Apparatus and method for determining the position and / or extent of a droplet

Abstract

Device for determining the position and / or extent of a droplet in a position determination space, wherein the device comprises a camera with an objective and a beam splitter in the recording area of ​​the camera, wherein the device is designed so that light from the position determination space can enter the objective of the camera along a first beam path and along a second beam path, wherein the light along the first beam path can be reflected at a first reflector element in the direction of the beam splitter and can be transmitted through the beam splitter towards the objective, and wherein the light along the second beam path can be reflected at a second reflector element in the direction of the beam splitter and can be reflected at the beam splitter towards the objective. The invention also relates to a measuring device with the device, a pipetting robot and a method for determining the concentration of a substance in a droplet, a method for keeping a droplet in the position determination space of a device, and a method for regulating the size of a droplet.

Description

Technical Field

[0001] The invention relates to a device for determining the position and / or extent of a droplet. The invention also relates to a measuring device comprising the device, a pipetting robot comprising the device, a pipetting robot comprising the measuring device, and a method for determining the concentration of a substance in a droplet, in particular for determining the concentration of nucleic acids such as RNA or DNA of proteins or other biological or organic substances. Furthermore, the invention relates to a method for holding a droplet in a position determination space of a device, and a method for adjusting the size of a droplet. Background Art

[0002] In the technical field of analysis and liquid handling, it is desirable to be able to analyze and handle small amounts of liquid, if possible without loss of liquid at the beginning due to the method applied. For example, the international publication WO 2014 / 114500 A1 discloses an optical measuring device that can be used to analyze drops hanging from the tip of a pipette. With the aid of this measuring device, measurements can be performed without pouring into another container or contact with a light guide or the like. Such measures will inevitably lead to liquid losses. However, a disadvantage of this method is that the accuracy or repeatability of the hanging droplet measurement results may be reduced compared to samples that have been placed in a container, such as a well of a microplate. Summary of the invention

[0003] The object of the present invention is to provide an alternative for manipulating the measurement of a liquid portion or a liquid portion to be placed in a container. A further object of the present invention is to improve the accuracy of the determination of the concentration of a substance in a droplet.

[0004] According to the invention, this object is achieved by a device according to claim 1 .

[0005] The device according to the invention is a device for determining the position and / or extent of a droplet in a position determination space. Instead of determining the position, the device can also be designed to determine the extent of the droplet, for example the diameter of the droplet. The device can be designed to determine the extent of the droplet in combination with the determination of the position.

[0006] The device according to the invention comprises a camera with an objective lens and a beam splitter in the recording area of ​​the camera. The recording area of ​​the camera is in front of the objective lens and comprises a spatial area that can be imaged from an object onto the image sensor of the camera.

[0007] The device is designed so that light from the position determination space can enter the objective of the camera along a first optical path and along a second optical path. On the one hand, the light can be reflected on the first reflector element in the direction of the beam splitter along the first optical path and can pass through the beam splitter to reach the objective. On the other hand, the light can be reflected on the second reflector element in the direction of the beam splitter along the second optical path and can be reflected at the beam splitter towards the objective.

[0008] This means that the light from the position determination space reaches the beam splitter on two separate paths via the first reflector element or the second reflector element. In the first case, at least part of the light reaches the camera due to the partial transmittance of the beam splitter through the beam splitter. In the second case, the light is at least partially reflected in the direction of the camera. The beam splitter thus forms a light branch. It partially transmits the light and partially reflects the light.

[0009] In this way, the camera can acquire images from two different viewing directions of the position determination space simultaneously or successively. If there is a droplet in the position determination space, its three-dimensional position can be determined by combining the images from different viewing directions. Furthermore, the extent of the droplet can be determined from these images, for example by the number of pixels occupied by the droplet image on the image sensor. The extent of the droplet can be, for example, the diameter of the droplet in the horizontal direction. The extent of the droplet can be, for example, the projected area of ​​the droplet in a projection along the horizontal direction.

[0010] A light path is a geometric path that light continues to follow when it is irradiated in the direction of the light path and on the light path, wherein reflection and refraction effects of the optical elements are taken into account. In the device according to the invention, the optical elements, i.e. the first reflector element and the second reflector element, the beam splitter and the objective of the camera define possible light paths even if no light is present in the device. In addition to the first light path and the second light path, the entire beam of each possible light path can be guided from the position determination space to the camera via the first reflector element and the second reflector element, respectively. It is important that from the position determination space to the camera, there is at least one first light path via the first reflector element and at least one second light path via the second reflector element.

[0011] The inventors have realised that, based on the device according to the invention, it is possible to determine the exact three-dimensional position with sufficient accuracy, depending on the requirements, additionally or alternatively, the extent of the droplet, thereby eliminating the need to keep the droplet in a container or to fix the droplet size between two plates. With the aid of the device according to the invention, a certain amount of liquid can be accurately irradiated at a predetermined position, for example in the plane of the maximum horizontal extent of the droplet, a predetermined diameter of the certain amount of liquid, etc. can be fixed, without the need to accommodate the certain amount of liquid in a container. This makes it possible to perform a large number of measurement or manipulation steps directly on the droplet. In particular, it can be a pendant droplet, for example a droplet pendant at the opening of a pipette tip. The device according to the invention, if suitably designed, also makes it possible to determine the position of a falling droplet at a certain point in time.

[0012] In particular, it is possible to determine the position relative to a beam path of an optical measuring device, as will be explained in detail below in conjunction with the measuring device according to the invention.

[0013] The position of other objects than the droplet is also possible, so that the relative position of the droplet with respect to another object can be determined with the aid of the device.

[0014] A first optical path length of a first optical path from the intersection in the position determination space to the beam splitter can differ from a second optical path length of a second optical path from the intersection in the position determination space to the beam splitter. When determining the size from the image, a conversion factor can be applied in this case, which depends on the respective path lengths and compensates for the resulting deviations, magnification factors or reduction factors.

[0015] Embodiments of the device are derived from the features of the dependent claims 2 to 7 .

[0016] In one embodiment of the device, the first light path and the second light path intersect at an intersection point in the position determination space. In particular, the first light path and the second light path may intersect at a right angle.

[0017] In one embodiment of the device, a first optical path length of the first optical path from the intersection in the position determining space to the beam splitter is equal to a second optical path length of the second optical path from the intersection in the position determining space to the beam splitter.

[0018] In this embodiment, two images from different viewing directions in the position determination space can be combined particularly easily, since there is no size distortion due to different path lengths from the droplet to the camera. In this embodiment, the sizes determined from the images can be compared directly, without the need for conversion between droplet or shadow sizes acquired via the first or second optical path.

[0019] In one embodiment of the device, the intersection of the first and second light paths in the position determination space and the section between the first reflector element and the second reflector element define a first plane, wherein the sections of the first and second light paths between the beam splitter and the first reflector element and the second reflector element define a second plane. In this case, the second plane is inclined relative to the first plane.

[0020] This means that the first plane and the second plane intersect at an angle greater than zero. The positions of the first plane and the second plane according to this embodiment can be obtained by tilting the first reflector element and the second reflector element relative to one plane of the beam splitter. If the plane of the beam splitter and the first reflector element and the second reflector element are arranged in planes parallel to each other, the first plane and the second plane defined above will be identical and will not be tilted to each other.

[0021] This embodiment allows placing the camera outside the first plane.As the inventors have appreciated, with this embodiment it is in particular possible to accommodate a position determination space of the device in a device or apparatus comprising the device at a position laterally adjacent to a position determination space which is limited.

[0022] In one embodiment of the device, the device further comprises a first light emitting element and a second light emitting element. The first light emitting element and the first reflector element are arranged on opposite sides of the position determination space. The second light emitting element and the second reflector element are arranged on opposite sides of the position determination space. The first light emitting element is adapted to illuminate the droplets in the position determination space in a direction parallel to the first light path in front of the first reflector element, and the second light emitting element is adapted to illuminate the droplets in the position determination space in a direction parallel to the second light path in front of the second reflector element.

[0023] With this embodiment of the device, the camera captures a shadow image. When the droplet is in the position determination space, it receives illumination along both the first optical path and the second optical path from the rear, i.e. from the side facing away from the camera as viewed along the optical paths. The first light emitting element and the second light emitting element may each have a light source, such as an LED (light emitting diode) or an incandescent lamp. The light source may emit monochromatic light, or it may also be a broadband light source, for example with white light. For example, the light emitting element may additionally have one or more lenses. For example, with a lens, light from the light emitting element may be guided into a larger cross-section parallel to the corresponding optical path, thereby increasing the light intensity available in the camera along the corresponding optical path. The light emitting element may include a diffuser element, such as a rough glass or plastic plate. In this embodiment, the first light emitting element and the second light emitting element may be arranged 90° offset from each other relative to the center of the position determination space.

[0024] In one embodiment of the device comprising the first and second light emitting elements, the first and second light emitting elements are designed to alternately not illuminate the position determination space at all or illuminate it alone in time. In particular, the first and second light emitting elements can have substantially the same construction.

[0025] During periods of time when there is no illumination at all, for example, optical measurements can be performed, where the light from the illumination source would interfere. When a single light emitting element is switched on, the camera can capture an image from the corresponding direction that is completely unaffected by possible reflections of light from the other of the first and second light emitting elements.

[0026] In one embodiment of the device, the beam splitter, the first reflector element, the second reflector element and the camera are located outside a region comprising: the position determination space and its imaginary continuation in the direction of gravity.

[0027] This arrangement of the elements of the device takes into account the possible falling directions of the droplets. In this way, wetting or soiling of the elements due to unintentional dripping of droplets can be avoided. For example, the elements of the device, in particular the first reflector element and the second reflector element, can be arranged transversely to the side of the position determination space with respect to the direction of gravity. In particular, the objective lens of the camera can be located outside the area into which the droplets may fall.

[0028] The embodiments of the device can be combined in any manner as long as they are not mutually exclusive.

[0029] In a further aspect, the invention relates to a measuring device according to claim 8 .

[0030] The measuring device according to the invention can be a measuring device for measuring the absorbance of electromagnetic radiation in a droplet. Alternatively or in combination with the device for measuring the absorbance, the measuring device can also be a measuring device for measuring fluorescence stimulated by electromagnetic radiation in a droplet. The measuring device comprises a device according to the invention for determining the position and / or range of a droplet in a position determination space. The measuring device also comprises an electromagnetic radiation source and a detector for electromagnetic radiation. In this case, the measuring device is designed so that the electromagnetic radiation can reach the detector along a radiation path starting from the radiation source via the droplet in the position determination space. The electromagnetic radiation can reach the droplet directly or indirectly from the radiation source. For an indirect path, for example, a radiation path through a monochromator or through a glass fiber light guide can be envisaged. The device according to the invention as part of the measuring device according to the invention is designed to determine the position of the droplet relative to the radiation path.

[0031] For example, the measuring device can be designed to evaluate the absorbance of electromagnetic radiation at several wavelengths, or to evaluate it over a wavelength spectrum. In the case of an absorbance measurement, the electromagnetic radiation passes through the droplet and then enters the detector, where the intensity of the wavelength impinging on the droplet is evaluated. In the case of a fluorescence measurement, the incident radiation excites the fluorescence of the substance in the droplet and generates electromagnetic radiation of another wavelength. Then, when the electromagnetic radiation in the ultraviolet range impinges on the droplet, the detector measures the intensity of another wavelength, such as visible light or infrared radiation. For example, in the case of a fluorescence measurement, the detector is positioned in a direction that deviates from the direction of incidence of the radiation impinging on the droplet, for example, in a direction that deviates by 90°. There may also be several detectors for fluorescence measurements, for example, on both sides of the direction of incidence of the irradiated electromagnetic radiation, each deviating by 90° in direction. In one embodiment of the measuring device, absorbance or fluorescence measurements can be combined. For example, the irradiation for absorbance and fluorescence measurements can be achieved by the same radiation source. A first detector in a straight line behind the droplet in the direction of illumination can be used for absorbance measurement, while a second detector with sensitivity in another wavelength range can be arranged perpendicular to the first detector direction in the direction originating from the droplet and used for fluorescence measurement. Another possibility for a combined measuring device for absorbance and fluorescence can be achieved by arranging the detectors and the radiation source in two different planes, wherein at least one plane is provided with a device according to the invention.

[0032] Such a measuring device may be referred to as a "droplet spectrometer". The inventors have recognised that for the operation of such a droplet spectrometer, precise control of the droplet position and droplet size is essential for accurate measurement results. Preferably, the positioning accuracy is better than 70 μm. Particularly preferably, the positioning accuracy is better than 50 μm. Due to the device according to the invention, the measuring device according to the invention is equipped with an optical system which is able to record position information about the droplets in three dimensions in an unexpectedly simple manner using a single camera. In addition, the optical system can be mounted in a very space-saving arrangement.

[0033] An exemplary embodiment of the measuring device results from the features of dependent claim 9 .

[0034] In an embodiment of the measuring device, the first optical path, the second optical path and the radiation path intersect at a common intersection point in the position determination space.

[0035] The determination of the position of the droplet relative to the radiation path is particularly accurate if the droplet is located at a common intersection point of the optical path and the radiation path. For example, the two optical paths and the radiation path may lie in a common plane in the region of the intersection point. For example, the two optical paths may intersect each other at an angle of 90°, while the radiation paths may intersect at an angle of 45°.

[0036] In a further aspect, the invention relates to a pipetting robot according to claim 10 or a pipetting robot according to claim 11 .

[0037] The pipetting robot according to the invention comprises a device according to the invention for determining the position and / or extent of a droplet in a position determination space. The pipetting robot is designed to:

[0038] - moving the pipette tip into the position determination space of the device;

[0039] - Formation of a droplet outside the opening of the pipette tip; and

[0040] - Determining the position of the formed droplet in the position determination space with the aid of at least one image recorded by the camera.

[0041] For example, a pipetting robot is a three-axis robot that can move a pipette tip along three coordinate directions of a Cartesian coordinate system. The pipetting robot comprises, for example, means for aspirating and / or dispensing liquids. The pipetting robot may also comprise a holder for a microplate.

[0042] Another pipetting robot according to the invention comprises a measuring device according to the invention for measuring the absorbance of electromagnetic radiation in a droplet and / or for measuring fluorescence stimulated by electromagnetic radiation in a droplet. The pipetting robot further comprises means for moving at least one pipette tip in at least one movement direction. The pipetting robot is designed to:

[0043] - moving at least one pipette tip into a position determination space of the device;

[0044] - Formation of a droplet outside the opening of the pipette tip;

[0045] - determining the position and / or extent of the formed droplet in the position determination space based on at least one image acquired by the camera; and

[0046] - Using a measuring device to measure the absorbance of electromagnetic radiation in the droplet and / or to measure fluorescence stimulated by electromagnetic radiation in the droplet.

[0047] The object of the invention is achieved by a method according to claim 12 .

[0048] It is a method for determining the concentration of a substance in a droplet by means of a measuring device according to the invention. The method comprises the following steps:

[0049] - introducing a droplet into the position-determined space or creating a droplet in the position-determined space;

[0050] - Determine the position of the droplet relative to the beam path of the measuring device;

[0051] - Determine the extent of the droplets;

[0052] - Determine the optical path length of the beam path in the droplet;

[0053] - measuring the absorption rate of electromagnetic radiation, wherein electromagnetic radiation starting from a radiation source along a radiation path passes through the droplet in the position determination space and reaches the detector, wherein the radiation intensity of the radiation source is related to the radiation intensity in the detector; and

[0054] - Calculate the concentration based on the measured absorbance and the determined optical path length in the droplet.

[0055] There are two alternatives for the way in which the droplet to be checked enters the position determination space. According to a first alternative, the droplet is introduced into the position determination space from outside the position determination space, which can occur, for example, by free fall of the droplet. It is also possible to move the pipette tip into the position determination space with the droplet suspended thereon. According to a second alternative, the droplet is generated in the position determination space, for example by dispensing from a pipette tip that was already in the position determination space before dispensing.

[0056] In order to determine the optical path length of the beam path, in the simplest case it is sufficient for the determination of the droplet diameter if the beam path passes through a circular droplet at its point of maximum diameter. In order to determine the optical path length of the beam path in the droplet, an optical beam path simulation can be used. The latter is useful if the optical path does not hit the droplet surface perpendicularly and refraction effects have to be taken into account. Consider the refractive index of the droplet at the wavelength of the electromagnetic radiation. For the droplet, the refractive index n of visible light is approximately n=1.33 for a wavelength of 600 nm. In the near-infrared range, the refractive index of water is smaller than in the visible range, while in the near-ultraviolet range it increases to as high as about n=1.5 for a wavelength of 200 nm. Simulations can be performed on the beamed light to take into account the influence of the geometry on the radiation intensity reaching the detector.

[0057] The relationship between the concentrations of absorbing materials can be determined using the Lambert-Beer law.

[0058] Under the Lambert-Beer law, the following relationship applies:

[0059] D = log 10 (I0 / I)=εCL

[0060] Where D is the absorptivity, I0 is the intensity of the incident radiation (at wavelength l), I is the intensity of the radiation after passing through the absorptive material (at wavelength λ), C is the concentration of the absorbing material, L is the optical path length in the absorbing material, and ε=ε(λ) is the (wavelength-specific) absorption coefficient.

[0061] Variants of the method result from the features of the dependent claims 13 and 14 .

[0062] In a variant of the method, a first extent of the droplets is determined before the measuring step and a second extent of the droplets is determined after the measuring step. An average of the first extent and the second extent of the droplets is used to determine the concentration of the substance.

[0063] This variant of the method is particularly suitable for small-volume droplets, where the surface area to volume ratio is so large that a significant portion of the droplet evaporates during the measurement process. By averaging, the effective range of the droplet during the measurement step can be measured more accurately without the risk of interfering with the measurement by determining the range of the droplet. In particular, the measurement can be performed without having to switch on the illumination light in order to record it with a camera.

[0064] A variant of the method is a method for determining the concentration of nucleic acids such as RNA (ribonucleic acid) or DNA (deoxyribonucleic acid), proteins or other biological or organic substances, wherein the wavelength of the electromagnetic radiation is characteristic of the substance and is optimized to determine the concentration. For example, the wavelength with the highest absorption of the substance is selected, or in the case of fluorescence measurements, the wavelength of maximum fluorescence is selected. In general, by adjusting the wavelength, the measuring method can be adapted to the substance whose concentration is to be determined.

[0065] The method can be carried out in particular by using a pipetting robot according to the invention.

[0066] A first example of application of the method according to the invention is to quantify the amount of RNA or DNA contained in a single droplet, wherein a pipetting robot is used for the measuring device. To this end, the absorbance of electromagnetic radiation passing through the droplet is measured at different wavelengths characteristic of RNA / DNA. For example, the absorption spectrum is recorded in a wavelength range from 230nm to 1000nm. One possibility for determining the concentration of RNA / DNA is to evaluate the absorbance at 260nm, optionally taking into account the turbidity of the sample by measuring the absorbance at a wavelength of 320nm. The absorbance at longer wavelengths, such as at 320nm, is mainly caused by larger particles that scatter light in the visible spectrum, in which case the particles are considered to be impurities. In addition, for example, the ratio of the absorbance measurements at wavelengths of 260nm and 280nm can be used, for example, to determine the measure of the purity of a DNA sample or an RNA sample contaminated with proteins relative to, for example, typical absorption of 280nm light. For example, by determining the ratio of the absorbance values ​​at 260nm and 230nm, information about possible contamination by phenolates, thiocyanates and other organic compounds can be provided.

[0067] A second example of application of the method according to the invention is the quantification of the amount of protein contained in a single droplet, wherein a pipetting robot is used for the measuring device. For this purpose, the absorbance of the electromagnetic radiation passing through the droplet is measured at 280 nm. The absorbance of the protein solution at 280 nm is due to the aromatic amino acids tryptophan and tyrosine as well as cysteine ​​and disulfide-bridged cysteine ​​residues. The protein concentration can be calculated from literature values ​​or by comparison with a calibration curve.

[0068] In these examples, the droplet will hang down on the pipette tip during the measurement. With the aid of the device according to the invention as part of the measuring device:

[0069] a) accurately determining the size of the droplet so that the optical path length extending in the droplet can be accurately determined; and

[0070] b) The position of the pipette tip is aligned so that the radiation path used for absorbance measurement hits the pendant droplet at the point of maximum horizontal range and in the middle.

[0071] By these measures, highly accurate and reproducible measurements can be achieved. The inventors have recognized that without these measures, which are only possible with the measuring device according to the invention, the radiation used for the absorbance measurement is refracted in directions that are difficult to control by the curvature of the non-centrally positioned droplet. Furthermore, the optical path length in this case does not correspond to the droplet diameter. Both effects lead to inaccurate measurements.

[0072] For example, using the method according to the invention the concentration of DNA in droplets can be determined with a lower detection limit of 2 ng / μl.

[0073] This method allows for a fast and accurate measurement of the concentration of a substance in a droplet without losing liquid during the measurement. For example, this method can be used as a preparatory step for further measurements, which should avoid any loss of sample liquid.

[0074] Furthermore, the present invention relates to a method according to claim 15.

[0075] This further method according to the invention is a method for keeping a droplet in a position determination space of a pipetting robot according to the invention. The method comprises at least repeatedly applying the following steps:

[0076] - determining the actual position of a drop formed at an opening of a pipette tip of the pipetting robot in a position determination space based on at least one image acquired by a camera;

[0077] - determining the deviation of the determined actual position from the predetermined target position; and

[0078] - Move the pipette tip in a direction that brings the actual position closer to the target position.

[0079] Furthermore, the present invention relates to a method according to claim 16.

[0080] The further method according to the invention is a method for adjusting the droplet size of a droplet in a position determination space of a pipetting robot according to the invention, wherein the method comprises repeatedly applying the following steps:

[0081] - determining a drop size of a drop formed at an opening of a pipette tip of the pipetting robot in the position determination space based on at least one image recorded by the camera;

[0082] - determining a deviation of the determined actual droplet size from a predetermined target droplet size; and

[0083] - Aspirating liquid into the pipette tip or ejecting liquid from the pipette tip, wherein aspiration is selected when the actual droplet size is larger than the target droplet size, and ejection is selected when the actual droplet size is smaller than the target droplet size.

[0084] The method enables the droplet diameter to be adjusted by determining the droplet diameter from the acquired image and by controlling a pressure changing device, such as a pump, of a pipetting robot so that aspiration is performed when the droplet diameter is too large and dispensing is performed when it is too small. In a variant of the method, the method can also be combined with the aforementioned method to keep the droplet at the actual position.

[0085] The adjustment of the droplet size can be performed, for example, as follows. The droplet diameter is selected as the droplet size to be adjusted. A circle is fitted to the shadow image acquired by the camera from two directions and its diameter is determined. The average of the two diameters determined by fitting is used as the actual diameter. For example, the actual diameter is adjusted to a target diameter of 1.5-1.6 mm. For example, the pipette will produce slightly larger drops at the beginning and then reabsorb some liquid as needed. For example, the adjustment of the droplet size is performed after positioning. For example, after positioning, a series of images are acquired with an exposure time of 2 ms. In addition, the droplet size can be determined before and after the measurement, i.e. the absorbance or fluorescence measurement, and the results can be compared to check the feasibility of an interference-free process. In this way, it is possible to prevent, for example, the use of measured values ​​in measurements in which the droplet has simultaneously undergone a strong deformation or the droplet has even fallen from the pipette tip.

[0086] The inventors have realised that droplets with diameters in the range of about 1.3-2.0 mm can generally be produced in a stable manner. A nominal diameter of 1.5-1.6 mm in the above range was found to be the best compromise between a large diameter favouring measurement accuracy and a small diameter reducing the risk of droplets falling off. For detecting lower concentrations, slightly larger droplets would be preferred. Starting from a 2.3 mm diameter, the droplets gradually break off.

[0087] The beam diameter of the irradiated electromagnetic radiation may be, for example, 0.2 mm. In combination with a droplet diameter of approximately 1.6 mm, the beam diameter is approximately one order of magnitude smaller than the droplet diameter. In this case, the effective optical path length can be approximated sufficiently accurately by the droplet diameter, which is determined as the actual droplet diameter as described above.

[0088] For example, the time scale of the control loop may be adapted to an image acquisition rate of 50 images per second. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Exemplary embodiments of the present invention are described in more detail below using the accompanying drawings, in which:

[0090] Figure 1 shows a schematic cross-sectional view of a device according to the invention;

[0091] Figure 2 A schematic cross-sectional view of an embodiment of the device is shown;

[0092] Figure 3 Figure 3.a ) shows a perspective view of an embodiment of a measuring device, in which Figure 3.b) and 3.c) The selected Figure 3.a ) is a schematic three-dimensional diagram of the geometric position of the elements in;

[0093] Figure 4 shows an arrangement of one embodiment of an apparatus associated with a portion of a pipetting robot;

[0094] Figure 5 shows a perspective view of a pipetting robot with a device according to the invention;

[0095] Figure 6 A schematic perspective view shows the situation in space for position determination of an exemplary embodiment of a measuring device or an exemplary embodiment of a pipetting robot. DETAILED DESCRIPTION

[0096] Figure 1A schematic cross-sectional view of a device 10 according to the invention is shown. The dotted circle shows the position of a position determination space 3. Within this position determination space, a smaller dotted circle shows a possible position of a droplet 5, the position and / or range of which can be determined with the aid of the device 10. The device comprises a camera 14 with an objective 15 and a beam splitter 13 in a recording area 4 of the camera. The recording area 4 of the camera in front of the objective is schematically marked with a dotted line. It corresponds to the area in which the incident light can reach the image sensor of the camera via the objective. Light from the position determination space 3 can reach the objective 15 along a first optical path 1, which is drawn as a dotted line with an arrow. The light is reflected at the first reflector element 11 and radiated through the beam splitter 13. Light from the position determination space 3 can also enter the objective along a second optical path 2, which is drawn as a dotted line with an arrow. In this case, the light is reflected at the second reflector element 12 and also at the beam splitter 13.

[0097] Figure 2 A schematic cross-sectional view of an embodiment of the device 10 is shown. In the embodiment shown here, a first light path 1 and a second light path 2 intersect at a right angle in the position determination space. In the illustration shown, there is a droplet 5, which is arranged directly at the intersection point 6 of the intersecting light paths. The intersection point 6 is located in the position determination space 3, and its position is indicated by a dashed line. In the embodiment shown, a first reflector element 11 and a second reflector element 12 are arranged parallel to the reflective surface of the beam splitter 13. The reflector elements 11 and 12 are also arranged symmetrically with respect to the reflective surface of the beam splitter 13. This results in a first path length of the first light path 1 from the intersection point 6 to the beam splitter 13 and a second path length of the second light path 2 from the intersection point 6 to the beam splitter, wherein the first path length and the second path length are equally large. In the embodiment shown here, a first light emitting element 16 and a second light emitting element 17 are also included. In the embodiment shown here, both light emitting elements include a light emitting diode (LED) 43 and a lens 42. The light along the first light path 1 and along the second light path 2 are drawn here as starting from the light emitting diode to the camera. A light-emitting element and a reflector element are each located on opposite sides of the position determination space. The droplet is illuminated by a first light-emitting element 16 parallel to the first light path 1 and a second light-emitting element parallel to the second light path 2. The illumination direction and the parallelism of the light paths are determined by the direction of the section of the corresponding light path in front of the first reflector element or the second reflector element. From the perspective of the camera, the droplet 5 is illuminated from behind along two light paths and is displayed as a shadow image. As an alternative to the lighting situation shown here, only one light-emitting element is turned on in a time-staggered manner, so that the position determination space and the droplet arranged therein are respectively illuminated by only one light-emitting element. The light-emitting elements shown here are basically the same in structure. Possible variations of the embodiment may include light-emitting diodes of different colors.

[0098] The cross in the circle indicates the gravity direction G, which in the case shown is perpendicular to the image plane of the figure. Therefore, the position determination space and its imaginary continuation in the gravity direction G are free of elements of the device. Therefore, a drop falling from the position determination space in the gravity direction does not hit any element in the beam splitter 13, the first reflector element 11, the second reflector element 12 or the camera 14.

[0099] Relay optics 41 are located between the beam splitter 13 and the camera 14. The camera may, for example, have a CMOS image sensor.

[0100] The arrangement shown can be constructed in a very compact manner. For example, the distance from the intersection point 6 to the first reflector element or the second reflector element can be approximately 25 mm, and the distance from the intersection point 6 to one of the LEDs can be less than 30 mm.

[0101] For example, with these dimensions and a camera with a pixel size of 10 micrometers, an imaging system can be realized which achieves an optical resolution of approximately 6 micrometers per pixel, i.e. a displacement of the real object in the position determination space of 6 micrometers results in a displacement of the image by one pixel. In this case, the optical system results in a reduction of approximately 1.7 times.

[0102] The optical layout of the embodiment shown is designed so that a device suitable for determining the position and extent of a droplet, for example in a measuring chamber of a spectrometer, is obtained with the aid of only two LEDs for illumination, two lenses, two mirrors, a beam splitter and a camera. The LEDs are used to illuminate the droplet from two directions, for example a horizontal x-direction and a vertical, also horizontal, y-direction. In this case, the camera can acquire images of the droplet from two different directions but from the same distance, wherein images of the droplet can be generated in the x-z plane and in the y-z plane. In this example, the z-direction is the vertical direction, which means that three-dimensional position information of the droplet can be obtained using a device without moving parts. For example, by sequentially illuminating the droplet in the position determination space with only one of the two LEDs at a time, two separate images can be obtained. Based on such a pair of images, the size of the droplet and also the position of the droplet can be adjusted in three dimensions by a control loop, which, for example, includes the control of a pipetting robot.

[0103] In this context, it is possible to temporarily deviate from conventional software architectures of control software which provide for control software controlling individual modules or devices (i.e. linear axes, pumps, incubators, oscillators, extraction modules, etc.) according to a predetermined flow chart, since in this case the measuring device determines how the pipette of the pipetting robot must be moved based on the position data determined by the device according to the invention.

[0104] Figure 3.a) shows a perspective view of an embodiment of a measuring device 20. The droplet 5 is located in the center of a position determination chamber, which is provided with a cylindrical wall with through holes for the optical paths 1 and 2 or the beam path 23. In this position, the droplet can, for example, hang on the tip of a pipette, not shown. The measuring device comprises a device according to the invention as described above. The device comprises a first reflector element 11, a second reflector element 12, a beam splitter 13 and a camera 14 with a C-mount 44 for attaching an objective lens. In the above-described manner, the first optical path 1 and the second optical path 2 lead in two different ways from the position determination space to the objective lens of the camera, which is not shown here for the sake of clarity, but whose position is determined by the position of the C-mount 44, which defines the position of the camera near the end of the objective lens. In the embodiment shown, the device also comprises a first light-emitting element 16 and a second light-emitting element 17. The light from the first light-emitting element 16 can be irradiated into the region of the droplet 5 through the through hole visible in the upper right corner of the cylindrical wall. From there, the light continues along a first optical path 1 via a first reflector element 11 through an invisible through hole, into the objective lens via a beam splitter 13. Light from a second light emitting element 17 can be irradiated into the region of the droplet 5 via a through hole visible in the upper left corner of the cylindrical wall. From there, the light continues along a second optical path 2 via reflection at the second reflector element 12 through an invisible through hole, and into the objective lens of the camera via reflection at the beam splitter 13. In the illustrated arrangement, the first optical path and the second optical path coincide in the region between the beam splitter 13 and the C-shaped mounting 44. The measuring device 20 further comprises a radiation source (not shown) for electromagnetic radiation and a detector 22 for electromagnetic radiation. The radiation source can be, for example, a flash lamp. A radiation path 23 is led from the radiation source via a monochromator and is deflected and focused via an ellipsoidal mirror 24. The radiation path passes through the through hole in the cylindrical wall, through the droplet 5 and through another invisible through hole to the detector 22, where the radiation intensity of the electromagnetic radiation can be measured. The first light path 1 and the second light path 2 and the radiation path 23 define a common first plane in the region of the droplet and intersect at a common intersection point. The two light paths intersect each other at an angle of approximately 90° and intersect the radiation path 23 at an angle of approximately 45°. Due to the inclined arrangement of the first reflector element 11 and the second reflector element 12, the first light path and the second light path leave the plane after being reflected at the respective reflector elements.

[0105] For example, the light emitting elements 16, 17 each include a light emitting diode and a lens arranged in a housing in front of the light emitting diode. The reflector elements 11, 12 are designed as plane mirrors, for example. With respect to the reflective side arranged behind the reflector elements 11, 12, a suspension for a mirror for precise alignment can be seen.

[0106] Figure 3.b ) to reduce but with Figure 3.a) shows the first beam path 1 and the second beam path 2 in a manner that is unchanged in orientation compared to the first plane E1. The first plane E1 is defined by the first beam path 1 and the second beam path 2 in the area between the intersection point 6 of the beam paths, the point 7 on the first reflector element at which the first beam path is deflected, and the point 8 on the second reflector element at which the second beam path is deflected. The first plane is shown as a dotted line. The first beam path 1 is shown as a dotted line and the second beam path 2 is shown as a dashed line. Starting from the points 7 and 8, the beam paths extend in a second plane E2 that is inclined relative to the plane E1. The point 9 on the beam splitter 13 is located in the plane E2. From this point, the two beam paths 1, 2 coincide.

[0107] Figure 3.c ) Figure 3.b ) are the same, only the angles are slightly different. In addition, the angle α at which the first plane E1 and the second plane E2 intersect is shown. The angle α can be, for example, about 90°. In one embodiment, the plane E1 can be a horizontal plane perpendicular to the gravity direction G. In this case, the light path goes down into the objective lens of the camera, which is arranged below the plane E1 to save space. In this arrangement, the camera is also outside the possible falling direction of the droplet.

[0108] exist Figure 3.a The compact design implemented in the embodiments of the devices shown in 3.a) (as part of the measuring device) and 3.c) allows the camera to be placed below a working surface parallel to the plane E1. The reflector element is inclined relative to the plane E1, i.e. also relative to the working surface. The reflector element is also inclined relative to the plane defined by the beam splitter. In this case, the plane defined by the beam splitter also forms a symmetry plane for the two reflector elements.

[0109] Figure 4 An exemplary arrangement of an embodiment of the device is shown, as it may be arranged as part of a pipetting robot. Only one storage area for microplates is shown, as well as some holders 32 for standard microplates. The device 10 has a circular access opening open at the top for inserting a pipette tip into a position determining space of the device, which is located inside the rectangular housing. A further housing 18 is located below the microplate storage area and contains a camera of the device, which is not visible in this view. The housing 18 may contain further components, such as a monochromator of a spectrometer. In this embodiment, for example, Figure 3.a ), the geometrical arrangement of the position determination space and the camera can be selected. In this way, a very space-saving arrangement is produced, wherein the device only has the space requirement for a single holder for a standard microplate.

[0110] By means of the compact design shown, the device according to the invention can be ideally integrated as a module into a platform for manipulating liquids, in particular in platforms where the upright surface of the module is critical. The embodiment shown saves space on the working surface.

[0111] Figure 5 A pipetting robot 30 with a device 10 according to the invention is shown in the context of an exemplary application. The pipetting robot comprises controllable linear axes which enable the pipette to be moved in three directions of a Cartesian coordinate system represented by arrows in the x, y and z directions. A pipetting channel 34 is connected to a pump device at the lower end where the pipette tip is located by a flexible tube. The pipette tip is located directly above the opening of the device 10. The pipette tip can be retracted into the device by lowering the pipette tip in the negative z direction. A microplate 33 is arranged on the working surface of the pipetting robot. The microplate shown has 4×6 holes, but a microplate with 8×12 holes or other common microplates can also be used. The space requirement of the device 10 on the working surface relative to the microplate is shown in practice. It shows a possible compact design of the device according to the invention.

[0112] Figure 6 The arrangement of the various elements of an embodiment of a measuring device or a pipetting robot that are spatially related to the position determination of the device during the measurement of a droplet 5 is schematically shown. The droplet 5 is suspended on a pipette tip 31, which can be moved in the x, y and z directions of the illustrated Cartesian coordinate system by the linear axes of the pipetting robot. The sections of each of the first optical path 1 and the second optical path 2 are drawn with dotted lines and dashed lines, respectively, so that the orientation of the other elements relative to the optical path of the device can be seen. The radiation path 23 of the electromagnetic radiation is emitted from the radiation source 21 and deflected in the direction of the droplet by a mirror 24, which can be, for example, an ellipsoidal mirror. Detectors for measuring the intensity of electromagnetic radiation are arranged in a straight line behind the droplet. At this time, the intensity reduced due to absorption in the droplet can be measured. Perpendicular to the direction of the beam is another detector 22'. At this time, the intensity of the electromagnetic radiation generated by the fluorescence in the droplet can be measured. The long dashed line and the short dashed line are sections 23', 23". of the radiation path between the droplet and the detectors for absorbance measurement and fluorescence measurement. In the embodiment shown here, the radiation source 21 can provide both the illumination intensity for absorbance measurement and the excitation of fluorescence. In the embodiment shown here, the first optical path 1, the second optical path 2, the section 23' of the radiation path leading to the detector 22' for absorbance, and the section 23" of the radiation path leading to the detector 22" for fluorescence are all in the same common plane.

[0113] Alternatively or additionally, the fluorescence can also be measured at another z position. Therefore, within the scope of the invention, the following procedure is also conceivable: the fall position in x and y is determined by the device for position determination and then a predetermined distance is covered in the z direction. This means that the measurement plane does not necessarily have to correspond to the position determination plane.

[0114] Reference numerals list

[0115] 1 First optical path

[0116] 2 Second optical path

[0117] 3 Position Determination Space

[0118] 4 Camera recording area

[0119] 5 Droplets

[0120] 6 Intersection Points

[0121] 7 Point on the first reflector element

[0122] 8 Point on the second reflector element

[0123] 9 Points on the beam splitter

[0124] 10. Installation

[0125] 11 First reflector element

[0126] 12 Second reflector element

[0127] 13 Beam splitter

[0128] 14 Camera

[0129] 15 Objective lens

[0130] 16. First light emitting element

[0131] 17 Second light emitting element

[0132] 18 Housing (to hold the camera)

[0133] 20 Measuring device

[0134] 21 Radiation Source

[0135] 22, 22', 22" detectors

[0136] 23 Radiation Path

[0137] 23', 23" Section of the radiation path in front of the detector

[0138] 24 Ellipsoid mirror

[0139] 30 Liquid handling robot

[0140] 31 Pipette Tips

[0141] 32 Holder for standard microplates

[0142] 33 Microplate

[0143] 34 pipetting channels

[0144] 41 Relay Optics

[0145] 42 Lens

[0146] 43 LED

[0147] 44 C-mount for cameras

[0148] D is the diameter of the droplet

[0149] E1 First plane

[0150] E2 Second Plane

[0151] α Angle between the first and second planes

[0152] G direction of gravity

Claims

1. A device (10) for determining the position and / or extent of a droplet (5) in a position determination space (3), wherein: The device comprises a camera (14) with an objective lens (15) and a beam splitter (13) in a recording area (4) of the camera; wherein the device is designed so that light from the position determination space (3) can enter the objective lens of the camera both along a first optical path (1) and along a second optical path (2); wherein the light along the second optical path (2) can be reflected at the second reflector element (12) in the direction of the beam splitter (13) and can be reflected at the beam splitter towards the objective lens (15); wherein light along the first optical path (1) can be reflected at a first reflector element (11) in the direction of the beam splitter and can be transmitted through the beam splitter (13) towards the objective lens (15); wherein the first optical path (1) and the second optical path (2) intersect at an intersection point (6) in the position determination space (3); and wherein the first optical path (1) and the second optical path (2) define a first plane (E1) in a section between the intersection point (6) in the position determination space and the first reflector element (11) and the second reflector element (12), wherein the first optical path and the second optical path define a second plane (E2) in a section between the beam splitter and the first reflector element (11) and the second reflector element (12), and wherein the second plane is arranged in an inclined manner relative to the first plane.

2. The device (10) according to claim 1, characterized in that The first light path and the second light path intersect at a right angle.

3. The device (10) according to claim 1, characterized in that A first optical path length of the first optical path (1) from the intersection point (6) in the position determination space to the beam splitter (13) is equal to a second optical path length of the second optical path (2) from the intersection point (6) in the position determination space to the beam splitter (13).

4. The device (10) according to any one of claims 1 to 3, characterized in that The device also includes a first light-emitting element (16) and a second light-emitting element (17), wherein the first light-emitting element (16) and the first reflector element (11) are arranged on opposite sides of the position determining space (3), wherein the second light-emitting element (17) and the second reflector element (12) are arranged on opposite sides of the position determining space, wherein the first light-emitting element is suitable for illuminating the droplets (5) in the position determining space in a direction parallel to the first optical path (1) in front of the first reflector element (11), and wherein the second light-emitting element is suitable for illuminating the droplets in the position determining space in a direction parallel to the second optical path (2) in front of the second reflector element (12).

5. The device (10) according to claim 4, characterized in that The invention comprises a first light-emitting element (16) and a second light-emitting element (17), wherein the first light-emitting element and the second light-emitting element are constructed substantially identically, wherein the first light-emitting element and the second light-emitting element are suitable for alternately not illuminating the position determination space (3) at all or illuminating it separately in terms of time.

6. The device (10) according to any one of claims 1 to 3, characterized in that The beam splitter (13), the first reflector element (11), the second reflector element (12) and the camera (14) are located outside a region comprising the position determination space and its imaginary continuation in the direction of gravity (G).

7. A measuring device (20) for measuring the absorption of electromagnetic radiation in a liquid droplet (5) and / or for measuring fluorescence stimulated by electromagnetic radiation in a liquid droplet (5), wherein: The measuring device comprises a device (10) according to any one of claims 1 to 6, wherein the measuring device further comprises a radiation source (21) of electromagnetic radiation and a detector (22, 22', 22") for electromagnetic radiation, wherein the measuring device is designed so that electromagnetic radiation can reach the detector via a droplet in the position determination space along a radiation path (23) from the radiation source, and wherein the device is suitable for determining the position of the droplet relative to the radiation path.

8. The measuring device (20) according to claim 7, characterized in that The first optical path (1), the second optical path (2) and the radiation path (23) intersect at a common intersection point (6) in the position determination space (3).

9. A pipetting robot (30) comprising a device (10) according to any one of claims 1 to 6, wherein: The pipetting robot is suitable for: - moving the pipette tip (31) into the position determination space (3) of the device; - forming a droplet (5) outside the opening of the pipette tip; and - Based on at least one image recorded by the camera (14), determining the position of the formed droplet in the position determination space.

10. A pipetting robot (30) having a measuring device (20) according to claim 7 or 8 and having a device for moving at least one pipette tip (31) in at least one direction of movement, wherein: The pipetting robot is suitable for: - moving the at least one pipette tip (31) into the position determination space (3) of the device (10); - forming a droplet (5) outside the opening of the pipette tip; - determining the position and / or extent of the formed droplet in the position determination space based on at least one image acquired by the camera (14); as well as - measuring the absorption of electromagnetic radiation in the droplets and / or measuring the fluorescence stimulated by electromagnetic radiation in the droplets by using the measuring device (20).

11. A method for determining the concentration of a substance in a droplet (5) by means of a measuring device (20) according to claim 7 or 8, the method comprising the following steps: - introducing a droplet (5) into the position determination space (3) or creating a droplet in the position determination space; - determining the position of the droplet relative to the beam path (23) of the measuring device; - Determine the extent of the droplets; - determining the optical path length of the light beam path in the droplet; - measuring the absorption rate of electromagnetic radiation, wherein electromagnetic radiation starting from the radiation source (21) along the radiation path passes through the droplet in the position determination space and reaches the detector (22, 22', 22"), wherein the radiation intensity of the radiation source is related to the radiation intensity in the detector; and - Calculate the concentration based on the measured absorbance and the determined optical path length in the droplet.

12. The method according to claim 11, characterized in that A first extent of the droplet (5) is determined before the measuring step, wherein a second extent of the droplet is determined after the measuring step, and wherein an average of the first extent and the second extent of the droplet is used to determine the concentration of the substance.

13. The method according to claim 11 or 12, characterized in that: The method is a method for determining the concentration of RNA, DNA or protein; Among them, the electromagnetic radiation has a wavelength characteristic of RNA, DNA or protein.

14. The method for holding a droplet (5) in the position determination space (3) of a pipetting robot (30) according to claim 9, wherein: The method comprises repeated application of the following steps: - determining the actual position of a drop formed at the opening of the pipette tip (31) of the pipetting robot in the position determination space (3) based on at least one image recorded by the camera (14); - Determine the deviation of the determined actual position from the predetermined target position; as well as - moving the pipette tip in a direction that brings the actual position closer to the target position.

15. Method for adjusting the droplet size of a droplet (5) in the position determination space (3) of a pipetting robot (30) according to claim 9, wherein: The method comprises repeated application of the following steps: - determining a droplet size of a droplet formed at an opening of a pipette tip (31) of the pipetting robot in the position determination space (3) based on at least one image recorded by the camera (14); - determining a deviation of the determined actual droplet size from a predetermined target droplet size; as well as - Aspirating liquid into the pipette tip or ejecting liquid from the pipette tip, wherein aspiration is selected when the actual droplet size is larger than the target droplet size, and ejection is selected when the actual droplet size is smaller than the target droplet size.

Images