Method and apparatus for spatially manipulating at least one particle in a fluid, as well as computer program product and computer-readable storage medium

Abstract

The invention relates to a method for spatially manipulating at least one particle in a fluid, in which one or more particles are spatially manipulated by a hydrodynamic flow generated in the fluid by dynamic local heating of the fluid. The method according to the invention is characterized in that at least one target spatial configuration of the particle(s) in the fluid is defined, and the following further steps are performed: a) imaging the actual spatial configuration of the particle(s), b) determining 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 the target configuration of the particle(s), c) applying the specific dynamic local heating determined in step c) at least once to the fluid, and d) repeating at least one or all of steps a) to c). The invention further relates to an apparatus, a computer program product and a computer readable storage medium for spatially manipulating at least one particle in a fluid by a hydrodynamic flow.

Description

[Technical Field]

[0001] A first aspect of the invention relates to a method for spatially manipulating at least one particle in a fluid according to the preamble of claim 1. A second aspect of the invention relates to an apparatus for spatially manipulating at least one particle in a fluid according to the preamble of claim 31. Further aspects of the invention relate to a computer program product and a computer readable storage medium. [Background technology]

[0002] The manipulation of micro-sized particles, especially their precise positioning, continues to be an active research topic in applications in life science, engineering, and manufacturing. Examples of techniques that have been successfully applied include optical, magnetic, electrokinetic, acoustic, and thermophoretic trapping, as well as self-propelled Janus particle positioning. However, some of these techniques are limited by the specific material properties of the particles or the requirements of the environments in which they can be applied. To overcome these limitations, hydrodynamic traps have been successfully implemented, making them particularly desirable in life science and lab-on-a-chip environments.

[0003] Hydrodynamic trapping methods can be divided into contact methods (Non-Patent Documents 1 and 2) and non-contact methods. In the former, dissolved particles are immobilized against walls, wells, pillars, or other obstacles by fluid flow. In the latter, particles can be trapped in stagnation flows (Non-Patent Documents 3 and 4), microvortex flows (Non-Patent Document 5), or microeddies (Non-Patent Document 6). It has been demonstrated that the position of stagnation flows can be controlled by dynamic feedback control of the flow in a chamber with multiple inlets. This allows users to manipulate particle position and counter particle displacement due to diffusion (Non-Patent Documents 3 and 4). This method allows the position of single colloidal particles to be controlled with an accuracy of 78 nm (Non-Patent Document 4). Hydrodynamic trapping methods reduce the limitations of the material properties of trapped particles (Non-Patent Document 7), but precise manipulation of particles requires the use of highly accurate and stable microfluidic pumps. Furthermore, hydrodynamic traps generally require specialized chambers and cannot be used to generate flow in closed systems such as cells. Furthermore, the flow generated is typically global, extending from the pump to the outlet, thus severely limiting the spatial resolution of the technique.

[0004] Another approach to positioning particles with high precision is to utilize thermoviscous flow. This has been described as the directed motion of an aqueous medium in response to a moving temperature field (Non-Patent Document 8), a sudden physical phenomenon caused by the thermal expansion of a fluid in a non-uniform viscosity field (compare Figure 1b). Specifically, thermoviscous flow has been used to transport aqueous solutions visualized by tracer particles or molecules along optically defined paths (Non-Patent Documents 8, 9). 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 placement of colloids immersed in the cytoplasm (Non-Patent Documents 10-12). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2008 / 077630A1

Non-licensed literature

[0006] [Non-licensed document 1] DDCarlo,LYWu and LPLee,Lab Chip,2006,6,1445-1449. [Non-licensed document 2] Q. Luan, C. Macaraniag, J. Zhou and I. Papautsky, Biomicrofluidics, 2020, 14, 031502. [Non-licensed document 3] M. Tanyeri, EM Johnson-Chavarria and CMSchroeder, Appl. Phys. Lett., 2010, 96, 224101.

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0007] For example, in a generalized method for spatially manipulating at least one particle in a fluid, known from US Pat. No. 5,629,999, particles are spatially manipulated within the fluid by hydrodynamic flows generated within the fluid by dynamic localized heating of the fluid.

[0008] For example, Patent Document 1 also discloses a general-purpose apparatus for spatially manipulating at least one particle in a fluid by hydrodynamic flow, the general-purpose apparatus comprising a vessel containing the fluid and one or more particles to be manipulated, and a heating device for generating hydrodynamic flow in the fluid by dynamic localized heating of the fluid. The dynamic localized heating is designed to cause spatial manipulation of the particles in the vessel by hydrodynamic flow. The general-purpose apparatus further comprises an imaging device for imaging at least a portion of the vessel, and a control unit for controlling the heating device and the imaging device and for evaluating image data from the imaging device.

[0009] It is seen as an object of the present invention to provide a method and apparatus of the above kind that is versatile in scope. [Means for solving the problem]

[0010] The object is achieved by a method having the features of claim 1, by an apparatus having the features of claim 31, and by a computer program product and a computer-readable storage medium having the features of claims 39 and 40, respectively.

[0011] According to the invention, the method for spatially manipulating at least one particle in a fluid as described above, together with the definition of at least one target spatial configuration of the particle(s) in the fluid, comprises the further steps of: 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 differential dynamic localized heating determined in step b) to the fluid at least once. d) Repeating at least one or all of steps a) through c).

[0012] According to the present invention, the above-mentioned general-purpose device further has the following features: The control section is designed as follows. A) activating an imaging device to capture an image of the actual spatial configuration of the particle(s) within 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) actuating 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).

[0013] According to the present invention, there is provided a computer program product or computer readable storage medium comprising instructions, which when executed by a control unit, cause the control unit to carry out a method comprising the steps of: A) operating an imaging device to capture an image of the actual spatial configuration of particles within the container; B) determining a control signal for a heating device suitable for a singular dynamic localized heating event applied to the fluid in response to at least one recent spatial configuration of the particles and a predefined target configuration of the particles; C) operating the heating device to apply 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).

[0014] 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.

[0015] The instructions on the computer program product and / or computer readable storage medium may specifically serve the purpose of operating the control unit to perform the method according to any one of claims 1 to 30.

[0016] The basic concept of the present invention is to use unique dynamic local heating events to spatially manipulate particles within a fluid, and to determine these unique dynamic local heating events in each case depending on a target configuration of particles and a recently imaged actual configuration of particles within the fluid. The unique dynamic local heating events serve the purpose of generating hydrodynamic flow sequences within the fluid. The spatial manipulation of particles can be performed within a closed system control, where the actual imaged particle configuration serves as feedback for determining the next applied unique dynamic local heating event.

[0017] A very important advantage of the present invention is that high precision manipulation, and in particular positioning, of a particle or particles is feasible, and that this manipulation can in principle be automated.

[0018] The device according to the invention can be designed to carry out the method according to the invention.

[0019] The term spatial manipulation of particles within a fluid generally means that the particle / fluid system within the vessel 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 vessel. However, spatial manipulation can also mean that the particles are held in a particular position relative to other behaviors, e.g., external forces acting on the particles.

[0020] In preferred embodiments of the method according to the invention, spatially manipulating the particle(s) may comprise at least one of the following: Move a specified particle or particles towards a specified target location within a fluid. Move a specified particle or particles along a specified path through a fluid. · Keeping a specified particle(s) at a specified target location within a fluid. · Pin a specified particle(s) to a specified target orientation within the fluid. Move specified particle(s) towards specified target orientation(s) within a fluid.

[0021] In general, the methods and devices of the present invention can be used to manipulate any suspended particle in a fluid that is at least partially freely movable 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 debris, metal particles, composite particles, polymer particles, microparticles, and nanoparticles.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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 sometimes 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 only on the optical and thermodynamic properties of the fluid used, e.g., water. Hydrodynamic flows generated by dynamic local heating of a fluid are also called thermoviscous flows.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] More specifically, the determination of the anomalous dynamic localized heating event determined for the fluid in step b) of claim 1 may include determining at least one of the following: Two-dimensional scanning path through the fluid, 3D scanning paths in the fluid, Laser intensity, Laser wavelength (if changeable), Laser scanning speed, Laser scanning frequency, - Number of scans along the scan path.

[0032] 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.

[0033] 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.

[0034] In general, the scan 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 scan path can be parallel to the connecting vector between the particle's destination and actual location. The scan path can be centered on the particle, end just before it, or start just after it. The laser scan can be applied along the path at a scan rate, for example, typically 1 to 3 kHz, which is slow enough to allow the temperature field to relax between successive scan periods. The scan rate can be variable along the scan path.

[0035] It is also 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 for a particular manipulation task.

[0036] 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 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] In a preferred embodiment according to the invention, the container comprises means for controlling the base temperature of the fluid.

[0041] 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.

[0042] 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.

[0043] 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).

[0044] 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.

[0045] Imaging the actual spatial configuration of the particle(s) may include at least one of the following: the one-dimensional position of the particle(s), the two-dimensional position of the particle(s), the 3D position of the particle(s), Measurement of the orientation of a particle or particles in a plane, · 3D orientation measurement of a particle or particles in space.

[0046] Defining a target spatial configuration means defining a predetermined configuration within which one or more particles are to be 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 a target configuration can be aided, for example, by image evaluation software, which recognizes predetermined structures in the measured image data. The target spatial configuration of the particle(s) within the fluid can include at least one of the following: · specified target position(s) for particle(s) within the fluid; one or more specified target velocities for the particle(s) in the fluid; specified target orientation(s) of particle(s) in the fluid, ·Specified target rotational velocity(s) for particle(s) in the fluid.

[0047] 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).

[0048] Additionally or alternatively, the target configuration further includes at least one of the following requirements: The specified particle(s) are not at the specified location, The designated particle(s) are as far away as possible from the designated location(s); The specified particle(s) are at least the specified distance(s) from the specified location(s); The designated particles are as close as possible to each other, -Specified particles must not touch each other; · Different types of particles are treated differently.

[0049] 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.

[0050] 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.

[0051] The overall concept of the present invention is realized as only one particle is spatially manipulated. However, in a particularly preferred embodiment of the method according to the present invention, at least two particles are spatially manipulated simultaneously. This means that the actual configuration of the particles is imaged. Thus, a specific dynamic local heating event can be determined as both (or more) particles are moved or manipulated, effectively manipulating or moving only one particle at a time.

[0052] 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.

[0053] 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 is useful to consider a predetermined type of particle. That is, the plurality of particles to be spatially manipulated can include at least a subset of equivalent and identical particles. Thus, the cost function can be invariant with respect to the replacement of equivalent or identical particles. In this case, the complexity of the underlying mathematical task can be reduced.

[0054] More specifically, the cost function may include at least one of the following arguments: the distance of a particular particle to a particular target location of that particle; Round-trip distance of a particular particle to a specified location, The distance of a given type of particle to a specific target position for each type of particle; The round-trip distance of a specified type of particle to a specified position specified for each type of particle, the angle between the actual particle orientation and the target orientation for each particle or type of particle; The difference between the actual particle velocity and the target velocity for each particle, or each type of particle.

[0055] 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: the actual spatial configuration in front of the particle(s), a previous dynamic local heating event applied to the fluid, determined based on at least each actual spatial configuration and the target configuration; · The change in the actual spatial configuration of the particle(s) caused by each dynamic local heating event applied to the fluid.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] In a further preferred embodiment of the method according to the present invention, the integrity of at least one particle can be altered before, during, or after spatial manipulation. For example, debris from a biological particle can be cut off by laser cutting. Thus, the apparatus according to the present invention can have at least one device for altering the integrity of the particle(s), such as a laser. The apparatus according to the present invention can further have a device for altering the orientation of at least one particle, such as optical tweezers.

[0061] Comparing the actual position of a particle with its expected position can be useful to predict forces on the particle. Detecting such forces can be particularly sensitive, as flow forces are typically weak. In some applications, it can be useful to be able to interactively control particles with hand gestures. For this, mixed reality devices provide a preferred and potentially compatible means. A force feedback interface can enable haptic interactive manipulation.

[0062] In a preferred embodiment of the method according to the invention, the displacement of the actual position of at least one particle to be manipulated is compared with the expected position of said particle and the force acting on the particle is determined depending on this displacement.

[0063] In this regard, it may be useful to orient at least two counter-flowing hydrodynamic flows toward the center and image the particle at the center where the two counter-flows meet. These counter-flows generate an effective potential for the imaged particle.

[0064] 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. The deviation of the particle's position from its equilibrium position of the effective potential can then be measured as a function of the magnitude of the external force. Thus, the magnitude of the force acting on the particle through the hydrodynamic flow can be determined.

[0065] Additionally or alternatively, calibration of the measured forces can be achieved by observing heat transfer, specifically the average distance of the particle from its equilibrium position 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.

[0066] Further features and advantages of the present invention will be described below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0067] [Figure 1] 1 is a schematic diagram of an apparatus according to the present invention; [Figure 2] 1 is a schematic diagram illustrating the overall principle of the method according to the invention; [Figure 3] 1 is a flow chart illustrating an example of a method according to the present invention. [Figure 4] FIG. 1 shows a simple example of manipulating two particles. [Figure 5] FIG. 10 shows a further example of manipulating six particles. [Figure 6] FIG. 1 shows two different possibilities for achieving a target configuration of three particles. [Figure 7] FIG. 10 shows a further example of achieving a target configuration of three particles. [Figure 8] FIG. 1 illustrates motivation for an exemplary cost function. [Figure 9] FIG. 1 shows an example of three particles, corresponding target positions, and visualized fluid hydrodynamic flow. DETAILED DESCRIPTION OF THE INVENTION

[0068] An embodiment of an apparatus 100 according to the invention will now be described with reference to Figure 1. Identical and equivalent components are essentially numbered the same in the figures. The apparatus 100 shown in Figure 1 is designed to carry out the method according to the invention.

[0069] 1 includes, as its main components, a vessel 10 containing a fluid 12 and particles p1, p2 to be manipulated, a heating device 20 for generating a hydrodynamic flow within the fluid 12 by dynamic localized heating of the fluid 12, an imaging device 40 for imaging at least a portion of the vessel 10, and a control unit 60 for controlling the heating device 20 and the imaging device 40 and for evaluating image data 52 from the imaging device 40. According to the present invention, the dynamic localized heating is designed to realize spatial manipulation of particles p1, p2 within the vessel 10 by the hydrodynamic flow. The fluid 12 and the particles p1, p2 contained therein are also referred to as the sample.

[0070] More specifically, in the example shown in FIG. 1 , the heating device 20 includes a laser 22, such as an infrared laser, for providing heating radiation 24. The heating radiation 24 is guided through an optical path into a vessel 10 containing the sample to be manipulated, i.e., fluid 12 and particles p1 and p2. In the example shown in FIG. 1 , the optical path includes a scanner 26, a beam shutter 28, a beam splitter 30, and a microscope objective 48. The scanner 26 can guide 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. 1 , the scanner 26 and the beam shutter 28 can send status information back to the controller and can be controlled by the controller 60.

[0071] It is clear that FIG. 1 is a schematic diagram, and in fact the optical beam path may include several additional components not shown in FIG. 1 . More specifically, the optical configuration may be as described in U.S. Pat. No. 6,275,999. The optical assembly of FIG. 1 is that of an inverted microscope. Of course, other configurations are possible. It is clear that the inverted configuration of this inverted microscope and the laser-assisted heating require that the vessel 10 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. 1 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. 1 . 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.

[0072] The imaging means 40 in the example of Fig. 1 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, p2 to be manipulated.

[0073] 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.

[0074] Target positions T1 and T2 are shown schematically within vessel 10 in Figure 1. Target positions T1 and T2 represent the positions to which particles p1 and p2, respectively, are to be spatially manipulated. That is, the spatial manipulation task in this example consists of moving particle p1 to position T1 and moving particle p2 to T2.

[0075] According to the present invention, the control unit 60 is designed as follows. A) The imaging device 40 is activated to capture the actual spatial configuration of the particles p1, p2 within the container 10. B) determining a control signal for a 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 particles p1, p2 and a predefined target configuration T1, T2 of the particles 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) Repeating at least a portion of steps A) through C).

[0076] 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.

[0077] 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.

[0078] The main features of the method for spatially manipulating particles p1, p2 within fluid 12 are described below, first generally with reference to FIG. 2 and then in more detail with reference to FIG.

[0079] The upper part of FIG. 2 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 target locations T1 and T2, respectively, as in FIG. 1. FIG. a1 shows an initial configuration in which particles p1 and p2 are significantly separated from their target locations T1 and T2. FIG. a2 shows a schematic representation of the hydrodynamic or thermoviscous flow generated within fluid 12, indicated by the dotted arrows. FIG. a3 shows the state after the application of a singular dynamic local heating event. As shown in FIG. 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 FIG. a1.

[0080] The bottom part of FIG. 2 shows the main steps of a method for spatially manipulating particles p1, p2 in a fluid 12 according to the present invention.

[0081] 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.

[0082] Next, in the example shown in Fig. 2, the positions of the particles to be manipulated are identified, i.e., the coordinates of each particle p1 and p2 are identified (Fig. b2).

[0083] After defining the target spatial configurations of particles p1 and p2, i.e., in the example of Fig. 2, 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).

[0084] According to step b) of the method of the present invention, a 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.

[0085] According to step c) of the method according to the invention, 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 2, 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).

[0086] Thus, in accordance with the method of the present invention, particles p1, p2 are spatially manipulated within the fluid 12 by hydrodynamic flows generated within the fluid 12 due to dynamic localized heating of the fluid 12.

[0087] According to step d) of the method of the present invention, at least one or all of steps a) to c) are repeated. In the example shown in Figure 2, steps a) to c) are cycled, for example at 30 Hz.

[0088] A more detailed example of the method according to the present invention will be described with reference to FIG. 3. 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. 3, the particles are then tracked in step S03, i.e., the particles present in the actual configuration captured in step S02 are identified with the 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 the particles p1, p2, e.g., the image captured in step S02, and the target configurations T1, T2 of the particles p1, p2, e.g., the target configurations or positions defined in step S01.

[0089] According to step b) of the method of the present invention, 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 a 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.

[0090] 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 according to the invention.

[0091] According to step d) of the method of the present invention, at least a part of steps a) to c) is repeated. In the flowchart shown in Fig. 3, a new image is acquired in step S08, i.e., step a) of the method of the present invention 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] In step S13, it is determined whether the error, ie, the cost function, has decreased compared to the value determined in step S05.

[0096] In fact, if the cost function is decreasing from the value determined in step S05, step c) realized in the example of FIG. 3 according to step S07 is repeated with the same singular dynamic local heating event determined in step S06.

[0097] On the other hand, if the cost function has increased from the value determined in step S05, step b) of the method according to the present invention is executed anew: 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.

[0098] Thus, closed feedback loop control and automated spatial manipulation of particles within a fluid is achieved.

[0099] Schematics 1 through 3 in Figure 4 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.

[0100] An example of an algorithm in which the correspondence of manipulated particles to specific target locations is reevaluated and, if necessary, modified after the application of a singular dynamic local heating event is described with reference to FIG.

[0101] The upper diagram of Figure 5 shows the same particles p1 to p6 and target positions T1 to T3. In each case, as indicated by the dotted arrows, particle p5 is associated with target position T1, particle p3 is associated with target position T2, and particle p6 is associated with target position T3. The principle of these associations is that in each case, for each target position, the particle closest to that position is selected. In a preferred embodiment, the so-called Munkres algorithm can be used.

[0102] Based on these correspondences and the initial configuration of particles p1 to p6 in the top diagram of Figure 5, a cost function and subsequently a singular dynamic local heating event are determined and applied. This heating event is schematically indicated in the top diagram of Figure 5 by the solid arrow "FLUCS."

[0103] The bottom diagram in Figure 5 shows the state after the application of the heating event. The application of the heating event moved particle p3 to position T2 as desired, but also changed the positions of particles p5 and p6, which were associated with target positions T1 and T3, respectively. Particle p6 is no longer the particle closest to position T3. A new evaluation of the target position associations results in particle p4 being associated with target position T3, rather than particle p6. Note also that this new evaluation does not consider particle p3 as the particle closest to target position T3, because particle p3 has already reached its destination T2. ​​Based on this new association, a cost function and the corresponding singular dynamic local heating event to be applied next are determined.

[0104] However, in some cases, it may be advantageous to include particles that have reached their destination in the new evaluation. This situation is illustrated with reference to Figure 6. The left diagram of Figure 6 shows a configuration with three identical particles p1, p2, and p3. Particles p1 and p3 have reached their target positions T1 and T3, respectively. That is, only particle p2 has not yet been placed at its assigned target position T2. ​​The right diagram of Figure 6 shows the algorithm for assigning the closest particle to the target position in each case. This leads to a modification of the assignment. Thus, particle p1, which has already reached its destination T1, is reassigned to target position T2, and particle p2 is assigned to target position T1. The cost function and the singular dynamic local heating event are determined accordingly. The approach shown in the right diagram of Figure 6 avoids the need to guide particle p2 between particles p1 and p3.

[0105] A further example of a method according to the present invention, in which multiple identical particles are spatially manipulated to their respective target locations, is described with reference to FIG. 7. In the example shown in FIG. 7, three particles p1, p2, and p3 are moved to their associated target locations T1, T2, and T3, respectively. In each case, the subsequently applied singular dynamic local heating event focuses on the particle furthest from its associated target location. FIG. 1 of FIG. 7 shows an initial state in which particle p1 is furthest from its associated target location T1. Determining and applying a singular dynamic local heating event results in the state shown in FIG. 2, where particle p1 is now significantly closer to its target location T1. The furthest target is now particle p2. Determining and applying a corresponding singular dynamic local heating event then results in the state shown in FIG. 3, where particle p1 is again furthest from its target location T2. As a further step in accordance with the present principles, in a configuration having multiple manipulated particles, in each case the particle manipulated in the next step is the particle furthest from its associated target position, as shown in Figures 4 to 6. In Figure 4, particle p3 is moved because it is furthest from its target position T3. In Figure 5, particle p2 is manipulated because it is furthest from its target position T2. ​​In the situation shown in Figure 6, particles p2 and p3 have reached their target positions T2 and T3, and only particle p1 has not yet moved to its target position T1.

[0106] An example of a cost function is provided and explained with reference to Figure 8. The top diagram of Figure 8 shows a situation with multiple particles, including two particle subsets A and B.

[0107] The goal of the spatial manipulation in this example is to position particle A close to each other in the center, and move particle B as far away from the group of particle A as possible.

[0108] A suitable cost function S that reflects these requirements is:

number

[0109] FIG. 9 shows a visualization of an exemplary two-dimensional flow field in which particles p1, p2, and p3 are moved to target locations T1, T2, and T3, respectively. The illustrated two-dimensional flow field can be thought of as being generated by the simultaneous application of three distinct unique dynamic localized heating events with three different scanning paths to a fluid, or by the sequential application of three distinct unique dynamic localized heating events with one scanning path in each case. Strictly speaking, the latter description is only appropriate if the three scanning paths are applied rapidly, i.e., faster than thermal relaxation within the fluid. As shown, for each particle, the hydrodynamic flow is oriented to carry each particle toward its associated target location.

[0110] The present invention demonstrates that by utilizing thermoviscous flow, pre-designated object(s) or particle(s) can be precisely positioned and even automated.

[0111] Furthermore, the present invention demonstrates that highly accurate hydrodynamic positioning of particles can be achieved entirely optically. This is achieved by combining laser-driven flow with a closed feedback loop that accounts for time-dependent and stochastic particle position. The inventors analyzed the physics of this novel method for controlling particle position. Optical refinements have enabled precision down to 24 nm, which has not been achieved with previous hydrodynamic traps. Unlike optical tweezers, the present method does not require specific materials or exposure of particles to a laser beam. Feedback-controlled thermoviscous flow is a desirable alternative to previous hydrodynamic trapping techniques and opens up opportunities for a wide range of new applications. [Explanation of symbols]

[0112] 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, A, B, p1 to p6 manipulated and / or positioned particles, T1 to T3 target positions, FLUCS cytoplasmic streaming by focused light.

Claims

1. A method for spatially manipulating at least one particle (p1, p2) in a fluid (12), comprising: the one or more particles (p1, p2) are spatially manipulated within the fluid (12) by hydrodynamic flows generated within the fluid (12) due to dynamic localized heating of the fluid (12); At least one target spatial configuration (T1, T2) of said particles (p1, p2) in said fluid (12) is defined; a) imaging the actual spatial configuration of said particle(s) (p1, p2); b) calculating a cost function representing the error between the spatial configuration of said particle(s) (p1, p2) and the target configuration (T1, T2) of said particle(s) (p1, p2) based on the most recent actual spatial configuration of said particle(s) (p1, p2) and the target configuration (T1, T2) of said particle(s) (p1, p2); c) determining a specific dynamic local heating event by a laser or a light emitting diode 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); d) applying the unique dynamic localized heating event determined in step c) to the fluid (12) at least once; e) recalculating the cost function based on the spatial configuration of the particles (p1, p2) after applying the idiosyncratic dynamic local heating event and the target configuration (T1, T2) of the particle(s) (p1, p2); f) repeating the idiosyncratic dynamic local heating event if the cost function calculated in step e) is decreased from the cost function calculated in step b), and determining a new idiosyncratic dynamic local heating event based on the cost function calculated in step e) if the cost function calculated in step e) is increased from the cost function calculated in step b); The method is characterized by the further step of:

2. The method of claim 1 , wherein the fluid is or comprises water.

3. 3. The method according to claim 1 or 2, characterized in that the particle(s) (p1, p2) to be manipulated are at least one of the following: cells, viruses, tissue fragments, metal particles, composite particles, polymer particles, nanoparticles.

4. Spatially manipulating the particle(s) (p1, p2) comprises: moving designated particle(s) (p1, p2) towards designated target locations (T1, T2) within said fluid (12); moving designated particle(s) (p1, p2) along a designated path within said fluid (12); - retaining designated particle(s) (p1, p2) at designated target locations (T1, T2) within said fluid (12); - trapping designated particle(s) (p1, p2) in a designated target orientation (T1, T2) within said fluid (12); moving designated particle(s) (p1, p2) towards designated target orientation(s) (T1, T2) within said fluid (12); 4. The method according to claim 1, further comprising at least one of the following steps:

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. 6. The method of claim 5, wherein the unique dynamic localized heating event in the fluid (12) is generated by repeatedly scanning the laser along a path within the fluid or a sample containing the fluid and the plurality of particles.

7. The determination of the specific dynamic local heating event applied to the fluid (12) in step c) 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 scan path; 7. The method according to claim 5, further comprising determining at least one of:

8. 7. A method according to claim 6, characterized in that the path scanned by the laser is chosen so as not to expose the particle(s) (p1, p2) to be manipulated to heating radiation.

9. 9. The method according to any one of claims 1 to 8, characterized in that the singular dynamic local heating event applied to the fluid (12) is also determined depending on the mobility of particles (one or more) within the fluid.

10. 10. The method according to claim 1, wherein the imaging device for taking the images is a microscope.

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

12. The target spatial configuration of the particles (p1, p2) in the fluid (12) is - designated target position(s) (T1, T2) of said particle(s) (p1, p2) within said fluid (12); one or more specified target velocities (T1, T2) of said particle(s) (p1, p2) in said fluid (12); - specified target orientation(s) (T1, T2) of said particle(s) (p1, p2) in said fluid (12); - specified target rotational speed(s) (T1, T2) of said particle(s) (p1, p2) in said fluid (12); 12. The method according to claim 1, further comprising at least one of the following steps:

13. The target spatial configuration of the particles (p1, p2) in the fluid (12) is the one-dimensional position of said particle(s) (p1, p2), the two-dimensional position of said particle(s) (p1, p2), or the three-dimensional position of said particle(s) (p1, p2); 13. The method according to claim 1, wherein

14. The target configuration must meet the following requirements: - the specified particle(s) are not at the specified location; The designated particle(s) are as far away as possible from the designated location(s); The specified particle(s) are at least the specified distance(s) from the specified location(s); - The designated particles are as close as possible to each other, The designated particles (p1, p2) must not touch each other. - Different types of particles are treated differently, 14. The method according to claim 1, further comprising at least one of the following steps:

15. 15. The method according to any one of claims 1 to 14, characterized in that at least two particles are spatially manipulated simultaneously.

16. 16. The method of claim 15, wherein the plurality of spatially manipulated particles comprises at least a subset of equivalent or identical particles.

17. 17. The method according to claim 15 or 16, characterized in that in a configuration in which at least two particles are manipulated, in each case the particle manipulated in the next step is the particle that is furthest from the target position and / or target orientation associated with each particle.

18. 18. The method according to any one of claims 1 to 17, characterized in that the anomalous dynamic local heating event determined in step c) is determined in dependence on the cost function.

19. 18. The method of any one of claims 1 to 17, wherein the cost function is invariant with respect to the exchange of equivalent or identical particles.

20. The cost function has the following arguments: - the distance of a particular particle to a particular target location of that particle; - Round trip distance of a particular particle to a specified location, - the distance of a specified type of particle to a specific target location for each type of particle; - The round trip distance of a specified type of particle to a specified position specified for each type of particle, the angle between the actual particle orientation and the target orientation for each particle, or each type of particle; The difference between the actual particle velocity and the target velocity for each particle or type of particle; 20. The method according to claim 1, further comprising at least one of:

21. Between steps a) and c), there is a further step (S03, S09): The particle(s) to be manipulated are associated with at least one of a target position and a target orientation.

21. The method according to claim 1, wherein the following is performed:

22. 22. The method according to any one of claims 1 to 21, characterized in that the tracking (S03, S09) of the particle(s) is performed by identifying particles present in a new photographed actual configuration that includes particles in a previous actual configuration.

23. 23. The method according to claim 22, characterized in that after tracking (S09) the particle, the target configuration is re-evaluated and, if the target configuration is changed to a new target configuration, the particle is then associated in each case with at least one of a new target position and a new target orientation.

24. The database contains the following data: the actual spatial configuration in front of said particle(s) (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) (p1, p2) caused by each dynamic localized heating event applied to the fluid (12); is memorized, 24. The method according to any one of claims 1 to 23, characterized in that future dynamic local heating events to be applied to the fluid (12) are calculated using at least a portion of the data stored in the database.

25. 25. The method of any one of claims 1 to 24, characterized in that future dynamic local heating events to be applied to the fluid (12) are calculated using machine learning.

26. 1. An apparatus for spatially manipulating at least one particle (p1, p2) in a fluid (12) by hydrodynamic flow, comprising: a vessel (10) containing said fluid (12) and said particle(s) (p1, p2) to be manipulated; a heating device (20) for generating a hydrodynamic flow in the fluid (12) by dynamic local heating with a laser or a light emitting diode, the dynamic local heating being designed to cause spatial manipulation of the particles (p1, p2) in the vessel (10) by the hydrodynamic flow; an imaging device (40) for imaging at least a portion of the container (10); 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); The control unit (60) A) activating the imaging device (40) to capture an image of the actual spatial configuration of the particles (p1, p2) in the container (10); B) calculating a cost function representing the error between the spatial configuration of the particle(s) (p1, p2) and the target configuration (T1, T2) of the particle(s) (p1, p2) based on the most recent actual spatial configuration of the particle(s) (p1, p2) and the target configuration (T1, T2) of the particle(s) (p1, p2); C) 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 particle(s) (p1, p2) and a predefined target configuration (T1, T2) of the particle(s) (p1, p2); D) operating the heating device (20) to apply the unique dynamic localized heating event determined in step C) to the fluid (12) at least once; E) re-calculating the cost function based on the spatial configuration of the particles (p1, p2) after application of the differential dynamic local heating event and the target configuration (T1, T2) of the particle(s) (p1, p2); F) repeating the idiosyncratic dynamic local heating event if the cost function calculated in step E) is decreased from the cost function calculated in step B); and determining a new idiosyncratic dynamic local heating event based on the cost function calculated in step E) if the cost function calculated in step E) is increased from the cost function calculated in step B). An apparatus characterized in that it is designed so as to

27. 27. Apparatus according to claim 26, designed to carry out the method according to any one of claims 1 to 25.

28. 28. Device according to claim 26 or 27, characterized in that the container (10) comprises means for controlling the temperature of the fluid (12).

29. 29. The apparatus of any one of claims 26 to 28, wherein the heating device comprises a laser that provides energy for the dynamic localized heating, and optical means including a scanner that relays the heating laser radiation to a variable location within the fluid.

30. 30. Apparatus according to any one of claims 26 to 29, characterized in that the imaging device (40) is a microscope.

31. 31. The apparatus of claim 30, 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).

32. 32. Apparatus according to any one of claims 26 to 31, characterized in that there is present at least one device for modifying the integrity of the particle(s).

33. 33. The apparatus of claim 32, wherein the device for altering the integrity of the particle(s) comprises at least one laser.

34. 1. A computer program product comprising instructions, When the program is executed by the control unit (60), the control unit (60) receives the instruction A) activating an imaging device (40) to capture an image of the actual spatial configuration of particles (p1, p2) in the container (10); B) calculating a cost function representing an error between the spatial configuration of the particles (p1, p2) and the target configuration (T1, T2) of the particles (p1, p2) based on the most recent actual spatial configuration of the particles (p1, p2) and the target configuration (T1, T2) of the particles (p1, p2); C) determining a control signal for a heating device (20) suitable for a specific dynamic local heating event by a laser or a light emitting diode applied to the fluid (12) depending on at least one recent spatial configuration of said particle(s) (p1, p2) and a predefined target configuration (T1, T2) of said particle(s) (p1, p2); D) operating the heating device (20) to apply the unique dynamic localized heating event determined in step C) to the fluid (12) at least once; E) recalculating the cost function based on the spatial configuration of the particles (p1, p2) after application of the unique dynamic local heating event and the target configuration (T1, T2) of the particles (p1, p2); F) repeating the idiosyncratic dynamic local heating event if the cost function calculated in step E) is decreased from the cost function calculated in step B), and determining a new idiosyncratic dynamic local heating event based on the cost function calculated in step E) if the cost function calculated in step E) is increased from the cost function calculated in step B); A computer program product for causing a computer to perform a method comprising:

35. A computer-readable storage medium containing instructions that, when executed by a control unit (60), cause the control unit (60) to: A) activating an imaging device (40) to capture an image of the actual spatial configuration of particles (p1, p2) in the container (10); B) calculating a cost function representing an error between the spatial configuration of the particles (p1, p2) and the target configuration (T1, T2) of the particles (p1, p2) based on the most recent actual spatial configuration of the particles (p1, p2) and the target configuration (T1, T2) of the particles (p1, p2); C) determining a control signal for a heating device (20) suitable for a specific dynamic local heating event by a laser or a light emitting diode applied to the fluid (12) depending on at least one recent spatial configuration of the particles (p1, p2) and a predefined target configuration (T1, T2) of the particles (p1, p2); D) operating the heating device (20) to apply the unique dynamic localized heating event determined in step C) to the fluid (12) at least once; E) recalculating the cost function based on the spatial configuration of the particles (p1, p2) after application of the unique dynamic local heating event and the target configuration (T1, T2) of the particles (p1, p2); F) repeating the idiosyncratic dynamic local heating event if the cost function calculated in step E) is decreased from the cost function calculated in step B), and determining a new idiosyncratic dynamic local heating event based on the cost function calculated in step E) if the cost function calculated in step E) is increased from the cost function calculated in step B); A computer-readable storage medium for causing a method to be performed, comprising:

Images