Patent Application
20240183771
The present invention is concerned in a first aspect with a method for spatially manipulating at least one particle in a fluid according to the preamble of claim 1. In a second aspect the invention is concerned with an apparatus for spatially manipulating at least one particle in a fluid according to the preamble of claim 31. In further aspects the invention is concerned with a computer program product and a computer-readable storage medium.
The manipulation of micron-size particles, particularly their high-precision positioning, remains an active research topic with applications in the life sciences, engineering, and manufacturing. Examples of successfully used technologies include optical, magnetic, electrokinetic, acoustic and thermophoretic trapping, and the positioning of self-propelled Janus particles. However, some of these techniques are limited by requirements for specific material properties of the particles or the environment that they can be applied in. To overcome this constraint, hydrodynamic trapping has been successfully deployed, and is particularly attractive for the life sciences and for a lab-on-a-chip environment.
Hydrodynamic trapping methods can be subdivided into contact-based methods [1,2], where dissolved particles are immobilized against walls, wells, posts or other obstacles by fluid flow, and non-contact methods, where particles can be confined in stagnation-point flows [3,4], microvortices [5] or microeddies [6]. Active feedback control of flows in multi-inlet chambers has been demonstrated to control the position of stagnation-point flows and allows users to manipulate the particle position and counteract particle displacements due to diffusion [3,4]. With this method, it is feasible to control the position of single colloidal particles with a precision of 78 nm [4]. Although hydrodynamic trapping methods pose reduced constraints on material properties of the trapped particle [7], high-precision manipulation of particles requires the use of extremely precise and stable microfluidic pumps. Furthermore, hydrodynamic trapping by definition requires special chambers and cannot be used to generate flows in closed systems such as a cell. Additionally, the generated flows are global, typically ranging between pumps and outlets, which strongly limits the spatial resolution of the technique.
Another way to position particles with high precision could be to make use of thermoviscous flows. These have been described as the directed motion of aqueous media in response to travelling temperature fields [8], an emergent physical phenomenon driven by the thermal expansion of fluids in non-homogenous viscosity fields (compare
In a generic method for spatially manipulating at least one particle in a fluid, which is known, e.g., from WO 2008/077630 A1, the particle is spatially manipulated in the fluid by hydrodynamic flows which are generated in the fluid by means of dynamic localized heating of the fluid.
A generic apparatus for spatially manipulating at least one particle in a fluid by means of hydrodynamic flows, which is also known, e.g., from WO 2008/077630A1, has a receptacle for receiving the fluid and the particle(s) to be manipulated and a heating device for generating hydrodynamic flows within the fluid by dynamic localized heating of the fluid. The dynamic localized heating is designed to bring about a spatial manipulation of the particles within the receptacle by hydrodynamic flows. The generic apparatus furthermore has an imaging device for imaging at least parts of the receptacle and a control unit for controlling the heating device and the imaging device and for evaluating image data from the imaging device.
It can be considered an objective of the invention to provide a method and an apparatus of the kind described above which have a wide range of applications.
This objective is achieved by the method having the features of claim 1, by the apparatus having the features of claim 31 as well as by the computer program product and the computer-readable storage medium having the features of claims 39 and 40, respectively.
According to the invention the method for spatially manipulating at least one particle in a fluid as described above comprises the definition of at least one target spatial configuration of the particle(s) in the fluid and the following further steps:
According to the invention, the generic apparatus as described above is further characterized in that the control unit is designed for:
According to the invention, a computer program product or a computer-readable storage medium comprises instructions which, when the program is executed by the control unit, causes the control unit to carry out a method with the steps of
Advantageous variants of the method according to the invention and preferred embodiments of the apparatus according to the invention will be described in the following, in particular with respect to the dependent claims and the attached figures.
The instructions of the computer program product and/or of the computer-readable storage medium can in particular serve the purpose of activating the control unit to carry out the method according to one of the claims 1 to 30.
The basic idea of the invention is to use specific dynamic localized heating events to spatially manipulate particles in a fluid and to determine these specific dynamic localized heating events, in each case, in dependence of a target configuration of the particles and a recently captured actual configuration of the particles in the fluid. The specific dynamic localized heating events serve the purpose of generating sequences of hydrodynamic flows within the fluid. The spatial manipulation of the particles can be carried out in a closed-loop control wherein the actual captured particle configurations serve as feedback for the determination of a specific dynamic localized heating event to be applied next.
An essential advantage of the invention is that a highly precise manipulation, in particular a positioning, of a particle or a plurality of particles can be achieved, and that the manipulation can, in principle, be automated.
The apparatus according to the invention can be designed for carrying out the method according to the invention.
The term spatial manipulation of a particle in the fluid generally means that the particle-fluid-system in a receptacle is influenced such that the particle is moved from one point to another and/or from one orientation to another as compared, e.g., to a reference frame in which the receptacle is stationary. Spatial manipulation can also mean, though, that a particle is kept at a certain position against the action of other, e.g., external forces acting on the particle.
In preferred embodiments of the method according to the invention, the spatial manipulation of the particle(s) can comprise at least one of:
Generally, the method and the apparatus according to the invention can be used to manipulate any suspended particle in a fluid which can at least partly move freely in the fluid. In preferred variants of the method according to the invention, the particle(s) to be manipulated is/are at least one of the following: a biological particle, a cell, a virus, a tissue fragment, a metal particle, a composite material particle, a polymer particle, a microparticle, a nanoparticle.
Generally, the method and the apparatus according to the invention can be used for any kind of fluid in which hydrodynamic flows can be generated by dynamic localized heating of the fluid. In preferred embodiments of the method according to the invention, the fluid is a liquid and, in particular, contains water or is water.
According to the invention, hydrodynamic flows are generated in the fluid by means of dynamic localized heating of the fluid and the manipulation of a particle is achieved by the particle being carried by the generated hydrodynamic flow.
It would in, principle, also be possible to generate hydrodynamic flows with specific dynamic localized heating events having a negative sign, i.e., events where hydrodynamic flows are introduced into the fluid by means of dynamic localized cooling of the fluid and the manipulation of a particle is again achieved by the particle being carried by the generated hydrodynamic flows.
In contrast to the phenomenon of thermophoretic motion which is strongly dependent on the specific types of particles and the specific liquid surrounding these particles, the general principle of the mechanism underlying the present invention is independent of the specific nature of the particles. In a thermophoretic motion, different particles will generally move differently, e.g., at different speeds and even in different directions. In hydrodynamic flows, as being used in the present invention, the particles will move essentially according to the physics of hydrodynamics. The mechanism of the present invention is governed essentially only by the optical, and thermodynamic properties of the used fluid, e.g., water. The hydrodynamic flows generated by dynamic localized heating of the fluid are also termed thermoviscous flows.
In principle, the dynamic localized heating of the fluid can be brought about by any energy deposition into the fluid which leads to the intended thermoviscous flows. E.g., the dynamic localized heating can be introduced by specific heating devices attached to the receptacle via heat conducting connections and which are selectively heated. In preferred embodiments of the method according to the invention, the dynamic localized heating of the fluid is brought about by a laser or an infrared laser.
In another preferred embodiments of the method according to the invention, the dynamic localized heating of the fluid his brought by light emitting diodes, e.g., infrared light emitting diodes.
Correspondingly, in a preferred embodiment of the apparatus according to the invention, the heating device has a laser for providing the energy for the dynamic localized heating and optical means such as a scanner, galvo-scanner, quasistatic scanner, a spatial light modulator, acousto-optical scanner, or any other suitable device for relaying heating laser radiation to variable, controlled locations in the fluid, and enabling the dynamic scanning of the heating laser beam across the sample.
An optical assembly as described in WO 2008/077630A1 can be used for the dynamic localized heating of the fluid. In this regard, the respective contents of WO 2008/077630A1 is included in the present disclosure.
In preferred embodiments of the method according to the invention, the dynamic localized heating events of the fluid are brought about by repetitive scanning of a focal volume of the laser along an especially selected path or a trajectory in the sample.
More specifically, the determination of the specific dynamic localized heating events to be determined to the fluid in step b) of claim 1 can comprise the determination of at least one of:
A specific dynamic localized heating event can comprise only one scan of a laser scan path or many, e.g., 100, scans of the same laser scan path.
Application of the specific dynamic localized heating as determined means that the determined dynamic heating pattern is applied to the fluid. This can be done only once or a plurality of times one after another. The heating device of the apparatus according to the invention will then correspondingly be activated by the control unit.
Generally, a scan path can be anywhere in the receptacle, and can consist of one or more straight or arbitrary shaped segments of arbitrary length, which need not necessarily be continuous. A scan path can be parallel to a connecting vector between a target destination and an actual position of the particle. The scan path can either be centered on the particle, ending slightly before it, or starting slightly after it. The laser scan can be applied along the path, with a typical scan rate of, e.g., 1-3 kHz, which is sufficiently slow to allow for the relaxation of temperature fields between successive scan periods. The scan rate can vary along the scan path.
It is also possible to vary and/or adapt a number of repetitions or a repetition rate of a loop containing at least some or all of steps a) to c) of claim 1 to a specific manipulation task.
In further preferred embodiments of the method according to the invention, the paths along which the laser is scanned can be chosen such that the heating radiation does not hit the particle(s) to be manipulated. I.e., the particle(s) is or are spatially manipulated essentially without being contacted by heating laser radiation. The danger of the particles as such, or the receptacle e.g., living cells or embryos, to be influenced or harmed by the heating radiation is thereby in minimized.
In further preferred embodiments of the method according to the invention, a scan rate of the repetitive scanning is chosen such that temperature fields in the sample can relax between successive scans. A general heating of the sample can thus be avoided.
The specific dynamic localized heating events to be applied to the fluid can further be individualized by being determined also in dependence of a mobility of particle(s) in the fluid. The accuracy of the manipulation, in particular the positioning, can thus be further improved. The mobility of particles to be manipulated can be derived from the observed data.
For the receptacle which receives the fluid and the particles to be manipulated components known in the art can be used. It is important that the receptacle allows the specific dynamic localized heating to be introduced into the fluid. E.g., the receptacle described in WO 2008/077630A1 can be used for carrying out the invention. In this regard, the respective contents of WO 2008/077630A1 is included in the present disclosure.
In a preferred embodiment of the according to the invention, the receptacle has means for controlling the basic temperature of the fluid.
The control unit for controlling the heating device and the imaging device and for evaluating image data from the imaging device can typically be a PC or equivalent devices, with peripheral components as known in the art.
The imaging device serves the purpose of capturing actual configurations of the particles to be manipulated in the fluid. It can be any, in particular optical, device with which at least the portions of the receptacle can be captured or imaged which contain particles to be manipulated. In preferred embodiments of the invention, the imaging device is a microscope. The microscope can be a computer-controlled microscope and does not need to allow a visual observation of the sample. Preferably, the acquisition of images is at least partly automated and image algorithms are being used for the evaluation of the captured configurations of the particles.
For example, the microscope can be designed for carrying out at least one of the following techniques: Fluorescence Microscopy, Multi-Photon-Fluorescence Microscopy, Widefield Microscopy, Scanning Microscopy, Darkfield Microscopy, Confocal Microscopy, Lightsheet Microscopy, Localisation Microscopy, Structured Illumination Microscopy, Photoactivated Localization Microscopy (FPALM), Stochastic Optical Reconstruction Microscopy (STORM), Stimulated Emission Depletion Microscopy (STED), Ground State Depletion Microscopy (GSD), Saturated Pattern Excitation Microscopy, Saturated Structured Illumination Microscopy (SSIM), Light Field Microscopy (LFM), Fourier Light Field Microscopy (FLFM), Oblique plan microscopy (OPM).
The microscope can relay imaging radiation onto and into the sample and can relay back radiation emitted from the sample in response to the imaging radiation, e.g., fluorescence radiation, via the same microscope objective that is also used for introducing the heating radiation into the sample.
The capturing of the actual spatial configuration of the particle(s) can comprise at least one of the following:
Definition of a target spatial configuration means that a certain configuration into which the particle or the particles to be manipulated is defined. This can, e.g., be done by a user at the computer screen on the basis of a measured actual configuration or an actual image of the particles. The definition of a target configuration can be assisted by image evaluation software which, e.g., recognizes certain structures in the measured image data. The target spatial configuration of the particle(s) in the fluid can comprise at least one of:
The target spatial configuration of the particle(s) in the fluid can furthermore be a 1-dimensional localisation of the particle(s), a 2-dimensional localisation of the particle(s) or a 3-dimensional localisation of the particle(s).
The target configuration can additionally or alternatively also include at least one of the following requirements:
A key feature of the present invention concerns, in step b), the determination of a specific dynamic localized heating event to be applied to the fluid in dependence of at least one recent actual spatial configuration of the particle(s) and a target configuration of the particle(s). In this regard, a cost function can be calculated on the basis of a recent, in particular the most recent, actual spatial configuration of the particles and a target configuration of the particles. The specific dynamic localized heating events can then be determined in dependence of the cost function. The cost function can be a scalar function of at least one recent actual spatial configuration of the particle(s) and a target configuration of the particle(s) and/or a description of the desired target configuration. A description of the target configuration can be that, e.g., all particles of a first kind should be moved to the left, and all particles of a second kind should be moved to the right side to enable an effective sorting.
For example, after an application or each application of the specific dynamic localized heating event in step c), the actual configuration can be captured and the cost function can then be calculated for the new configuration and, if the cost function has decreased from the most recent value, step c) can be repeated with the same specific dynamic localized heating event, or, if the cost function has increased from the most recent value, step b) can carried out anew.
The general idea of the invention is realized where only one particle is spatially manipulated. In particularly preferred embodiments of the method according to the invention, though, at least two particles are simultaneously spatially manipulated. This means that the actual configuration of a plurality particles is captured. The specific dynamic localized heating events can then be determined such that both (or more) particles are moved or manipulated but also such that essentially only one particle is manipulated or moved at a time.
E.g., in a configuration with at least two particles to be manipulated, the particle being mainly manipulated, in each case, in the next step can be the particle which is farthest away from at least one of a target position and a target orientation associated with the respective particle.
In principle, each particle to be manipulated can be considered individually. E.g., each particle can individually be moved to a certain location or to a certain orientation. For certain applications, though, it is useful to consider certain types of particles. I.e., a plurality of particles to be spatially manipulated can comprise at least one subset of equivalent are identical particles. The cost function can then be invariant with regard to exchange of equivalent or identical particles. The complexity of the underlying mathematical task can thus be reduced.
More specifically, the cost function can contain at least one of the following arguments:
In further preferred embodiments of the method according to the invention, at least some of the following data can be stored in a database:
Future dynamic localized heating events to be applied to the fluid can be calculated using at least parts of the data stored in the database, in particular making use of machine learning/artificial intelligence.
In further preferred embodiments of the method according to the invention, between steps a) and b) the following further step can be carried out: the particle(s) to be manipulated is or are associated with at least one of a target position and a target orientation. Association of a particle with a specific target position means that the target position is attributed to the respective particle.
In further preferred embodiments of the method according to the invention, a tracking of the particle(s) can be carried out by identifying particles present in a captured new actual configuration with particles in the most recent actual configuration. Thus, trajectories of individual particles can be derived from a sequence of actual configurations.
In further preferred embodiments of the method according to the invention, after the tracking of the particles, a target configuration can be reassessed and, if the target configuration is changed to a new target configuration, the particles can then be associated, in each case, with at least one of a new target position and a new target orientation. Thus, the system can dynamically react to a change of the target configuration and can change the attribution of the particles to specific target configurations on the fly.
In further preferred embodiments of the method according to the invention, the integrity of at least one particle can be changed prior or during or after the spatial manipulation. E.g., a fragment from a biological particle can be cut off by laser cutting. The apparatus according to the invention can correspondingly have at least one device, e.g., one laser, for changing the integrity the particle(s). The apparatus according to the invention can also have devices for changing and orientation of at least one particle, e.g., an optical tweezer.
Comparing the actual positions of particles to their expected positions might be useable to infer forces acting on the particles. Given that flow-induced forces are typically weak, such force sensing might be particularly sensitive. For some applications it might be beneficial to be able to control particle interactively via hand gestures, for which mixed reality devices offer an attractive and likely compatible solution. Force feedback interfaces could enable tactile interactive manipulations.
In a preferred embodiment of the method according to the invention, a deviation of an actual position of at least one particle to be manipulated is compared with an expected position for this particle and a force acting on the particle is determined in dependence of this deviation.
In this regard, it can be useful to have at least two hydrodynamic flows directed in opposite directions to a center and to capture the particle in the center, at the point where the two opposite flows meet. By such flows in opposite directions an effective potential is generated for the captured particle.
For a calibration of the measured forces, it can be useful to impose external forces, e.g., electrostatic, or magnetic forces, of a known magnitude on the fluid containing the particles. The deviation of the position of the particle from an equilibrium position in the effective potential can then be measured in dependence of the magnitude of the external force and, thus, the magnitude of the force acting on the particle through the hydrodynamic flows can be determined.
A calibration of the measured forces can alternatively or additionally also be achieved by observing thermal movements, in particular a mean distance from an equilibrium position of the particle in the effective potential. With the assumption that the mean amount of energy in each degree of freedom is kBT (kB=Boltzmann constant, T=temperature) the force acting on the particle can be determined.
Further features and advantages of the invention will be described in the following with respect to the attached figures. Therein shows:
An embodiment of an apparatus 100 according to the invention will be described in the following with reference to
As essential components, the apparatus 100 as shown in
More specifically, in the example shown in
It is clear that
The imaging means 40 in the example of
Fluorescence radiation radiated back from the sample, e.g., from dyes with which, e.g., the particles to be manipulated are prepared, or autofluorescence light travels back through the microscope objective 48, beam splitter 30, and beam splitter 46 and reaches an optical detector 50 and is detected there. The optical detector 50 can be a camera which can record images of a field of view as propagated by the optical beam path, i.e., the camera can capture an actual spatial configuration of the particles p1, p2 within the receptacle 10. Both, the light source 42 and the optical detector 50 are controlled by the control unit 60 and can, in each case, sent back status data to control unit 60.
Target positions T1, T2 are shown schematically in the receptacle 10 of
According to the invention, the control unit 60 is designed for:
Further devices for manipulating the sample, and specifically the particles p1, p2, to be spatially manipulated, e.g., further lasers can be present in the apparatus 100 of
The control unit 60 can be a PC or a similar computing device with peripheral components as generally known in the art. The control unit 60 can have both of a computer program product and a computer-readable storage medium according to the invention.
The essential features of the method for spatially manipulating particles p1, p2 in a fluid 12 will now be described, first more generally with regard to
The upper portion of
The lower portion of
First (drawing b1), an actual spatial configuration of the particles p1, p2 is captured (step a)), e.g., an image is recorded with the camera 50 of the microscope 40 in
Then, in the example shown in
After definition of a target spatial configuration of the particles p1, p2, i.e., in the example of
According to step b) of the method according to the invention, a specific dynamic localized heating event to be applied to the fluid 12 is then determined in dependence of at least one recent actual spatial configuration of the particles p1, p2, e.g., in dependence of at least the image recorded in step a) (drawing b1) and the target locations T1, T2 of the particles p1, p2.
According to step c) of the method according to the invention, the specific dynamic localized heating event as determined in step b) is applied at least once to the fluid 12. In the example shown in
Thus, with the method according to the invention, the particles p1, p2 are spatially manipulated in the fluid 12 by hydrodynamic flows which are generated in the fluid 12 by means of dynamic localized heating of the fluid 12.
According to step d) of the invention, at least one or all of the steps a) to c) is or are repeated. In the example shown in
A more detailed example of a method according to the invention will be described with reference to
According to step b) of the method according to the invention, a specific dynamic localized heating event to be applied to the fluid 12 will then be determined in step S06 “compute new FLUCS vector”, e.g., in dependence of the cost function calculated in step S05. Thus, the specific dynamic localized heating event will be dependent of at least one recent actual spatial configuration of the particles and a target configuration of the particles. It is also possible, though, that the specific dynamic localized heating event to be applied to the fluid 12 is determined irrespectively of the cost function value, e.g., by selecting the furthermost particle and pushing it towards its target.
In step S07 “apply FLUCS vector”, the specific dynamic localized heating event determined in step S06 is applied to the sample, i.e., to the fluid containing the particles to be manipulated. This corresponds to step c) of the method according to the invention.
According to step d) of the method according to the invention, at least some of steps a) to c) are repeated. In the flowchart depicted in
Step S10 is a query whether or not a target configuration is to be updated. In preferred embodiments the software decides whether or not the target configuration will be updated.
In the case where the target configuration is left unchanged, the query S10 is followed by step S11 where, as in step S04, the particles to be manipulated are associated, in each case, with a target position. In the case where in response to the query in step S10 the target configuration is to be updated, a new target configuration, e.g., new target locations are defined in step S14 “define new targets” and the program continues with step S11.
Step S11 is followed by step S12 in which, as in step S05, the cost function is calculated anew for the new actual configuration of the particles as captured in step S08 and, if applicable, for the new target configuration as defined in step S14.
In step S13 it is decided whether or not the error, i.e., the cost function, has decreased as compared to the value determined in step S05.
If the cost function has, in fact, decreased from the value determined in step S05, step c) being realized in the example of
If, on the other hand, the cost function has increased from the value determined in step S05, step b) of the method according to the invention is carried out anew. I.e., a new specific dynamic localized heating event to be applied to the fluid 12 will be determined in step S06 “compute new FLUCS vector” in dependence of the cost function calculated in step S12.
Thus, a closed-feedback-loop-control and an automated spatial manipulation of particles in a fluid are realized.
An example of an algorithm where the association of the particles to be manipulated to specific target locations is reassessed and, if need be, changed after a specific dynamic localized heating event is applied will be described in connection with
The upper diagram in
On the basis of these associations and the initial configuration of the particles p1 to p6 in the upper diagram in
The lower diagram in
In some cases, it is advantageous, though, to include even particles which have reached their destination in a new assessment. Such a situation will be described with reference to
A further example of the method according to the invention where a plurality of identical particles are to be spatially manipulated to respective target locations will be described with reference to
Determination and application of a specific dynamic localized heating event leads to the situation shown in diagram 2 where particle p1 has considerably approached its target location T1. The target farthest away from its target location is now particle p2. Determination and application of a corresponding specific dynamic localized heating event then leads to the situation in diagram 3 where, again, particle p1 is farthest away from its target location T2. The further proceeding according to the principle that in a configuration with a plurality particles to be manipulated, the particle being manipulated, in each case, in the next step is the particle being farthest away from the target position associated with the respective particle is depicted in diagrams 4 to 6. In diagram 4 particle p3 is farthest away from its target location T3 and, accordingly, particle p3 is moved. In diagram 5, particle p2 is farthest away from its target location T2 and is accordingly manipulated. In the situation depicted in diagram 6, particles p2 and p3 have reached their target locations T2 and T3, and only particle p1 is left to be moved to its target location T1.
An example of a cost function will be motivated and explained with reference to
The objective for the spatial manipulation in this example shall be that particles A are to be positioned in close proximity to each other in the center and that particles B are to be moved to positions as far away as possible from the agglomeration of particles A.
A suitable cost function S reflecting these requirements is:
where ra and ra are the distances of a particle of the type A and the type B, respectively, from the center. N and M are the numbers of particles of the type A and the type B, respectively.
The invention shows that thermoviscous flows can be used to enable the precise positioning of (a) pre-specified object(s) or particle(s) and that positioning can even be automated.
Furthermore, the invention shows that precision hydrodynamic positioning of particles can be achieved all-optically by combining laser-induced flows with a closed feedback loop that considers time-dependent and stochastic particle positions. The inventors have analyzed the physical characteristics of this novel way to control particle position. While optically facilitated, a precision of up to 24 nm was achieved, that is unprecedented even by classic hydrodynamic trapping. In contrast to optical tweezers, the method according to the invention requires neither specific materials nor the exposure of particles to the laser beam. Feedback-controlled thermoviscous flows are an attractive alternative to classic hydrodynamic trapping techniques, while generating opportunities for a wide range of novel applications.
[10] M. Mittasch, P. Gross, M. Nestler, A. W. Fritsch, C. Iserman, M. Kar, M. Munder, A. Voigt, S. Alberti, S. W. Grill and M. Kreysing, Nat Cell Biol, 2018, 20, 344-351.
[11] M. Mittasch, V. M. Tran, M. U. Rios, A. W. Fritsch, S. J. Enos, B. Ferreira Gomes, A. Bond, M. Kreysing and J. B. Woodruff, Journal of Cell Biology, DOI:10.1083/jcb.201912036.
[12] N. T. Chartier, A. Mukherjee, J. Pfanzelter, S. Fürthauer, B. T. Larson, A. W. Fritsch, M. Kreysing, F. Jülicher and S. W. Grill, bioRxiv, 2020, 2020.05.30.125864.
| Number | Date | Country | Kind |
|---|---|---|---|
| EP21182612.8 | Jun 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/071437 | 7/30/2021 | WO |
