PROBING MECHANICAL PROPERTIES OF BIOLOGICAL MATTER

Abstract

A method for probing mechanical properties of cellular bodies includes: providing a plurality of particles in a fluid medium contained in a holding space of a sample holder, each of the plurality of particle being attached to a cellular body; generating a resonant bulk acoustic wave in the holding space, the resonant bulk acoustic wave exerting an acoustic force on each of the plurality of particles, each of the plurality of particles having an acoustic contrast factor and a size, the acoustic contrast factor and the size being selected such that the force exerted on a particle is larger than the force exerted on the cellular body to which the particle is attached; measuring a displacement of a particle in response to the exertion of the force on the particle, the measured displacement being associated with a mechanical property of the cellular body attached to the particle.

Claims

1. A method for probing mechanical properties of cellular bodies, the method comprising steps of: providing a plurality of particles and cellular bodies in a fluid medium contained in a holding space of a sample holder and fixating the cellular bodies to a surface of the holding space, wherein each of the plurality of particles is attached to one of the cellular bodies; generating a resonant bulk acoustic wave in the holding space, the resonant bulk acoustic wave exerting an acoustic force on each of the plurality of particles in a direction away from the surface of the holding space or in a direction towards the surface of the holding space, each of the plurality of particles having an acoustic contrast factor and a size, the acoustic contrast factor and the size being selected such that the force exerted on one said particle is larger than the force exerted on the cellular body to which the one said particle is attached; and, for at least part of the particles which are attached to a cellular body, measuring, one or more displacements of one said particle in response to the exertion of the force on the one said particle, the measured one or more displacements being associated with at least one mechanical property of the cellular body.

2. The method according to claim 1 wherein the particles have an acoustic contrast factor that is larger than 0.5 or smaller than −0.5.

3. The method according to claim 1, wherein the particles include hollow particles, which hollow articles may be filled with a gas, a gas mixture or a liquid.

4. The method according to claim 3 wherein the hollow particles have a low compressibility, the hollow particles comprising a shell material having a Young's modulus selected between 1 and 1000 GPa.

5. The method according to claim 3, wherein the hollow particles have a shell thickness between 0.1 and 5 microns.

6. The method according to claim 1, wherein a material of one said particle has an optical refractive index that differs at least 80% from a refractive index of the cellular body to which the particle is attached.

7. The method according to claim 1, wherein the size of the particles is selected to be 20% of the size of the cellular body or smaller; and/or, wherein the size of the particle is selected to be between 0.2 and 20 micron and wherein the size of the cellular body is selected to be between 1 and 100 micron.

8. The method according to claim 1, wherein at a first acoustic resonant frequency the force exerted on one said particle is in a direction away from the surface of the flow cell and wherein at a second acoustic resonant frequency the force exerted on the one said particle is in a direction towards the surface of the holding space of the sample holder.

9. The method according to claim 1, wherein at least a portion of the particles and/or at least a portion of the surface of the holding space is/are functionalized using one or more primers comprising one or more interaction moieties type(s) for adhesion to at least part of the cellular body, wherein said interaction moieties type(s) include at least one type selected from the group consisting of: viruses, virus particles, antibodies, peptides, biological tissue factors, biological tissue portions, antigens, proteins, ligands, lipid layers, fibronectin, cellulose, nucleic acids, RNA, small molecules, allosteric modulators, biofilms, and specific atomic or molecular surface portions.

10. The method according to claim 1 further comprising steps of: providing a plurality of the cellular bodies in the medium of the holding space of the sample holder, one said particle being attached to each of the plurality of the cellular bodies; controlling the resonant bulk acoustic wave in the sample holder in order to exert the force on the particles, the acoustic force being selected to be smaller than a gravitational force that pulls cellular bodies towards the surface of the holding space; the gravitational force depositing the cellular bodies onto the surface of the holding space, wherein the acoustic force acting on one said particle attached to one said cellular body ensures that the on said cellular body will land onto the surface of the holding space with the one said particle on top of the one said cellular body.

11. The method according to my claim 1, further comprising steps of: providing a plurality of the cellular bodies in the medium of the holding space of the sample holder, one said particle being attached to each of the plurality of cellular bodies, wherein the particles have a density lower than tea density of the medium of the holding space; a gravitational force depositing the cellular bodies onto the surface of the holding space, wherein a buoyancy force of one said particle attached to one said cellular body ensures that the one said cellular body will land onto the surface of the holding space with the one said particle on top of the one said cellular body.

12. The method according to claim 1, further comprising steps of: providing a plurality of the cellular bodies and a plurality of the particles in the medium of the holding space of the sample holder; controlling the resonant bulk acoustic wave in the sample holder in order to exert the force on the particles, the acoustic force being selected to be smaller than a gravitational force that pulls the plurality of the cellular bodies towards the surface of the holding space and the force being selected such that the particles are trapped in a node or an antinode of the resonant bulk acoustic wave, the gravitational force depositing the cellular bodies onto the surface of the holding space; controlling another resonant bulk acoustic wave in the sample holder to release the particles from the node or antinode, the gravitational force on the particles depositing the particles onto the cellular bodies for attaching the particles to the cellular bodies.

13. The method of claim 1 further comprising the steps of: for at least part of the particles attached to the cellular bodies, measuring displacements of said at least part of the particles as a function of time; and classifying each of the cellular bodies on the basis of the measured displacements.

14. A system for probing mechanical properties of cellular bodies, the system comprising: a sample holder comprising a holding space for holding a sample, the sample comprising a plurality of particles and cellular bodies in a fluid medium contained in the holding space of the sample holder, the cellular bodies being fixated to a surface of the holding space, wherein each of the plurality of particles is attached to one of the cellular bodies; an acoustic wave generator connectable or connected with the sample holder to generate an acoustic wave in the holding space for exerting a force on the sample; a detector for detecting a response of the sample to the acoustic wave; and, a controller module for controlling the acoustic wave generator and the detector, the controller module including; a computer readable storage medium having computer readable program code embodied therewith, and a processor coupled to the computer readable storage medium, wherein responsive to executing the computer readable program code, the processor is configured to perform executable operations comprising: controlling the acoustic wave generator to generate a resonant bulk acoustic wave in the holding space, the resonant bulk acoustic wave exerting an acoustic force on each of the plurality of particles in a direction away from the surface of the holding space or in a direction towards the surface of the holding space, each of the plurality of particles having an acoustic contrast factor and a size, the acoustic contrast factor and the size being selected such that the force exerted on a particle is larger than the force exerted on the cellular body to which the particle is attached; and controlling the detector to measure for at least part of the particles attached to a cellular body, one or more displacements of one said particle in response to the exertion of the force on the one said particle, the one or more measured displacements being associated with at least one of the mechanical properties of the cellular body.

15. The system according to claim 14, the executable operations further comprising: computing at least one of the mechanical properties of the cellular body based on the one or more measured displacements.

16. The method according to claim 1, wherein the particles are microparticles or nanoparticles and the measuring step is carried out by an optical detector.

17. The method according to claim 2 wherein the particles have an acoustic contrast factor that is larger than 0.6 or smaller than −0.6.

18. The method according to claim 3, wherein the particles include hollow inorganic particles and/or the hollow particles including hollow organic particles.

19. The method according to claim 4, wherein the hollow particles have a low compressibility, the hollow particles comprising a shell material having a Young's modulus selected between 50 to 90 GPa.

20. The method according to claim 6, wherein a material of one said particle has an optical refractive index that differs at least 20-25% from a refractive index of the cellular body to which the particle is attached.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The above-described aspects will hereafter be explained with further details and benefits with reference to the drawings showing a number of embodiments by way of example.

[0080] FIG. 1 schematically depicts an acoustic force spectroscopy system according to an embodiment of the invention;

[0081] FIGS. 2A and 2B schematically depict details of a sample holder for use in an acoustic force spectroscopy system according to an embodiment of the invention;

[0082] FIG. 3A-3F schematically depicts a process for probing mechanical properties of cells according to an embodiment of the invention;

[0083] FIG. 4 depicts mechanical responses of cellular bodies measured using an acoustic force spectroscopy system according to an embodiment of the invention;

[0084] FIG. 5 depicts experimental data of high acoustic contrast particles according to an embodiment of the invention;

[0085] FIG. 6 schematically depicts a process for probing mechanical properties of cells according to another embodiment of the invention;

[0086] FIG. 7 schematically depicts part of a flow cell for an acoustic force spectroscopy system according to an embodiment of the invention;

[0087] FIG. 8 schematically depicts acoustic resonant modes of part of a flow cell for an acoustic force spectroscopy system according to an embodiment of the invention;

[0088] FIG. 9 depicts a process for preparing cells for a mechanical probing process according to an embodiment of the invention;

[0089] FIG. 10 schematically depicts a process of classifying cells on the basis of mechanical according to an embodiment of the invention.

DETAILED DESCRIPTION OF EMBODIMENTS

[0090] It is noted that the drawings are schematic, not necessarily to scale and that details that are not required for understanding the present invention may have been omitted. The terms “upward”, “downward”, “below”, “above”, and the like relate to the embodiments as oriented in the drawings, unless otherwise specified. Further, elements that are at least substantially identical or that perform an at least substantially identical function are denoted by the same numeral, where helpful individualised with alphabetic or subscript numeric suffixes.

[0091] While the embodiments and examples hereunder are described with reference to cellular bodies, it is appreciated that these embodiments and examples are not limited thereto and also include systems and methods for probing mechanical properties of biological soft matter layers such as a tissue layers, lipid bilayers, organ on chip, etc.

[0092] FIG. 1 schematically depicts an acoustic force spectroscopy system according to an embodiment of the invention. FIG. 2A schematically depicts a cross section of a sample holder and FIG. 2B is a detail of the sample holder of FIG. 2A as indicated with “IIA”.

[0093] The system 100 comprises a sample holder 102 comprising a holding space 104 for holding a sample 106 comprising one or more biological cellular bodies in a fluid medium wherein each of (at least part of) the cellular bodies is attached to a particle, a microparticle or in some cases a nanoparticle. These particles, microparticles and nanoparticles are hereafter referred to in short as particles. The fluid preferably is a liquid or a gel. The system further comprises an acoustic wave generator 108, e.g. a piezo element, connected with the sample holder 102 to generate an acoustic wave in the holding space exerting a force on the particles in the sample. The acoustic wave generator may be connected to a controller 110.

[0094] As shown in FIG. 2B, the sample holder 202 comprising the holding space 206 may comprise a wall 204 comprising a surface for supporting cells. In an embodiment, the surface may be provided with a functionalised surface portion 208 to be contacted, in use, by part of a plurality of samples, each sample including a cellular body 210 attached to a particle 212. Hence, on side of the cellular body is connected (fixated) to the functionalized surface of the sample holder and another side of the cellular body is connected to the particle.

[0095] The manipulation system of FIG. 1 may further comprise a microscope 112 with an adjustable objective 114 and a camera 116 connected with a computer 118 comprising a controller and a memory. The computer may also be programmed for tracking one or more of the particles and/or cellular bodies based on signals from the camera and/or for performing microscopy calculations and/or for performing analysis associated with super-resolution microscopy and/or video tracking, which may be sub-pixel video tracking. The computer or another controller (not shown) may be connected with other parts of the system (not shown) for controlling at least part of the microscope and/or another detector (not shown). In particular, the computer may be connected with one or more of the acoustic wave generator and the controller thereof, as shown in FIG. 1.

[0096] The system may further comprise a light source 120 for illuminating the sample using any suitable optics (not shown) to provide a desired illumination intensity and intensity pattern, e.g. plane wave illumination, Kahler illumination, etc., known per se. Here, the system light 122 emitted from the light source may be directed through the acoustic wave generator 108 to (the sample in) the sample holder 106 and sample light 124 from the sample is transmitted through the objective 114 and through an optional ocular 126 and/or further optics (not shown) to the camera 116. The objective and the camera may be integrated. In an embodiment, two or more optical detection tools, e.g. with different magnifications, may be used simultaneously for detection of sample light, e.g. using a beam splitter.

[0097] In another embodiment, not shown but discussed in detail in WO2014/200341, the system may comprise a partially reflective reflector and light emitted from the light source is directed via the reflector through the objective and through the sample, and light from the sample is reflected back into the objective, passing through the partially reflective reflector and directed into a camera via optional intervening optics. Further embodiments may be apparent to the reader.

[0098] The sample light may comprise light affected by the sample (e.g. scattered and/or absorbed) and/or light emitted by one or more portions of the sample itself e.g. by chromophores attached to the cellular bodies.

[0099] Some optical elements in the system may be at least one of partly reflective, dichroic (having a wavelength specific reflectivity, e.g. having a high reflectivity for one wavelength and high transmissivity for another wavelength), polarisation selective and otherwise suitable for the shown setup. Further optical elements e.g. lenses, prisms, polarizers, diaphragms, reflectors etc. may be provided, e.g. to configure the system 100 for specific types of microscopy.

[0100] The sample holder 102 may be formed by a single piece of material with a channel inside, e.g. glass, injection moulded polymer, etc. (not shown) or by fixing different layers of suitable materials together more or less permanently, e.g. by welding, glass bond, gluing, taping, clamping, etc., such that a holding space 106 is formed in which the fluid sample is contained, at least during the duration of an experiment.

[0101] FIG. 2A schematically depicts a flow cell for use in an acoustic force spectroscopy system according to an embodiment of the invention. The sample holder 212 may comprise a part that has a recess being, at least locally, U-shaped in cross section and a cover part to cover and close (the recess in) the U-shaped part providing an enclosed holding space 206 in cross section.

[0102] Further, the sample holder 212 may be connected to an optional fluid flow system 214 for introducing fluid into the holding space 206 of the sample holder and/or removing fluid from the holding space, e.g. for flowing fluid through the holding space (see arrows in FIG. 2A). The fluid flow system may be comprised in a manipulation and/or control system. The fluid flow system may comprise one or more of reservoirs 216, pumps, valves, and conduits 218 for introducing and/or removing one or more fluids, sequentially and/or simultaneously. The sample holder and the fluid flow system may comprise connectors, which may be arranged on any suitable location on the sample holder, for coupling/decoupling. The sample holder may further include an acoustic wave generator 222, e.g. in the form of a (at least partially transparent) piezoelectric element, connected to a controller 224.

[0103] FIG. 3A-3E schematically depicts a process for probing mechanical properties of cells according to an embodiment of the invention. As shown in FIG. 3A an acoustic force spectroscopy system as e.g. described with reference to FIG. 1-2 may be used in order to examine mechanical properties of cellular bodies in the holding space of a flow cell. FIG. 3B depicts a first state of the sample under examination, including a plurality of cellular bodies 302.sub.1, in the example Red Blood Cells (RBCs), may be attached on one side to a (bottom) surface of the flow channel of the flow cell. The surface of the flow channel may be functionalized, e.g. using Poly-L-lysine or the like, in order to fixate the RBCs to the surface. A particle 304.sub.1, e.g. a Concanavalin A functionalized silica microsphere (approx. 6.8 micron in diameter), may be attached to each of the cellular bodies. In this state, the acoustic wave generator may be switched off so that no force is applied to the samples.

[0104] When the acoustic wave generator is switched on, a bulk acoustic standing wave will be generated at a predetermined resonant frequency in the holding space of the sample holder. This way, acoustic nodes and antinodes appear at predetermined heights in the flow cell. The acoustic field will act upon the cellular bodies and the particles, causing—in this case—a force away from the surface of the flow channel towards a node of the acoustic standing wave in the holding space.

[0105] In order to quantify the response of a body of a certain material to the acoustic field, the so-called acoustic contrast factor (Φ) is used. The acoustic contrast factor is a well-known parameter in the field as e.g. described in the article by Lenshof, A., et al, J. Acoustofluidics 5: Building microfluidic acoustic resonators. Lab Chip 12, 684 (2012). The acoustic contrast factor for a spherical object of a certain volume is given by the following expression:

[00001]$\Phi =\frac{{\rho }_p+2/3\left({\rho }_p-{\rho }_m\right)}{2{\rho }_p+{\rho }_m}-\frac{1}{3}\frac{{\rho }_m{c}_m^2}{{\rho }_p{c}_p^2}$

wherein ρ.sub.p and ρ.sub.m are the densities, and c.sub.p and c.sub.m are the speed of sound of the particle and the medium, respectively. The acoustic contrast factor Φ may be positive. In that case, a particle will experience a force in the direction of a node in an acoustic force field. If the acoustic contrast factor Φ is negative, the particle will experience a force in the direction of an antinode in an acoustic force field.

[0106] The acoustic contrast factor of a particle may be selected such that it is substantially higher (in an absolute sense) than the acoustic contrast factor of the cells, in this exemplary case, red blood cells which have an acoustic contrast factor of around 0.05 in a medium similar to water. Therefore, for a certain acoustic force field and a certain (average) particle size, the particles will experience a force, while the cellular bodies will experience a force that is negligible with respect to the force experience by the particles. This way, when the acoustic field is generated, the particles will effectively pull at the cell or push on the cell, causing the cellular body or a part thereof to deform. This is schematically shown in FIG. 3C wherein an acoustic force field will force the particles upwards, thereby pulling on the membrane of the RBCs. In this experiment, silica microspheres of relatively large dimensions (approx. 7 micron) were selected in order to achieve an effective pulling force as well as a high imaging contrast for 3-dimensional tracking compared to the RBCs. As shown in FIG. 3D, the camera captures an optical image of the sample holder comprising the RBCs. Tracking software is configured to analyse (image process) the captured images, to track selected samples (an RBC connected to a particle) and to determine displacements of the microbeads when applying a force to the samples. Known optical tracking techniques may be used as described in WO2014/200341, which is hereby incorporated by reference into this application.

[0107] In a typical experiment, a constant force is applied to the micro particles and their position is tracked over time. FIG. 3E shows an example of the viscoelastic response of a red blood cell to an applied force F of 500 pN. As shown in this figure, cells exhibit a three-phase creep response: an instantaneous elastic response I, then a retarded elastic response II, followed by viscous flow behavior III. This type of response can be described by a simple viscoelastic model consisting of springs and dashpots with stiffness's k1 and k2 and damping coefficients μ1 and μ2 (as illustrated by FIG. 3F), a four-parameter model termed Burger's viscoelastic model as described in the article by Rand et al., Biophysical Journal 4, 115-135:

[00002]$L\left(t\right)=L_0+L_{cross}.Math.\left[1-e^{\left(-\frac{t-t_0}{\tau }\right)}\right]+L_v^{\prime }\left(t-t_0\right)$

Here, L.sub.0 (corresponding to F/k1) is the instantaneous elastic elongation, L.sub.cross (corresponding to F/k2; is the retarded elastic behavior, τ (corresponding to μ1/k2) is the characteristic time constant of the retarded elastic behavior and L′v (corresponding to F/μ2) is the long-term viscous flow. Modeling compliance by a simple combination of elastic and viscous elements, denoted by springs and dashpots, was previously used in a range of experiments, such as for lipid vesicles in fluid flow [Guevorkian et al., Biophys J 109, 2471-2479.], micro-rheological measurements on cells [Bausch et al. Biophys J 75, 2038-2049] [Bausch et al., Biophys J 76, 573-579.] and AFM studies of cell mechanics [Wu et al., Scanning 20, 389-397]. Based on this analysis, the viscoelastic behavior of cells can be described by the above-mentioned four parameters.

[0108] FIG. 4 depicts mechanical responses of cellular bodies measured using an acoustic force spectroscopy system according to an embodiment of the invention. In particular, FIG. 4 shows distributions of fitting parameters for red blood cell populations treated with different chemicals and or vesicles. Healthy red blood cells where compared to cells treated with 5-Cholesten-3β-ol-7-one (7KC)—a cholesterol analogue that is expected to soften the cell membrane or with formaldehyde (FA), a well-known crosslinking agent that is expected to stiffen cells. The distribution of fitting parameters L0 (A), Lcross (B), L′v (C) and τ (D) for the different treatments are shown. The difference detected was significant: in the figure samples associated with a *** reference mark correspond to a P value <0.005 compared to healthy RBCs as determined by a two-samples Kolmogorov-Smirnov test (KS test). This is a non-parametric test which quantifies the distance between the empirical distributions of two data sets, where the null hypothesis states that the two samples are drawn from the same distribution.

[0109] Besides the chemical treatments, red blood cells were also treated with red blood cell derived extracellular vesicles (EVs) by first introducing cells and microspheres into the flow cell and then exchanging the buffer solution with a buffer containing EVs derived from other RBCs (10 microliter at a concentration of 1012 particles/ml). The mechanical properties of the RBCs were probed immediately after vesicle introduction. It was expected that vesicles in the vicinity of RBC might be taken up by the RBC, and thereby change cell deformability. As RBCs do not have internal organelles, such as ER and Golgi, it was expected that uptake of vesicles might increase the surface area of the cell membrane, and thereby alter the mechanical response.

[0110] Based on the measurements it was found that such treatment indeed induces a change in RBC mechanical response: lower elastic coefficient values are obtained after vesicle treatment. A possible explanation for this increased deformability is that there is simply more available membrane to be pulled, due to incorporation of the vesicle lipids into the cell membrane. Interestingly, the long-term viscous flow (L′v) following vesicle treatment is significantly larger than for untreated RBCs similarly to the effect of 7KC, thus further supporting the explanation of increased membrane surface area.

[0111] These results show that the acoustic force spectroscopy system can be used to study the mechanical properties of cellular bodies, such as RBCs, in a multiplexed fashion, providing insights into cell mechanics. Mechanical properties of cells are essential for their function and response to the environment. Differences in stiffness can be related to several diseases, such as cancer, anemia or malaria. The embodiments in this disclosure thus provide substantial advantages over current methods for studying mechanical properties of cells, like atomic force spectroscopy, fluid flow experiments or optical tweezers. These techniques lack data throughput making it a tedious process to distinguish mechanical properties in a heterogeneous population.

[0112] When probing mechanical properties of cellular bodies using an acoustic force spectroscopy system as described in this disclosure, it is required that the particles experience a larger acoustic force than the cellular bodies. The force exerted by a given acoustic field on the particles depends on the acoustic contrast factor which quantifies the strength of the acoustic interaction of a material in a specific medium. The acoustic force further scales with the volume of the object.

[0113] The acoustic contrast factor depends on the difference in density and speed of sound of the material used compared to the medium. Cellular bodies may have an a relatively low acoustic contrast factor between −0.2 and 0.2. For example, Augustsson, P. et al. reported values between 0.03-0.11 in their article Measuring the Acoustophoretic Contrast Factor of Living Cells in Microchannels. Cell 1337-1339 (2010).

[0114] For example, red blood cells have an acoustic contrast factor of around 0.07), while materials that are commonly used for the particles have an acoustic contrast factor between 0.20 and 0.55 (e.g. the acoustic contrast factor for polystyrene is approximately 0.22, while the acoustic contract factor for silica 0.54). Hence, in order to achieve a situation in which a silica particle experiences an acoustic force that is substantially higher than the acoustic force experienced by a red blood cell it is attached to, the silica particle needs to have a size that is comparable to the size of the cellular body, e.g. around 7 micron. Therefore local probing of specific parts of a cell is not possible. Additionally, the optical tracking of the silica particle will be affected by the cell it is attached to and in more general by the cells in the optical background.

[0115] Hence, when probing mechanical properties of cells using an acoustic force spectroscopy system as described with reference to the embodiments in this application, high acoustic contrast particles may be used. Such particles enable pulling at cellular bodies with higher forces than particles of the same size but having a relatively low acoustic contrast factor. Additionally, high acoustic contrast particles allow reduction of the size of the particles thus providing higher localization accuracy.

[0116] Further, in some embodiments, the mechanics of a cell may be probed using a frequency dependent method. For example, a frequency dependent rheology method may be used. This method is a spectral method where a periodic (or sinusoidal) acoustic force signal may be applied to the particles that are attached to the cells and the responses of the particles are tracked. The response may be tracked over a range of frequencies to determine a spectral response. Smaller particles have a faster response time and thus may be advantageous for use in such spectral methods. Smaller particles enable examining the response of the cells over a broader frequency spectrum.

[0117] In an embodiment, high acoustic contrast factor particles may include hollow particles that are filled with a gas or air. FIG. 5A-F depict a comparison between hollow air-filled polyvinyl alcohol (PVA) particles and silica (glass) particles. In order to determine the acoustic contrast factor of the air-filled particles, the acoustic response of the air-filled microspheres is compared to the microspheres with a well-known acoustic contrast factor (silica microspheres with approx. 6.8 micron in diameter). To make a correct comparison, both microspheres are calibrated using with the same resonance frequency in the same chip. To this end, a resonance frequency is used that pushes the air-filled microspheres downwards to the acoustic anti-node and the silica microspheres upwards to the acoustic node. This is schematically shown in FIG. 5A.

[0118] Air-filled microspheres and silica microspheres can be forced in a controlled fashion to the acoustic anti-node and node, respectively as shown in FIG. 5B. The force on the microspheres at each height location is determined from the velocity with which they move from the surface to the node (or anti-node). The force profiles for different applied voltages are fitted with sine functions (FIGS. 5C and 5D) and the force/voltage.sup.2 ratio is calculated for a population of silica and air-filled microspheres (FIG. 5E).

[0119] A large spread in the force/voltage.sup.2 ratio for the air-filled microspheres is observed. Therefore, the upward velocity of the air-filled microspheres is used to calibrate each individual microsphere as a function of the radius. To this end, a force balance of all the forces experienced by the microsphere: the buoyance (Fb), gravitation (Fg) and the stokes drag force (F.sub.Stokes) may be determined and solved for the velocity:

[00003]$F_b+F_g+F_{\mathrm{Stokes}}=0$$F_g=\mathrm{Vpg}=\frac{4}{3}\pi g\left(\left(R_2^3-R_1^3\right){\rho }_{\mathrm{PVA}}+R_1^3{\rho }_{\mathrm{air}}\right)$$F_{\mathrm{Stokes}}=-6\pi \eta R_{2}v$$v=\frac{2_g}{9\eta R_2}(\left(R_2^3-{\left(R_2-d\right)}^3{\rho }_{\mathrm{PVA}}+{\left(R_2-d\right)}^3{\rho }_{\mathrm{air}}-R_2^3{\rho }_{\mathrm{water}}\right)$

here V represents the volume of the microsphere, ρ the density, R.sub.1 and R.sub.2 the inner and the outer radius of the microsphere, respectively, η the viscosity of the medium and d the shell thickness (R.sub.2−R.sub.1=300 nm).

[0120] Since the acoustic force scales with the volume of the particle, the force/V.sup.2 ratio is plotted against the inner radius and fitted with a third power function (FIG. 5F). Here, V is the voltage applied over the piezo element used to control the acoustic force, as expected, the force scales quadratically with the applied voltage. When extrapolating this function to the radius of the silica microspheres (approx. 3.4 micron), it was found that the air-filled microspheres experience 170±14 fold higher force than the silica microspheres, but in the opposite direction. As a result, it was found that the acoustic contrast factor is −170.Math.0.54=−92±7.

[0121] Thus, compared to polystyrene microspheres (a commonly used particle material) the increase in force is about 400-fold, which means at least 7 times smaller microspheres can be used and still exert the same force on the microspheres. This way, smaller microspheres or even sub-micron spheres (nanospheres) can be used to exert forces on a cell. Furthermore, microspheres can be selected that are (substantially) smaller than the typical dimensions of a cell to probe specific parts of the cell instead of the mechanical response of the whole cell. The use of particles smaller than the cells which are probed also may have an advantage for throughput: using smaller particles, more particles can be tracked within the same field-of-view.

[0122] Hence, high acoustic contrast particles referred to in the embodiments of this disclosure may include hollow organic particles, e.g. hollow polymer-based particles as described with reference to FIGS. 5A-5F, and inorganic hollow particles, e.g. hollow glass particles.

[0123] In an embodiment, the particles may include hollow particles, which may be filled with a gas, a gas mixture (including air) or a liquid. The hollow particles may include hollow inorganic particles, e.g. oxide-based, e.g., silicon oxide-based hollow particles, glass-based hollow particles or ceramic-based hollow particles. Alternatively, the hollow particles may include hollow organic particles. Such hollow particles may include polymer-based hollow particles, e.g. polyvinyl alcohol (PVA) based hollow particles.

[0124] In yet another embodiment, the hollow particles may have an acoustic contrast factor (in a water-type fluid medium) that is larger than 0.5 or smaller than −0.5. In another embodiment, the hollow particles may have an acoustic contrast factor (in a water-type fluid medium) that is larger than 0.6 or smaller than −0.6.

[0125] Further, in an embodiment, hollow particles with a low compressibility (e.g. a ‘hard’ shell) may be selected. Low-compressibility particles are desired because highly compressible particles as e.g. used as ultrasound contrast agents generate a strong local acoustic field around themselves when placed in an acoustic field. This extra acoustic field distorts the force applied to the cell to which it is attached to and/or influences other nearby particles. Hence, preferably the hollow particles may have a shell of a material with a high Young's modulus, preferably a Young's modulus selected between 1 and 1000 GPa. For example, in an embodiment, the shell may include a glass material having a Young's modulus between 50-90 GPa. In another embodiment, the shell may include a polyvinyl alcohol material having a Young's modulus between 1-10 GPa. In yet a further embodiment, the shell may include a ceramic material having a Young's modulus of more than 50 GPa.

[0126] The air-filled microspheres further provide the advantage of high optical contrast. As shown in FIG. 3D, cells are imaged using an optical microscope. For accurate measurements, the microspheres that are placed on top of the cells, need to be tracked in three dimensions, in particular in the direction of the z-axis (the direction perpendicular to the surface of the sample holder). Because z-tracking relies on precise analysis of radial ring patterns that appear around a particle, the image of the cells can interfere with the ring patterns.

[0127] Additionally, for accurate optical tracking against a background of cells, the particles need to have an optical contrast that is higher, preferably substantial higher, than the optical contrast of the cells. The optical contrast depends on the difference between the refractive index between the object and the medium. Water (the medium) has a refractive index of 1.33 and glass has 1.5 (13% difference), while red blood cells have a refractive index of −1.4 (5% difference). Hence, the microbeads have a higher optical contrast compared to the red blood cells, thus optical tracking of the microbeads in the z-direction is possible. Nevertheless, improvements in the optical contrast are desirable, especially since the particles need to be tracked against a cellular background.

[0128] The optical contrast may be optimized by choosing particles with a sufficiently different refractive index. This may be realized selecting a material with a high refractive index, or at least a high real part of the refractive index in the visible range. For example, for diamonds, the real part of the refractive index is 2.4-2.5 in the visible range, thus providing a refractive index difference between 80% and 88%.

[0129] Similarly, this may be realized selecting a material with a low refractive index, or at least a low real part of the refractive index in the visible range. For example, air in air-filled hollow particles has a refractive index of 1 (25% difference), thus providing a substantial higher difference in refractive index when compared with glass (silica) particles. The air-filled hollow particles thus not only provide a high acoustic contrast factor but also high optical visibility when compared with solid silica particles.

[0130] In a further embodiment, solid particles of a high acoustic contrast factor material may be selected. For example, in an embodiment, diamond particles may be selected. Diamond particles will have an acoustic contrast factor that is approximately three times higher than polystyrene particles.

[0131] The (average) size of the particles (microparticles and nanoparticles) and the contrast factor may be selected on the basis of a particular application. For example, for locally probing mechanical properties of a cellular body, the size of a particle may be substantially smaller than the dimensions of the cellular body. For example, in an embodiment, for probing local mechanical parameters, the size of the particles may be selected to be 20% of the cell size or smaller. In another embodiment, for probing global parameters the size of the particles may be selected to be 50% of the cell size or larger.

[0132] Taken into account application specific conditions (e.g. measuring global or local mechanical parameters, measuring cells with a relatively high acoustic contrast factor and/or optical refraction index, applying a relatively large force, etc.), the (average) size of the particle may be selected 0.2 and 20 micron wherein—depending on the cell type—the size of the cells may vary between 3 and 100 micron.

[0133] The surface of the particles described in this disclosure may be functionalized in order to adhere to a cell and/or a specific part of a cell. Known materials may be used to functionalize the surface of the particles. For example, in an embodiment, a particle is functionalized using one or more primers comprising one or more interaction moieties for adhesion to at least part of the cellular body, preferably an interaction moiety including at least one of: viruses, viral particles, antibodies, peptides, biological tissue factors, biological tissue portions, antigens, proteins, ligands, lipid (bi)layers, fibronectin, cellulose, nucleic acids, RNA, small molecules, allosteric modulators, (bacterial) biofilms, and specific atomic or molecular surface portions (e.g. a gold surface).

[0134] In an embodiment, hollow particles PVA particles may be functionalized using antibodies in order to adhere particles to certain cell types, such as cancer cells as e.g. described by Faridi et al, in their article MicroBubble Activated acoustic cell sorting Biomed Microdevices. 2017 June; 19(2):23.

[0135] FIG. 6 schematically depicts a process for probing mechanical properties of cells according to another embodiment of the invention. In this embodiment, forces can be applied in two directions by changing the applied resonance frequency. A sample holder (a chip) has a specific configuration of layers leading to a set of resonance frequencies associated with a specific force profile in z direction of the sample holder. As shown in FIG. 6, cell 604 may be bound to a substrate 602 and a particle 606 may be bound to the cell. A force indicated by an arrow can be applied to the particle in a direction away from the substrate 608a which means the particle pulls on the cell. Alternatively, it can be applied in a direction towards the substrate 608b which means that the particle pushes on the cell. Pushing and pulling can probe different aspects of the cell mechanics: while pulling probes both the mechanics of the membrane and the cytoskeleton, pushing applies direct load on the cytoskeleton of the cell, which dominates the mechanical properties of the membrane of the cell.

[0136] FIG. 7 schematically depicts a cross-section of part of a sample holder for an acoustic force spectroscopy system according to an embodiment of the invention. The sample holder may include an objective 702 that is positioned underneath part of a sample holder (a chip) wherein the sample holder may comprise a capping layer 706, a matching layer 710, a fluid 708 contained in the holding space formed by the capping and the matching layer, and a piezo element 712. By applying an AC voltage V to the piezo element at the appropriate frequency, a resonant bulk acoustic standing wave can be generated in the sample holder which optionally has a node 716 in the fluid layer. Particles that have a positive acoustic contrast factor with respect to the fluid medium will be attracted to the acoustic node. In an embodiment, an immersion fluid 704 between the objective and the capping layer may be used to improve the optical NA of the imaging system.

[0137] FIG. 8 schematically depicts acoustic resonant modes of part of a sample holder for an acoustic force spectroscopy system according to an embodiment of the invention. In particular, FIGS. 8A and 8B depict two (non-limiting) examples of force profiles created by the acoustic standing wave over the height of the fluid channel of the sample holder. Different force profiles may be shaped in the sample holder by tuning the frequency of the applied wave or by changing the material properties and dimensions of elements that form the sample holder, including the piezo, the matching layer, the fluid layer and/or the capping layer (see FIG. 7).

[0138] The figure shows a part of the capping layer 802, a part of the fluid layer with a resonant standing wave 804a,804b. As shown in these figures, the fluid layer contains regions of negative force 806 and positive force 808 as would be experienced by a particle with a positive acoustic contrast. For example, in the first example of FIG. 8A, the fluid layer includes two regions of negative force 806.sub.1,2 and two regions of positive force 808.sub.1,2. Similarly, the second example of FIG. 8B depicts two regions of positive force 808.sub.3,4 and one region of a negative force 806.sub.3. The first acoustic node as seen from the bottom surface 810 is also indicated. In the regions of negative force a particle of positive acoustic contrast would experience a force away from the bottom surface. In the regions of positive force a particle of positive acoustic.

[0139] FIG. 9 depicts a method for preparing cells for a mechanical probing process according to an embodiment of the invention. The method provides an increased yield of cell-particle constructs for a mechanical probing process.

[0140] In order to measure many cells in parallel and gather enough data for proper statistical analysis, it may be advantageous to create a sample substrate that has many cells in the field-of-view of the microscope wherein a particle is attached to the top of each cell. In some embodiments, it may be advantageous that the particle is nicely centred on top of the cell. FIG. 9 depicts the preparation of a sample substrate wherein cells are positioned on the surface of the sample holder 902 having a particle positioned on top of the cell. The preparation of the sample substrate may include the steps of: mixing particles 906 with cells 904 and flushing them into the sample chamber. During this process, a particle that has a functionalized surface may adhere to a surface of a cell. Thereafter, the gravitational force F.sub.gravity 908 may cause the cells to sink to the bottom surface where they can attach to the substrate.

[0141] In an embodiment 900b, particles may have a density higher than the density of the medium. In that case, a gentle acoustic force 910b may be applied that acts on the particle, while the gravity force acts on the cell. These forces will align the cell—particle construct with the axis perpendicular to the surface of the sample holder. In the aligned position, the particle is on top of the cell as depicted in the figure. The acoustic force generator may be controlled to generate an acoustic force F.sub.acoustic onto the particles that is smaller than the gravitational force on the particles. The acoustic force may be slowly lowered while the particles sink to the surface as required. This way, cell particle constructs may be deposited in a controllable way onto the surface of the holding space of the sample holder wherein the particles are positioned on top of the cellular bodies.

[0142] In an embodiment 900a, the particles may have a density lower than the density of the medium, e.g. hollow gas or air filled particles. In that case, the particles may generate a buoyancy force F.sub.bouyancy 910a. The buoyancy force that acts on the particle and the gravitational force that acts on the cell may cause the cell—particle construct to align with the axis perpendicular to the surface of the sample holder. In the aligned position, the particle is on top of the cell as depicted in the figure. The gravity force will gently pull the cells towards the surface of the sample holder, while the buoyancy force keeps the particle on top of the cell. This way, cells are deposited in a controllable way onto the surface of the holding space of the sample holder wherein the particles are positioned on top of the cellular bodies. This way, a high trough put sample substrate is realized having many cell—particle constructs that are suitable for use in mechanical probing experiments.

[0143] FIG. 10 schematically depicts a process of classifying cells on the basis of mechanical parameters according to an embodiment of the invention. In particular, FIG. 10 illustrates a method for classification of different cell populations based on multi-dimensional analysis. Graphs 1002 and 1004 represent histograms of mechanical parameters L0 and L′v of a simulated dataset based on two cell populations where these parameters are differently correlated for the two populations. Based on the histograms, it is not obvious that this dataset contains to different cell populations. When the data is plotted on a 2D graph 2006 with the two parameters on the two axes, a clear separation between the two cell populations, a first cell population 1008 associated with one or more first cell parameters and a second cell population 1010 associated with one or more cell parameters becomes apparent. This way, cells can be classified (assigned to either one of the populations) based on whether the data points are located above or below a separation line 1012. This method can obviously be extended to any number of dimensions.

[0144] Hence, using for example the burgers model (as is explained above with reference to FIG. 3E) a number of fit parameters can be obtained using the mechanical probing techniques as described with reference to the embodiments of this disclosure. Analyzing the fit parameters in 2D, 3D or even 4D space allows classifying differences in responses between groups of cells. Such classification scheme may be used to distinguish between healthy and diseased cells.

[0145] The disclosure is not restricted to the above described embodiments which can be varied in a number of ways within the scope of the claims as explained supra. Elements and aspects discussed for or in relation with a particular embodiment of the method or system may be suitably combined with elements and aspects of other embodiments of the system or method, unless explicitly stated otherwise.