission light, which changes its angle, will be reflected back from the plane mirror's mirror surface through the transparent base and the sample toward the light entrance of the concave mirror. The emission path then leads from the light exit side below the sample plane, through the sample again, and up through the concave mirror to the first detector.The first detector can then receive both second emission light emitted directly in the backward direction and second emission light emitted initially in the forward direction, which, after reflection at the plane mirror, passes again backward through the sample in the direction of the first detector.

[0051] In some embodiments, the first detector has a sensitive area that is larger than the light exit opening of the concave mirror. When positioned close to the light exit opening, this ensures that all of the light reflected by the concave mirror's mirror surface reaches the detector surface, as does a larger portion of the light emerging directly from the concave mirror (without reflection by the concave mirror's mirror surface). Furthermore, it is possible to ensure that first emission light, which is reflected by the parabolic mirror but does not emerge from the parabolic mirror in parallel because it does not originate from its focal point, can still be detected by the detector at an oblique angle.

[0052] As a first detector, a photomultiplier tube or a semiconductor-based detector, such as a silicon photomultiplier (SiPM) with a correspondingly large sensitive area, can be used.

[0053] In some embodiments, it is provided that the light which has passed through the sample without a change in direction, in particular without scattering (second emission light), strikes a light absorber or a light trap or is directed by suitable deflection into non-critical areas of the device in such a way that interference with the measurements on the light deflected by scattering is avoided.

[0054] Other embodiments are characterized by a second detector for detecting light that has passed through the sample without changing direction or angle (e.g., due to scattering and / or fluorescence) (second emission light). Using the photosensitive second detector, it is possible to generate second signals that represent the intensity of the light that has passed through the sample without changing angle after passing through the sample. These second signals can be used for further measurement methods and / or to correct measurement results from the measurement methods performed using the first detector. Some measurement methods that can be performed using the second detector are explained in more detail elsewhere.

[0055] Although the sensitive surface of the second detector can be oriented perpendicular to the optical axis of the emission path at the location of the second detector, preferred embodiments provide for the second detector to have a sensitive surface oriented obliquely to the optical axis of the emission path at the location of the second detector. This ensures that the light, possibly incident at relatively high intensity and passing through the sample without changing its angle, is not reflected back into the emission path, where it could potentially cause interference, but is coupled out into non-critical regions outside the emission path.

[0056] There are different ways to design the emission path so that a first detector and a second detector can be used simultaneously.

[0057] In some embodiments, the emission path has a deflection mirror arranged on the optical axis of the concave mirror for deflecting the second emission light toward the second detector. This allows the second detector to be arranged laterally with respect to the main axis of the device, which runs along the passage direction from the excitation path to the emission path. This allows the installation space immediately adjacent to the light exit opening of the concave mirror to be used for other components, if necessary.

[0058] Although the deflecting mirror can be arranged outside the reflection chamber of the concave mirror, it is preferably located inside the reflection chamber, i.e., in the area between the light entrance and exit openings of the concave mirror, which, among other things, saves space along the optical axis. In this case, the concave mirror can have a lateral light exit opening in the mirror surface or in the mirror substrate.

[0059] Preferably, the deflecting mirror is arranged in an opaque angled tube having a first tube section for receiving the light that passes through the sample without changing the angle (second emission light) and a second tube section arranged at an angle to the first tube section for directing the light reflected by the deflecting mirror toward the second detector. Stray light, which may occur during reflection from the deflecting mirror, can be kept away from the detectors in this way. The angled tube can simultaneously serve as a mounting unit for the deflecting mirror in the concave mirror. Light trap structures can be provided on the inner wall of the angled tube to further reduce the level of stray light.

[0060] Alternative designs are also possible. For example, the emission path can be designed such that the second emission light passes through without deflection to an absorber, a light trap, or a second detector, while the first emission light, whose propagation direction has been altered by scattering and / or other means within the sample, is deflected towards the first detector via a flat deflection mirror. This arrangement can save space on the light exit side of the sample plane. For example, a photomultiplier with a relatively large sensitive area can be arranged laterally next to the main axis of the device in order to detect scattered light after reflection from the flat deflection mirror.The directly transmitted light can be captured in a tube located in the optical axis with light traps near the light entrance opening of the concave mirror and then pass through an opening in the flat deflecting mirror to the second detector.

[0061] The device is suitable for nephelometric measurements because the emission path is angle-selective in the sense that, for example, scattered light emerging from the sample volume at an angle to the light passing through without a change in angle can be geometrically separated from the sample volume, specifically guided to the first detector, and detected there. The inventors have recognized that the optical arrangement is therefore fundamentally also suitable for use for fluorescence measurements in the forward direction. This is due to the fact that in fluorescence measurements the sample volume excited by excitation light essentially emits isotropically fluorescent light, so that a portion is also emitted in the forward direction. This can be viewed as a change in angle or direction with respect to the excitation light, since a significant portion of the fluorescent light propagates in a different direction than the generating excitation light.The optical arrangement makes it possible to guide a significant portion of the fluorescent light possibly emerging from a sample volume in the forward direction directly and / or via reflection from the concave mirror surface to the first detector and detect it there. The resulting initial signals can thus also represent fluorescent light intensity.

[0062] In order to be able to use the device for fluorescence measurements in the forward direction, some embodiments provide an emission filter device that can be switched between several operating positions for selectively spectrally filtering first emission light before it enters the first detector, wherein the emission filter device preferably has a filter-free operating position in which scattered light (i.e. light deflected due to scattering effects) can pass through without filtering. In this operating position, filter-free nephelometry can be operated. In another operating position with spectral filtering, the emission filter device can be designed as a wavelength-selective device for blocking light at the excitation wavelength (i.e. light with the wavelength of the excitation light) and for allowing the passage of fluorescent light that is shifted to longer wavelengths compared to the excitation light.

[0063] In some embodiments, the device has a concave mirror, preferably designed as a parabolic mirror, on the light entry side of the sample plane. The concave mirror has a light entry opening on its side facing the sample and widens to form a light exit opening with a larger diameter. This allows fluorescence to be measured in the backward direction, for example with the aid of a third detector optically arranged downstream of this concave mirror. If detection is possible on both the light entry side and the light exit side, fluorescence can be measured simultaneously at two different wavelengths, for example. The concave mirror, in particular a parabolic mirror, provided for collecting divergent backward radiation can be provided in addition to a concave mirror, in particular a parabolic mirror, arranged on the light exit side, for collecting divergent forward radiation.

[0064] There are various options for the design of the light source. In some embodiments, a light source arrangement has a single laser light source, which can be, for example, a laser (e.g., a laser diode). A laser light source has the advantage of immediately generating highly parallelized excitation light, which can then be directed through the sample volume in a laser beam of defined beam width, for example, essentially perpendicular to the sample plane. Relatively small-area devices may be sufficient to capture the light that has passed through the sample without scattering or to redirect it to a second detector.

[0065] The light source arrangement can also comprise multiple light sources emitting different wavelengths, as well as a light source selection device for selecting one of the light sources. For example, a group of light-emitting diodes (LEDs) emitting different wavelengths can be used, each of which can be positioned at the desired light source position in the excitation path.

[0066] Alternatively, it is also possible for the light source to be a polychromatic light source, and for an adjustable wavelength-selective device to be arranged in the excitation path between the light source and the measurement position, with which a narrower spectral range can be selected from the broad spectrum of the polychromatic light source. The adjustable wavelength-selective device can, for example, be a dispersive monochromator (e.g., a single monochromator or double monochromator) or a filter changing device with several filters of different transmission wavelengths, whereby the filter changing device can be designed, for example, as a filter wheel or filter slider. Tunable filter monochromators with linearly variable filters can also be used as an adjustable wavelength-selective device.

[0067] If devices are provided for continuously or stepwise adjustment of the wavelength of the excitation light before it enters the sample, the wavelength dependence of the scattered light signal or of other signals detected with the device can be determined if required. For example, for a specific sample type, the spectral dependence of the scattered light signal determined in a nephelometric measurement can be determined in order to obtain information about the sample properties. Scattering can also be measured as a function of wavelength in order to find optimal parameters for a series of measurements. Since the scattered light intensity is strongly dependent on the wavelength, it is also possible to obtain information about the size distribution of the scattering particles within the sample through wavelength-selective scattered light measurement. It is also possible that different adapted wavelengths are optimal depending on the sample type.These can also be selected by adjusting the wavelength of the excitation light.

[0068] There are various options for selecting a suitable first detector. In some embodiments, a photomultiplier tube is provided as the first detector, which can have a relatively large sensitive area and high sensitivity. In particular, if the first detector has a relatively large sensitive area, it can be arranged in the emission path without the interposition of collecting optical elements, for example, directly behind a concave mirror. However, the use of other detector types is not excluded. For example, a semiconductor-based detector, such as a simple photodiode or an avalanche diode, can be used as the first detector. If necessary, an array with a large number of Si-PMTs can also be used as the first detector.

[0069] In some embodiments, a collecting lens optic is arranged in the emission path between the concave mirror and the detector, which makes it possible to concentrate the scattered light onto a relatively small-area first detector.

[0070] For a second detector, if present, the same options are essentially available. However, since the excitation light passing through the sample without scattering is usually significantly more intense than the scattered light (or fluorescence light), a relatively simple detector, such as a photodiode, may suffice.

[0071] The device can be designed as a pure nephelometer, i.e., a device intended exclusively for nephelometric measurements. Only scattered light is evaluated.

[0072] The device can also be designed as a pure fluorometer, i.e., a device intended exclusively for fluorescence measurement. In this case, only fluorescent light is evaluated.

[0073] Preferably, the device is configured to allow two or more different operating modes from which a user can select. It may therefore be a device for a multi-technology device.

[0074] For this purpose, an evaluation device for evaluating first signals of the first detector and / or second signals of the second detector can be provided, wherein the evaluation device is configured to carry out different evaluation methods by processing the first signals and / or the second signals, wherein different evaluation methods can be selected.

[0075] In particular, two or more of the following operating modes can be optionally provided.

[0076] A first operating mode in which initial signals (i.e., signals from the first detector) are processed to determine a first measured value representing the intensity of the scattered light. This mode can also be referred to as "nephelometry mode."

[0077] A second operating mode, in which a wavelength-selective device is arranged between the measuring position and the first detector, capable of largely blocking light with the wavelength of the excitation light (excitation wavelength) and allowing the passage of fluorescent light that is shifted to longer wavelengths relative to the excitation light. In this operating mode, first signals can be processed to determine a second measured variable representing the intensity of fluorescent light. Since this fluorescent light generally leaves the sample in a forward direction relative to the direction of incidence of the excitation light, this second operating mode can also be referred to as "forward fluorescence mode."Compared to the conventional measurement of fluorescence in the backward direction—i.e., in the direction from which the excitation light is radiated—measuring fluorescence in the forward direction has the advantage, among other things, that excitation light reflected from the sample surface cannot enter the emission channel. With a concave mirror on the light exit side, i.e., below the sample, the excitation light can be coupled out after passing through the sample, for example, in an angled tube.

[0078] A third operating mode, in which second signals are processed to determine a third measured value, which represents the intensity of the light that passed through the sample without scattering. This third operating mode can also be referred to as "light loss mode." The light loss mode can essentially detect all intensity losses that occur when a portion of the excitation light does not pass through the sample without deflection, but rather undergoes deflection or a change in direction upon passing through the sample. This change in direction can have several causes. If scattering centers are present in the sample, for example, in the form of scattering particles, molecules, or the like, the light loss due to scattering can be detected in the light loss mode.If excitation light within the sample generates fluorescence, in which fluorescent light is emitted isotropically in all directions, this also leads to a loss of intensity due to fluorescence, which causes the light arriving at the second detector to have a lower intensity than the light entering the sample. Finally, the third operating mode can also detect intensity losses caused by absorption within the sample. Such light losses can also occur, for example, in samples that do not have scattering centers, i.e., do not scatter light, and therefore do not produce fluorescence. In this case, the third operating mode can also be referred to as absorption mode.

[0079] A particular advantage of embodiments with a first detector and a second detector is that the device can be configured such that, in a fourth operating mode, first signals (i.e., signals from the first detector) and second signals (i.e., signals from the second detector) are processed together. This mode can be referred to as a combination mode.

[0080] The combined processing can be performed independently of each other, so that, for example, a first measurement value representing the scattered light intensity can be determined based on the first signals (nephelometry mode), while a third measurement value representing the light loss can be determined from the second signals. Thus, two different measurement values ​​can be determined simultaneously.

[0081] Particular advantages arise when, in a variant of the fourth operating mode, first signals and second signals are processed together in such a way that the measured variable determined on the basis of the second signals is corrected using the first signals and / or the measured variable determined on the basis of the first signals is corrected using the second signals.

[0082] For example, it is possible to correct an absorption measurement or light loss measurement using a nephelometric measurement. To do so, the second signals can be used to determine the extent to which light loss has occurred in the excitation light. At the same time, the first signals can be used to determine what proportion of the intensity loss results from scattering, i.e., deflection of the excitation light from its original propagation direction. Assuming that only absorption and scattering are considered causal processes within the sample, the correction described can be used to determine the extent to which light loss actually occurs due to absorption in the sample.Such measurement results are more precise and meaningful than those of conventional absorption measurements because they take into account that intensity loss within a sample can be caused not only by absorption but also by scattering. This ability to correct one measurement parameter with another measured using a different detector can significantly contribute to a better understanding of processes within the sample.

[0083] When measuring the fluorescence of cells that typically settle at the bottom of the sample well, a nephelometry measurement could be used to pre-test the extent to which cells or cell clumps have actually formed at the bottom. Their scattering behavior can provide information about cell size and number, and an independent subsequent fluorescence measurement can then assess the actual binding ability of fluorescein or another fluorescent dye to the cell complex. This is based, among other things, on the knowledge that dead cells do not bind, but scatter. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Further advantages and aspects of the invention emerge from the claims and from the following description of preferred embodiments of the invention, which are explained below with reference to the figures. Fig. 1 schematically shows a first embodiment of a device suitable, among other things, for nephelometric measurements for determining the optical properties of samples, having a parabolic mirror on the light exit side of the sample plane; Fig. 2 schematically shows an embodiment with two parabolic mirrors; Fig. 3 schematically shows an embodiment with a laser as the light source; Fig. 4 schematically shows an embodiment with LED light sources and a horizontally arranged photomultiplier; Fig. 5 schematically shows an embodiment with a converging lens behind a parabolic mirror; Fig. 6 schematically shows an embodiment for a luminescence measurement; Fig. 7 schematically shows a variant not belonging to the claimed invention for a top / top nephelometry measurement; and Fig. 8 schematically shows a variant not belonging to the claimed invention of a device in which scattered light falls directly onto a photomultiplier without being collected by a converging lens. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0085] In Fig. 1 An embodiment of a device 100 for determining the optical properties of samples is shown schematically. The device can be operated in several different modes for different measurement methods (including nephehlometry, fluorescence measurement, absorption measurement, etc.). Fig. 1 shows the device 100 in a configuration in which the optical and other components are adjusted so that a nephelometric measurement can be performed in a first operating mode.

[0086] In the description, relative positions of optical elements are described using prepositions such as "between," "before," "behind," or the like. These prepositions, or expressions such as "downstream," refer to positions along an optical path between a light source, through a measurement position, and finally to a detector. Terms such as "first," "second," "third," etc., serve solely to uniquely identify the corresponding elements and do not generally describe the order in which elements are arranged in an optical path.

[0087] The samples to be measured are filled into sample containers, which in the example are formed by wells in a microtiter plate or microplate (MPL). The microplate is held by a holding plate (not shown), which can be moved by a motor parallel to a horizontal sample plane (PE) in two mutually orthogonal horizontal directions. The sample containers shown each have a bottom (B) transparent to visible light. They are therefore suitable, among other things, for nephehlometric measurements and / or absorption measurements and / or fluorescence measurements in the forward direction.

[0088] In the example, the microplate MPL is arranged such that a sample container PB containing a sample P is located in a measuring position MP, where a nephelometric measurement can be performed. A vertical optical main axis HA of the device runs perpendicular to the sample plane PE through the measuring position MP.

[0089] The device 100 has a primary light source LQ in the form of a xenon lamp. Depending on the application, this can be a flash lamp or a continuous-wave lamp. Alternatively, an incandescent lamp (e.g., a halogen lamp for continuous-wave operation) or a white-light LED can be used as the light source. The polychromatic light source LQ has a broad emission spectrum in the visible spectral range ("white light").

[0090] An excitation path AP serves to transmit light from the light source LQ as excitation light AL to the measurement position MP, where the sample P is located. A measurement volume of the sample P is illuminated by the excitation light. The measurement volume is thus the intersection volume between the excitation light and the sample.

[0091] In this example, the excitation path comprises a wavelength-selective device (WLS) in the form of a dispersive double monochromator (MO), followed by a converging lens (L1). The side of the sample plane from which the excitation light is incident is referred to here as the light entrance side (LES).

[0092] An emission path EP leads below the sample plane PE from the measurement position MP to a first detector DET1. The emission path EP contains devices for transmitting light influenced by the sample from the sample to the first detector DET1. In this example, the first detector DET1 is a photomultiplier with a relatively large photosensitive area SF. The first detector DET1 is connected to the control device SE, which also contains an evaluation unit for evaluating the electrical first signals generated by the first detector DET1.

[0093] In addition to the first detector DET1, the device 100 comprises a photosensitive second detector DET2, which is also connected to the evaluation unit of the control device SE. The second detector DET2 is formed by a photodiode whose flat sensitive surface is aligned obliquely to the optical axis AX at the location of the second detector, so that light reflected by the sensitive surface is not reflected back into the emission path.

[0094] The emission path EP is designed as an angle-selective emission path. This means that light rays emerging from the irradiated measurement volume in a forward-downward direction are guided along different sub-paths depending on their propagation direction or their angle relative to the direction of the excitation light passing through without changing the angle. This division is achieved by geometric beam splitting.

[0095] For further understanding and terminology, the following should be noted: The excitation light interacts with the sample as it passes through the sample within the measurement volume. The light resulting from the interaction of the excitation light with the sample is referred to in this application as "emission light" or "emitted light."

[0096] Depending on the type of interaction, different types of emission light can be distinguished. The interaction can be such that the resulting emission light propagates in the same direction or at the same beam angles as the excitation light. Thus, there is no change in the angle of the light rays as they pass through the sample. This type of emission light is also referred to here as "non-angle-changed emission light" or "second emission light."

[0097] There are also interactions that cause the resulting emission light to propagate in a different direction than the excitation light that caused it. Such angle-changing interactions include scattering and the generation of fluorescent light. Emission light whose propagation direction is angularly altered compared to the excitation light—that is, which propagates at different angles or directions than excitation light that passed through the sample without any angle-changing interaction—is also referred to here as "first emission light" or "angle-altered emission light."

[0098] For the purpose of geometric separation of first and second emission light, a concave mirror PS in the form of a downwardly opening parabolic mirror PS and a flat deflection mirror US inclined by 45° relative to the main axis HA, which is arranged on this main axis, are provided in the emission path EP.

[0099] The concave mirror, designed as a parabolic mirror, has a mirror surface SPF that is rotationally symmetrical to its optical axis for collecting radiation that emerges divergently downwards from the irradiated sample volume in the forward direction. The optical axis of the parabolic mirror coincides with the vertical main axis HA of the device. The rotationally symmetric mirror surface SPF is a section of a paraboloid of revolution and extends from a circular upper light entrance opening LEO facing the sample plane PE to the circular light exit opening LAO further down, which is larger in diameter. The focal point of the paraboloid of revolution defining the mirror surface SPF lies above the level of the transparent base B of the microtiter plate in the region of the sample plane, i.e. during measurement inside the sample sub-volume irradiated by the excitation light AL.

[0100] In other words, there is a section of a concave mirror arranged rotationally symmetrically to the optical axis of the emission path EP. Its collecting area (focal point) lies within the sample measurement volume and its aperture points toward the further course of the emission path EP. However, its apex is sufficiently separated from the concave mirror surface to prevent collision with the sample and the sample support. The remaining ring collects the emission light over a large solid angle range and directs it in a direction approximately parallel to the optical axis of the emission path as it continues.

[0101] The optical devices of the excitation path AP are designed so that the excitation light is focused essentially in the area of ​​the mirror surface of the deflection mirror US. Light that passes through the irradiated sample volume without changing direction (second emission light) strikes the inclined, flat mirror surface of the deflection mirror US and is reflected through a lateral opening in the mirror body of the concave mirror onto the sensitive surface of the second detector DET2. In order to couple out a section of the incident radiation that is rotationally symmetrical to the main axis, the mirror surface of the deflection mirror is not circular, but elliptical, so that it is circular in horizontal projection (onto a projection surface parallel to the sample plane PE).

[0102] A large portion of the light which changes its propagation direction within the sample, for example due to scattering, and which emerges from the sample divergently downwards at an angle to the main axis HA (ie first emission light), reaches the sensitive surface SF of the first detector DET1.

[0103] This light, deflected, for example, by scattering (second emission light in the form of scattered light), can be divided into two components. Those rays whose beam angles lie between a first critical angle W1 and a second critical angle W2 are reflected once by the concave-parabolically curved mirror surface of the concave mirror and then travel more or less parallel to the main axis of the device through the light exit opening LAO to the sensitive surface SF of the first detector DET1. Those rays whose beam angles (measured relative to the optical axis of the concave mirror) are smaller than W2 but can pass the deflecting mirror US travel directly, i.e., without interaction with an optical surface that changes the beam direction, to the sensitive surface SF of the first detector DET1.

[0104] The first detector DET1 generates first signals S1 based on the light intensity incident on the sensitive surface SF. These signals represent, for example, the total intensity of the light incident on the sensitive surface SF. The same applies to the second detector DET2, whose second signals S2 represent the intensity of the light passing through the sample without deflection.

[0105] In the embodiment of Fig. 1 An emission filter device EF, switchable between several operating positions, is inserted between the light exit opening LAO of the concave mirror PS and the first detector DET1 for selectively filtering (first) emission light before it enters the first detector DET1. The emission filter device can, for example, comprise a filter slide with several receptacles arranged in a row for different emission filters or a filter wheel. The emission filter device has a filter-free operating position, which is characterized in that all light exiting downwards from the light exit opening LAO of the concave mirror reaches the first detector DET1 directly without optical filtering. This configuration is shown in Fig. 1 shown and can be selected, for example, if the device is to be used for nephelometric measurements. The scattered light intensity of the light passing through the sample can be determined from the first signals S1 of the first detector DET1.

[0106] The device 100 is also suitable for performing fluorescence measurements in the forward direction. This utilizes the fact that fluorescent light generated in a sample irradiated by excitation light AL radiates isotropically, i.e., in all spatial directions. The fluorescent light emitted in the forward-directed solid angle that can be detected by the concave mirror PS can be detected by the first detector DET1.

[0107] Since fluorescent light has a wavelength that is shifted to longer wavelengths compared to the wavelength of the excitation light AL (excitation wavelength), the emission filter device EF can be adjusted so that an emission filter EFT is positioned between the concave mirror and the first detector. This filter efficiently blocks wavelength-selective light of the excitation wavelength, but allows the fluorescent light shifted to longer wavelengths to pass more or less unhindered to the first detector. This allows the fluorescence intensities of the fluorescent light leaving the sample P in the forward direction to be determined from the first signals S1.

[0108] Due to the fact that the wavelength of the excitation light in the device 100 can be continuously adjusted using the tunable monochromator, it is also possible to measure the wavelength dependence of the scattered light intensity on samples with the device 100.

[0109] The second signals S2 generated by the second detector DET2 can be used, for example, for a turbidimetric measurement (in turbidimetry mode). In this turbidity measurement, the portion of the primary radiation (excitation light) that is scattered is determined from the difference between the incident and transmitted light. In the example case, the intensity of the excitation light before passing through the sample is thus calculated using the second signal S2. The intensity of the excitation light can be determined, for example, by a measurement without a sample (with an empty sample container) using the second detector. Alternatively, a measurement is possible using a reference diode or another detector, which in some embodiments is provided for intensity control or intensity stabilization of the excitation light coming from the light source. Thus, in addition to the first and second detectors, at least one further detector can be provided.If this is used to measure the intensity of the excitation light, the first and second detectors can be used for other measurements at the same time.

[0110] It can be seen that, with the aid of device 100 and the use of suitable emission filtering, fluorescence can be measured in the forward direction. For several reasons, fluorescence measurement in the forward direction can be advantageous over the conventionally used top-top arrangement, in which the excitation light comes from above and the fluorescence radiation generated in the sample enters an emission path backwards and upwards. In these conventional designs, the excitation path and emission path are on the same side of the sample plane. However, with the top-top arrangement, it should be noted that in addition to the fluorescence radiation, scattered light generated at the well edge and in the sample can also be emitted upwards into the emission path.Furthermore, intense excitation light reflected from the sample surface can enter the emission path. This light is insufficiently blocked by the emission filter and can generate a false signal that is not due to long-wavelength fluorescence light. To eliminate interference, these components should be filtered before entering the detector. However, experience has shown that achieving sufficiently effective blocking is difficult.

[0111] In the device presented here, however, fluorescent light emitted in the forward direction, i.e. in the general direction of the excitation beam, is measured. Fluorescence emission is essentially isotropic, but fluorescence is measured at the beam angle that reaches the downwards emission path EP. The suppression of the excitation radiation (or the second emission light) required for sensitive measurement is achieved in this arrangement by two measures: firstly, by geometric beam splitting, in which light with the same propagation direction as the excitation light (i.e., second emission light) is blocked out and only emission light with other propagation directions is directed to the detector, and secondly, by filtering (emission filtering) upstream of the first detector in such a way that essentially only the fluorescent radiation of interest is transmitted.The novel arrangement offers the potential for the solid angle for the fluorescent radiation emerging downwards from the well to be significantly larger than for upward emission, especially when the well is only slightly filled. Finally, there may be the advantageous effect that the excitation radiation is at least partially absorbed by the transparent bottom, while the longer-wavelength emission radiation is not absorbed or is absorbed to a lesser extent. For these reasons, among others, the forward fluorescence measurement possible with the device appears superior to the conventional top-to-top measurement. In addition, it offers the possibility of combining other operating modes, such as nephelometry and / or turbidimetry, with the fluorometry results.

[0112] Further advantages of this arrangement are that, unlike top-to-top measurements, fluorescence measurements in the forward direction are not subject to interference from the sample surface (a lens-shaped surface, due to surface tension, emits fluorescent light and reflects excitation light at different angles than a flat liquid surface). The light entrance of the parabolic mirror is always facing a flat surface, namely the bottom of the well. Even varying sample fill levels thus produce less variance in the measurement result than with the top-to-top arrangement.

[0113] In the following description of other embodiments, for reasons of clarity, the same reference numerals are used for functionally and / or structurally identical or similar elements as in Fig. 1 .

[0114] The device 200 in Fig. 2 has a very similar structure to the first embodiment in the area of ​​the sample plane PE and below the sample plane in the emission path. One difference is that the deflecting mirror US, which deflects the light that has passed through the sample without a change in direction (second emission light) towards the second detector DET2, is arranged in the knee region of a light-tight angled tube WR, which has a first tube section arranged coaxially to the main axis HA for receiving the second emission light and a second tube section arranged at a 90° angle to the first tube section for guiding the second emission light reflected by the deflecting mirror US towards the second detector DET2. The angled tube WR simultaneously serves as a holder for the deflecting mirror US, which is arranged on the main axis HA. Light trap structures are formed inside the first tube section on the entrance side.

[0115] Furthermore, the device 200 has a further concave mirror PSO in the form of an upper parabolic mirror PSO on the light entrance side of the sample plane PE. This has a small light entrance opening on the lower side facing the sample and widens parabolically upwards to a light exit opening with a larger diameter. Analogous to the concave mirror PS arranged on the light exit side, the further concave mirror PSO serves to collect light that emerges from the sample P in a divergent upward direction. Immediately behind the light exit opening, i.e., above the further concave mirror PSO, a further emission filter device EF1 is arranged for selectively spectrally filtering the light emerging upwards from the further concave mirror.Above this, a third detector DET3 is arranged in the form of a photomultiplier or in the form of several planar silicon PMTs, whose photosensitive area is slightly larger than the upper light exit opening of the concave mirror PSO and the filter following it.

[0116] The excitation path AP leads horizontally from the light source LQ via the wavelength-selective device WLS to another deflection mirror US1 arranged on the main axis HA. This deflection mirror is angled at 45° to the main axis and deflects the excitation light AL, focused toward the sample volume, coaxially to the main axis downwards into the measurement position MP. The other deflection mirror US1 is also housed in an angled tube attached to the concave mirror PSO. Light trapping structures are attached to the exit-side tube section.

[0117] Although the wavelength-selective device WLS can also be formed here, for example, by a dispersive monochromator, in the example case a variant with interchangeable spectral filters is provided, which are arranged in the parallel beam path between an entrance-side lens and an exit-side lens of an overall collecting lens group.

[0118] Using this configuration, in addition to a possible nephelometric measurement or a fluorescence measurement on the forward radiation traveling downwards toward the first detector DET1, the portions of fluorescent light (and / or scattered light) emitted upwards from the sample volume can also be captured and detected using the third detector DET3. The arrangement is therefore suitable for both a bottom fluorescence measurement (in the forward direction) and a top fluorescence measurement, i.e., a measurement in the area above the sample. A scattered light measurement in the backward direction is also possible in principle.

[0119] The arrangement can be described as follows: a multilabel reader section for nephelometry, turbidimetry, absorption, and forward fluorescence is arranged below the sample plane (PE). Above the sample plane, for example, time-resolved fluorescence measurement (TRF), homogeneous time-resolved fluorescence measurement (HTRF), simple fluorescence measurement, or luminescence measurement can be realized.

[0120] A key advantage of this arrangement is that, for example, fluorescence can be measured simultaneously with the third detector DET3 (top detector) and the first detector DET1 (bottom detector) located below, thus obtaining a double signal. The third signals generated by the third detector DET3 can be evaluated together with the first signals and / or the second signals of the second detector DET2 and optimized, for example, by means of mutual correction.

[0121] Dual simultaneous fluorescence measurement allows not only upstream and downstream fluorescence measurements at the same wavelength, but also at different wavelengths if needed. Different fluorescence technologies, such as HTRF or bioluminescence resonance energy transfer (BRET) measurements, can also be used for such dual-filter measurements.

[0122] A variant of the device 200, which is particularly suitable for measuring fluorescence polarization upwards, has a lens arrangement instead of the upper concave mirror PSO, for example with two converging lenses for collecting the divergent fluorescent light emerging upwards.

[0123] The device 300 in Fig. 3 can be constructed in the area of ​​the sample plane PE and below it in a similar or identical manner to the first exemplary embodiment. Here, too, a concave mirror PS is provided below the sample plane, which opens downwards towards the first detector DET1, and a second detector DET2 is provided. One difference from the first exemplary embodiment is that here a laser or a laser diode is used as the light source LQ, which emits a laser beam entering the sample from above coaxially to the main axis HA. The laser light source LQ generates ideally parallel excitation radiation. After passing through the measuring container, this is deflected laterally by an encapsulated angle mirror US and, with the help of the second detector DET2, can be used, for example, for turbidimetry measurements.Of the scattered light generated within the sample, which diverges downwards, i.e., in the forward direction, the laterally directed portion is directed essentially vertically downwards by the mirror surface SPF of the parabolic reflector PS to the first detector DET1, allowing nephelometry to be performed in this arrangement. Scattered radiation with a smaller scattering angle can, if necessary, reach the sensitive surface SF of the first detector DET1 without reflection by the parabolic mirror surface SPF. This ensures that the majority of the scattered radiation entering the light entrance of the parabolic mirror PS actually reaches the first detector DET1. Except for those portions that are covered by the deflection mirror US and its encapsulation.

[0124] At the Fig. 4 In the embodiment of a device 400 shown, light-emitting diodes (LEDs) are used as the light source. A white light LED can be used in conjunction with a downstream wavelength-selective device WLS, analogous to the embodiments of Fig. 1 or 2 In the example of Fig. 4 The light source arrangement consists of four separate, relatively narrow-band color LEDs (LQ1, LQ2, etc.). This also features a wavelength-selective device (WLS) with a converging lens and a downstream replaceable filter (excitation filter). One of the optical advantages is the relatively small luminous surface of the light-emitting diodes. The downstream lens collects the excitation light and sends it through the sample container as a slender, convergent beam.

[0125] In order to save construction height, the variant of Fig. 4 The first detector DET1, designed as a photomultiplier, is arranged horizontally. To ensure that the light emerging downwards from the parabolic mirror PS reaches the sensitive surface SF of the first detector, a flat deflecting mirror US3 is arranged in the emission path immediately behind the concave mirror, inclined at 45° to the main axis HA. The relatively large mirror surface of the deflecting mirror US3, in horizontal projection, is preferably somewhat larger than the light exit opening of the parabolic mirror PS. The light that passes through the sample without changing the angle (second emission light), on the other hand, strikes the second detector DET2 without deflection, which in this design is arranged on the main axis HA. For this purpose, the angled mirror USP3 has a passage opening in the area of ​​the main axis, in which a light-tight tube is located. The tube is provided with light trap structures on the entrance side and carries the second detector DET2 at its light exit located below.

[0126] In the embodiment of the device 500 in Fig. 5 The excitation path AP is folded by 90° using a deflection mirror US4 tilted at 45° to the main axis HA. The light from the white light source LQ, after spectral filtering in the wavelength-selective device WLS and reflection at the deflection mirror US4, hits the detector similar to the embodiment of Fig. 1 from above slightly convergent into the sample, with the focus area of ​​the excitation light beyond the sample volume below the sample plane PE. As in the embodiment of Fig. 1 The concave mirror PS, located below the sample plane, with its paraboloidal mirror surface SPF, serves to collect the radiation that emerges downwards, i.e., in the forward direction, from the sample volume. A smaller portion of this radiation emerging downwards from the sample emerges from the light exit aperture at a relatively small beam angle close to the main axis HA without reflection from the concave mirror.

[0127] Immediately behind the light exit opening LAO of the parabolic mirror PO there is a collecting lens optic LO with a single collecting lens with a light entrance surface convex towards the concave mirror and a light exit surface that is essentially flat downwards. With the help of this collecting lens optic LO between the concave mirror PS and the first detector DET1, the radiation (light) of interest for the measurement, which emerges divergently downwards from the sample, can be collected towards a relatively small-area sensitive area SF of the first detector DET1. Such a solution with light collection in the emission path EP behind the concave mirror can be useful, for example, if a first detector DET1 is to be used for the scattered light measurement, the sensitive area of ​​which is smaller than the light exit opening of the parabolic mirror PS.The first detector DET1 can be, for example, an avalanche diode or a silicon PMT with high sensitivity.

[0128] The embodiment of a device 600 in Fig. 6 is configured for luminescence measurement from below. Since the luminescence measurement does not require excitation light, the corresponding components present in the overall device are Fig. 6 not shown. A light-tight cover plate APL is arranged on top of the microtiter plate MPL. This cover plate preferably has a reflective coating on the side facing the sample in order to reflect the luminescence light emitted isotropically by the sample back towards the detector in order to achieve an even higher luminescence signal. In the middle, a passage opening for reagents is provided in order to be able to introduce reagents into the sample with the help of an injection device PP in order to trigger a chemiluminescence reaction. Below the sample plane PE there is again a concave mirror PS designed as a parabolic mirror, which is raised in the area of ​​its light entrance opening located at the top to just below the level of the transparent base. In order to avoid crosstalk during the luminescence measurements, i.e.To minimize crosstalk with neighboring wells, the upside-down paraboloid mirror can be gently pressed against the bottom of the microplate MPL. The cover plate APL also serves to prevent crosstalk via the top opening of the well. Otherwise, the setup below the sample plane is similar or identical to the first embodiment, so please refer to the description there.

[0129] Here, too, the emission filter device EF is shown in a filter-free position, so that luminescence light strikes the sensitive surface SF of the first detector DET1 unfiltered. Only a small portion of the luminescence light, which passes through essentially parallel to the main axis HA or at a very small beam angle to it downwards, is shaded by the inclined deflection mirror US.

[0130] In Fig. 7 7 shows a device 700 not belonging to the claimed invention, which is suitable, among other things, for performing nephelometry in a top / top / arrangement. In this arrangement, the excitation path AP runs similarly to the embodiment of Fig. 2 starting from the light source LQ horizontally through a wavelength-selective device WLS equipped with lenses via a deflection mirror US1 arranged on the main axis HA from above into the sample, which is located in the measuring position MP.

[0131] A concave mirror in the form of an upper parabolic mirror PSO is arranged at the light entrance side LES of the sample plane PE. This has a light entrance opening on the lower side, facing the sample, and widens parabolically upwards to a larger diameter light exit opening. The upper parabolic mirror serves to collect the first emission light, which emerges from the sample P in an upward divergent direction, i.e., in the reverse direction relative to the direction of incidence of the excitation light. Optically immediately behind the light exit opening, above the parabolic mirror PSO, an emission filter device EF is arranged for selectively spectrally filtering the first emission light emerging upwards from the parabolic mirror. Above this is the first detector DET1 in the form of a photomultiplier.

[0132] In the operating position shown, a plane mirror PLS is located below the sample plane PE and inserted into the emission path EP. This plane mirror can be moved either into the position shown directly below the transparent base of the well or into an adjacent, retracted position outside the emission path. On the side facing the sample plane PE or the sample, the plane mirror PLS has a flat mirror surface that runs orthogonal to the main axis HA, which corresponds to the optical axis of the emission path. The plane mirror PLS has a central aperture for transmitting second emission light, i.e. emission light that, as an extension of the excitation light coming from above, passes through the sample without changing the angle and exits downwards. This second emission light is deflected to the second detector DET2 by a deflection mirror US2 arranged inside an angle tube.

[0133] Those portions of the emission light whose propagation direction differs from the direction of the excitation light (first emission light) are reflected upwards by the mirror surface of the plane mirror PLS and exit from below through the transparent base and the sample toward the first detector DET1. The plane mirror PLS thus serves as a geometric beam splitter in the emission path and ensures that the emission path for the first emission light leads through the concave mirror to the first detector DET1.

[0134] This setup allows nephelometry to be performed by collecting the scattered light emitted by scattering particles (first emission light) using the parabolic mirror PSO and directing it to the first detector DET1 located above it. This portion of the scattered light points backward relative to the direction of the excitation light (i.e., at an angle of more than 90° to 180° to the optical axis) and is referred to as backscattering.

[0135] The arrangement can also be used simultaneously for absorption measurement or turbidimetry by evaluating the second signals generated by the second detector DET2. To ensure that only the emission light attenuated by absorption and / or scattering is detected, the aperture formed by the plane mirror PLS is used below the transparent base. Its aperture is slightly larger than the beam diameter of the second emission light. If, as shown in the illustrations, this aperture is designed as an upward-facing plane mirror with a central bore for the non-angularly altered, continuous second emission light (base mirror), this mirror will reflect the emission light that strikes the mirror surface outside the aperture toward the upper opening of the sample container and toward the first detector.

[0136] It should be noted that the portion of the first emission light which is reflected at the mirror surface of the plane mirror PLS and then reflected back through the sample towards the first detector can be regarded as forward scattering generated at an angle of less than 90° to the optical axis.

[0137] Alternatively, the second detector DET2 can also be positioned directly below the PLS floor mirror, i.e., without a second deflection mirror. Using the same arrangement, fluorescence measurements can also be performed in combination with turbidimetry and / or absorption measurements by inserting excitation filters in the excitation beam path and emission filters or blocking filters in the emission beam path upstream of the first detector DET1.

[0138] In Fig. 8An example of a device 800 not belonging to the claimed invention with a relatively simple construction is shown. This device does not comprise a concave mirror. A laser is used as the light source LQ, whose parallel light beam is irradiated as excitation light from above into the sample parallel to the main axis HA. In this exemplary embodiment, there is no collecting optics for collecting the scattered light emerging downwardly from the sample volume. Rather, the first detector DET1, in the form of a photomultiplier, is moved so close to the underside of the sample plane that all scattered light generated in the sample volume and then emerging downwardly through the transparent bottom directly or immediately strikes the sensitive surface SF of the first detector DET1. The scattered light is therefore directly measured using the detector input arranged near the sample well.

[0139] The geometric separation of excitation light (laser radiation) passing directly through the sample and scattered light is achieved using a reflector REF with a conical reflector surface located on the main axis HA directly in front of the detector window. Due to the conical shape, the excitation radiation reflected by the reflector surface is not directed toward the well located in the measurement position MP, but rather into non-critical areas to the side of this well. This arrangement does not require a second detector or concave mirrors and is primarily intended for nephelometric measurements.

Claims

1. Apparatus for determining optical properties of samples arranged in a matrix arrangement in wells of a microtiter plate (MPL), comprising: a light source arrangement having at least one light source (LQ); an excitation path (AP) for transferring light from the light source as excitation light from a light entrance side (LES) into a measurement position (MP) which lies in a sample plane (PE) and in which is arranged or arrangeable a sample (P) accommodated in a well (PB) of a microtiter plate (MPL), said well being equipped with a transparent base (B), a first detector (DET1); an angle-selective emission path (EP) which leads from a light exit side (LAS) of the measurement position, said light exit side being situated opposite the light entrance side (LES), to the first detector (DET1) and which is embodied in such a way that a portion of first emission light, which has resulted from interaction between excitation light and sample and which is angularly altered relative to the excitation light, is incident on a sensitive surface of the first detector (DET1) and second emission light, which is not angularly altered relative to the excitation light, does not reach the first detector (DET1), wherein there is arranged in the emission path (EP) a concave mirror (PS) having a mirror surface (SPF), which is rotationally symmetrical with respect to an optical axis of the emission path, for collecting first emission light emerging divergently from the sample, wherein the concave mirror has a light entrance opening (LEO) on a light entrance side facing the sample plane and widens towards a light exit opening (LAO) optically facing the first detector (DET1), wherein the concave mirror (PS) is arranged below the sample plane (PE) in such a way that the light entrance opening (LEO) lies above the light exit opening (LAO) of the concave mirror (PS).

2. Apparatus according to Claim 1, wherein the concave mirror is designed as a parabolic mirror (PS) or as an approximate parabolic mirror.

3. Apparatus according to Claim 2, wherein the parabolic mirror is designed and arranged such that a focal point of the parabolic mirror lies in the region of the measurement volume in the sample above the transparent base (B) of the well.

4. Apparatus according to any of the preceding claims, wherein the first detector (DET1) has a sensitive surface (SF) which is larger than the light exit opening (LAO) of the concave mirror (PS).

5. Apparatus according to any of the preceding claims, comprising a second detector (DET2) for detecting second emission light, wherein preferably the second detector (DET2) has a sensitive surface which is oriented in a manner inclined with respect to the optical axis of the emission path at the location of the second detector.

6. Apparatus according to Claim 5, wherein the emission path (EP) has a deflection mirror (US) arranged on the optical axis of the concave mirror and serving for deflecting the second emission light in the direction of the second detector (DET2).

7. Apparatus according to Claim 6, wherein the deflection mirror (US) is arranged in the interior of a reflection chamber of the concave mirror between the light entrance opening (LEO) and the light exit opening (LAO) of the concave mirror, wherein preferably the deflection mirror (S) is arranged in a light-nontransmissive angled tube (WR) having a first tube section for receiving the second emission light and a second tube section, arranged at an angle to the first tube section, for enabling the second emission light reflected at the deflection mirror to be guided out in the direction of the second detector (DET2).

8. Apparatus according to any of the preceding claims, wherein the emission path is designed such that the second emission light passes through without deflection to an absorber, a light trap or a second detector (DET2), while the first emission light is deflected via a plane deflection mirror (US3) in the direction of the first detector (DET1).

9. Apparatus according to any of the preceding claims, wherein there is arranged in the emission path (EP) an emission filter device (EF) switchable between a plurality of operating positions and serving for the selective filtering of first emission light before entry into the first detector (DET1), wherein the emission filter device (EF) preferably has a filter-free operating position in which scattered light can pass through without filtering.

10. Apparatus according to any of the preceding claims, wherein the apparatus has, on the light entrance side (LES) of the sample plane (PE), a concave mirror (PSO) preferably embodied as a parabolic mirror and serving for collecting divergent backward radiation, wherein the concave mirror has a light entrance opening on its side facing the sample plane (PE) and widens towards a light exit opening of larger diameter, wherein preferably a third detector (DET3) is disposed downstream of the concave mirror.

11. Apparatus according to any of the preceding claims, comprising devices for the continuously variable or stepwise adjustment of the wavelength of the excitation light, wherein preferably one of the following conditions holds true: the light source (LQ) is a polychromatic light source and an adjustable wavelength-selective device (WLS) is arranged in the excitation path (AP) between the light source (LQ) and the measurement position (MP); a light source arrangement has a plurality of light sources (LQ1, LQ2) for emitting different wavelengths and also a light source selection device for selecting one of the light sources.

12. Apparatus according to any of the preceding claims, wherein the light source (LQ) is a laser.

13. Apparatus according to any of the preceding claims, wherein a lens optical unit (LO) having a converging effect is arranged in the emission path (EP) between the concave mirror (PS) and the detector.

14. Apparatus according to any of the preceding claims, wherein the apparatus is configured such that at least two different operating modes from the following group are usable: a first operating mode, in which first signals for determining a first measurement variable representing the intensity of the scattered light are processed (nephelometry mode); a second operating mode, in which a wavelength-selective device for blocking excitation light and for transmitting fluorescent light shifted to longer wavelengths relative to the excitation light is arranged between the measurement position (MP) and the first detector (DET1) and first signals for determining a second measurement variable representing the intensity of fluorescent light are processed (forward fluorescence mode); a third operating mode, in which second signals for determining a third measurement variable representing the intensity of the second emission light are processed (light loss mode); a fourth operating mode, in which first signals and second signals are processed jointly (combination mode).

15. Apparatus according to Claim 14, wherein in the fourth operating mode first signals and second signals are processed jointly in such a way that a correction of the measurement variable determined on the basis of the second signals is carried out with the aid of the first signals and / or a correction of the measurement variable determined on the basis of the first signals is performed using the second signals.