Method and microscope for determining flow characteristics in sample chamber through which medium flows

Abstract

To provide a method and a microscope for determining flow characteristics in a sample chamber through which a medium flows.SOLUTION: Provided is a method for determining flow characteristics in a sample chamber through which a medium flows, the method including: a step of, at a plurality of time points over a measurement period, detecting detection radiation from the confocal volume generated in a sample chamber by using a plurality of detector elements of a detector in a form of a detector array, and a step of selecting at least one pair of the detector elements. The detector is arranged on a plane conjugate to a rear image surface of an objective lens, and the measurements from the plurality of detector elements may be individually analyzed. The cross-correlation between the measurements obtained from at least one pair of detector elements is generated and analyzed in two directions. The present invention is characterized in that the confocal volume is generated at least at two positions different from each other in the sample chamber, and speed and optionally a moving direction are determined as the flow characteristics of a medium at each of the positions. These are used to create a flow profile over at least one area of the sample chamber.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for determining flow characteristics in a sample chamber through which a medium flows The present invention further relates to a microscope suitably configured for carrying out said method. [Background technology]

[0002] In the field of microscopy, especially confocal laser scanning microscopy, it is of interest to identify the movements of objects in a sample chamber and to determine their characteristics, such as speed and direction. Of particular interest here is the provision of data on flow profiles to evaluate the effect of the design of a sample chamber, for example a tube, a reaction chamber or a miniaturized reaction chamber (cavity), for example in a cell culture chamber ("Lab-on-a-chip", "organ-on-a-chip").

[0003] Patent document 1 discloses a method for investigating molecular motion. The method of fluorescence correlation spectroscopy (FCS) is used here, for example, to investigate the dynamics of molecular behavior inside cells. This method, also called FCS for short, is available in various variants (e.g., Non-Patent Document 1 and Non-Patent Document 2). One variant is known as "spot-variation FCS" (e.g., Non-Patent Document 3). Here, excitation radiation is focused and directed into a sample chamber. As a result of the focusing and broadening of the focus of the focused beam of excitation radiation both transversely (x or y direction) and in the propagation direction (z direction) to the propagation direction (usually the optical axis), what is known as a confocal volume (also called "focus" for simplicity in this context) is illuminated in the sample chamber, in conjunction with confocal detection. Brightness information (measurements) can be obtained for confocal volumes of different sizes. The brightness information is obtained by a photodetector (e.g., a photodiode).

[0004] A variant of the spot variation FCS is known from the publication by Scipioni et al. (Non-Patent Document 4). For detection purposes, a spatially resolved surface detector is used, whose detector elements can be read and evaluated individually and independently of each other. In particular, a detector type called "Airyscan detector" is used by Scipioni et al., which is arranged in an intermediate image plane of the detection beam path, each detector element of which acts as an individual pinhole (Non-Patent Document 5).

[0005] US Patent No. 5,399,633 describes the targeted readout of measurements from selected pairs of such two-dimensional detectors. Cross-correlation can be used to determine the speed and direction of moving molecules that emit detection radiation in the form of fluorescent radiation within biological cells. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] DE 102023201620 [Non-patent literature]

[0007] [Non-Patent Document 1] Lenne et al.,EMBO J.25,2006:3245-3256 [Non-Patent Document 2] Wenger et al., Biophys. J.92, 2007:913-919 [Non-Patent Document 3] Wawreznieck et al.,Biophys. J.89,2005:4029-4042 [Non-Patent Document 4] Scipioni et al.,Nature Communications,2018,DOI:10.1038 / s41467-018-07513-2 [Non-Patent Document 5] Huff, Nature Methods, Application Notes, December 2015 Summary of the Invention

[0008] The object of the invention is to propose the possibility of determining the flow characteristics in sample chambers through which a medium flows, in particular in sample chambers of small dimensions. This object is achieved by the subject matter of the independent and dependent claims, which relate to advantageous developments.

[0009] The method according to the invention for determining the flow characteristics in a sample chamber through which a medium flows comprises the following steps: In step A, detection radiation originating from a confocal volume generated in the sample chamber is detected. Detection is carried out at multiple time points over a measurement period (also called measurement period, measurement interval, for example 10 seconds to several minutes per measurement duration in each case) with a plurality of detector elements of a detector array, the detector array being arranged in a plane conjugate to the rear image plane of the objective lens. This conjugate plane is also called the "pinhole plane". The measurement values ​​from the detector elements can each be read out and analyzed individually. It is also possible to combine the measurement values ​​from each selected detector element with one another ("binning").

[0010] Furthermore, at least one pair of detector elements is selected, each of the two partners of the pair being formed by at least one detector element, the pair being formed by the at least one selected detector element and a further selected detector element.

[0011] In a further method step (step B), a cross-correlation of the acquired measurements is made between them from the measurements for at least one pair. In this process, in a first sub-step B1, the cross-correlation is calculated starting from one of the selected detector elements in a first direction up to a further selected detector element. This first direction may also be called the forward direction.

[0012] In a second substep B2, further cross-correlations are calculated starting from the further selected detector element in a second direction (backward) to the initially selected detector element. In step C, the cross-correlation or its results are analyzed for occurrences of changes in intensity of measurements from selected detector elements.

[0013] The method according to the invention is characterized in that confocal volumes are generated in at least two mutually different positions of the sample chamber, whereby in order to accommodate the different positions, the confocal volumes can here optionally overlap by up to 50% of their extent in the z direction or in their cross sections in the x and / or y directions relative to the z direction.

[0014] In this document, the direction of the main flow of the medium in the sample chamber is referred to as direction x, and the direction perpendicular thereto is referred to as direction y, according to the Cartesian coordinate system. The z-axis (direction z) is perpendicular to both directions, along which the detection radiation is usually detected.

[0015] The positions at which the measurements are taken can be selected manually, for example using a suitable user interface, or automatically. For example, the user can specify rules according to which the number and location of the positions are determined. Here, it is possible to select, for example, absolute positions in the sample chamber or positions respectively relative to the sample located in the sample chamber. The positions can also be selected using previously acquired overview images of the sample chamber and / or the sample. The sample referred to here is an artificial or biological structure located in the sample chamber and towards which a medium flows and / or which is surrounded by a medium. The artificial structure is mainly composed of technical materials such as metals, alloys and plastics, but may optionally be composed of biological materials, for example vascular implants. The biological structure includes, for example, cells, cell layers, spheroids, organoids and tissues of organisms or whole organisms.

[0016] At each of the locations, the velocity and optionally the direction of movement of the medium are determined based on the cross-correlations, in particular by calculating multiple cross-correlations across different orientation pairs and fitting all the curves thus obtained with direction and / or velocity as parameters.

[0017] The velocities per position and, where appropriate, the movement directions are assigned to one another and retrievably stored to create a velocity profile, or, if the respective movement directions are added, a flow profile, over at least one spatial region of the sample chamber.

[0018] In this document, the term direction is used in the sense of a vector with a starting point and a virtual propagation line in relation to the calculation of the cross-correlation. This means that in the horizontal, for example, a first direction (forward) runs from the starting point towards 0° and a second direction (reverse) runs from the starting point towards 180° (see also FIG. 4). This is also true for (polar) coordinate systems of different orientations.

[0019] The essence of the invention is to obtain information of the flow in a flowing medium, for example by the movement of a particular object, using measurements from selected, in particular individually readable detector elements, resulting in the generation of a flow profile over at least one region of the sample chamber. Here, for each measurement point (position), the velocity currently reached there and, optionally, the direction of movement of the object are determined. To simplify matters, in the following text, reference is made to a flow profile, even if only a velocity profile may be created. The flow profile is generated from measurements for a number of different measurement points. The measurements combined in the flow profile are advantageously determined within a defined measurement period in order to ensure their interaction or their simultaneous presence. Within the scope of this description, objects refer to molecules, for example proteins, nucleic acids and their derivatives, in particular aggregates, complexes, agglomerates and chelates.

[0020] The object used to determine the flow properties is preferably labeled with a fluorescent dye or is itself capable of emitting fluorescent radiation (fluorochrome). The emitted fluorescent radiation that is detected as detection radiation is caused in particular by excitation radiation emitted into the sample chamber.

[0021] In order to preserve the sample in the sample chamber and / or the objects in the medium, the excitation radiation is in each case shaped and directed only into a selected region of the sample chamber, the local excitation being advantageously synchronized with the detection of the detection radiation at the respective position.

[0022] Advantageously, existing configurations of optical devices suitable for generating confocal volumes, for example microscopes, can be used in embodiments of the invention to verify the data without additional equipment costs. Here, the situation is exploited that each of the detector elements images a slightly different part of the confocal volume. Thus, a number of mutually different detection foci are generated and their measurements are analyzed, the very high precision of the manufacture and positioning of the detector elements and their upstream optical elements, for example microlens arrays in so-called Airy Scan detectors (Non-Patent Document 5) and arrays of SPAD (Single Photon Avalanche Diode) detectors, advantageously requiring very precise knowledge of the distance of the respective confocal volume. Compared to solutions from the prior art, precise knowledge of the position and nature of the respective confocal volume advantageously makes it possible to dispense with its calibration, for example using known dye molecules as references.

[0023] The basic procedure according to the invention is illustrated by the following example, based on an Airyscan detector (5) with 32 channels. Further detectors suitable for implementing the invention are, for example, SPAD arrays and other surface detectors with individually readable or combined ("binned") detector elements.

[0024] As can be seen by way of example from figure 4, by using an Airyscan detector, six cross-correlations can be calculated (see below) if a spacing of one detector element remains between the paired detector elements and the outer detector elements are not used. In this case, the difference between the cross-correlations calculated for opposite directions indicates the movement of the object causing or emitting the detected radiation.

[0025] To determine the speed and direction of a moving object, it is possible to make an experimental cross-correlation of the form specified in equation [1] or to make an experimental cross-correlation by a more detailed model.

[0026] The formula for [1] is as follows:

[0027]

number

[0028] where v is the flow velocity (a fit parameter), α is the angle between the flow direction and the vector connecting the detector elements (a fit parameter), and r 0 is the distance between the foci of a (extended) pair of detector elements, and w0 is 1 / e 2 is the radius, D is the diffusion coefficient, and G diff (τ) is the diffusion term (2D or 3D). The latter represents the autocorrelation in the absence of detector element motion.

[0029] Parameter r 0 cannot be calculated directly from the spacing between the detector elements, but must be determined empirically or theoretically. The reason for this can be found in the situation where the PSF (point spread function) of the illuminating radiation and the PSF of the detected radiation are not the same. On the one hand, the parameter r 0 can be determined by measuring a static sample, e.g., a color solution, and r 0 is inserted as a fitted parameter and the flow velocity is set to v = 0. Alternatively, r 0can be derived from the theoretical PSF, for example, by using cross-correlation with the theoretical PSF and adjusting ("fitting") them according to equation [1].

[0030] Fitting multiple cross-correlations for different directions improves the stability of the fit with respect to the angle α. In contrast to prior art methods using two foci, all cross-correlations can also be determined from a single set of measurements.

[0031] In a further method configuration, it is possible to combine several detector elements to form each one of the parts or partners of a pair, referred to below as an extended pair, in which case the measurements acquired for each partner of the extended pair are mathematically combined, for example averaged, before the cross-correlation between the thus mathematically combined measurements of the two partners of the extended pair is determined.

[0032] The method according to the invention can be implemented using only one pair of detector elements or one extended pair. To improve the signal-to-noise ratio, in a further configuration, a group of several pairs of detector elements and / or several extended pairs can be defined. In this case, the virtual connecting lines of the center points of each pair or extended pair run substantially parallel to each other. The virtual connecting lines may start, merely by way of example, at the center points of different detector elements, but they all run, for example, towards 0° (see also FIG. 5). After the measurements acquired per pair are averaged, equation [1] can be applied again to the measurements from the pairs thus selected. The same applies for the second direction, i.e. the reverse direction, for example towards 180°. In these cases, the forward direction can also be called the common first direction and the reverse direction can also be called the common second direction.

[0033] In particular, for more detailed analysis and / or calculation, average values ​​of the cross-correlation in the first direction and / or the second direction generated from measurements from the pairs or extended pairs may in each case be formed and, optionally, stored for further use.

[0034] An advantage of the method according to the invention is the situation that several pairs or extended pairs can be analyzed simultaneously, whereby groups of pairs or extended pairs are advantageously analyzed and the respective common directions of the virtual connection lines between each (extended) pair of the group are arranged differently from one another.

[0035] Based on the cross-correlation results, the current concentration of the object excited for the emission of fluorescent radiation can also be advantageously determined. In an advantageous configuration of the method according to the invention, multiple cross-correlations can be calculated, for example, over multiple pairs / multiple extended pairs of different orientations, for example over at least two different groups, or average values ​​thereof (see above). Optionally, all curves thus obtained can be fitted parametrically with the corresponding directions and / or velocities (global fit).

[0036] When selecting detector elements of each pair / extended pair, it is possible to map sufficient diffusion paths or flow paths by having the selected pairs each spatially separated by at least one detector element that does not belong to the pair in question. Thus, the detector elements belonging to an extended pair can be selected to be separated from each other by at least a distance corresponding to the width of the detector elements.

[0037] As explained below, detector elements can be specifically selected that have sufficiently similar radii of the confocal volume and are the same distance from each other, however, results obtained using measurements from peripheral detector elements for local velocity and direction of movement have also proven to be valid and robust.

[0038] In order to improve the efficiency of the method according to the present invention, it is possible for the point spread function (PSF) of the illumination radiation used to generate the confocal volume and directed along the illumination beam path into the sample chamber and / or the detected detection radiation to be provided as a pair or extended pair partner for subsequent analysis or to be shaped so that suitable detector elements acquire measurements.

[0039] For example, the cross section of the illumination beam path can be stretched laterally for each beam path so that it has an elongated elliptical cross section. Such beam shaping can be achieved by a suitable slit diaphragm or by the effect of a suitably controlled spatial light modulator (SLM).

[0040] Optionally, further properties of the moving object can be derived based on the data ascertained by the method according to the invention, in particular based on the cross-correlation results. It is thus advantageously possible to determine the local temperature (micro-temperature) of the medium, for example the matrix or solvent of the sample, at the origin of the detected radiation.

[0041] Furthermore, it is possible to determine the viscosity of the medium at the origin of the detected radiation, or more precisely, the viscosity of each of the components of the detected radiation, for example on the basis of the so-called Einstein-Stokes equations [2].

[0042]

number

[0043] Where: D is the diffusion coefficient, kB is the Boltzmann constant, T is the absolute temperature, η is the dynamic viscosity of the medium, r is the hydrodynamic radius (Stokes radius) of the object.

[0044] The temperature dependence of several of the components of this relationship is evident from equation [2]. The viscosity η or its change can be determined, for example, when a pressure change occurs at constant temperature T. The invention may be advantageously used in flow-through sample chambers such as cavities of what are known as tubes, cell culture chambers, biochips, reaction chambers, flow chambers, etc., where the sample chamber is preferably a component of a microfluidic device or system.

[0045] For example, in one possible use, the influence of the walls of the respective sample chamber in the region of measurement on at least one velocity profile can be investigated by setting the wall properties in relation to the (relevant) determined flow properties. In addition to the dimensions, the material of the wall and / or the macro- and / or micro-structure of its surface can also be a wall property.

[0046] The high spatial resolution of the measurement, which can be advantageously achieved by the present invention, allows flow characteristics to be determined in very small sample chambers and allows flow profiles of correspondingly small scale to be created. The spatial resolution of the method according to the present invention is determined by the diffraction-limited range of the illumination volume. The resolution achievable by the method according to the present invention is less than 100 nm, in particular about 40 nm, in the direction x and in the direction y. In the direction z, a resolution of about 10 nm can be achieved.

[0047] Measurements in the direction z, i.e. the detection direction, can currently be measured with sufficient quality up to a depth of a few millimeters, for example less than 3 mm, for example about 1 mm, in particular up to about 500 μm, depending on the optical properties of the walls of the sample chamber, the medium and the fluorophores. For example, confocal volumes can be generated with a PSF that is qualitatively suitable for evaluation up to a depth (direction z) of 150 μm to 200 μm (for example at a working distance of 400 μm). The manifestation of the PSF here depends on the optical properties of the components involved as well as on the wavelengths of the excitation radiation and the emitted detection radiation and is of particular importance for the achievable quality of the acquired data. With further improvements and adjustments of the optical elements it is possible to achieve even greater depths.

[0048] In a further possible application of the present invention, both the flow behavior of objects located in the medium, such as moving or (freely) suspended cells, and their influence on the flow behavior in the sample chamber can be investigated. For example, if a cell or tissue is capable of generating a flow itself, for example by its cilia, then the generated flow and / or its interaction with further flows present in the sample chamber can also be investigated.

[0049] The flow characteristics, particularly the flow profile, can also be determined as a function of the presence and / or specific dimensions of objects serving as samples, such as spheroids, tissues, organoids and / or other three-dimensional structures.

[0050] A further application of the present invention is the measurement of flow properties in vivo within living blood vessels or vasculature, for example in the case of zebrafish, as long as they are optically transparent to the excitation and detection radiation during their development.

[0051] Due to the high achievable spatial resolution, for example, in the application of the method according to the invention, the influence of modifications of the wall of the sample chamber can be investigated. It is thus possible to determine the flow properties of the medium in a replica of a blood vessel in an untreated state and to compare them with the flow properties in a treated state, for example after the insertion of a vascular implant (stent).

[0052] A further field of application of the method according to the invention is fundamental biomedical research. Thus, for example, the distribution of pharmacologically active substances in the sample chamber and their supply to biological samples can be investigated and described. The method can also be used in material research, in particular in biomedical material research, but also in quality control. On the one hand, it is also advantageous that the flow properties of the medium and the transport behavior of pharmacologically active substances can be determined by the method according to the invention, and, moreover, using the same microscope, the absorption and location of active substances can be detected and investigated in biological structures, for example using the method described in WO 2005 / 023363. To do so, the sample chamber has the advantage that it only has to remain on the sample stage of the microscope.

[0053] The method according to the invention can additionally or alternatively be used to provide measurements as input data for models and / or to validate existing simulations. In addition to mathematical simulations and models, such input data can also be used, for example, for machine learning and artificial intelligence methods (collectively referred to as simulations and models). Both approaches can be implemented for both general and individual situations in the sample chamber. For example, simulations and models can be developed, refined, and applied with or without a sample in the sample chamber.

[0054] A fundamental advantage of the invention is that interference with the observed sample or with the sample chamber can be kept very low: no additional hardware is required, but existing systems can be used, only their control and evaluation are adapted to the method according to the invention.

[0055] The invention is typically practiced with wavelengths of excitation radiation in the range of about 440 nm to 640 nm, In further configurations, excitation wavelengths up to, for example, 1600 nm are possible when using multiphoton excitation.

[0056] To carry out the method according to the invention, a microscope can be used to determine the flow characteristics in the sample chamber through which the medium flows. For example, a microscope suitable for carrying out a fluorescence-based method such as fluorescence correlation spectroscopy (FCS) or FRAP (fluorescence recovery after photobleaching) microscopy can be used. Such a microscope can be, for example, a point-scanning or line-scanning microscope.

[0057] In further embodiments of the invention, the illumination with excitation radiation can be, for example, in the form of a thin light sheet, such that only objects in the regions that are illuminated layer-by-layer in the light sheet are excited to emit fluorescent radiation (see, for example, Singh, AP et al., "The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy", Optics Express, 21(7) (2013) 8652-8668).

[0058] In an advantageous embodiment, the microscope comprises a sample chamber, an objective lens for detecting detection radiation coming from a confocal volume generated in the sample chamber, and a detection beam path in which the detected detection radiation is directed to a detector array with a number of detector elements, the detector array being arranged in a plane conjugate with the rear image plane of the objective lens (image plane, pinhole plane), the measurement values ​​from the detector elements can be read out individually. The plane is in particular an intermediate image plane in which at least one pinhole is arranged in the confocal detection beam path. In the microscope according to the invention, the function of the pinhole is realized by individual detector elements of the detector.

[0059] As detector there may advantageously be one of the already mentioned Airyscan detectors or a SPAD array. For example, optical elements such as microlens arrays may be arranged in front of the detector elements. Other two-dimensional detectors with individually readable detector elements are also possible.

[0060] A microscope according to the invention can have a zoom optical unit, which can be used to vary the extent of the detected beam of detection radiation in a controlled manner, for example to adapt it to the size of the detection surface of the spatially resolved detector.

[0061] The microscope according to the invention also comprises an analysis unit configured to perform the method according to any of the method claims. Such an analysis unit may for example be a computer, a microcontroller or an FPGA.

[0062] Furthermore, control units are available which can be designed not only for controlling the components of the microscope but also for the selection of a position and / or for approaching a selected position. The selected position is approached by placing the sample chamber and / or the objective lens in a relative position in which the focus of the detection beam path of the microscope is directed towards the selected position.

[0063] The analysis unit and the control unit can be individual components of a microscope, in further embodiments they are correspondingly configured compartments of a computing device such as a computer or control circuitry.

[0064] The method according to the invention can be advantageously supported by adapting the beam shape of the illumination or detection radiation according to the current requirements. For this purpose, an iris diaphragm that can be set in a controlled manner can be arranged, for example, in the illumination beam path or the detection beam path of the microscope as a beam shaping optical unit. For example, a reduction in the diameter of the illumination beam path leads to an underfill on the input side of the objective pupil and thus to an increase in the confocal volume.

[0065] In an embodiment of the microscope according to the invention, a controllably settable (laser) beam expander can be arranged in the illumination beam path or in the detection beam path of the microscope as a beam shaping optical unit. The beam expander is typically based on a telescopic structure. The confocal volume can likewise be increased or decreased by the effect of the setting of the motorized variable beam expander.

[0066] In another embodiment of the microscope according to the invention, a slit diaphragm can be arranged in the illumination beam path or in the detection beam path as a beam shaping optical unit, so that a PSF that is elongated transversely to the beam path is generated. Similarly, the presence of optical elements that spatially and / or temporally modulate the PSF, for example a spatial light modulator (SLM) or a phase mask that can be set in a correspondingly controllable manner, and / or that can be controllably inserted and removed from the beam path, is also possible.

[0067] The abovementioned diaphragms or optical elements can advantageously be controlled and their optical effects influenced in a targeted manner. The invention is explained below on the basis of exemplary embodiments and drawings. [Brief description of the drawings]

[0068] [Figure 1] FIG. 1 is a schematic diagram of a first example of a detector array. [Diagram 2] FIG. 2 is a schematic diagram of a second example of a detector array. [Diagram 3] FIG. 3 is a schematic diagram of an exemplary embodiment of a microscope according to the invention. [Figure 4] FIG. 4 is a schematic diagram of a detector array and detector element pairs selected as an example. [Diagram 5] FIG. 5 is a schematic diagram of a detector array and a group of selected pairs of detector elements. [Figure 6] FIG. 6 is a schematic diagram of a detector array and an extended pair of detector elements selected as an example. [Figure 7] FIG. 7 is a schematic diagram of a sample chamber through which a medium flows, a microscope according to the invention, and components of a device for transporting the medium. [Figure 8] FIG. 8 is a diagram of the implementation of the method according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] In the exemplary embodiments and figures, identical technical elements are provided with the same reference numerals. The figures are not to scale. In FIG. 1, a detector 414 in the form known as an Airyscan detector can be seen in a plan view of its detection plane or detection surface. The detector 414 in this embodiment comprises 32 detector elements 1-32 and can be used as a spatially resolved detector 414. The detected brightness information (measurements) of the detector elements 1-32 can be read out individually and can also be combined with each other if necessary ("binned"). The spread of the beam of detection radiation (see also FIG. 3) can be selected to be incident on the detection plane with, for example, 1.25 Airy units (AU). For example, each of the detector elements 1-32 can cover a portion of 0.2 AU. The central detector element, indicated by the reference "1", is located on the optical axis (oA) of the detection beam path 410 (see FIG. 3).

[0070] 2 shows a further embodiment of a spatially resolved detector 414 in a matrix arrangement of detector elements 1-31, as may be realized for example in a SPAD array, a CMOS chip or an sCMOS chip. Again, the brightness information from detector elements 1-31 can be read out individually or combined as desired.

[0071] Fig. 3 shows a schematic representation of a microscope M according to the invention. It comprises a light source 41, for example a laser light source, from which a beam of excitation radiation (illumination radiation) is emitted and guided along an illumination beam path 42 (excitation beam path 42). Optional optical elements for shaping and / or collimation of the excitation radiation are not shown. In the illumination beam path 42, a beam-shaping optical unit 44 for controlled modification of the beam spread is optionally arranged, which in an exemplary embodiment is in the form of a diaphragm that can be set in a controlled manner. In another embodiment, a turret or slide can also be provided, by means of which different diaphragms can be introduced into the illumination beam path 42. Alternatively, the beam-shaping optical unit 44 can be a telescope, a beam expander, a zoom optical unit, an acousto-optical element or an SLM that is set in a controlled manner.

[0072] The beam shaping optical unit 44 can be moved (shown in dashed lines) by a drive 416 out of the illumination beam path 42 to provide different numerical apertures, within their respective angular ranges, to direct the excitation radiation towards the sample 48 to be imaged. The beam shaping optical unit 44 may in further embodiments be configurable with regard to its transmittance for the excitation radiation, in particular with regard to the hole diameter (pinhole, iris diaphragm) or the length and width of the slit or transparent area (configurable slit aperture, acousto-optical element) or the angular alignment and phase information.

[0073] After passing through the beam shaping optical unit 44, the excitation radiation is incident on the main color splitter 43, which is transparent to the excitation radiation and passes the excitation radiation. Downstream of the main color splitter 43, the excitation radiation passes through a part of the beam path of the microscope M, called the common beam path 4210, along which the excitation radiation and the detection radiation (see below) are or can be co-guided.

[0074] By means of a subsequently arranged scanner 46, the beam of excitation radiation previously redirected by the mirror 45 can be controllably deflected and directed towards the entrance pupil EP of the objective lens 47. The mirror 45 allows for a compact design and may be omitted in further embodiments of the device if redirection of the illumination beam path 42 is not required or envisaged.

[0075] The excitation radiation, the lateral extent of which is set by the action of a beam shaping optical unit 44 and which is controllably deflected by a scanner 46, is focused by the action of an objective lens 47 into a sample chamber in which the sample 48 to be imaged may be present on a sample stage 49. In combination with confocal detection, the thus focused and emitted excitation radiation results in a confocal excitation volume.

[0076] Detection radiation brought into the sample 48 by the excitation radiation in the confocal excitation volume is captured by the objective lens 47 and guided along a detection beam path 410 (shown as an all-dashed line) that coincides with the excitation beam path 42 to the main color splitter 43 .

[0077] In a further embodiment of the device according to the invention, the detection radiation can be captured by a further objective lens (not shown). In such a case, the excitation beam path 42 and the detection beam path 410 can be completely separated from each other or they are recombined, for example by a further color splitter (not shown), to form a common beam path 4210.

[0078] In the illustrated exemplary embodiment, the detection radiation is transformed into a stationary beam as a result of passing through the scanner 46 ("descanned") and reaches the main color splitter 43, which is reflective for wavelengths of the detection radiation different from those of the excitation radiation. The detection radiation reflected at the main color splitter 43 reaches a zoom optical unit 411, which is optionally provided in the detection beam path 410. The zoom optical unit can be controllably set by a zoom drive 412. In a further embodiment, the transmission and reflectance of the main color splitter 43 may be implemented inversely, so that the excitation radiation is reflected and the detection radiation can pass. The beam paths 42 and 410 must therefore be designed accordingly.

[0079] The beam shaping optical unit 44, the scanner 46, the zoom drive 412, the drives 415 and 416, and optionally the light source 41, are suitably connected to an analysis and control unit 413 for exchanging data and control commands. The analysis and control unit 413 is, for example, a computer or a suitable control circuit, configured to execute the method according to the invention. In particular, it is configured to perform and evaluate cross-correlation of image data from the above-mentioned pairs, extended pairs and / or groups, and optionally configured to determine the diffusion coefficient, flow velocity and flow direction, local temperature and / or local viscosity of the moving object.

[0080] The zoom optical unit 411 is a means for a controlled change of the beam spread of the detected detection radiation, whereby the beam spread of the detected detection radiation can be adapted in the intermediate image ("pinhole plane") to the size of the detection surface (detection plane) of a spatially resolved detector 414, which is also arranged in the detection beam path 410. The aim here is to illuminate the detection plane as completely as possible or to adapt it according to the respective requirements. The detection radiation is therefore directed by the zoom optical unit 411 to the detector 414 and adapted in terms of its beam spread by the zoom drive 412.

[0081] Optionally, the control unit 413 and the detector 414 may be interconnected, for example, to enable the control unit 413 to generate and / or verify control commands based on brightness information obtained from the detector 414. By way of example, these control commands serve to control the light source 41, the means 44, the scanner 46, the zoom drive 412, and / or the optional drive 415 of the sample stage 49.

[0082] Figures 4 to 6 respectively show the technical situation based on detector elements 1 to 32 of a detector 414 of the Airyscan detector type. The same applies to a detector 414 of the type shown in Figure 2. For the numbering of the relevant detector elements in the following specification please refer to Figure 1. In the following Figures 4 to 6 the central detector element 1 is shown without pattern filling for the sake of clarity.

[0083] FIG. 4 again shows a plan view of the detection surface of the detector 414 (rotated about 30° counterclockwise with respect to FIG. 1). For the purposes of explanation, the directions are defined in the direction proceeding from detector element 1 and are indicated by angle values ​​between 0° and 180° and between −60° and −120° as examples. Arrows are used to indicate three pairs of detector elements chosen as examples, the start and end of each arrow indicating one partner of the associated pair. The arrow directions indicate the forward and reverse directions of the six cross-correlations performed overall on the basis of the intensity values ​​(brightness values, measurements) recorded by the partners of each pair. The filling of different patterns highlights three rings arranged around the central detector element 1.

[0084] If only the pairs and directions shown as an example in connection with Fig. 4 are used for the determination of the cross-correlation, the efficiency of the method is low, since measurements obtained from other detector elements remain unaccounted for. However, if the situation is exploited in which the radius of the confocal volume w0 for the central detector element 1 and the two inner rings of detector elements 2-19 is approximately constant, it is possible to select and define nine pairs of detector elements for each direction, as shown as an example in Fig. 5 for the direction 180 ° → 0 °. The nine curves obtained therefrom can be averaged and fitted using equation [1]. The use of multiple curves improves the signal-to-noise ratio.

[0085] Cross-correlations for the other five directions (-120°→60°, -60°→120°, 0°→180°, 60°→-120° and 120°→-60°, not shown) can be calculated similarly.

[0086] In another method configuration, depending for example on the selected imaging ratio of the zoom optical unit 411 (see FIG. 3) and the resulting signal strength of the individual channels (detector elements), it is also possible to use the measured values ​​from detector elements 20-32 or from detector elements 19-31 according to FIG. 2. For example, the measured values ​​from detector elements 1-19 or 1-18 can be used for the actual FCS calculation and thus the measured values ​​from detector elements 20-32 or 19-31 can be used for the determination of the flow characteristics and thus for the creation of a flow profile.

[0087] In an alternative configuration of the method according to the invention, several detector elements are combined to form an extended pair. In Fig. 6 this is shown as an example with detector elements 5, 6, 7, 16, 17 and 18 as elements of one partner of the extended pair and detector elements 2, 3, 4, 10, 11 and 12 as elements of the other partner of the extended pair. In this example, the cross-correlation can be calculated for the direction 180° → 0° (common first direction, forward) and for the direction 0° → 180° (common second direction, reverse), the measurements from the elements of each partner of the extended pair being previously averaged. The center points of the respective partners of the extended pair are separated from each other by more than the range of one of the detector elements 1 to 32.

[0088] In Fig. 7 a sample chamber 419 in the form of a tubular cavity, for example a bioreactor or cell culture chamber, is shown diagrammatically. The sample chamber 419 is bounded laterally by a wall 419.1. In further embodiments, this wall may be partially planar or curved. The wall 419.1 may be made of different materials and may optionally have a surface structure on its side facing the sample chamber 419.

[0089] In the wall 419.1 there may optionally be a window 420 transparent for the excitation radiation and / or the detection radiation. This is advantageous if the wall 419.1 is not transparent or if the manifestation of the detection PSF during the passage through the wall 419.1 is very adversely affected. The window 420 allows a reverse or lateral arrangement of the beam paths for excitation and detection, so that for example the installation space above the sample chamber 419 is still available for other technical elements.

[0090] Alternatively, the sample chamber 419 is open to the top, and excitation and detection are performed through corresponding openings in the sample chamber 419. The medium flowing through the sample chamber 419 (shown as a dot pattern) may be freshly provided and / or stored in a container 417. A pump 418 allows the medium from the container 417 to be directed through a line (shown in simplified form) into the sample chamber 419 and back from there. The pump 418 is controllable by a control unit 413. Optionally, the medium is returned to the container 417 to be used again. In other embodiments, the medium may be disposed of or otherwise used after exiting the sample chamber 419.

[0091] The vessel 417 can be connected to other components that serve to treat the spent medium for reuse, for example, nutrients, hormones, growth factors, pharmacological agents and / or gases such as oxygen can be added thereto.

[0092] The sample chamber 419 contains as sample 48, for example cell clusters, here indicated by black dots. In the sample chamber 419, selected locations L1-L5 are marked with + as an example. The current respective flow direction and respective flow velocity at each location L1-L5 are indicated by arrows. As shown in the example of location L3, multiple spatially separated measurements are required in the vicinity of location L3 to determine a turbulent flow that is stable over at least the measurement period.

[0093] For each of the positions L1 to L5, the exact spatial position is known and available, for example in the form of associated coordinates in a Cartesian coordinate system. In order to record the flow characteristics at some or all of these positions L1-L5, the detection area, in particular the focal point F, of the used microscope M, represented here in simplified form by the objective lens 47, can be directed to the respective positions L1-L5. For this purpose, the sample chamber 419 and / or the microscope M can be moved in a controlled manner in at least one of the directions x, y and z (represented by a double-headed arrow), for example using the sample stage 49 (FIG. 3). The focal position F of the microscope M can advantageously be set in the direction z, for example by the microscope M being equipped with a zoom optical unit (not shown) and / or a facility for adjusting the objective lens 47 in the direction z.

[0094] The control unit 413 optionally comprises a data memory or is suitably connected to such a memory for the transmission of data. The obtained measurements can be stored in the data memory. Metadata such as the coordinates of the source of the measurements, and optionally also information regarding the properties of the medium, the sample 48, the fluorescent dyes used, and also information regarding, for example, temperature, duration, light regime, etc., can be assigned to the measurements and stored. In addition, properties of the sample chamber 419, such as its dimensions and / or the properties of the walls 419.1, can be assigned to the measurements and retrievably stored.

[0095] Fig. 8 is used to show a possible configuration of the method according to the invention. First, at least one pair of detector elements 1 to 32, whose measurements are intended to be used in the following steps, is selected and defined. The same applies to groups of pairs or extended pairs (see above). The detection radiation coming from preselected and approached positions L1 to L5 from the sample chamber 419 is detected and the resulting measurements from the selected pairs can be read out and provide them for the calculation of the corresponding cross-correlation in the forward and reverse directions, respectively. The cross-correlation results are then analyzed, the flow characteristics are determined and at least one flow profile is created.

[0096] Following the calculation of the cross-correlation, or in response to the results of the analysis, it is optionally possible to acquire further data and / or reselect corresponding pairs of locations L1-L5 and detector elements 1-32.

[0097] The actual implementation of the method according to the invention can be supported by menu navigation ("wizards"), which in each case proposes to the user suitable options for the individual method steps.

[0098] The use of menu navigation means that some technical requirements are specified by the user. For example, the objective lens to be used, the laser lines supported, and the correct beam path (illumination and / or detection) are specified. Additionally or alternatively, the dyes or fluorescent dyes to be used can be specified by the user. The appropriate laser lines, beam paths and / or control parameters are suggested or set accordingly by the menu navigation. Furthermore, a precise alignment of the detectors 414 used is necessary in order to subsequently obtain good quality measurements and thus to be able to calculate multiple cross-correlations.

[0099] In a further step, reference images of the sample can be recorded. For example, images of the sample at different wavelengths ("tracks") can be recorded at one or more locations and displayed. The user and / or suitable programming (e.g. image evaluation methods, use of artificial intelligence) can select at least one region of interest (ROI) based on the displayed data. The advantage of using multiple wavelengths lies in the higher information density for the selection of the region of interest to be performed. Subsequently, an FCS measurement of the selected locations is performed. This is carried out using only one wavelength.

[0100] The user can use the reference image as a map on which he can define, for example, up to ten positions for one spot each (step 3). A corresponding symbol (e.g. "spot selection icon") reproduces the linked relationship between the selected spot position and the image coordinates of the inner ring with the associated detector elements, in particular detector elements 1 to 19. The quantity and / or quality of the acquired measurements can be displayed graphically, optionally for each spot, for example by count data displayed with different color codes. This allows the user to select positions differently if desired, optionally with the aid of a second track (see above). To optimize the CMP (counts per molecule), it is possible to update the intensity of the laser used to excite the detected radiation, the gain (e.g. high voltage of the PMT) and the observed z-position.

[0101] The fourth step of the menu navigation is only available if a reference image exists. The measurements are stored in a metafile at each step. If the menu navigation is started again, all values ​​can be retrieved. [Explanation of symbols]

[0102] 1 to 32: Detector elements 41...Light source 42...Illumination beam path, excitation beam path 43…Main color splitter 44...Beam shaping optical unit 45…Mirror 46…Scanner 47…Objective lens 48...Sample 49...Sample stage 410...detection beam path 411...Zoom optical unit 412...Zoom drive unit 413…Control unit 414…Detector 415…Drive unit 416…Drive unit 4210…Common beam path 417...Container 418…Pump 419... Sample chamber 419.1...wall (of sample chamber 419) 420…Window F…Focus position L1~L5…Position 1~Position 5 M…Microscope

Claims

1. 1. A method for determining flow characteristics in a sample chamber (419) through which a medium flows, comprising: A step A of detecting, at multiple time points over a measurement period, detected radiation from a confocal volume generated in the sample chamber (419) using multiple detector elements (1-32) of a detector (414) in the form of a detector array, the detector (414) being arranged in a plane conjugate to a rear image plane of an objective lens (47), measurements from the multiple detector elements (1-32) can be analyzed individually, selecting at least one pair of detector elements (1-32), each partner of a pair being formed by at least one detector element (1-32); A step B of generating a cross-correlation of measurements obtained from the at least one pair of the detector elements (1-32), In a first substep B1, a cross-correlation is calculated starting from one of the selected detector elements in a first direction to a further selected detector element, A step B, in which in a second sub-step B2 a cross-correlation is further calculated starting from said further selected detector element in a second direction (backward) to the first selected detector element; step C, in which the cross-correlation is analyzed for occurrences of changes in intensity of the measurements from selected detector elements (1-32); Including, The confocal volume is generated at at least two mutually distinct positions (L1-L5) of the sample chamber (419); A number of cross-correlations are calculated over different orientation pairs, and all the curves thus obtained are fitted with direction and / or velocity as parameters, thereby determining at each of said locations (L1-L5) a velocity and optionally a direction of movement as a flow characteristic of said medium based on said cross-correlations; The velocities and, optionally, directions of movement for each position (L1-L5) are assigned to one another and retrievably stored to generate a velocity profile over at least one region of the sample chamber (419).

2. The method of claim 1, wherein the plurality of detector elements (1-32) are combined to form respective ones of extended pair partners.

3. 3. The method according to claim 1 or 2, wherein a group of a plurality of pairs or a plurality of extended pairs of the detector elements (1 to 32) is defined, and imaginary connecting lines of center points of the plurality of pairs or the plurality of extended pairs run parallel to each other.

4. The method of claim 3 , further comprising forming an average value of cross-correlations in the first direction and / or the second direction made from measurements from the plurality of pairs or the plurality of extended pairs.

5. The method according to claim 3 or 4, wherein a plurality of groups of detector elements (1-32) are determined, the respective virtual connecting lines between said plurality of groups being oriented differently from one another.

6. The method according to any one of claims 3 to 5, wherein a cross-correlation of measurements from a plurality of pairs or a plurality of extended pairs of at least two different groups is formed.

7. Method according to any one of claims 2 to 4, wherein the partners of a pair are each spatially separated by at least one detector element (1-32) which does not belong to the pair.

8. A method according to any one of the preceding claims, wherein the point spread function of the illumination radiation and / or the detected detection radiation used to generate the confocal volume and directed into the sample chamber (419) along an illumination beam path (42) is elongated in a direction transverse to the respective beam path such that it has an elliptical cross-section.

9. 10. The method according to any one of the preceding claims, wherein the sample chamber (419) is bounded by a wall (419.1) in at least one direction x, y and z, and starting from said wall (419.1) and over at least a part of a cross-section of the sample chamber (419), a plurality of positions (L1-L5) are selected, at each of said plurality of positions a confocal volume is generated.

10. 13. Use of the method according to any one of the preceding claims for determining the influence of the wall (419.1) of the sample chamber (419) on at least one velocity profile by setting the properties of the wall (419.1) in relation to the determined flow properties.

11. 10. Use of the method according to any one of claims 1 to 9, using a biological structure as a sample chamber (419), wherein the influence of a wall (419.1) of the sample chamber (419) modified with an implant on at least one velocity profile is determined by setting the properties of said modified wall (419.1) in relation to determined flow characteristics.

12. 13. Use of the method according to any one of the preceding claims for providing measurements as input data for a model and / or for validating existing simulations.

13. A microscope (M) for determining flow characteristics in a sample chamber (419) through which a medium flows, comprising: an objective lens (47) for detecting detected radiation from a confocal volume generated within said sample chamber (419); a detection beam path (410) along which the detected radiation is guided to a detector (414) in the form of a detector array having a plurality of detector elements (1-32), the detector (414) being arranged in a plane conjugate with a rear image plane of the objective lens (47), and measurements from the detector elements (1-32) can each be read individually; An analysis and control unit (413) configured to carry out the method according to any one of claims 1 to 11; A microscope (M).

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