METHOD FOR OPTICAL DETECTION OF MOVEMENT IN A SPATIALLY EXTENSIVE BIOLOGICAL SAMPLE

Description

[0001] The invention relates to a device for the optical in vitro detection of movement in a biological sample with spatial extent.

[0002] Practical experience has shown that there is a need to monitor the active dynamics of closed and three-dimensional cell and tissue cultures in areas such as developmental biology, toxicity testing, and pharmaceutical research.

[0003] For example, tissue samples grown from embryonic stem cells that have differentiated into muscle tissue are used in toxicity tests to assess the harmfulness of a substance under investigation. These tests examine whether a substance applied to the muscle tissue affects muscle contractions, which can be an indicator of the substance's toxicity. This requires measurement techniques to detect movement, such as contraction, within such a three-dimensional biological sample in the form of a cell cluster. Typical diameters of these cell clusters range from 100 to 400 µm, although diameters in the millimeter range are also possible.

[0004] These studies are currently conducted primarily through visual observations and only rarely through video microscopy with subsequent image analysis. The former are time-consuming and always involve subjective assessment. The latter has the disadvantage of requiring complex imaging optics and sophisticated image analysis, coupled with considerable computational effort. Furthermore, its inherent sensitivity to minor shifts is a disadvantage.

[0005] Non-optical methods such as impedance measurements only work in contact with the sample. However, if the sample shape deviates from a plane (adherent monolayer) or is even three-dimensional, and is also freely floating in a medium, the aforementioned techniques are not applicable.

[0006] Automated imaging methods have the disadvantage that, for example, with a depth of field of 10 µm and the aforementioned typical cell cluster size of 100 to 400 µm, 10 to 40 image planes of the sample would have to be measured. With a typical minimum measurement time of approximately 10 seconds to detect movement, and the additional time required for repositioning or refocusing, such methods are not suitable for quickly monitoring a large number of samples.

[0007] Serial measurement of large numbers of samples is generally not advisable due to the relatively long observation times inherent in the timescales of biological dynamics. In practice, however, there is a need to perform such motion detection on a large number of separated samples, for example, in a multiwell plate, also known as a microtiter plate, with 96 or 384 wells. Imaging techniques are unsuitable for the parallel measurement of a large number of such samples because, for geometric and spatial reasons, it is difficult to position an imaging optical device at each well of the multiwell plate.

[0008] The article: JA COLE ET AL: "Laser speckle spectroscopy-a new method for using small swimming organisms as biomonitors", BIOIMAGING, Vol. 4, No. 4, December 1996, pages 243-253 discloses a point-by-point measurement of a speckle pattern with subsequent Fourier transformation (FFT) for the analysis of the movement of micro-organisms.

[0009] It is therefore an object of the invention to provide a device for detecting movement in a biological sample with spatial extent, which avoids the disadvantages of conventional devices.

[0010] This problem is solved by the device with the features of the independent claim. Advantageous embodiments and applications of the invention are set forth in the dependent claims and are explained in more detail in the following description with partial reference to the figures.

[0011] The invention is based on the technical insight that optical methods are most suitable for contactless monitoring and that, since the exact location of any movement within the sample is not known in advance, it is necessary to illuminate the entire sample. Because the sample typically has a considerable depth, i.e., greater than the depth of field of an imaging optic, the interactions of the light with the sample must be measured cumulatively along its entire path through the sample. Transmission methods are therefore unsuitable, as they would measure precisely the non-interacting portion of the light, and the expected fluctuations would be accompanied by a high level of background noise, rendering the technique insensitive.The inventive approach is therefore based on detecting the scattered, polarized, and / or diffraction radiation of the exposed sample, searching for fluctuations caused by sample movement in the portion of the light that has been altered in its beam direction, polarization state, and / or diffraction pattern by interaction with the sample. For this purpose, it is advantageous to separate the transmitted light from the scattered, polarized, and / or diffraction radiation using suitable filters. Within the scope of the present invention, the term "light" is used instead of "radiation." Both terms are to be considered synonymous within the scope of the present invention and encompass electromagnetic radiation in the visible, IR, and UV ranges.

[0012] Outside the scope of the invention, a method for the optical in vitro detection of movement in a biological sample with spatial extent is provided.

[0013] The aforementioned method comprises providing a fixture for the sample, a light source, optics, and a detector. The optics are configured to illuminate the entire sample within the fixture with radiation emitted from the light source and to direct at least a portion of the radiation from the light source—which is altered at any point within the sample by interaction with the sample in terms of its beam direction, polarization state, and / or diffraction pattern—onto a detection surface of the detector. This allows the interactions of the radiation emitted by the light source with the sample to be measured cumulatively along its entire path through the sample.The detector is designed to generate a measurement signal depending on the detected radiation, the temporal profile of which indicates a temporal profile of the intensity of the detected radiation and / or from which the temporal profile of the intensity of the detected radiation can be derived.

[0014] The aforementioned method further includes illuminating the sample with radiation from the light source and detecting movement in the biological sample as a function of a temporal change in the measurement signal.

[0015] A particular advantage of this approach is that the sample dynamics can be directly derived from the measured quantity, since fluctuations caused by sample movement in the portion of the light that has been altered by interaction with the sample in terms of beam direction, polarization state, and / or diffraction pattern are directly visible as fluctuations in the measurement signal. Thus, the complex processing of the measurement data required in imaging techniques can be avoided.

[0016] For example, the method can be designed to detect movement in the sample if a change in the measurement signal exceeds a predetermined threshold. When monitoring contractions in muscle tissue, movement can also be detected if the temporal change in the measurement signal exhibits periodicity.

[0017] The method can be performed using relatively simple optical elements. Optics for focusing on individual image planes and for successively scanning the sample volume are not necessary. Therefore, the device for carrying out the method can be inexpensive and compact.

[0018] Due to the simple evaluation of the measurement signal and the compact design, the method is also suitable for the parallel monitoring of a large number of samples and can be integrated efficiently into screening environments or automated high-throughput processes, e.g. high-throughput screening methods.

[0019] According to a particularly preferred embodiment, the detector is single-channel or outputs a single-channel measurement signal. The measurement signal preferably indicates only the radiation intensity incident on the detector surface per unit of time. Movement in the sample can thus be detected directly based on a temporal change or fluctuation of the signal.

[0020] The detector is preferably a non-imaging detector or a detector with a non-spatially resolved measurement signal, e.g., a non-spatially resolved photodetector, such as a photodiode. Such detectors are compact and cost-effective.

[0021] A biological sample with spatial extent is understood to be a three-dimensional biological sample, e.g. in the form of a three-dimensional cell and / or tissue culture or a cell cluster.

[0022] The diameter of the biological sample can be at least 50 micrometers (µm) in at least one spatial direction, and preferably at least 50 micrometers in all spatial directions, and is often more than 100 µm in all spatial directions.

[0023] The diameter of the sample is preferably in the range of 100 µm to 5 mm, more preferably in the range of 100 µm to 1 mm. The biological sample can in particular be a sample of muscle tissue and / or of living cells, i.e., cells that exhibit active dynamics, i.e., cells that can initiate movement.

[0024] The detection of movement in a biological sample refers specifically to movement within the sample itself or movement of a component of the biological sample. In other words, it generally means being able to detect dynamic phenomena in or within biological samples with spatial extent. In cell cultures of muscle cells, such movements can be triggered, for example, by the contraction of individual muscle cells.

[0025] The biological sample can also be a sample of free-swimming microorganisms, such as sperm. In this case, the method can be used to detect the movement of the free-swimming microorganisms, for example, in the case of sperm, to determine sperm motility.

[0026] The optics can include an illumination optic arranged on the illumination side, by means of which the radiation from the light source is directed onto the entire sample in order to illuminate the sample completely and as uniformly as possible.

[0027] The optics can further include a detection optic by means of which the light emitted by the sample, which is changed in its beam direction, polarization state and / or diffraction pattern by an interaction with the sample, is directed onto a detection surface of the detector.

[0028] This functional property of the optics can be realized using one or more suitably arranged and designed known optical components, such as filters, lenses, apertures, refractive elements, etc., which will be explained below using further examples.

[0029] An advantageous embodiment provides that the optics are designed and / or the detector is arranged relative to the illumination beam path and the sample in such a way that no beam paths exist in which radiation from the light source transmitted through the sample hits the detector and / or in which light from the light source hits the detector by bypassing the sample.

[0030] According to this design variant, only the light from the light source that has been altered in its beam direction, polarization state, and / or diffraction pattern by an interaction with the sample reaches the detector, while the optics prevent transmitted radiation or radiation bypassing the sample from reaching the detector's detection surface. This reduces interfering background signals and increases the sensitivity of the measurement.

[0031] In the device of the invention, movement within the sample is detected by means of a change in the diffraction pattern. According to the invention, the light source generates coherent light. Furthermore, the optics are configured, e.g., by means of a spatial filter, to image a boundary region of a diffraction pattern, which is generated by light diffracted through the sample from the light source, onto the detector. Movement within the sample causes a change in the diffraction pattern, for example, a speckle pattern. Investigations within the scope of the invention have shown that the change in the diffraction pattern at its center is difficult to measure because the relative change in radiation intensity is small. At the boundary, however, the change can be reliably detected and can, for example, lead to a momentary change from a local diffraction maximum to a local diffraction minimum or vice versa in the diffraction pattern.In a diffraction pattern, each point of the pattern contains the diffraction information of the entire sample.

[0032] In an advantageous variant of this design, the optics comprise a pinhole aperture which is arranged between the sample and the detector such that a hole of the pinhole aperture is located at the edge of the diffraction pattern produced by the sample.

[0033] A pinhole aperture is understood to be a hole-shaped opening, preferably a small one, and preferably without a lens. Pinhole apertures serve to collect light in a localized manner. For the same purpose, the end faces of optical fibers have long been used, particularly in confocal microscopes.

[0034] Preferably, the boundary region of the diffraction pattern encompasses all observation angles at which no transmitted light is received. The diffraction pattern is caused by positive and negative interference of light waves diffracted by objects, in this case, the sample. Whether interference is positive or negative depends on the object size, the wavelength of the light, and the observation angle. Light passing through the sample without interaction, i.e., transmitted light (ballistic photons), has an observation angle of 0° and strikes the center of the diffraction pattern. The diffraction pattern is overexposed by the transmitted light, and the signal-to-background (S / B) and signal-to-noise (S / N) ratios decrease drastically. Therefore, preferably, the boundary region of the diffraction pattern encompasses all observation angles at which no transmitted light is received.Since the entire sample is irradiated here and the illumination is not collimated, the "reception area" of the transmitted radiation is larger than just a point.

[0035] According to another variant of this design, the aperture can also have several holes arranged relative to the diffraction pattern such that they are located in an edge region of the diffraction pattern. In this case, the holes must be arranged so that the superposition of the diffracted radiation passing through the holes onto the detector enhances the diffraction effect and does not diminish it.

[0036] According to another variant of the design, which detects movement in the sample based on a change in the diffraction pattern, the optics can include an aperture positioned between the light source and the sample. This aperture is designed such that radiation from the light source exiting through its opening does not directly strike the aperture, i.e., it bypasses the sample. Furthermore, the optics can include a refractive optical element, i.e., a radiation-refracting element such as a convex lens or prism, positioned between the light source and the sample. This refractive element is designed such that radiation deflected by it does not directly strike the aperture. These variants represent a cost-effective and easily adjustable example of an optics system that directs only the diffracted radiation to the aperture.

[0037] According to a further embodiment not according to the invention, movement in the sample is detected by means of a fluctuation in polarized light caused by the movement. According to this embodiment, the optics comprise a first polarization filter and a second polarization filter having different polarization directions, wherein the first polarization filter is arranged between the light source and the sample and the second polarization filter is arranged between the sample and the detector. Movement in the sample leads to a change in the interaction of the polarized light with the sample and to a change in the polarization states, which results in a fluctuation in the detector signal.

[0038] The detector and the second polarization filter can be positioned on the opposite side of the sample, to the side of the sample, or on the same side as the light source, relative to the light source.

[0039] According to a further embodiment not in the invention, movement in the sample is detected by means of a fluctuation in the light scattered by the sample caused by the movement. In this case, the detector for detecting scattered light from the sample can be arranged on the same side of the sample (1) as the light source. Alternatively, the detector for detecting lateral scattered radiation from the sample can be arranged obliquely and / or laterally to the sample with respect to the direction of the illumination beam.

[0040] These variants offer the advantage that no optical components such as apertures are necessary to prevent transmission radiation from reaching the detector.

[0041] Furthermore, the detector for transmittal detection of scattered light from the sample can be arranged in the direction of transmission towards the sample. According to this variant, the optics include an aperture arranged between the light source and the sample, configured to block light rays in the illumination beam path that would otherwise reach the detector as transmitted rays through the sample. Alternatively or additionally, the optics can include a refractive optical element arranged between the light source and the sample, configured to alter the direction of the illumination beam path so that light rays transmitted through the sample do not reach the detector.

[0042] Furthermore, it is advantageous if the optics have a bandpass filter arranged in front of the detector, which is designed to suppress ambient light. This can further increase the sensitivity of the detection.

[0043] According to another advantageous variant, the optics, particularly the illumination optics, include an axicon. The use of an axicon offers the advantage of more homogeneous illumination of spatially "deeper" samples compared to convex lenses, since the focus of an axicon extends along the optical axis—rather than at a single point. A further advantage of an axicon is the simple spatial filtering of the incident (unaffected) light, as it is refracted at a constant angle to the optical axis.

[0044] One possible implementation involves the sample being located on a support matrix. The sample receptacle can thus comprise a support matrix, preferably a biopolymer. Alternatively, the sample can be contained within a suspended droplet. Furthermore, the sample receptacle can be configured as a support matrix, preferably a biopolymer such as alginate, contained within a suspended droplet.

[0045] For use of the device of the invention in screening environments, the sample receptacle can be a cavity of a multiwell plate (microtiter plate). Alternatively, the receptacle can be a cavity of a multiwell plate designed to form a hanging droplet at each cavity (so-called hanging-drop multiwell plate). Such hanging-drop multiwell plates are offered, for example, by Insphero AG, CH-8952 Schlieren, under the name "GravityPLUS™ 3D Culture and Assay Platform". Patent EP 2342317 B1 also discloses such a plate.

[0046] It has already been mentioned above that the device of the invention is suitable for the parallel monitoring of a large number of samples, e.g. samples that are to be examined as part of automated high-throughput procedures.

[0047] The invention therefore provides for the parallel optical detection of movement in several separate biological samples. The sample holder is preferably a multiwell plate with a plurality of cavities arranged in rows and columns. The detector is designed as a detector array, preferably a photodiode array, wherein the grid spacing of the individual detectors corresponds to the grid spacing of the cavities of the multiwell plate.

[0048] The light source is designed to illuminate the individual cavities. According to the invention, the light source for illuminating the samples in the cavities is designed as a laser diode array, wherein the grid spacing of the individual laser diodes corresponds to the grid spacing of the cavities of the multiwell plate. A laser diode array represents a space-saving and energy-efficient illumination source.

[0049] To ensure efficient heat dissipation, the mounting of the laser diode array can be made of a thermally conductive material, preferably aluminum. Furthermore, according to this variant, the optics can comprise a lens array, e.g., a microlens array, wherein each lens of the lens array is assigned to one of the laser diodes and the lenses direct the light from the laser diodes into the cavities.

[0050] Instead of a laser diode array as the light source, the light source can be a conventional light source (laser, arc lamp, etc.) outside the scope of the invention. The light from the light source is coupled into the individual cavities containing the samples via a fiber optic bundle. Each fiber optic strand is assigned to one cavity. This offers the advantage that the light source can be operated at a sufficient distance from the sample to avoid excessive heat generation near the sample.

[0051] Furthermore, according to another alternative approach, the multiwell plate can be illuminated across its entire surface with a light source. This is a simple implementation option, but it has disadvantages in terms of energy efficiency, as the light source must be correspondingly powerful. It is advantageous here if the light is parallelized and directed precisely onto the cavities using appropriately designed condenser optics and, if necessary, angle-dependent transmission filters.

[0052] According to the invention, a device for the contactless in vitro detection of movement in a biological sample with the spatial extent of claim 1 is provided. The device comprises a receptacle for the biological sample, a light source, a detector, and optics configured to illuminate the entire sample in the receptacle with radiation emitted by the light source and to direct at least a portion of the radiation from the light source, which has been altered in its diffraction pattern at any point within the sample by interaction with the sample, onto a detection surface of the detector. The detector is configured to generate a measurement signal, depending on the detected radiation, which indicates the temporal profile of the intensity of the detected radiation and / or from which the temporal profile of the intensity of the detected radiation can be derived.

[0053] The device also includes an evaluation unit designed to detect movement in the biological sample by displaying and / or evaluating temporal changes in the detected radiation.

[0054] To avoid repetition, features disclosed purely by way of procedure shall also be deemed disclosed by way of device. The aforementioned aspects and features of the invention, in particular with regard to the design of the optics, the detector, the recording (e.g., as a multiwell plate, hanging drop or hanging-drop multiwell plate), and the light source, therefore also apply to the device.

[0055] The preferred embodiments and features of the invention described above can be combined in any way desired. Further details and advantages of the invention are described below with reference to the accompanying drawings. These show: Figure 1 is a highly schematic representation of a method and apparatus according to an embodiment for understanding the invention; Figures 2A-2E are embodiments outside the invention that use scattered light from the sample for motion detection; Figures 3A-3C are embodiments outside the invention that use polarized light for motion detection; Figures 4A-4C are embodiments for understanding the invention that use a diffraction pattern from the sample for motion detection; Figure 5A is a sample in the form of a cardiac muscle tissue model on a support matrix; Figure 5B shows a speckle pattern at two successive time points; and Figure 5C shows a time course of an exemplary measurement signal.

[0056] Identical parts are marked with the same reference symbols in the figures and are not described separately.

[0057] Figure 1shows a highly schematic representation of a method and a device according to one embodiment for understanding the invention.

[0058] To carry out the procedure, a device 100 for optical in vitro detection of movement in a biological sample 1 with spatial extent is provided.

[0059] The device 100 comprises a receptacle (not shown) for the three-dimensional sample 1, a light source 6, optics 7, 8 and a detector 2.

[0060] The uptake is not limited to a specific type of uptake, but can be appropriately designed depending on the application and type of sample, e.g. as a support, support plate, vessel, cavity of a multiwell plate, or as a support matrix in the form of a biopolymer, e.g. alginate, on which the sample is grown.

[0061] The light source 6 can be, but does not have to be, a coherent light source, e.g., a laser. Only those implementation variants that generate a diffraction pattern of the sample for motion detection (see below) require a coherent light source. Figures 4A to 4C , 5A to 5C Those who use coherent radiation require it.

[0062] The representation of optics 7, 8 in Figure 1 The diagram is merely schematic and is intended to illustrate the functional properties of optics and not to represent specific optical elements, since in principle a large number of concrete optical design variants are possible, some of which are described as examples in the following figures.

[0063] This functional property of optics can be realized using one or more suitably arranged and designed known optical components, such as filters, lenses, apertures, refractive elements, etc.

[0064] The optics 7, 8 comprise an illumination optic 7 arranged on the illumination side, by means of which the radiation 10 from the light source 6 is directed onto the entire sample 1 in order to illuminate the sample completely and as uniformly as possible. The illumination optic can include suitable optical elements or components for beam shaping, such as apertures, lenses, and / or filters. The use of an axicon is particularly advantageous.

[0065] The optics 7, 8 further comprise a detection optic 8, by means of which the light 11 emitted by the sample, i.e. light from the light source 6, which has been altered in its beam direction, polarization state and / or diffraction pattern by an interaction with the sample 1, is directed onto a detection surface 2a of the detector 2. Figure 1For example, a scattering process is shown at point P1, in which light 11 is scattered towards detector 2 and focused onto detector 2 by means of the detection optics 8. Since the entire sample 1 is illuminated uniformly, the interaction of the incident light 10 can take place at any point within the sample 1, so that the interactions of the light with the sample are cumulatively measured by detector 2 along its entire path through the sample.

[0066] The detection optics 8 can further include optical elements that ensure that light which has not been altered in its beam direction, polarization state, and / or diffraction pattern by interaction with the sample does not reach the detector 2. For example, the detection optics 8 can have a bandpass filter arranged upstream of the detector input that filters out or suppresses ambient light but allows light with a wavelength from the light source 6 to pass through. This requires the use of a monochromatic light source or the placement of a suitable filter at the output of the light source to illuminate the sample with only a specific wavelength of light.

[0067] Furthermore, the detection optics 8 can block beam paths by means of apertures, lenses, etc., in which radiation from the light source 6 transmitted through the sample 1 would hit the detector and / or in which light from the light source 6 would hit the detector 2 by bypassing the sample.

[0068] Additionally or alternatively, the detector can also be arranged so that no transmitted light 12 hits its detector surface 2a, e.g. by arranging the detector 2 laterally relative to the illumination beam path 10, as in Figure 1 illustrated.

[0069] The detector 2 is designed to generate a measurement signal 9 depending on the detected radiation 11, the temporal course of which indicates a temporal course of an intensity of the detected radiation 11.

[0070] The detector signal 9 thus corresponds to a full-volume measurement of the sample. The detector 2 is preferably single-channel, so that only one measurement 9 is generated, which corresponds to the fluctuation of the light intensity detected by the detector per unit time. The detector 2 is, for example, a conventional photodiode 2.

[0071] With the in Figure 1 In the illustrated device 100, the sample 1 is illuminated and the corresponding measurement signal 9 is evaluated.

[0072] If movement occurs in sample 1, e.g., a contraction in the case of cultured muscle tissue, then the interaction of sample 1 with the light incident on the sample also changes due to the movement in the sample; that is, the proportion of the light incident on the sample 11 that is altered in its beam direction, its polarization state and / or diffraction pattern by an interaction with sample 1 changes and generates a change in the detector signal 9. According to the method, movement in the biological sample can thus be detected by means of the fluctuation of the detector signal 9.

[0073] The following describes some exemplary embodiments, which represent specific configurations of the [design / method]. Figure 1The solution approach shown is illustrated. In Figures 2A to 3C, the light source 6 is not shown, but is located above the optics and detector arrangement shown, as can be seen from the beam path 10.

[0074] The Figures 2A to 2E do not show embodiments according to the invention that use scattered light from the sample for motion detection. In the Figure 2A and 2B Detector 2, for the transmission detection of scattered light 11 from sample 2, is arranged in the direction of transmission towards sample 1. To prevent rays transmitted through sample 2 from reaching detector 2, a Figure 2AAn aperture 3 is arranged to block light rays that would otherwise reach detector 2 as rays transmitted through sample 2 or that could reach detector 2 laterally past the sample. The illumination optics in the form of the aperture 3 thus only allow radiation 12 transmitted through the sample that does not reach detector 12.

[0075] The special feature of the in Figure 2B The illustrated embodiment is that instead of a large aperture 3, a smaller aperture 3 is used, to which a refractive optical element, e.g. a lens, an axicon, etc., is arranged, which changes the direction of the illumination beam path 10 passed through the aperture 3 in such a way that rays 12 transmitted through the sample 2 cannot hit the detector 2.

[0076] The special feature of the in Figure 2CIn the illustrated embodiment, the detector 2 for detecting scattered light in the form of backscatter from sample 1 is arranged on the same side of sample 1 as the light source 6, with the detector surface 2a again facing sample 2. This offers the advantage that no aperture is necessary to block transmitted light. However, an aperture surrounding detector 2 is provided here to reduce the effects of interfering light sources.

[0077] The special feature of the in the Figures 2D and 2E The difference in the illustrated embodiment is that lateral scattered light 11 is detected. For this purpose, the detector 2 is arranged laterally to the direction of illumination. Figure 2DA refractive optical element, e.g. a lens or prism 4, is arranged between the light source and sample 1, which changes the direction of the beam path so that light from the illumination beam path 10 cannot directly, i.e. bypassing sample 1, hit the detector 2.

[0078] The in Figure 2E The depicted version differs from the version in Figure 2E by using an aperture 3a instead of the refractive optical element 4, which has an opening to narrow the beam path 10, so that in turn light from the illumination beam path 10 cannot directly, i.e. bypassing the sample 1, hit the detector 2.

[0079] The Figures 3A to 3CFigures do not show embodiments according to the invention that use polarized light for motion detection. The illumination optics comprise a first polarization filter 5a, which is arranged between the light source and the sample 1. The detection optics comprise a second polarization filter 5b, which has a different polarization direction compared to the first polarization filter 5a and is arranged between the sample 1 and the detector 2, preferably at the detector input.

[0080] The detector 2 and the second polarization filter 5b can be arranged on the opposite side of sample 1, to the side of sample 1, or on the same side as the light source, as indicated by the different variants in the Figures 3A to 3C is shown.

[0081] Due to the different polarization directions of the two polarization filters 5a and 5b, polarization filter 5b only transmits light that has been "depolarized" by an interaction with the sample. The arrangement of the two polarization filters 5a and 5b thus ensures that no light transmitted through the sample or light that has bypassed the sample is detected.

[0082] Movement in the sample changes the proportion of depolarized light and leads to a fluctuation in the detector signal, so that movement in sample 1 can be directly detected from the fluctuation in the detector signal.

[0083] To reduce scattering effects, a refractive optical element 4, e.g. a convex lens or a prism, can be arranged in front of the first polarization filter 5a, which focuses the illumination beam path 10 onto the sample ( Figure 3A and 3C ).

[0084] In the version of the Figure 3B The detector 2, for the epidetection of scattered light in the form of backscatter from the sample 1, is arranged on the same side of the sample 1 as the light source 6. An aperture 3 surrounding the detector 2 can be provided here, which can reduce the influence of interfering light sources.

[0085] The Figures 4A to 4C Figure 1 shows embodiments for understanding the invention, which use a diffraction pattern of the sample, e.g., in the form of a speckle pattern, for motion detection. The light source 6 is a coherent light source, e.g., a laser diode. The light from the light source 6, diffracted by the sample, produces a diffraction pattern with a center Z of high intensity and a border region R of low intensity.

[0086] The detection optics comprise a pinhole aperture 3b arranged in front of the detector 2, which is positioned between the sample 1 and the detector 2 such that a hole 3c of the pinhole aperture 3b is located in the edge region R of the diffraction pattern produced by the sample.

[0087] According to the variant of Figure 4A The illumination optics comprise an aperture 3a arranged between the light source and the sample, which is designed such that radiation from the light source 6 exiting through an aperture opening 3d of the aperture 3a does not directly hit the hole 3c of the aperture 3b.

[0088] According to the variants of Figure 4B and 4CThe illumination optics comprise a refractive optical element 4a, e.g. a convex lens, arranged between the light source 6 and the sample 1, which is designed such that radiation diffracted by the refractive optical element 4a does not directly, i.e. bypassing the sample, hit the hole 3c of the aperture 3b.

[0089] In the variant of Figure 4C Furthermore, a bandpass filter 13 is arranged between sample 1 and aperture 3b, which filters out ambient light that does not correspond to the wavelength of the light source 6. With the Figure 4C The setup shown currently allows heart muscle beats to be detected with a sensitivity comparable to visual observation (under a microscope).

[0090] Figure 5A Figure 1 shows an example of sample 1 in the form of a cell cluster. The cell cluster is located in a hanging droplet, from which in Figure 5AOnly a portion is visible, formed in a cavity of a hanging-drop multititer plate. The sample consists of a stem cell-derived cardiac muscle tissue model adhered to alginate carrier beads 15. The in Figure 5A The sample shown has a diameter of approximately 1 millimeter.

[0091] Sample 1 is fully illuminated with a laser diode emitting light with a wavelength of 650 nm. The textured sample 1, structured on different size scales, diffractes the coherent light in a variety of ways, generating a complex diffraction pattern (so-called "speckle pattern") in the transmitted light direction. The slight deformations of these tissue structures caused by local cardiac muscle contractions lead to a change in the overall speckle pattern.

[0092] Figure 5BFigures 17a and 17b illustrate two different states of a speckle pattern at minimum and maximum contraction displacement. Figures 17a and 17b are for illustrative purposes only and are from a different experiment; they do not depict the speckle pattern of sample 1. Figure 5A The measurement principle, however, is the same. A spatial filter, e.g., the pinhole 3b, images a point in the edge region R of the diffraction pattern 17a, 17b onto the detector 2. The change in the pattern 17a, 17b now leads to a fluctuating amount of light transmitted by the pinhole 3b, and thus to a fluctuating detector signal 9, which in Figure 5C The periodic fluctuation of signal 9 corresponds to the periodic contractions in the muscle tissue. The representation in Figure 5CThis serves only to illustrate the point, but again does not show a measurement signal that was measured when the sample shown in Figure 1A was illuminated.

[0093] Although the invention has been described with reference to specific embodiments, it is apparent to a person skilled in the art that various modifications can be made and equivalents used as replacements without departing from the scope of the invention. Furthermore, many modifications can be made without departing from the relevant scope. Consequently, the invention is not intended to be limited to the disclosed embodiments but is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A device for non-contact in vitro detection of a movement in a biological sample with spatial expansion in the configuration of a three-dimensional cell and / or tissue culture or a cell cluster or a sample of free-floating microorganisms, wherein the device is configured for parallel optical detection of a movement in a plurality of biological samples separated from each other, comprising: - a receptacle (15, 16) for the biological sample (1), wherein the receptacle for the samples is a multi-well plate or a hanging-drop multi-well plate comprising a plurality of cavities arranged in rows and columns for receiving the samples, - a light beam source (6) which is configured as a laser diode array for illuminating the samples in the cavities, wherein a grid spacing of the individual laser diodes corresponds to the grid spacing of the cavities of the multiwell plate and lenses guide the light of the laser diodes into the cavities, - a detector (2), wherein the detector is configured as a detector array, preferably as a photodiode array, wherein a grid spacing of the individual detectors corresponds to a grid spacing of the cavities of the multiwell plate, - optics (7, 8) which is configured to illuminate the entire sample in the image with radiation emanating from the light beam source and to direct at least a portion of the radiation (11) from the light beam source (6), the diffraction pattern of which has been altered at any point within the sample (1) by an interaction with the sample (1), onto a detection surface (2a) of the detector (2), wherein the detector (2) is configured to generate a measurement signal (9) dependent on the detected radiation (11), the time characteristic of which indicates a time characteristic of the intensity of the detected radiation and / or from which the time characteristic of the intensity of the detected radiation can be derived, wherein the device further comprises an evaluation unit which is configured to detect a movement in the biological sample by displaying and / or evaluating a temporal change in the detected radiation.

2. Device according to claim 1, characterised in that the optics (7, 8) are configured and / or the detector (2) is arranged relative to the illumination beam path (10) and the sample (1) in such a way that no beam paths exist in which radiation (12) from the light beam source (6) transmitted through the sample (1) strikes the detector (2) and / or in which light from the light beam source strikes the detector while bypassing the sample.

3. Device according to one of the preceding claims, characterised in that the optics comprise a bandpass filter (13) arranged in front of the detector (2), which is configured for spatial light suppression.

4. Device according to one of the preceding claims, characterised in that the receptacle for the sample is a hanging-drop multi-well plate.

5. A device according to any one of the preceding claims, characterised in that, a) that the holder of the laser diode array is provided from a thermally conductive material, preferably aluminium; and / or b) that the optics comprises a microlens array, wherein each lens of the lens array is associated with one of the laser diodes.