Method and apparatus for measuring a force on at least one particle in a fluid, and computer program product and computer-readable storage medium

Abstract

The present invention relates to a method for measuring a force on at least one particle in a fluid, where a singular dynamic localized heating event generates a non-uniform field of hydrodynamic flow in the fluid, the hydrodynamic flow spatially manipulates the particles, a spatial configuration of the particle(s) in the fluid is imaged, and at least one force on the particle(s) is determined by evaluating the imaged spatial configuration of the particle(s). The present invention further relates to an apparatus, a computer program product, and a computer readable storage medium for measuring a force on at least one particle in a fluid.

Description

[Technical Field]

[0001] The present invention relates generally to a method and apparatus for measuring a force on at least one particle in a fluid. In a further aspect, the present invention provides a computer program product and a computer-readable storage medium for assisting in measuring a force on at least one particle in a fluid. [Background technology]

[0002] The use of optical tweezers to measure forces on mesoscopic particles has revolutionized fields ranging from materials science to cell biology (Non-Patent Documents 1-9). However, the application of this technique imposes certain prerequisites regarding the refractive index of the probe (Non-Patent Document 10). Furthermore, concerns about its impact on vital processes may limit its use in biological systems (Non-Patent Documents 11, 12).

[0003] Thermoviscous flow can be utilized for particle positioning. Thermoviscous flow has been described as the directed motion of an aqueous medium in response to a moving temperature field (Non-Patent Document 13), a sudden physical phenomenon caused by the thermal expansion of a fluid in a non-uniform viscosity field. Specifically, thermoviscous flow has been used to transport aqueous solutions visualized by tracer particles or molecules along optically defined paths (Non-Patent Documents 13, 14). Recently, it has been shown that such flows can also be generated in cells and developing embryos, inducing cytoplasmic flow, albeit with limited ability to control the positioning of colloids immersed in the cytoplasm (Non-Patent Documents 15-17). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2008 / 077630 [Non-patent literature]

[0005] [Non-Patent Document 1] Gomez, GA, McLachlan, RW & Yap, ASProductive tension: force-sensing and homeostasis of cell-cell junctions. Trends Cell Biol. 21, 499-505 (2011).

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[0006] A device for spatially manipulating particles in a fluid by hydrodynamic flow is known, for example, from US Pat. No. 5,629,999.

[0007] It may be seen as an object of the present invention to provide a new method and a new device for measuring forces on particles in a fluid.A further object of the present invention is to provide suitable software means to assist in measuring forces on particles in a fluid. [Means for solving the problem]

[0008] The problem is solved by a method having the features of claim 1 and by a device having the features of claim 33. For software means, the problem of the invention is solved by a computer program product according to claim 41 and a computer-readable storage medium according to claim 42.

[0009] In a method of the present invention for measuring a force on at least one particle in a fluid (claim 1), a unique dynamic localized heating event generates a non-uniform field of hydrodynamic flow in the fluid, the particles are spatially manipulated by the hydrodynamic flow, the spatial configuration of the particle(s) in the fluid is imaged, and at least one force acting on the particle(s) is determined by evaluating the imaged spatial configuration of the particle(s).

[0010] The apparatus for measuring a force on at least one particle in a fluid according to the present invention (claim 32) has the following features: a vessel for containing the fluid and particles, a heating device for generating a non-uniform field of hydrodynamic flow in the fluid by a singular dynamic local heating event, a device for imaging at least a portion of the spatial configuration of the particle(s) in the vessel, and a control unit for controlling the heating device and the device for imaging at least a portion of the spatial configuration of the particle(s), evaluating data from the device for imaging at least a portion of the spatial configuration of the particle(s), and determining at least one force on the particle by evaluating the spatial configuration of the particle.

[0011] The computer program product according to the present invention (claim 40) and the computer readable storage medium according to the present invention (claim 41) in each case comprise instructions which, when the program is executed by the control unit, cause the control unit to carry out the method according to the present invention, comprising the steps of: A) Activating a device for imaging at least part of the spatial configuration of the particle(s), in particular an imaging device, in order to image the actual spatial configuration of the particles in the container. B) determining a control signal for a heating device appropriate for a singular dynamic localized heating event applied to the fluid in response to at least one recent spatial configuration of the particle(s) and a predefined target configuration of the particle(s); C) activating the heating device to apply the specific dynamic localized heating event determined in step B) to the fluid at least once. D) Repeat at least one or all of steps A) through C). E) Determining the force on the particle(s) depending on the imaged spatial configuration of the particle(s).

[0012] Advantageous variants of the method according to the invention and preferred embodiments of the device according to the invention are explained in particular below with reference to the dependent claims and the accompanying drawings.

[0013] The device according to the invention can be designed to carry out the method according to the invention.

[0014] The instructions on the computer program product and / or computer readable storage medium may specifically serve the purpose of operating a control unit to perform the method according to any one of claims 1 to 32.

[0015] The basic concept of the present invention is to utilize singular dynamic local heating events to spatially manipulate particles within a fluid to generate a non-uniform hydrodynamic flow field within the fluid, thereby observing or imaging the position of at least one particle in the non-uniform flow field, and deriving a force on the at least one particle by evaluating the observed particle configuration.

[0016] The unique dynamic local heating event may be determined in each case in response to a target configuration of particle(s) and a recently captured actual configuration of particle(s) within the fluid. The unique dynamic local heating event serves the purpose of generating a hydrodynamic flow sequence within the fluid. The spatial manipulation of particles may be performed within a closed system control. In that case, the actual captured particle configuration may serve as feedback for determining the next applied unique dynamic local heating event.

[0017] In general, it should be understood that the use of the term "target" does not necessarily imply that the particle being manipulated and / or analyzed will, for example, arrive at a target location. In a typical situation, the target location serves the purpose of calculating a desired specific localized heating event. The particle, for example, continues to be pushed by the specific localized heating event toward the target location against an applied external force.

[0018] It should also be understood that, in general, the term spatial configuration as used herein does not necessarily imply that the complete coordinates of the particle(s) being manipulated and / or analyzed are known. Rather, spatial configuration as used herein may be embodied in at least one parameter or coordinate that characterizes the particle(s) being manipulated and / or analyzed.

[0019] A key advantage of the present invention is that, because it is a non-contact trapping method based on optically induced hydrodynamic flow, the method can be applied to sensitive particles. The inventors were able to demonstrate a linear relationship between force and extension, detecting forces in the femtonewton range near the thermal sensitivity limit. The technique of the present invention eliminates the need for laser contact with the particle, essentially eliminating material limitations on the particles that can be analyzed. Furthermore, the method can be performed with a standard optical microscope, eliminating the need for specialized chambers. This allows for the investigation of localized forces within more complex materials. Optically induced hydrodynamic flow therefore facilitates sensitive, non-invasive force measurements with fewer limitations on sample properties.

[0020] The term spatial manipulation of particles within a fluid generally means that the particle / fluid system within the container is affected, e.g., so that the particles move from one point to another and / or from one orientation to another relative to a fixed reference frame of the container. However, spatial manipulation can also mean that the particles are held in a particular position despite other behaviors, e.g., external forces acting on the particles.

[0021] In a preferred embodiment of the method according to the present invention, spatially manipulating the particle(s) comprises at least one of: pushing or moving the designated particle(s) towards a designated target location within the fluid; moving the designated particle(s) along a designated path within the fluid; imprisoning the designated particle(s) at a designated target location within the fluid; imprisoning the designated particle(s) in a designated target orientation within the fluid; pushing or moving the designated particle(s) towards a designated target orientation(s) within the fluid.

[0022] In a preferred embodiment of the method according to the invention, the hydrodynamic flow field is at least partially decreased along the field direction in the region where the particles to be analyzed are present, and it has been confirmed that at least in such a state the particles to be analyzed can reach a steady state.

[0023] In general, the method and apparatus according to the present invention can be used with any type of fluid in which hydrodynamic flow can be generated by dynamic localized heating of the fluid. In a preferred embodiment of the method according to the present invention, the fluid is a liquid, in particular comprising or being water.

[0024] In general, the methods and devices of the present invention can be used to manipulate any particle suspended in a fluid that is at least partially free to move within the fluid. In preferred variations of the methods of the present invention, the particle(s) to be manipulated are at least one of biological particles, cells, viruses, tissue fragments, metal particles, composite particles, polymer particles, nanoparticles, spherical beads, magnetic beads, organelles, phase-separated droplets that themselves contain proteins, RNA, or other biomolecules, and tethered molecules.

[0025] According to the present invention, dynamic localized heating of a fluid generates hydrodynamic flows within the fluid, and particles are transported by the generated hydrodynamic flows, thereby achieving particle manipulation.

[0026] In principle, hydrodynamic flows can also be generated by negative singular dynamic local heating events, i.e., events in which hydrodynamic flows are induced within a fluid by dynamic local cooling of the fluid. Again, particle manipulation can be achieved by transporting particles within the generated hydrodynamic flows.

[0027] In contrast to the phenomenon of thermophoretic motion, which is strongly dependent on the specific type of particles and the specific liquid surrounding them, the overall concept of the mechanism on which the present invention is based does not depend on the specific properties of the particles. In the case of thermophoretic motion, different particles generally move differently, for example, at different speeds and possibly in different directions. In the case of hydrodynamic flows used in the present invention, particles move fundamentally according to the physical properties of fluid dynamics. The mechanism of the present invention is fundamentally based solely on the thermodynamic properties of the fluid used, e.g., water. When electromagnetic radiation, e.g., a laser, is used to heat the sample, the optical properties of the fluid also become important. Hydrodynamic flows generated by dynamic local heating of a fluid are also called thermoviscous flows.

[0028] In principle, dynamic local heating of a fluid can be achieved by any energy storage in the fluid that results in the desired thermoviscous flow. For example, dynamic local heating can be introduced by a specific heating device that is attached to the container via a thermally conductive connection and selectively heated. In a preferred embodiment of the method according to the invention, dynamic local heating of the fluid is achieved by a laser or an infrared laser.

[0029] In another preferred embodiment of the method according to the invention, the dynamic local heating of the fluid is achieved by means of light emitting diodes, for example infrared light emitting diodes.

[0030] Therefore, in a preferred embodiment of the apparatus according to the invention, the heating device comprises a laser providing energy for dynamic local heating and optical means such as a scanner, galvanometer scanner, quasi-static scanner, spatial light modulator, acousto-optical scanner, or any other suitable device that relays the heating laser radiation to a variable, controlled location in the fluid and allows dynamic scanning of the heating laser beam over the sample.

[0031] An optical assembly such as that described in US Patent No. 6,269,999 can be used for dynamic localized heating of fluids. Therefore, the present disclosure incorporates the contents of US Patent No. 6,269,999.

[0032] In a preferred embodiment of the method according to the invention, a dynamic localized heating event of the fluid is generated by repeatedly scanning the focal volume of the laser along a specially selected path or trajectory within the sample.

[0033] More specifically, determining the anomalous dynamic localized heating event determined for the fluid may include determining at least one of the following: a two-dimensional scan path within the fluid, a three-dimensional scan path within the fluid, a laser intensity, a laser scan speed, a laser scan frequency, and a number of scans of the scan path.

[0034] The unique dynamic localized heating event may include a single scan of the laser scan path, or multiple scans, for example 100 scans, of the same laser scan path.

[0035] Applying the determined differential dynamic localized heating means applying the determined dynamic heating pattern to the fluid, which may be performed once or multiple times sequentially, in which case the control unit activates the heating device of the apparatus according to the present invention accordingly.

[0036] In general, the scanning path can be anywhere within the container and can consist of one or more straight lines or segments of any shape and length, not necessarily continuous. The scanning path can be parallel to the connecting vector between the particle's destination and actual location. The scanning path can be centered on the particle, terminate just before it, or begin just after it. Many other variations in this regard are possible. The laser scan can be applied along the path at a scanning rate, for example, typically 1 to 3 kHz. This is slow enough to allow the temperature field to relax between successive scanning periods. The scanning rate can be variable along the scanning path.

[0037] In a further preferred embodiment of the method according to the invention, the path along which the laser is scanned can be selected so that the particle(s) to be manipulated and analyzed are not exposed to the heating radiation. That is, the particle(s) are spatially manipulated without being substantially contacted by the heating laser radiation. Thus, the risk of such particles or containers, such as living cells or embryos, being affected or harmed by the heating radiation is minimized.

[0038] In a further preferred embodiment of the method according to the invention, the scan rate of the repeated scans is selected such that the temperature field within the sample can relax between successive scans, thus avoiding global heating of the sample.

[0039] The specific dynamic localized heating event applied to the fluid can be further personalized by also determining the mobility of the particle(s) within the fluid, which can further improve the precision of manipulation, particularly placement. The mobility of the manipulated particles can be derived from observational data.

[0040] Components known in the art can be used for the vessel containing the fluid and the particles to be manipulated. It is important that the vessel be capable of inducing specific dynamic localized heating in the fluid. For example, the vessel described in Patent Document 1 can be used to implement the present invention. Therefore, the present disclosure incorporates the contents of Patent Document 1.

[0041] In a preferred embodiment of the invention, the container has means for controlling the base temperature of the fluid.

[0042] A control unit that controls the heating device and the imaging device and evaluates image data from the imaging device may typically be a PC or equivalent device, with peripheral components known in the art.

[0043] The device for imaging at least a portion of the spatial configuration of the particle(s) may be any device capable of obtaining a signal encoding at least a portion of the spatial configuration of the particles, and may specifically be an optical device, such as an imaging device, a lensless camera, or at least one of a quadrant photodiode.

[0044] The imaging device serves the purpose of photographing the actual configuration of the manipulated particles in the fluid. At least a part of the vessel containing the manipulated particles can be photographed or imaged by any device, particularly an optical device. In a preferred embodiment of the invention, the imaging device is a microscope. The microscope can be a computer-controlled microscope and does not necessarily allow visual observation of the sample. Preferably, image acquisition is at least partially automated and an imaging algorithm is used to evaluate the configuration of the photographed particles.

[0045] For example, the microscope may be designed to perform at least one of the following techniques: fluorescence microscopy, multiphoton fluorescence microscopy, widefield microscopy, scanning microscopy, darkfield microscopy, confocal microscopy, light sheet microscopy, localization 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 plane microscopy (OPM).

[0046] The microscope may relay imaging illumination, e.g., fluorescent illumination, onto and into the sample, and may relay back illumination emitted from the sample in response to the imaging illumination through the same microscope objective lens that was also used to introduce heating illumination into the sample.

[0047] Imaging the actual spatial configuration of the particle(s) may include at least one of the following: one-dimensional position of the particle(s), two-dimensional position of the particle(s), three-dimensional position of the particle(s), measuring the orientation of the particle(s) in a plane, measuring the three-dimensional orientation of the particle(s) in space.

[0048] In particular, in a preferred embodiment of the method according to the invention, the spatial organization of the particles being manipulated and analyzed is assessed over time.

[0049] The practice of the present invention requires that the hydrodynamic flow field be non-uniform. Therefore, equivalently, the gradient of at least one vector component of the hydrodynamic flow field is non-zero, or simply, the hydrodynamic flow field is not constant within at least a predetermined volume of the sample in which the particle(s) to be manipulated and analyzed are located. However, in a particularly preferred embodiment, the non-uniform hydrodynamic flow field has at least one stagnation point. This allows the particle(s) to be at least temporarily trapped near the stagnation point. In this condition, the hydrodynamic flow generates a quasi-potential that allows the force on the particle(s) to be determined.

[0050] In the case of a hydrodynamic field having a stagnation point, imaging the configuration of particles being manipulated and analyzed can be observing the deviation of the actual position of at least one particle from the stagnation point.

[0051] The force on the particle can therefore be determined according to the observed displacement.

[0052] For example, a non-uniform field of hydrodynamic flow having at least one stagnation point can be generated by at least two hydrodynamic flows in opposite directions toward the stagnation point, which can be rotated in a plane around the stagnation point in an attempt to achieve a metastable state.

[0053] In another preferred embodiment of the method according to the invention, stagnation points can be generated in only one hydrodynamic flow, especially when the fluid is subjected to an external force, e.g. gravity, or when a physical barrier already confines the particles in one direction, e.g. trapping them against a plane.

[0054] More specifically, depending on the imaged spatial configuration of the particles, in particular at least one of the measured azimuthal and radial coordinates of the particle(s) relative to a stagnation point, azimuthal directions in which at least two opposing hydrodynamic flows are applied can be selected, thereby generating a two-dimensional quasi-potential for the particle(s) to be manipulated and analyzed.

[0055] In a further preferred embodiment of the method according to the invention, at least one external force is applied to the particle. The external force may be at least one of magnetic force, electrostatic force, gravity, frictional force due to additional flows in the system, force exerted on the particle by a tether, in particular a molecular tether, force generated by an optical trap, in particular optical tweezers, or force exerted by a tethered molecule, for example a tethered polymer. The external force allows the force exerted on the particle to be quantitatively determined. In a preferred variant of the invention, the external force is time-dependent or constant, at least over a specified period of time. It is also possible to include a variable external force, which allows the mechanical parameters of the system fluid / particle to be determined.

[0056] To calibrate the measured forces, it may be useful to apply an external force, such as an electrostatic or magnetic force, of known magnitude to the fluid containing the particles to be manipulated and analyzed. The deviation of the particle's position from its equilibrium position in the effective potential, e.g., a stagnation point, can then be measured as a function of the magnitude of the external force. Thus, the magnitude of the force exerted on the particle through hydrodynamic flow can be determined.

[0057] In a further preferred embodiment of the method according to the invention, the particle to be manipulated and analysed is a tethered molecule, a flow field having at least two stagnation points is generated in the fluid, and at least two portions of the tethered molecule, e.g. the terminal particles of the tethered molecule, are held within the stagnation points by the hydrodynamic flow.

[0058] The forces within the tethered molecule, particularly between the end particles, can then be measured, particularly as the tethered molecule is extended.

[0059] Additionally or alternatively, calibration of the measured force can be achieved by observing the fluctuating displacement, specifically the average distance from the equilibrium position of the particle in the effective potential. The average amount of energy in each degree of freedom is k B T(k B = Boltzmann's constant, T = temperature), the force on the particle can be determined. In this respect, a preferred embodiment of the method according to the invention is characterized in that the force on the particle is determined by evaluating the statistical distribution of the lateral position of the particle near the stagnation point and the temperature of the fluid.

[0060] In particular, the present invention allows the velocity and dynamics of trapped particles to be studied as they move back to a stagnation point in a hydrodynamic flow field in the absence of external forces.

[0061] In determining a single force on a single particle, the method of the present invention is implemented. It is also possible to determine the torque on each particle by evaluating the actual spatial configuration of the particles, e.g., their orientation relative to the surrounding hydrodynamic flow field.

[0062] In this regard, it may be useful for the fluid to contain particles, such as fluorescent particles, eg tracer particles, that allow imaging of the hydrodynamic flow field.

[0063] The anomalous localized heating event may be determined as a function of at least one of a recently captured spatial configuration of the particle(s) and a recently captured hydrodynamic flow field.

[0064] The specific dynamic local heating events applied to the fluid can be determined in a feedback loop with respect to the spatial manipulation of the particle(s) to be analyzed. More specifically, in a preferred embodiment of the method according to the invention, at least one target spatial configuration of the particle(s) in the fluid is defined, and the following further steps are performed: a) Photographing the actual spatial configuration of the particle(s). b) determining a singular dynamic local heating event to be applied to the fluid in response to at least one recent actual spatial configuration of the particle(s) and a target configuration of the particle(s); c) applying the unique dynamic localized heating event determined in step b) to the fluid at least once. d) Repeating at least one or all of steps a) through c).

[0065] For a particular manipulation task, it is possible to vary and / or adapt the number of iterations or the iteration rate of the loop comprising at least some or all of steps a) to c) of claim 1.

[0066] A preferred embodiment of the device according to the invention is therefore characterized in that the control unit is designed as follows. A) Activating a device for imaging at least part of the spatial configuration of the particle(s), in particular an imaging device, in order to image the actual spatial configuration of the particle(s) in the container. B) determining a control signal for a heating device appropriate for a singular dynamic localized heating event applied to the fluid in response to at least one recent spatial configuration of the particle(s) and a predefined target configuration of the particle(s); C) activating the heating device to apply the specific dynamic localized heating event determined in step B) to the fluid at least once. D) Repeat at least one or all of steps A) through C).

[0067] Defining a target spatial configuration means defining a predetermined configuration within which one or more particles are manipulated. This can be performed by a user, for example, on a computer screen based on a measured actual configuration or an actual image of the particles. Defining the target configuration can be supported, for example, by image evaluation software that recognizes predetermined structures in the measured image data. The target spatial configuration of particle(s) in the fluid can include at least one of the following: specified target position(s) of particle(s) in the fluid, in particular stagnation points, one or more specified target velocities of particle(s) in the fluid, specified target orientation(s) of particle(s) in the fluid, specified target rotational velocity(s) of particle(s) in the fluid.

[0068] The target spatial configuration of the particle(s) within the fluid may further be a one-dimensional position measurement of the particle(s), a two-dimensional position measurement of the particle(s), or a three-dimensional position measurement of the particle(s).

[0069] For force measurements, the target position may in particular be realized by a stagnation point in the flow field.

[0070] Additionally or alternatively, the target configuration may further include at least one of the following requirements: the designated particle(s) are not at the designated locations; the designated particle(s) are as far away as possible from the designated location(s); the designated particle(s) are at least a designated distance(s) from the designated location(s); the designated particles are as close as possible to each other; the designated particles must not touch each other; different types of particles are treated differently.

[0071] A key feature of the present invention relates to determining, in step b), a specific dynamic local heating event to be applied to the fluid depending on 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 based on the recent, particularly the most recent, actual spatial configuration of the particles and the target configuration of the particles. Thus, the specific dynamic local heating event can be determined depending on the cost function. The cost function can be a scalar function of at least one recent actual spatial configuration of the particle(s) and the target configuration of the particle(s), and / or a description of the desired target configuration. The description of the target configuration can be, for example, that all particles of a first type should be moved to the left and all particles of a second type should be moved to the right to enable effective sorting.

[0072] For example, after the application, or each application, of the unique dynamic local heating event in step c), the actual configuration may be photographed, after which a cost function may be calculated for the new configuration, and if the cost function has decreased from the most recent value, step c) may be repeated with the same unique dynamic local heating event, or if the cost function has increased from the most recent value, step b) may be performed anew.

[0073] The method of the present invention is realized when the force on one particle is determined. However, it is also possible for at least two particles to be spatially manipulated simultaneously and / or for the forces on at least two particles to be determined simultaneously. This means that the actual configuration of the particles is imaged. Thus, a unique dynamic local heating event can be determined such that both (or more) particles are moved or manipulated, effectively manipulating or moving only one particle at a time. Simultaneously in this context means that the forces on the multiple particles are determined from one and the same observed spatial configuration of the particles.

[0074] In principle, each particle to be manipulated can be considered individually. For example, each particle can be moved individually to a predetermined position or a predetermined orientation. However, for a given application, it may be useful to consider a predetermined type of particle. That is, the plurality of particles to be spatially manipulated may include at least a subset of equivalent or identical particles. Thus, the cost function may be invariant with respect to the replacement of equivalent or identical particles. In this case, the complexity of the underlying mathematical task may be reduced.

[0075] For example, in a configuration in which at least two particles are manipulated, in each case the particle primarily manipulated in the next step may be the particle furthest from the target position and / or target orientation associated with each particle.

[0076] More specifically, the cost function may include at least one of the following arguments: distance of a particular particle to a particular target location of that particle, round trip distance of a particular particle to a specified location, distance of each particle of a specified type of particle to a specific target location, round trip distance of a specified particle type to a specified location specified for each particle of a type of particle, angle between actual particle orientation and target orientation for each particle or type of particle, difference between actual particle velocity and target velocity for each particle or type of particle.

[0077] In a further preferred embodiment of the method according to the invention, between steps a) and b), a further step may be performed: the particle(s) to be manipulated are associated with a target position and / or a target orientation. Associating a particle with a specific target position means that a target position is associated with each particle.

[0078] In a further preferred embodiment of the method according to the invention, tracking of particle(s) can be performed by identifying particles present in a new photographed real configuration that includes particles in the most recent real configuration, and thus trajectories of individual particles can be derived from a sequence of real configurations.

[0079] In a further preferred embodiment of the method according to the invention, after tracking of the particle, the target configuration can be re-evaluated and, if the target configuration is changed to a new target configuration, the particle can then be associated with a new target position and / or a new target orientation in each case. Thus, the system can dynamically react to changes in the target configuration and quickly change the binding of the particle to a particular target configuration.

[0080] In a further preferred embodiment of the method according to the invention, at least some of the following data may be stored in the database: previous actual spatial configuration of the particle(s), previous dynamic local heating events applied to the fluid determined based on at least each actual spatial configuration and the target configuration, and changes in the actual spatial configuration of the particle(s) caused by each dynamic local heating event applied to the fluid.

[0081] Future dynamic localized heating events applied to the fluid may be calculated using at least a portion of the data stored in the database, particularly utilizing machine learning / artificial intelligence.

[0082] Further features and advantages of the present invention will be described below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0083] [Figure 1] 1 is a schematic diagram illustrating the measurement principle of the present invention. [Figure 2] 1 is a schematic diagram of an apparatus according to the present invention; [Figure 3] 1 is a schematic diagram showing aspects of spatial manipulation in a method according to the present invention. FIG. [Figure 4] 1 is a flow chart further illustrating spatial manipulation aspects of the method of the present invention. [Figure 5] FIG. 1 shows a simple example of manipulating two particles. [Figure 6] 1 is an example of a hydrodynamic flow field with a stagnation point. [Figure 7]FIG. 7 illustrates particle trapping near a stagnation point in a hydrodynamic flow field similar to that shown in FIG. 6. [Figure 8] FIG. 1 shows the radial displacement of a particle from a stagnation point over time with the hydrodynamic trap being cycled on and off. [Figure 9] This is a histogram of the phase space in which a trapped particle moves around. [Figure 10] FIG. 1 shows the power spectral density function, or mean square displacement, of a particle obtained from raw probe particle position data. [Figure 11] The particles being analyzed are magnetic spherical beads, and the images were taken in an example where an external magnetic force was applied. [Figure 12] FIG. 10 shows the displacement of a probe particle bead away from a stagnation point over time as the magnitude of the applied external magnetic force is varied. [Figure 13] FIG. 8 shows an estimate of the robustness of the trap shown in FIG. 7. [Figure 14] 1 is a schematic diagram of a further preferred embodiment for carrying out the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0084] The same and equivalent components are generally numbered the same in the figures.

[0085] The principle on which the present invention is based will be explained with reference to FIG.

[0086] Figure 1 shows the inhomogeneous field of hydrodynamic flow caused by a local heating event.

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[0087] For spherical, incoherent particles with uniform configuration, smooth surfaces, and laminar flow, the friction force acting on the particle is given by Stokes' equation, specifically:

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[0088] Returning now to Figure 1, for identical particles p1 and p2, the force F2 holding particle p2 at position x2 is smaller than the force F1 holding particle p1 at position x1. More generally, in the example shown in Figure 1, each force decreases with increasing values ​​of x.

[0089] The essence of the present invention is to generate a suitable non-uniform field of hydrodynamic flow within a fluid by using a series of specific localized heating events applied to the fluid and then using the position / force relationship to measure the force on the particles.

[0090] An embodiment of an apparatus 100 according to the invention will now be described with reference to Figure 2. The apparatus 100 shown in Figure 2 is designed to carry out the method according to the invention. Details of the spatial manipulation of particles in a fluid will now be described with reference to Figures 3 to 5. Then an embodiment of the method according to the invention will be described with reference to Figures 6 to 13.

[0091] As main components, the apparatus 100 shown in Figure 2 has a container 10 containing a fluid 12 and particles p1 and p2 to be manipulated, a heating device 20 that generates a hydrodynamic flow within the fluid 12 by dynamic local heating of the fluid 12, an imaging device 40 that images at least a portion of the container 10, and a control unit 60 that controls the heating device 20 and the imaging device 40 and evaluates image data 52 from the imaging device 40.

[0092] Dynamic localized heating is designed to achieve spatial manipulation of particles p1, p2 within a vessel 10 by hydrodynamic flow. The fluid 12 and the particles p1, p2 contained therein are also referred to as the sample.

[0093] More specifically, in the example shown in FIG. 2 , the heating device 20 includes a laser 22, such as an infrared laser, for providing heating radiation 24. The heating radiation 24 is directed through an optical path into a vessel 10 containing a sample to be manipulated and analyzed, i.e., a fluid 12 and particles p1 and p2. In the example shown in FIG. 2 , the optical path includes a scanner 26, a beam shutter 28, a beam splitter 30, and a microscope objective 48. The scanner 26 can direct the heating radiation 24 to different positions within the vessel 10. The beam shutter 28 serves the purpose of preventing the heating radiation 24 from reaching the vessel 10. The beam splitter 30 can be, for example, a dichroic mirror that directs the heating radiation toward the microscope objective 48. In the example shown in FIG. 2 , the scanner 26 and the beam shutter 28 can send status information back to the controller and can be controlled by the controller 60.

[0094] It is clear that FIG. 2 is a schematic diagram, and that in practice the optical beam path may include several additional components not shown in FIG. 2 . More specifically, the optical configuration may be as described in U.S. Pat. No. 6,275,999. The optical assembly of FIG. 2 is that of an inverted microscope. Of course, other configurations are possible. It is clear that due to the inverted configuration of this inverted microscope and the laser-assisted heating, the vessel 10 must have a window that allows the heating and imaging radiation 44 to be incident on the sample. More generally, devices or vessels with local heaters and / or upright microscopes that do not require such windows are also possible. The vessel 10 of FIG. 2 may be, for example, a Petri dish. The vessel 10 may have means for controlling the temperature of the fluid 12, which is also not shown in FIG. 2 . The means for controlling the temperature of the fluid are also described in U.S. Pat. No. 6,275,999, and reference is made to that document in this regard.

[0095] The imaging means 40 in the example of Fig. 2 is realized by a microscope, for example, a wide-field fluorescence microscope. As mentioned above, many other imaging and microscopy techniques are possible. The microscope, also shown only diagrammatically, comprises a light source 42, a beam splitter 46, and a microscope objective 48. The light source 42, for example, a laser, provides imaging radiation 44, which is directed towards the microscope objective 48 by the beam splitter 46, for example, a dichroic beam splitter. The imaging radiation passes through the beam splitter 30 and enters the microscope objective 48, where it is focused by the microscope objective into the sample, i.e., into the fluid 12 containing the particles p1 and p2 to be manipulated in the diagrammatic example.

[0096] For example, fluorescent illumination reflected from the sample, e.g., a dye, in which the manipulated particles are made, or autofluorescence, passes back through the microscope objective 48, the beam splitter 30, and the beam splitter 46 to the photodetector 50, where it is detected. The photodetector 50 may be a camera capable of recording an image of the field of view propagated by the light beam path. That is, the camera can capture the actual spatial configuration of the particles p1, p2 within the container 10. Both the light source 42 and the photodetector 50 are controlled by the controller 60, and in either case can send status data back to the controller 60.

[0097] In the schematic example of Figure 2, target positions T1 and T2 are shown schematically within the vessel 10 of Figure 2. Target positions T1 and T2 may represent locations to which particles p1 and p2, respectively, are to be spatially manipulated. That is, the spatial manipulation task in this example consists of pushing or moving particle p1 toward position T1 and moving particle p2 toward T2.

[0098] According to the present invention, the control unit 60 is designed as follows. Controlling the heating device 20 and the imaging device 40. Evaluating the image data 52 from the imaging device 40 with respect to at least the spatial configuration of the particles p. Determine at least one force acting on particle p by evaluating the spatial configuration of particle p.

[0099] The control unit 60 can further be designed as follows. A) The imaging device 40 is activated to capture the actual spatial configuration of the particle(s) p1, p2 within the container 10. B) determining a control signal for the heating device 20 suitable for a singular dynamic local heating event to be applied to the fluid 12 depending on at least one recent spatial configuration of the particle(s) p1, p2 and a predefined target configuration T1, T2 of the particle(s) p1, p2. C) activating the heating device 20 to apply the unique dynamic localized heating event determined in step B) to the fluid 12 at least once. D) Repeat at least one or all of steps A) through C).

[0100] Further devices for manipulating the sample, in particular the spatially manipulated particles p1, p2, such as for example further lasers, may be present in the apparatus 100 of FIG.

[0101] The control unit 60 may be a PC or equivalent computing device, with peripheral components known in the art. The control unit 60 may include both a computer program product and a computer-readable storage medium according to the present invention.

[0102] Aspects of the present invention relating to spatial manipulation using feedback from a captured spatial configuration will now be described with reference to Figures 3 to 5. It is important to note, however, that feedback from a captured actual spatial configuration is not a required feature of the present invention for force measurement.

[0103] An embodiment of the present invention will now be described with reference to Figures 6 to 13. Key features of the spatial manipulation of particles p1, p2 in a fluid 12 will now be described, first more generally in relation to Figure 3 and then in more detail with reference to Figure 4. More specifically, with reference to Figures 3 and 4, aspects of an embodiment of a method according to the present invention relating to the spatial manipulation of particles will now be described. Now, having defined at least one target spatial configuration T1, T2 of particle(s) p1, p2 in the fluid 12, the following further steps are performed: a) Photograph the actual spatial configuration of the particle(s) (p, p1, p2). b) determining a singular dynamic local heating event to be applied to the fluid 12 depending on at least one recent actual spatial configuration of the particle(s) p1, p2 and a target configuration T1, T2 of the particle(s) p1, p2. c) applying the unique dynamic localized heating event determined in step b) to the fluid 12 at least once. d) Repeating at least one or all of steps a) through c).

[0104] The upper part of Figure 3 shows three schematic diagrams a1, a2, and a3 of a vessel 10 containing particles p1 and p2 that are being spatially manipulated, i.e., moved from their actual locations to or pushed toward target locations T1 and T2, respectively, as in Figure 1. Figure a1 shows an initial configuration in which particles p1 and p2 are significantly separated from their target locations T1 and T2. Figure a2 shows a schematic representation of the hydrodynamic or thermoviscous flow generated within fluid 12, indicated by the dotted arrows. Figure a3 shows the state after the application of a singular dynamic local heating event. As shown in Figure a3, particle p1 has been moved to its corresponding target location T1, and the distance between particle p2 and its target location T2 has been at least reduced compared to the initial state of Figure a1.

[0105] The bottom part of FIG. 3 shows the main steps of the spatial manipulation of particles p 1 , p 2 within fluid 12 .

[0106] First (Fig. b1), the actual spatial configuration of particles p1, p2 is photographed (step a). For example, the image is recorded by the camera 50 of the microscope 40 of Fig. 1.

[0107] Next, in the example shown in Fig. 3, the positions of the particles to be manipulated are identified, i.e., the coordinates of each particle p1 and p2 are identified (Fig. b2).

[0108] After defining the target spatial configurations of particles p1 and p2, i.e., in the example of Fig. 3, after defining the target positions T1 and T2, the paths for both particles p1 and p2 to reach their respective target positions T1 and T2 are calculated (Fig. b3).

[0109] According to step b) of the method variant according to the invention, the specific dynamic local heating event to be applied to the fluid 12 is then determined depending on at least one recent actual spatial configuration of the particles p1, p2, e.g. at least the image recorded in step a) (Figure b1), and the target positions T1, T2 of the particles p1, p2.

[0110] According to step c) of this variant, the specific dynamic local heating event determined in step b) is applied at least once to the fluid 12. In the example shown in Figure 3, the specific dynamic local heating event is applied to the fluid 12 by suitably scanning the heating radiation 24 through the sample, for example with the scanner 26 of Figure 1 (Figure b4).

[0111] Thus, dynamic localized heating of the fluid 12 causes particles p1, p2 to be spatially manipulated within the fluid 12 by hydrodynamic flows generated within the fluid 12.

[0112] According to step d), at least one or all of steps a) to c) are repeated. In the example shown in Figure 3, steps a) to c) are cycled, for example at 30 Hz.

[0113] A more detailed example of a method for spatially manipulating particles is described with reference to FIG. 4. In step S01 "target initialization," at least one target spatial configuration of the particle(s) to be manipulated in the fluid is defined. Next, in step S02, an image of the sample is acquired. This corresponds to step a) of capturing the actual spatial configuration of the particle(s) to be manipulated. This can be achieved, for example, by capturing a microscope image. See the description of FIG. 1. In the example of FIG. 4, the particles are then tracked in step S03. That is, particles present in the actual configuration captured in step S02 are identified with particles in the most recent actual configuration. In step S04, in each case, the manipulated particles are associated with a target position. In step S05 "error calculation," a cost function is calculated depending on the most recent actual spatial configuration of particles p1, p2, e.g., the image captured in step S02, and the target configurations T1, T2 of particles p1, p2, e.g., the target configurations or positions defined in step S01.

[0114] According to step b) of the method, a specific dynamic local heating event to be applied to the fluid 12 is then determined in step S06 "Calculate new FLUCS vector", for example, depending on the cost function calculated in step S05. The specific dynamic local heating event thus depends on at least one recent actual spatial configuration of the particles and on the target configuration of the particles. However, it is also possible to determine the specific dynamic local heating event to be applied to the fluid 12 independently of the cost function value, for example, by selecting the farthest particle and pushing it towards the target.

[0115] In step S07 "Apply FLUCS Vectors", the specific dynamic local heating events determined in step S06 are applied to the sample, i.e., the fluid containing the particles to be manipulated, which corresponds to step c) of the method above.

[0116] According to step d) of the method, at least a part of steps a) to c) is repeated. In the flowchart shown in FIG. 4, a new image is acquired in step S08, i.e., step a) of the method is repeated. Then, in step S09, the particles are further tracked, i.e., the particles present in the actual configuration photographed in step S08 are identified, i.e., the path of each particle is obtained.

[0117] In step S10, a query is made as to whether the target configuration is to be updated. In a preferred embodiment, software determines whether the target configuration is to be updated.

[0118] If the target configuration remains unchanged, query S10 is followed by step S11, in which, as in step S04, the manipulated particles are associated with target positions in any case. If, in response to the query of step S10, the target configuration is to be updated, a new target configuration, e.g., a new target position, is defined in step S14 "Define New Target", and the program continues with step S11.

[0119] In step S12 following step S11, the cost function is calculated anew, as in step S05, for the new actual configuration of particles imaged in step S08 and, if applicable, for the new target configuration defined in step S14.

[0120] In step S13, it is determined whether the error, ie, the cost function, has decreased compared to the value determined in step S05.

[0121] In fact, if the cost function is decreasing from the value determined in step S05, step c) realized in the example of FIG. 4 according to step S07 is repeated with the same singular dynamic local heating event determined in step S06.

[0122] On the other hand, if the cost function has increased from the value determined in step S05, step b) of the method is executed anew, i.e., in step S06 "Calculate new FLUCS vector", a new singular dynamic local heating event to be applied to the fluid 12 is determined according to the cost function calculated in step S12.

[0123] Thus, closed feedback loop control and automated spatial manipulation of particles within a fluid is achieved.

[0124] Schematics 1 through 3 in Figure 5 show how two particles p1 and p2 are brought close to each other using thermoviscous flow. Figure 1 shows the initial state. A first singular dynamic local heating event is determined and applied, moving particle p2 closer to the center. Next, a second singular dynamic local heating event is determined and applied, moving particle p1 closer to the center and adjacent to particle p2, as shown in Figure 2. Figure 3 shows the final configuration of particles p1 and p2.

[0125] Next, an embodiment of the present invention will be described with reference to FIGS.

[0126] To realize optically induced hydrodynamic trapping, two counter-directional thermoviscous flows were generated by splitting the relay laser scan line into two counter-directional paths. Figure 6 shows a schematic of the generated inhomogeneous hydrodynamic flow field u(x). With this approach, no intersecting micropaths are required. The direction of the laser scan determines the direction of the induced flow, and a stagnation point S is formed between arbitrarily selected laser scan paths (the horizontal arrows in Figure 6 point to the stagnation point S), where the particles can be trapped.

[0127] The confinement in this case is weakly metastable, since the restoring properties of the trap are only observed along the compression axis, i.e., the transverse direction in Fig. 6, and therefore any positional fluctuations along the vertical axis (vertical arrow in Fig. 6) lead to particle ejection.

[0128] To avoid such ejection, active feedback control is utilized, which allows dynamic rotation of the counterflow and rapid readjustment of the two in-plane axes (Fig. 7).

[0129] With respect to claim terminology, the non-uniform field of hydrodynamic flow shown in FIG.

number

number

[0130] Figure 7a) shows the deviation δr of the actual position of particle p from the stagnation point S. This deviation δr is observable, and the force acting on particle p can be determined depending on this deviation δr. Figures 7b), 7c), and 7d) show in each case different directions of the applied thermoviscous flow, the position of particle p, and the stagnation point S. In Figures 7b) and 7d), particle p is very close to the stagnation point S, while in the situation in Figure 7c), particle p is far from the stagnation point S.

[0131] The robustness of the trap is adjustable, as it depends on the intensity of the scanning laser, the frequency of the scan, and the update rate of the scan path, as well as many other user-specified parameters.

[0132] Further properties of the trap can be evaluated by intermittently switching the trap on and off. The results of such measurements are shown in the diagram of Figure 8, which shows the radial displacement of a particle from the stagnation point S over time.

[0133] As soon as the laser is turned on, particle p is dragged towards the stagnation point S, which corresponds to the solid line in the diagram of Figure 8. On the other hand, when the laser is turned off, particle p diffuses again in a Brownian motion fashion, which corresponds to the dotted line in the diagram of Figure 8.

[0134] Figure 9 shows a histogram of the phase space in which trapped particles move within the water, which indirectly shapes the trap shape contour. The histogram, also called a heat map, shows the number of times (count, vertical axis) that a particle is observed at a given distance (horizontal axis) from the stagnation point S.

[0135] The histogram in Figure 9 corresponds to the profile of the trapping potential. The potential exhibits symmetry, which indicates that the restoring force realized by the dynamic rotation of the laser scanning path (see Figure 7) is independent of the direction of the particle's displacement from the stagnation point. This essentially creates a quasi-one-dimensional trapping state, where the particle is always displaced along the compression axis.

[0136] This can be confirmed by analyzing the mean-squared displacement (MSD) of particle p, shown in Figure 10. Figure 10 shows the power spectral density function of the mean-squared displacement of particle p, obtained from the raw position data of particle p. Similar to optical tweezers, a power spectral density (PSD) roll-off analysis can be used to obtain a Lorentzian fit to the Fourier-transformed flow trap data. Therefore, the robustness of the trap along each abscissa can be accurately estimated. Figure 10 shows the data for the x-coordinate. The symmetry of the trap is evident from the agreement of the robustness of the two in-plane traps with the x-coordinate, denoted kx, which overlap within the calculated uncertainty. Data for the y-coordinate is not shown.

[0137] The transition between short-term and long-term diffusion occurs at a break frequency f c Therefore, the trap robustness k along each orthogonal axis can be accurately estimated via

number

[0138] Sensitive force measurements require that the displacement from the trapping point can be used as a measure of the force on the particle. This allows us to investigate the force-extension relationship exhibited by optically induced hydrodynamic traps. Stokes resistance calibration can be used to determine the velocity-distance relationship, a technique also used to validate the near-high-frequency trapping potential generated by optical tweezers.

[0139] It is also possible to displace a trapped particle from the stagnation point and track its relaxation behavior. The inventors observed an exponential approach of the particle to the stagnation point, suggesting a linear relationship between velocity and displacement: the particle that is further away from the trap is dragged faster towards the stagnation point. This is reminiscent of a Hookean spring. The trapping time in an optical tweezers experiment can be derived as follows:

number

[0140] External forces can be used to further characterize the trap and serve calibration purposes. This is illustrated with reference to FIGS. 11 through 13. For the experiments underlying the data in FIGS. 11 through 13, an external custom-made magnetic needle M and magnetic particle p were used to balance the forces and quantify the counter-flow trapping force. The magnetic force can be adjusted by steadily changing the current applied to the electromagnet. In the absence of any flow, dynamic magnetic actuation of particle p exhibited a favorable attraction effect, as expected. This can be verified by ballistic motion (not shown) over a long time lag.

[0141] Increasing the current allows the particle to move over a larger region of space in the same acquisition time, reflecting an increased magnetic attraction. In either case, extracting the particle's long-term (steady-state) velocity allows the magnetic force to be calibrated through a force balance with the known Stokes drag force, as follows:

number

[0142] A driving current is applied to electromagnet M, and the resulting magnetic field pulls the particle away from the stagnation point. This is shown in Figure 11b). The trap is still in operation, and the external electromagnetic force Fext, realized by electromagnet M, is turned on. As shown, particle p is dragged away from the stagnation point S by a distance δr. Therefore, the minimum of the potential energy for the particle is shifted toward the magnetic field source.

[0143] The magnitude of the displacement depends on the strength of the magnetic force: the greater the current, the greater the displacement from the stagnation point.

[0144] This is illustrated with reference to Figure 12, which shows the displacement over time for various magnitudes of current through the electromagnet. From curve s to curve k, the current through electromagnet M is increased in increments of 0.2 A.

[0145] By mapping the calculated magnetic force to the applied current, a linear relationship between force and extension can be fitted. Thus, a further estimate of the robustness of the backflow trap is obtained. This is shown in Figure 13, which shows the particle displacement δr under the influence of an external force plotted against the calculated magnetic force. The trap robustness obtained here is very close in magnitude to the two previous estimates, differing by no more than two standard deviations. Importantly, the backflow force determined by the application of a well-controlled magnetic force can be used to quantify any other unknown, externally applied forces in the configuration.

[0146] The explicit application of an external force confirms that equilibrium thermodynamics can indeed be used to accurately characterize relaxation dynamics after position perturbation. Furthermore, histograms reflecting the magnetic particle's fluctuations around its steady-state position, where the countercurrent is precisely balanced with the magnetic force, demonstrate that the measurements are close to the thermal limit. Smaller force detection entails a broader range of potential applications, enabling trapping over a larger phase space than is currently achievable with many point-trap optical tweezers configurations, where the focal spot is typically diffraction-limited. Furthermore, our optically generated hydrodynamic traps are highly tunable, allowing for further optimization by increasing the laser intensity, increasing the scan path distance, or reducing the countercurrent update frequency.

[0147] A further embodiment of the method according to the invention will now be described with reference to Figure 14, which shows how a hydrodynamic flow field with two distinct stagnation points S1 and S2 is generated by a suitable laser scanning pattern.

[0148] More specifically, by scanning the laser beam in the directions of arrows s1 and s2, hydrodynamic flows are generated in the directions of arrows f1 to f4. A first stagnation point S1 is generated at the position where flows f1 and f4 collide with each other.

[0149] Further scanning of the laser beam in the directions of arrows s3 and s4 generates hydrodynamic flows in the directions of arrows f5 to f8. A second stagnation point S2 is generated at the position where flows f5 and f8 join.

[0150] The object of manipulation and analysis in this example is a tethered molecule having two terminal particles p1 and p2 and a molecular tether C, shown schematically, connecting p1 and p2. As described above, particle p1 is trapped near stagnation point S1. Particle p2 is trapped near stagnation point S2. The forces that pull particles p1 and p2 back to stagnation points S1 and S2, respectively, when no force is applied by the molecular tethers can be measured as described above. For this purpose, either flows f1 through f4, which generate stagnation point S1, or flows f5 through f8, which generate stagnation point S2, are activated.

[0151] The forces F1 and F2 that molecular chain C exerts on particles p1 and p2 can then be measured by varying the distance from stagnation point S1 to stagnation point S2 and observing the displacement of particles p1 and p2 from stagnation points S1 and S2, respectively, as a function of the distance from S1 to S2.

[0152] A particular advantage of the configuration of FIG. 14 is that the entire region R is not exposed to the heating radiation from the laser on the fluid 12, and therefore the tethered molecules p1-C-p2 under investigation are not exposed to the heating radiation from the laser.

[0153] Overall, we disclose a highly sensitive, tunable, non-contact trap generated by two counter-directional optically induced thermoviscous flows. This novel approach may be highly relevant for the application of optical trapping in in vivo systems and for addressing emerging concerns regarding heating effects and potential photodamage due to the geometric limitations of microfluidic traps. The arbitrarily defined scanning path and resulting stagnation points enhance the localization and flexibility of this approach. The induced level of heating by laser scanning is moderate and easily tolerated in in vivo systems. Therefore, the inventive approach may have diverse applications in life sciences, from cell biology to embryonic development. In terms of materials science, the method is highly advantageous for determining the viscoelastic properties of complex fluids. Finally, the ability of counter-flow traps to detect femtonewton forces on the micrometer scale may be particularly advantageous in the field of mechanobiology for the detection of local mechanical indicators that drive key cellular processes, such as differentiation and proliferation.

[0154] In this disclosure, we present a novel non-contact trapping method based on optically induced hydrodynamic flow. We demonstrate a linear force-extension relationship that allows femtonewton force detection near the thermal sensitivity limit. The presented technique eliminates the need for laser contact with the particle, eliminating material limitations on the particles that can be analyzed. Furthermore, the method can be performed with a standard optical microscope without the need for specialized chambers, enabling the investigation of local forces within more complex materials. Therefore, optically induced hydrodynamic flow contributes to sensitive, non-invasive force measurements within a variety of samples. [Explanation of symbols]

[0155] 10 container, sample chamber, 12 fluid, e.g., water, 20 heating device, 22 irradiation source, e.g., infrared laser, 24 heating irradiation, 26 scanner, 28 beam shutter, 30 means for combining light beams, e.g., dichroic mirror, 40 microscope, e.g., fluorescence microscope, 42 imaging light source, 44 imaging irradiation, 46 e.g., filter cube consisting of excitation, dichroic, and emission filters, 48 ​​objective lens, 50 detector for imaging irradiation, e.g., camera, 52 image data, 60 control unit, e.g., PC, 100 device according to the invention, C molecular chain, p, p1 to p6 manipulated and / or positioned particles, f, f1 to f8 hydrodynamic flow direction, F force on particle, F1 force on particle p1, F2 force on particle p2, Fext external force on particle, FLUCS cytoplasmic streaming by focused light, R Regions where heating radiation does not enter the fluid, S, S1, S2: stagnation point, target position, T1 to T3: target position, u, u(x), u(x, y): inhomogeneous field of hydrodynamic flow, δr: deviation of actual particle position from stagnation point.

Claims

1. 1. A method for determining a force on at least one particle in a fluid, comprising: A non-uniform field of hydrodynamic flow within a fluid (12) due to a specific dynamic local heating event by a laser or light emitting diode. [Equation 1] and said hydrodynamic flow spatially manipulating said particles (p); imaging the spatial configuration of said particle(s) (p) within said fluid (12); and evaluating the imaged spatial configuration of the particle(s) (p) to determine at least one force (F) acting on the particle(s) (p).

2. The hydrodynamic flow field [Equation 2] is the case [Equation 3] 2. The method of claim 1, wherein the value of the saturation voltage decreases in the direction of

3. 3. A method according to claim 1 or 2, characterized in that the fluid (12) is or contains water.

4. 4. The method according to any one of claims 1 to 3, characterized in that the particle(s) (p) to be manipulated are at least one of a biological particle, a cell, a virus, a tissue fragment, a metal particle, a composite particle, a polymer particle, a nanoparticle, a spherical bead, a magnetic bead, a tethered molecule, an organelle, a phase-separated droplet containing a biomolecule, the biomolecule itself including a protein or RNA, a tethered molecule.

5. 5. A method according to any one of claims 1 to 4, characterized in that the dynamic local heating of the fluid (12) is achieved by means of a laser or an infrared laser or at least one infrared light emitting diode.

6. The method of claim 5, wherein the dynamic localized heating event in the fluid (12) is caused by repeatedly scanning the laser along a path within the fluid (12).

7. The determination of the specific dynamic local heating event applied to the fluid (12) comprises: - a two-dimensional scanning path through the fluid; a three-dimensional scanning path within the fluid; laser intensity, - laser scanning speed, the scanning frequency of the laser; The number of scans of the laser, 7. The method according to claim 1, further comprising determining at least one of:

8. 8. Method according to any one of claims 5 to 7, characterized in that the path along which the laser is scanned is selected so that the particle(s) (p, p1, p2) to be manipulated are not exposed to heating radiation.

9. 9. The method according to claim 6, wherein the scan rate of the repeated scans is selected so as to reduce the temperature field within the sample of the fluid and the particles between successive scans.

10. Spatially manipulating the particle(s) (p, p1, p2) comprises: - pushing or moving designated particle(s) (p, p1, p2) towards designated target locations (S, T1, T2) within said fluid (12); moving designated particle(s) (p, p1, p2) along a designated path within said fluid (12); - retaining designated particle(s) (p, p1, p2) at designated target positions (S, T1, T2) within said fluid (12); - trapping designated particle(s) (p, p1, p2) in a designated target orientation (S, T1, T2) within said fluid (12); - pushing or moving designated particle(s) (p, p1, p2) towards designated target orientation(s) (S, T1, T2) within said fluid (12); 10. The method according to claim 1, further comprising at least one of the following steps:

11. The image obtained by capturing the actual spatial configuration of the particle(s) (p, p1, p2) is the one-dimensional position of said particle(s) (p, p1, p2); the two-dimensional position of said particle(s) (p, p1, p2); the three-dimensional position of said particle(s) (p, p1, p2); - measuring the orientation of said particle(s) (p, p1, p2) in a plane; - a three-dimensional orientation measurement of said particle(s) (p, p1, p2) in space; 11. The method according to claim 1, further comprising at least one of the following steps:

12. the non-uniform field of the hydrodynamic flow [Equation 4] 12. The method according to claim 1, wherein the at least one particle (p) is at least temporarily trapped in the vicinity of the stagnation point (S).

13. 13. The method of claim 12, wherein a deviation (δr) of an actual position of the at least one particle (p) from the stagnation point (S) is observed and a force acting on the particle (p) is determined as a function of the deviation (δr).

14. 14. The method according to claim 12 or 13, characterized in that the non-uniform field of hydrodynamic flow comprising at least one stagnation point (S) is generated by at least two hydrodynamic flows (f) in opposite directions towards the stagnation point (S).

15. 15. A method according to claim 14, characterized in that the at least two hydrodynamic flows (f) of opposite directions are rotated in a plane around the stagnation point (S).

16. 16. The method according to claim 15, characterized in that the azimuthal directions in which the at least two opposite hydrodynamic flows (f) are applied are selected depending on the imaged spatial configuration of the particles (p), in particular depending on at least one of the measured azimuthal and radial coordinates of the particle(s) (p) with respect to the stagnation point (S).

17. 17. Method according to any one of claims 1 to 16, characterized in that at least one external force (Fext) is applied to the particles (p).

18. The external force (Fext) is ・Magnetism, electrostatic forces, ·gravity, - Forces generated by optical traps, especially optical tweezers; - Forces exerted by tethered molecules, including tethered polymers; 18. The method of claim 17, wherein the method is at least one of:

19. 19. A method according to claim 17 or 18, characterized in that the external force (Fext) is time-dependent or constant, at least for a specified period of time.

20. 20. A method according to any one of claims 17 to 19, characterized in that the force on the particle (p) is calibrated by comparison with the external force (Fext).

21. 21. The method according to any one of claims 1 to 20, characterized in that the force (F) acting on the particle (p) is determined, for example, by evaluation of the statistical distribution of the lateral position of the particle (p) near a stagnation point and the temperature of the fluid (12).

22. 22. The method according to any one of claims 1 to 21, characterized in that at least two particles (p1, p2) are simultaneously spatially manipulated and / or forces on at least two particles are simultaneously determined.

23. 23. A method according to any one of claims 1 to 22, characterized in that for at least one particle (p), the torque acting on each particle (p) is determined.

24. 24. A method according to any one of the preceding claims, characterized in that the fluid (12) contains fluorescent particles that allow imaging the hydrodynamic flow field.

25. The specific local heating event is a recently photographed spatial configuration of said particle(s); Recently photographed hydrodynamic flow fields and 25. The method of claim 24, wherein the temperature is determined in response to at least one of:

26. At least one target spatial configuration (T1, T2) of said particle(s) (p, p1, p2) in said fluid (12) is defined, a) imaging the actual spatial configuration of said particle(s) (p, p1, p2); b) determining a specific dynamic local heating event to be applied to said fluid (12) depending on at least one recent actual spatial configuration of said particle(s) (p, p1, p2) and a target configuration (T1, T2) of said particle(s) (p, p1, p2); c) applying the unique dynamic localized heating event determined in step b) to the fluid (12) at least once; d) repeating at least one or all of steps a) to c); and 26. The method according to any one of claims 1 to 25, characterized in that the further step of:

27. The target spatial configuration of the particles (p, p1, p2) in the fluid (12) is: - specified target position(s) (T1, T2) of said particle(s) (p, p1, p2) in said fluid (12), in particular stagnation points; one or more specified target velocities (T1, T2) of said particle(s) (p, p1, p2) in said fluid (12); - specified target orientation(s) (T1, T2) of said particle(s) (p, p1, p2) in said fluid (12); - specified target rotational speed(s) (T1, T2) of said particle(s) (p, p1, p2) in said fluid (12); 27. The method of claim 26, comprising at least one of:

28. The target spatial configuration of the particles (p, p1, p2) in the fluid (12) is: - one-dimensional position measurement of said particle(s) (p, p1, p2), - measuring the two-dimensional position of said particle(s) (p, p1, p2), or - three-dimensional position measurement of said particle(s) (p, p1, p2) 28. The method according to claim 26 or 27, characterized in that:

29. Based on the recent actual spatial configuration of the particles (p, p1, p2) and the target configuration (T1, T2) of the particles (p, p1, p2), a cost function is calculated (S05, S12) that represents the error between the spatial configuration of the particles (p1, p2) and the target configuration (T1, T2) of the particles (p1, p2), in particular:

29. The method according to any one of claims 26 to 28, characterized in that the anomalous dynamic local heating event determined in step b) is determined in response to the cost function.

30. The database contains the following data: the actual spatial configuration in front of said particle(s) (p, p1, p2), - a previous dynamic local heating event applied to said fluid (12), determined based on at least each actual spatial configuration and target configuration (T1, T2); - the change in the actual spatial configuration of the particle(s) (p, p1, p2) caused by each dynamic localized heating event applied to the fluid (12); is memorized, 30. The method of any one of claims 26 to 29, characterized in that future dynamic local heating events to be applied to the fluid (12) are calculated using at least part of the data stored in the database.

31. 31. The method of any one of claims 1 to 30, characterized in that future dynamic local heating events to be applied to the fluid (12) are calculated using machine learning.

32. the particle to be manipulated and analyzed is a tethered molecule; A flow field is generated within the fluid (12) having at least two stagnation points; 32. The method according to any one of claims 1 to 31, characterized in that the end particles (p1, p2) of the tethered molecule are held within the stagnation points (S1, S2) by the hydrodynamic fluid.

33. 1. An apparatus for determining a force on at least one particle in a fluid, comprising: a container (10) containing the fluid (12) and the particles (p); a heating device (20) for generating a non-uniform hydrodynamic flow field within the fluid (12) by a specific dynamic local heating event (12) using a laser or a light emitting diode; a device (40) for imaging at least a portion of the spatial configuration of said particle(s) (p) within said container (10); A control unit (60), - controlling said heating device (20) and said device (40) for imaging at least a part of the spatial configuration of said particle(s) (p); evaluating data (52) from said device (40) imaging at least a portion of the spatial configuration of said particle(s) (p); determining at least one force on said particle (p) by evaluating said spatial configuration of said particle; A control unit (60); An apparatus comprising:

34. the device for imaging at least a portion of the spatial configuration of the particle(s), - imaging device, - a camera without a lens, - Quadrant photodiode, 34. The device of claim 33, wherein the device is at least one of:

35. 35. Apparatus according to claim 33 or 34, designed to carry out a method according to any one of claims 1 to 31.

36. 36. Device according to any one of claims 33 to 35, characterized in that the container (10) comprises means for controlling the temperature of the fluid (12).

37. 37. Apparatus according to any one of claims 33 to 36, characterized in that the heating device (20) comprises a laser (22) that provides energy for the dynamic localized heating, and optical means including a scanner (26) that relays the heating laser radiation to a variable location within the fluid.

38. 38. Apparatus according to any one of claims 33 to 37, characterized in that the imaging device (40) is a microscope.

39. 39. The apparatus of claim 38, wherein the microscope is designed to perform at least one of the following techniques: fluorescence microscopy, multiphoton fluorescence microscopy, widefield microscopy, scanning microscopy, darkfield microscopy, confocal microscopy, light sheet microscopy, localization 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 plane microscopy (OPM).

40. The control unit (60) A) activating the device for imaging at least a part of the spatial configuration of the particles(s), in particular the imaging device (40), in order to image the actual spatial configuration of the particles(s) (p, p1, p2) in the container (10), B) determining control signals for the heating device (20) suitable for a singular dynamic localized heating event to be applied to the fluid (12) depending on at least one recent spatial configuration of the particle(s) (p, p1, p2) and a predefined target configuration (T1, T2) of the particle(s) (p, p1, p2); C) operating the heating device (20) to apply the unique dynamic localized heating event determined in step B) to the fluid (12) at least once; D) Repeat at least one or all of steps A) through C).

40. Device according to any one of claims 33 to 39, characterized in that it is designed to

41. 1. A computer program product comprising instructions, When the program is executed by the control unit (60), the control unit (60) is instructed to: A) operating an imaging device (40) for imaging at least a portion of the spatial configuration of the particles (p, p1, p2) in the container (10) to image the actual spatial configuration of the particles (p, p1, p2) in the container (10); B) determining a control signal for the heating device (20) suitable for a singular dynamic localized heating event to be applied to the fluid (12) depending on at least one recent spatial configuration of the particle(s) (p, p1, p2) and a predefined target configuration (S, T1, T2) of the particle(s) (p, p1, p2); C) operating the heating device (20) to apply the unique dynamic localized heating event determined in step B) to the fluid (12) at least once; D) repeating at least one or all of steps A) through C); and E) determining the forces on the particles (p, p1, p2) as a function of the imaged spatial configuration of said particle(s) (p, p1, p2); 33. A computer program product causing the computer to carry out the method of any one of claims 1 to 32, comprising:

42. A computer-readable storage medium containing instructions that, when executed by the control unit (60), cause the control unit (60) to: A) operating an imaging device (40) for imaging at least a portion of the spatial configuration of the particles (p, p1, p2) in the container (10) to image the actual spatial configuration of the particles (p, p1, p2) in the container (10); B) determining a control signal for the heating device (20) suitable for a specific dynamic localized heating event to be applied to the fluid (12) depending on at least one recent spatial configuration of the particles (p, p1, p2) and a predefined target configuration (S, T1, T2) of the particles (p, p1, p2); C) operating the heating device (20) to apply the unique dynamic localized heating event determined in step B) to the fluid (12) at least once; D) repeating at least one or all of steps A) through C); and E) determining the forces on the particles (p, p1, p2) as a function of the imaged spatial configuration of said particle(s) (p, p1, p2); 33. A computer-readable storage medium causing execution of the method of any one of claims 1 to 32, comprising:

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