Method and system for determining mechanical properties of biological cells or biological cell-like particles

Abstract

The present application provides a method for determining the mechanical properties of biological cells or biological cell-like particles, comprising the following steps: allowing a fluid containing the biological cells or biological cell-like particles to pass through a microfluidic channel along a first direction; in the process of the fluid passing through the microfluidic channel, using optical tweezers to capture at least a portion of the biological cells or biological cell-like particles in a second direction transverse to the first direction; collecting an image of the biological cells or biological cell-like particles passing through the microfluidic channel; and determining the mechanical properties of the biological cells or biological cell-like particles based on the deformation amount of the biological cells or biological cell-like particles captured by the optical tweezers in the image. The present application also provides a system for implementing the above method.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 918,234, filed on January 23, 2019, entitled “An Optofluidic Stretcher for Biological Cells and Soft Particles,” the contents of which are incorporated herein by reference in their entirety. Field of the Invention

[0003] The present application generally relates to the fields of biomechanics, optics and fluid mechanics. Specifically, the present application provides a method and system for determining the mechanical properties of biological cells or biological cell-like particles by combining optical tweezers technology and microfluidics technology. Background of the Invention

[0004] The mechanical properties of biological cells (also known as mechanical properties) can be used as intrinsic, label-free biomarkers that can indicate physiological and pathological changes in cells [1]. Changes in cell deformability are associated with changes in the underlying cytoskeleton [2] and have been found to be associated with a wide range of cellular functional changes, including differentiation [3], apoptosis [4], disease transformation [5], and drug response [6]. In particular, there is increasing evidence that reduced deformability of human red blood cells (RBCs) is a symptom of various human diseases, such as malaria infection [7], diabetes [8], and sickle cell anemia [9]. For Plasmodium falciparum malaria, recent experiments have shown that the membrane stiffness of parasitized RBCs can increase by up to 10-fold

[10] . These findings suggest that characterization of cell deformability has the potential to provide useful information for distinguishing healthy from unhealthy cells, and thus become a consideration for disease diagnosis.

[0005] The mechanical properties of cells have attracted extensive research interest over the past few decades in order to investigate the pathophysiological importance of cell deformability. Several techniques have been developed to assess the mechanical deformability of single cells. Reported characterization techniques include micropipette aspiration

[11] , atomic force microscopy

[12] , and optical tweezers

[13] that require high refractive index microbeads (e.g., silica or polystyrene microbeads) attached to both ends of the cell for mechanical loading. These techniques involve direct physical contact between the solid surface and the cell under test, which may alter the natural behavior of the cell or even damage the cell during the measurement. Another practical problem is that these techniques are based on static test conditions, which means that the characterization throughput is low. In fact, high-throughput testing is more valuable to quickly obtain characterization or screening results of multiple cells in order to obtain statistically relevant results given the inherent heterogeneity of biological cells.

[0006] Applying optical tweezers directly on cells without the attachment of microbeads is an ideal method for non-contact, non-invasive and light-induced cell deformation. Standard optical tweezers consist of a highly focused Gaussian laser beam that can exert piconewton-level optical forces on micro-sized transparent objects for optical trapping and manipulation

[14] . The optical force of optical tweezers can be decomposed into two components, namely, the optical gradient force that attracts high-refractive index objects to the center of the beam waist where the light field intensity is highest, and the scattering force that pulls the object along the longitudinal beam propagation direction. For a highly focused laser beam, the optical gradient force component in the longitudinal direction can balance the scattering force, thus forming a conventional single laser beam tweezer.

[0007] Cells can be deformed using dual optical tweezers, where two focused laser beams propagating in parallel are applied directly to the cell’s ends, mechanically stretching the cell in the direction of the two beams’ divergence. Another type of optical cell stretching involves two counter-propagating diverging beams emitted from two optical fibers, where the cell is optically trapped and stretched in the longitudinal direction of the two laser beams

[16] .

[0008] Microfluidics has gained increasing research interest as an attractive tool to study the mechanical properties of cells. Microfluidic devices contain microfluidic channels, typically on the micrometer and millimeter scale, with inlets and outlets to connect to the external environment, in which fluids and biological samples can be precisely controlled and studied

[17] . One advantage of microfluidic devices for biological cell analysis is the high characterization throughput achieved by continuous cell sample delivery using fluids. Another attractive feature of microfluidic devices is the small volume required for analysis, which is attractive for lab-on-a-chip applications. Other advantages of microfluidic devices are mature manufacturing processes and materials. Most microfluidic devices are fabricated using mature photolithography methods used by the microelectronics industry, which allows for precise and reproducible manufacturing processes. The materials used to fabricate microfluidic channels are usually transparent, such as polydimethylsiloxane (PDMS) or glass, which allows for optical imaging for cell analysis.

[0009] Microfluidic devices have been developed to study cell deformation using fluid dynamics induced by fluid flow. A typical approach is to study cell deformation when cells flow at high speed through a straight and narrow microfluidic channel with a cross-sectional dimension close to the cell size

[18] . When interacting with the non-uniform distribution of fluid in the narrow cross-section of the microfluidic channel, the cell deforms under the action of fluid stress.

[0010] The inventors of the present application also considered the following documents when developing the various inventions of the present application:

[0011] 1. CT Lim, M. Dao, S. Suresh, CHSow, and KTC Hew in "Large deformation of living cells using laser traps," Acta Materialia 52 (7), 1837-1845 (2004), demonstrated the stretching of red blood cells (RBC) for silica microbead attachment. In this work, each test cell was specially prepared with two small silica beads attached to both ends of the cell. During the test, the two silica beads were used for mechanical loading. Two optical tweezers were used to capture and manipulate the two silica beads separately. When the two optical tweezers increased the distance between the two beads, cell stretching was achieved.

[0012] 2. S. Rancourt-Grenier, M.T. Wei, J.J. Bai, A. Chiou, P.P. Bareil, P.L. Duval, and Y. Sheng in “Dynamic deformation of red blood cell in dual-trap optical tweezers,” Opt. Express 18(10), 10462–10472 (2010), demonstrating dual optical tweezers that can stretch osmotically swollen red blood cells by applying optical forces directly at both ends of the cell without the need for microbeads.

[0013] 3. DS Moura, DC Silva, AJ Williams, MA Bezerra, A. Fontes, and RE de Araujo in "Automatic real time evaluation of red blood cell elasticity by optical tweezers," Rev. Sci. Instrum., 86(5), 053702 (2015), demonstrated cell stretching using a single optical tweezer controlled by a motorized stage to capture RBCs in a fluid chamber. In this work, RBC deformation was achieved by using optical tweezers to drag the captured cells through a static fluid in a fluid chamber at a defined speed. The captured cells deformed due to fluid stress.

[0014] 4. US Patent 6067859 discloses optical cell stretching based on two counter-propagating divergent laser beams from two optical fibers for deforming cells.

[0015] 5. B. Lincoln, F. Wottawah, S. Schinkinger, S. Ebert, and J. Guck in “High-throughput rheological measurements with an optical stretcher,” Methods Cell Biol. 83, 397-423 (2007) further developed their previous technology in U.S. Patent 6067859 by combining optical cell stretching with microfluidic channels for continuous cell sample delivery.

[0016] 6. KB Roth, KB Neeves, J. Squier, and DW Marr in “High-throughput linear optical stretcher for mechanical characterization of blood cells,” Cytometry Part A 89(4), 391-397 (2016), demonstrated linear optical cell stretching using a line beam in a microfluidic channel. In this method, a linear diode rod laser is used as the laser source, and a cylindrical lens is used to project the pattern into the microfluidic channel. When the cells overlap with the linear beam, they stretch along the line due to the outward optical stress that aligns the two ends of the cells with the linear beam pattern.

[0017] 7.Z.Yao and AWPoon in "Optical lattice-based cell guiding and stretching using integrated vertical multimode-interference waveguides," CLEO: Science and Innovations, STh3J.6 (2018), reported a beam shaping technique using SU8-filled vertically embedded multimode interference (MMI) waveguides for cell stretching. In this work, RBCs were guided and deformed as they passed through the optical lattice landscape generated by the MMI waveguides in a microfluidic channel.

[0018] 8. O. Otto, P. Rosendahl, A. Mietke, S. Golfier, C. Herold, D. Klaue, S. Girardo, S. Pagliara, A. Ekpenyong, A. Jacobi, and M. Wobus in “Real-time deformability cytometry: on-the-fly cell mechanical phenotyping,” Nat. Methods, 12(3), 199 (2015), demonstrated microfluidics-based cell deformability counting that can be used for continuous cell mechanical characterization. In this work, a bottleneck microfluidic channel design was used, and the cross-sectional dimensions of the cells were comparable to the cell size when the cells flowed through the narrow part of the microfluidic channel at high speed, causing the cells to deform. When interacting with the non-uniformly distributed fluid in the cross-section of the narrow microfluidic channel, the cells deform under the action of the fluid stress. The characterization throughput is reported to be approximately 100 cells / second.

[0019] 9.DR Gossett,TK Henry,SALee,Y. Ying,AG Lindgren,OO Yang,J. Rao,AT Clark,and D. Di Carlo in "Hydrodynamic stretching of single cells for large population mechanical phenotyping,"Proc. Natl. Acad. Sci. USA 109 (20), 7630-7635 (2012), demonstrated a microfluidic-based cell stretching technique, in which cells were decelerated and deformed by fluid forces at the stagnation point of the fast extension flow in a cross-shaped microfluidic channel. The cell deformation was imaged for a few seconds at a speed of 100,000 frames / second using a high-speed camera. A characterization throughput of about 2000 cells / second was demonstrated. SUMMARY OF THE INVENTION

[0020] In a first aspect, the present application provides a method for determining the mechanical properties of biological cells or biological cell-like particles, comprising the following steps:

[0021] Passing a fluid containing the biological cells or biological cell-like particles through a microfluidic channel in a first direction;

[0022] During the process of the fluid passing through the microfluidic channel, capturing at least a portion of the biological cells or biological cell-like particles in a second direction transverse to the first direction using optical tweezers;

[0023] capturing an image of the biological cell or biological cell-like particle passing through the microfluidic channel; and

[0024] Based on the deformation amount of the biological cell or biological cell-like particle captured by the optical tweezers in the image, the mechanical properties of the biological cell or biological cell-like particle are determined.

[0025] The term "biological cell-like particles" as used in this application refers to particles having physical properties similar to those of biological cells, for example, soft, insulating, micron-sized particles. Examples of biological cell-like particles include, but are not limited to, phospholipid membrane vesicles.

[0026] In some embodiments, the medium of the fluid has a lower optical refractive index than the biological cells or biological cell-like particles.

[0027] In some embodiments, the cross-section of the microfluidic channel is large enough so that the biological cells or biological cell-like particles do not physically contact the wall of the microfluidic channel to generate deformation force when passing through the microfluidic channel.

[0028] In some embodiments, the optical tweezers are a highly focused laser beam with a Gaussian distribution.

[0029] In some embodiments, the spectral window of the optical tweezers is transparent to one or more of the following:

[0030] The biological cell or biological cell-like particle,

[0031] the fluid medium,

[0032] a wall of the microfluidic channel.

[0033] In some embodiments, the optical tweezers are periodically modulated to periodically capture and release the biological cells or biological cell-like particles. In some embodiments, the optical tweezers are periodically activated and deactivated.

[0034] In some embodiments, the modulation is performed by a modulator.

[0035] In some embodiments, the frequency of the modulation is at the Hertz level, such as 1-5 Hz.

[0036] In some embodiments, the method uses multiple optical tweezers.

[0037] In some embodiments, the multiple optical tweezers are generated by an interferometer configured with a single laser source.

[0038] In some embodiments, the multiple optical tweezers are generated by multiple laser sources.

[0039] In some embodiments, the plurality of optical tweezers are arranged into an optical tweezers array, such as a one-dimensional optical tweezers array or a two-dimensional optical tweezers array.

[0040] In some embodiments, the optical tweezers array is generated using a multimode interference waveguide with a single laser source.

[0041] In some embodiments, the optical tweezers array is generated using a spatial light modulator with a single laser source.

[0042] In some embodiments, the focus of the optical tweezers is set so that the cells captured by the optical tweezers are away from the wall of the microfluidic channel.

[0043] In some embodiments, images are acquired using a microscope objective and a digital camera, such as a long working distance microscope objective and a CCD digital camera.

[0044] In some embodiments, the image is acquired in a third direction transverse to the first direction.

[0045] In some embodiments, images of the biological cell or biological cell-like particle are collected before, during, and after being captured by the optical tweezers.

[0046] In some embodiments, the dimension (eg, path length) of the biological cell or biological cell-like particle captured by the optical tweezers in the image along the first direction and perpendicular to the first direction is measured, and the ratio of the two is used to characterize the deformation amount.

[0047] In a second aspect, the present application provides a system for determining the mechanical properties of biological cells or biological cell-like particles, comprising:

[0048] A microfluidic device comprising a microfluidic channel for passing a fluid containing the biological cells or biological cell-like particles in a first direction;

[0049] Optical tweezers provide means for capturing at least a portion of the biological cells or biological cell-like particles using optical tweezers in a second direction transverse to the first direction during the passage of the fluid through the microfluidic channel;

[0050] an image acquisition device for acquiring images of the biological cells or biological cell-like particles passing through the microfluidic channel; and

[0051] An analysis device is used to determine the mechanical properties of the biological cells or biological cell-like particles based on the deformation amount of the biological cells or biological cell-like particles captured by the optical tweezers in the image.

[0052] In some embodiments, the medium of the fluid has a lower optical refractive index than the biological cells or biological cell-like particles.

[0053] In some embodiments, the cross-section of the microfluidic channel is large enough so that the biological cells or biological cell-like particles do not physically contact the wall of the microfluidic channel to generate deformation force when passing through the microfluidic channel.

[0054] In some embodiments, the optical tweezers are a highly focused laser beam with a Gaussian distribution.

[0055] In some embodiments, the spectral window of the optical tweezers is transparent to one or more of the following:

[0056] The biological cell or biological cell-like particle,

[0057] the fluid medium,

[0058] a wall of the microfluidic channel.

[0059] In some embodiments, the optical tweezers providing device further comprises a component for periodically modulating the optical tweezers, thereby periodically capturing and releasing the biological cells or biological cell-like particles. In some embodiments, the component is used to periodically activate and deactivate the optical tweezers.

[0060] In some embodiments, the component that periodically modulates the optical tweezers is a modulator.

[0061] In some embodiments, the frequency of the modulation is at the Hertz level, such as 1-5 Hz.

[0062] In some embodiments, the optical tweezers provide a device capable of using multiple optical tweezers.

[0063] In some embodiments, the multiple optical tweezers are generated by an interferometer configured with a single laser source.

[0064] In some embodiments, the multiple optical tweezers are generated by multiple laser sources.

[0065] In some embodiments, the plurality of optical tweezers are arranged into an optical tweezers array, such as a one-dimensional optical tweezers array or a two-dimensional optical tweezers array.

[0066] In some embodiments, the optical tweezers array is generated using a multimode interference waveguide with a single laser source.

[0067] In some embodiments, the optical tweezers array is generated using a spatial light modulator with a single laser source.

[0068] In some embodiments, the focus of the optical tweezers is set so that the cells captured by the optical tweezers are away from the wall of the microfluidic channel.

[0069] In some embodiments, the image acquisition device includes a microscope objective and a digital camera, such as a long working distance microscope objective and a CCD digital camera.

[0070] In some embodiments, the image acquisition is configured to acquire images in a third direction transverse to the first direction.

[0071] In some embodiments, the image acquisition is configured to acquire images of the biological cell or biological cell-like particle before, during, and after being captured by the optical tweezers.

[0072] In some embodiments, the analysis device is configured to measure the dimension (e.g., path length) of the biological cells or biological cell-like particles captured by the optical tweezers in the image along the first direction and perpendicular to the first direction, and use the ratio of the two to characterize the deformation amount.

[0073] As non-limiting examples, the present application provides the following embodiments:

[0074] 1. A method for determining the mechanical properties of biological cells or biological cell-like particles, comprising the following steps:

[0075] Passing a fluid containing the biological cells or biological cell-like particles through a microfluidic channel in a first direction;

[0076] During the process of the fluid passing through the microfluidic channel, capturing at least a portion of the biological cells or biological cell-like particles in a second direction transverse to the first direction using optical tweezers;

[0077] capturing an image of the biological cell or biological cell-like particle passing through the microfluidic channel; and

[0078] Based on the deformation amount of the biological cell or biological cell-like particle captured by the optical tweezers in the image, the mechanical properties of the biological cell or biological cell-like particle are determined.

[0079] 2. A method as described in embodiment 1, wherein the optical refractive index of the medium of the fluid is lower than that of the biological cells or biological cell-like particles.

[0080] 3. A method as described in embodiment 1 or 2, wherein the cross-section of the microfluidic channel is large enough so that the biological cells or biological cell-like particles do not physically contact the wall of the microfluidic channel to generate deformation force when passing through the microfluidic channel.

[0081] 4. A method as described in any of embodiments 1-3, wherein the optical tweezers is a highly focused laser beam with a Gaussian distribution.

[0082] 5. The method of any one of embodiments 1-4, wherein the spectral window of the optical tweezers is transparent to one or more of:

[0083] The biological cell or biological cell-like particle,

[0084] the fluid medium,

[0085] a wall of the microfluidic channel.

[0086] 6. A method as described in any one of embodiments 1-5, wherein the optical tweezers are periodically modulated, for example, the optical tweezers are periodically turned on and off, thereby periodically capturing and releasing the biological cells or biological cell-like particles.

[0087] 7. A method as described in embodiment 6, wherein the modulation is implemented by a modulator.

[0088] 8. A method as described in embodiment 6, wherein the frequency of the modulation is at the Hertz level, such as 1-5 Hz.

[0089] 9. A method as described in any of embodiments 1-8, wherein multiple optical tweezers are used.

[0090] 10. A method as described in embodiment 9, wherein the multiple optical tweezers are generated by an interferometer configured with a single laser source.

[0091] 11. A method as described in embodiment 9, wherein the multiple optical tweezers are generated by multiple laser sources.

[0092] 12. A method as described in embodiment 9, wherein the multiple optical tweezers are arranged into an optical tweezers array, such as a one-dimensional optical tweezers array or a two-dimensional optical tweezers array.

[0093] 13. A method as described in embodiment 12, wherein the optical tweezers array is generated using a multi-mode interference waveguide with a single laser source.

[0094] 14. A method as described in embodiment 12, wherein the optical tweezers array is generated using a spatial light modulator with a single laser source.

[0095] 15. A method as described in any of embodiments 1-14, wherein the focus of the optical tweezers is set so that the cells captured by the optical tweezers are away from the wall of the microfluidic channel.

[0096] 16. A method as described in any of embodiments 1-15, wherein the image is collected using a microscope objective and a digital camera, such as a long working distance microscope objective and a CCD digital camera.

[0097] 17. A method as described in any of embodiments 1-16, wherein the image is acquired in a third direction transverse to the first direction.

[0098] 18. A method as described in any of embodiments 1-17, wherein images of the biological cell or biological cell-like particle are collected before, during and after being captured by the optical tweezers.

[0099] 19. A method as described in any one of embodiments 1-18, wherein the dimension (e.g., path length) of the biological cells or biological cell-like particles captured by the optical tweezers in the image along the first direction and perpendicular to the first direction is measured, and the ratio of the two is used to characterize the deformation amount.

[0100] 20. A system for determining mechanical properties of biological cells or biological cell-like particles, comprising:

[0101] A microfluidic device comprising a microfluidic channel for passing a fluid containing the biological cells or biological cell-like particles in a first direction;

[0102] Optical tweezers provide means for capturing at least a portion of the biological cells or biological cell-like particles using optical tweezers in a second direction transverse to the first direction during the passage of the fluid through the microfluidic channel;

[0103] an image acquisition device for acquiring images of the biological cells or biological cell-like particles passing through the microfluidic channel; and

[0104] An analysis device is used to determine the mechanical properties of the biological cells or biological cell-like particles based on the deformation amount of the biological cells or biological cell-like particles captured by the optical tweezers in the image.

[0105] 21. A system as described in embodiment 20, wherein the optical refractive index of the medium of the fluid is lower than that of the biological cells or biological cell-like particles.

[0106] 22. A system as described in embodiment 20 or 21, wherein the cross-section of the microfluidic channel is large enough so that the biological cells or biological cell-like particles do not physically contact the wall of the microfluidic channel to generate deformation force when passing through the microfluidic channel.

[0107] 23. A system as described in any of embodiments 20-22, wherein the optical tweezers is a highly focused laser beam with a Gaussian distribution.

[0108] 24. The system of any one of embodiments 20-23, wherein the spectral window of the optical tweezers is transparent to one or more of:

[0109] The biological cell or biological cell-like particle,

[0110] the fluid medium,

[0111] a wall of the microfluidic channel.

[0112] 25. A system as described in any of embodiments 20-24, wherein the optical tweezers providing device also includes a component for periodically modulating the optical tweezers, for example, for periodically starting and shutting down the optical tweezers, thereby periodically capturing and releasing the biological cells or biological cell-like particles.

[0113] 26. A system as described in embodiment 25, wherein the component that periodically modulates the optical tweezers is a modulator.

[0114] 27. A system as described in embodiment 25, wherein the frequency of the modulation is at the Hertz level, such as 1-5 Hz.

[0115] 28. A system as described in any of embodiments 20-27, wherein the optical tweezers providing device can use multiple optical tweezers.

[0116] 29. A system as described in embodiment 28, wherein the multiple optical tweezers are generated by an interferometer configured with a single laser source.

[0117] 30. A system as described in embodiment 28, wherein the multiple optical tweezers are generated by multiple laser sources.

[0118] 31. A system as described in embodiment 28, wherein the multiple optical tweezers are arranged into an optical tweezers array, such as a one-dimensional optical tweezers array or a two-dimensional optical tweezers array.

[0119] 32. A system as described in embodiment 31, wherein the optical tweezers array is generated using a multi-mode interference waveguide with a single laser source.

[0120] 33. A system as described in embodiment 31, wherein the optical tweezers array is generated using a spatial light modulator with a single laser source.

[0121] 34. A system as described in any of embodiments 20-33, wherein the focus of the optical tweezers is set so that the cells captured by the optical tweezers are away from the wall of the microfluidic channel.

[0122] 35. A system as described in any of embodiments 20-34, wherein the image acquisition device includes a microscope objective and a digital camera, such as a long working distance microscope objective and a CCD digital camera.

[0123] 36. A system as described in any of embodiments 20-35, wherein the image acquisition is configured to acquire images in a third direction transverse to the first direction.

[0124] 37. A system as described in any of embodiments 20-36, wherein the image acquisition is configured to acquire images of the biological cell or biological cell-like particle before, during and after being captured by the optical tweezers.

[0125] 38. A system as described in any of embodiments 20-37, wherein the analysis device is configured to measure the dimension (e.g., path length) of the biological cells or biological cell-like particles captured by the optical tweezers in the image along the first direction and perpendicular to the first direction, and use the ratio of the two to characterize the deformation amount. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1 : (a) shows a schematic diagram of the working principle of optofluidic tweezing and drag-stretching. Biological cells or biological cell-like particles are captured by optical tweezers near the focused beam portion, and are dragged and stretched by the microfluidic flow along the flow direction (x direction) transverse to the beam (z direction). The beam is periodically blocked to allow the stretched cells or particles to escape from the optical tweezers; (b) shows a top view of optical tweezers and fluid drag-stretching; (c) shows a schematic diagram of the characterization scheme, where a stiffer (unhealthy) cell (cell 2) has a smaller deformation (smaller change in the dx / dy ratio) than a normal (healthy) cell (cell 1) under the same test conditions.

[0127] Figure 2 : (a)-(b) show schematic diagrams of (a) cross-sectional view and (b) top view of the fabricated microfluidic chip having inlets and outlets for continuous cell or particle delivery and collection; (c) shows a schematic diagram of the experimental setup, MO: microscope objective, LED: light emitting diode.

[0128] Figure 3 : (a) shows the extracted trajectories of multiple osmotically swollen rabbit RBCs flowing through the field of view; (b) shows a scatter plot of cell velocity as a function of x position in the region indicated by the black line in (a), the position of the optical tweezers is indicated by the gray background column, and the total number of cells N and the number of trapped cells N' are marked as (N, N'); (c) shows a scatter plot of cell velocity as a function of x position in the region indicated by the white line in (a).

[0129] Figure 4 :(a)-(e) show optical micrographs of representative cells across the region of interest; (f)-(h) show the extracted (f) dx, (g) dy and (h) dx / dy ratios as Figure 3The black wireframe in (a) shows the trend of the x position of the cell in the region of interest. The position of the optical tweezers is indicated by the gray background column in the three figures. (i)-(k) show the extracted (i) dx, (j) dy and (k) dx / dy ratio as Figure 3 (a) The changing trend of the x position of the cells in the area indicated by the white line frame.

[0130] Figure 5 : (a)-(e) show the dx / dy ratio of untreated (healthy) cells extracted as the trend of the x position of cells at different flow rates from about 3.0 μl / hour to about 1.0 μl / hour; (f)-(j) show the dx / dy ratio of glutaraldehyde-treated cells extracted (unhealthy) as the trend of the x position of cells at different flow rates from about 3.0 μl / hour to about 1.0 μl / hour; the position of the optical tweezers is indicated by a gray background column. The total number of cells N and the number of cells captured N' are marked as (N, N').

[0131] Figure 6 : Comparison of the maximum dx / dy ratios of untreated (left column) and chemically treated (right column) RBCs at different flow rates is shown. The total number of cells N and the number of captured cells N' are labeled (N, N').

[0132] Figure 7 : A process schematic diagram of an implementation scheme of a parallel optical tweezers and fluid dragging process based on two spatially multiplexed optical tweezers utilizing two sufficiently spaced optical tweezers in a microfluidic channel is shown.

[0133] Figure 8 : Schematic diagram of the experimental setup showing an implementation of parallel optical tweezers and fluid dragging process based on spatial multiplexing using two sufficiently spaced optical tweezers in a microfluidic channel, where two independent laser sources are used to generate the two light beams.

[0134] Fig. 9 : A schematic diagram of the experimental setup of an implementation scheme of parallel optical tweezers and fluid dragging processes based on spatial multiplexing using two sufficiently spaced optical tweezers in a microfluidic channel is shown, wherein an interferometer configuration including two beam splitters and two mirrors is used to generate two displaced light beams from one laser source.

[0135] Fig.10 : Schematic diagram of the process for an implementation scheme of spatially multiplexed parallel optical tweezers and fluid dragging processes in a microfluidic channel based on utilizing optical tweezers arrays in (a) one dimension and (b) two dimensions.

[0136] Fig.11: Schematic diagram of the experimental setup of an implementation of spatially multiplexed parallel optical tweezers and fluid dragging processes in a microfluidic channel based on utilizing a one-dimensional optical tweezers array formed by interference between two light beams, where the interferometer configuration includes two beam splitters and two mirrors to generate two overlapping light beams from a single laser source.

[0137] Fig.12 : Schematic diagram of the experimental setup showing another embodiment based on spatially multiplexed parallel optical tweezers and fluid dragging processes in microfluidic channels using an optical tweezer array generated by shaping the laser beam using an MMI waveguide. The inset schematically shows a 3×3 optical lattice generated from the waveguide output facet as an example.

[0138] Fig.13 : A schematic diagram of the experimental setup of another embodiment is shown, which is based on an optical tweezers array generated by shaping an expanded laser beam using a spatial light modulator, spatially multiplexed parallel optical tweezers and fluid dragging processes in a microfluidic channel. Specific implementation plan

[0139] As mentioned above, optical tweezers technology and microfluidics technology have been used to evaluate the mechanical properties of biological cells, but the inventors of the present application believe that they each potentially have certain disadvantages. For example, most cell stretching methods based only on optical forces rely on static conditions during the test to minimize the impact of fluid stress on cell deformation, which is not conducive to high-throughput characterization. Since methods based on microfluidics usually require cells to move at high speed in microfluidic channels, cells can only be deformed in an extremely short time scale of 1μs or 1ms, resulting in the measured cell elasticity being unable to be compared with static cell elasticity; in addition, the requirements for the imaging system are high, requiring an exposure time of 1μs and a frame rate of 4000fps. Therefore, the inventors of the present application, through a lot of research and exploration, provide a method and system for measuring the mechanical properties of biological cells or biological cell-like particles that combines optical tweezers technology and microfluidics technology. The various inventions of the present application can at least partially overcome the above-mentioned shortcomings.

[0140] Without being bound by any particular theory, the working principles of the inventions of the present application include: based on optical tweezers technology, optical tweezers are used to capture cells or particles in a fluid flow near a focused light beam, and the captured cells or particles are dragged and stretched by the fluid flow transverse to the light beam to produce deformation, thereby evaluating the mechanical properties of the cells or particles according to the deformation amount. In some cases, the light beam can be blocked periodically (for example, at a frequency of Hz level) to allow temporary capture of cells or particles and allow stretched cells or particles to escape from the optical tweezers. The fluid flow enables cells or particles to be continuously delivered to the optical tweezers, while taking away the cells or particles after capture and evaluation. In some cases, it is advantageous to capture cells or particles by optical tweezers at a position away from the walls (including the substrate and side walls) of the microfluidic channel, so as to avoid physical contact between the cells and the solid surface of the microfluidic channel. Some inventions of the present application can achieve non-contact, continuous characterization (high throughput) of the mechanical properties of various biological cells or particles. Some inventions of the present application can be implemented in a variety of ways, including but not limited to, using multiple optical tweezers or optical tweezers arrays in a microfluidic flow in a spatially multiplexed form to achieve higher characterization throughput. Some inventions of the present application are applicable to the characterization of mechanical properties of biological cells and particles, and are not limited by the shape or cell type of the original cell or particle.

[0141] Compared to the cell mechanical property characterization methods based solely on optical technology described above, the inventions of the present application can reflect one or more advantages of integrated microfluidic technology, such as (i) using fluid force to stretch and deform captured cells or particles, (ii) the fluid can continuously transport cells or particles to optical tweezers, and (iii) the fluid can carry cells or particles away. Therefore, some inventions of the present application allow continuous fluid flow during measurement, so dynamic test conditions can achieve greater characterization throughput compared to optical-based techniques that rely on static test conditions.

[0142] Compared with the cell mechanical property characterization methods based solely on microfluidics described above, the inventions of the present application can embody one or more advantages of integrated optical tweezers technology, for example, (i) the optical tweezers can generate a sufficiently large optical trapping force at the pN level to optically trap cells or particles in a fluid flow, so that the fluid resistance can stretch the cells or particles; (ii) by deforming the cells over a longer time scale (e.g., 0.1s), the measured cell elasticity can be compared with the static cell elasticity; (iii) the requirements for the imaging system are reduced. In some cases of the present application, operating the optical tweezers at a low power (tens of mW or less) at a wavelength of 1064nm that is transparent to biological cells can avoid any light damage or thermal damage to the cells.

[0143] The following describes in detail a number of exemplary embodiments of the present application in conjunction with the accompanying drawings. However, it should be understood that the various embodiments described are not intended to limit the invention of the present application in any aspect, and the technical features of the various embodiments may also be combined within the scope that can be understood by those skilled in the art.

[0144] Figure 1 Figure (a) in the figure schematically illustrates the working principle of the optofluidic optical tweezers and fluid drag stretching according to an embodiment of the present application. The figure takes osmotic swelling of red blood cells as an example. The optical tweezers and fluid dragging process includes using optical tweezers to capture cells near a focused Gaussian beam portion, and the microfluidic flow drags and stretches the captured cells along the flow direction (x direction) transverse to the beam (z direction). Under dynamic equilibrium, the stretched cells experience balanced optical gradients and fluid forces.

[0145] Figure 1 The embodiment shown in (a) of FIG. 1 also blocks the light beam periodically (at a Hz horizontal frequency) to allow the stretched cells to escape from the optical tweezers and move with the fluid when the light beam is blocked. The fluid flow continuously transports the cells to the optical tweezers and stretches the trapped cells. Figure 1 In the embodiment shown in FIG. 5 (a), in order to avoid physical contact between the test cells and the solid surface, the optical tweezers are focused above the substrate and away from the microfluidic channel wall so that the cells are trapped at a position away from any solid surface, for example, see Figure 1 (a) is a cross-sectional view and (b) is a top view.

[0146] Figure 1 Figure (c) schematically illustrates the characterization scheme of cell mechanical properties. In this embodiment, a conventional optical microscope is used with a bright field imaging system to record top view images of cells through the top surface of the microfluidic channel. The system includes a long working distance microscope objective and a digital charge coupled device (CCD) camera. The recorded videos and images are analyzed using an image processing program.

[0147] The deformation of cells under the action of stretching force depends on their hardness or elasticity. Under the same force, a cell with greater stiffness (cell 2 in the diagram) has a smaller deformation than a cell with less stiffness (cell 1 in the diagram). Therefore, in cases where the disease state may affect the mechanical properties of the cell, by measuring the deformation of the cell under a certain stretching force, it can provide some indication of the difference between healthy (elastic) cells and diseased (less elastic) cells.

[0148] To quantify the deformation of cells, the cell dimensions (e.g., path length) along the flow direction (x direction) and transverse to the flow direction (y direction) can be measured from the top view images, marked as dx and dy, respectively, and the dx / dy ratio can be extracted to represent the cell shape. The dx / dy ratio of osmotic swollen spherical cells before stretching is 1, and the dx / dy ratio of cells stretched along the flow direction exceeds 1. Considering that the cells are not ideally spherical before stretching, the dx / dy ratios during and after stretching can be normalized to the ratios before stretching in the analysis.

[0149] During the development phase of this application, cell stiffness was qualitatively compared by comparing the dx / dy ratio between healthy and chemically treated (non-healthy cell modeling) cells under the same test conditions. A more quantitative data analysis was performed under the assumption that the linear spring model and fluid forces dominate cell extension, which allowed an estimate of cell elasticity (by measuring the change in dx under estimated fluid drag forces) to a first order approximation.

[0150] Experimental verification

[0151] The following describes a verification experiment of the inventive concept of the present application.

[0152] Microfluidic channel fabrication

[0153] Standard soft lithography techniques were used to fabricate microfluidic channels in this experiment. Briefly, contact lithography and deep reactive ion etching (DRIE) were used to define the microfluidic channel pattern on a silicon chip. The patterned silicon chip was used as a mold to transfer the pattern to a PDMS layer of about 3 mm thickness. After peeling off the PDMS from the silicon mold, a punch was used to form an inlet and an outlet with a diameter of about 1 mm at both ends of the microfluidic channel in the PDMS layer.

[0154] The patterned PDMS layer was bonded to a thin coverslip to form microfluidic channels. The PDMS layer and the glass slide were treated with oxygen plasma before bonding them together. The bonded PDMS-glass interface was sufficiently stable at different flow pumping rates from 1 μl / hour to 3 μl / hour.

[0155] Figure 2 Figures (a) and (b) schematically show a cross-sectional view and a top view of a microfluidic chip with an inlet and an outlet for cell delivery. In this experiment, a PDMS microfluidic channel with a width of about 90 microns and a height of about 40 microns was used.

[0156] Experimental setup

[0157] Figure 2Figure (c) schematically shows the experimental setup. A continuous wave fiber laser with a wavelength of 1064 nm was used as the light source. This wavelength is compatible with biological applications, causes very little light damage to biological cells (cells are transparent to 1064 nm wavelength), and has very little absorption by water (α = 0.61 cm -1 ), so that the laser beam does not heat the fluid medium. The linearly polarized laser power is controlled using a rotatable half-wave plate and a fixed polarization beam splitter (PBS).

[0158] The laser beam was focused into the microfluidic channel using a microscope objective with a high numerical aperture (NA) of about 0.85 in air. The focused beam waist diameter inside the fluid medium was estimated to be about 1.1 microns, and the depth of focus was about 2.6 microns. The position of the beam waist relative to the channel substrate was calibrated by the divergence of the back-reflected beam from the glass-fluid interface. For this experiment, the optical tweezers were set at a distance of about 6 μm above the substrate to avoid contact between the trapped cells (theoretical diameter is about 7 μm) and the substrate.

[0159] The laser is periodically blocked using a mechanical shutter so that the stretched cells can escape from the optical tweezers after reaching a dynamic equilibrium state, thereby achieving continuous characterization of multiple incoming cells brought by the fluid. The blocking frequency is set to about 2 Hz (corresponding to a laser exposure time of about 0.25 s) to provide a sufficiently long time for capturing and stretching cells. Other embodiments based on this embodiment include adjusting and optimizing the blocking frequency to stretch the cells to a state that statistically allows the most sensitive distinction between different cell elasticities.

[0160] Cells were imaged in the microfluidic channel using a long-distance microscope objective with a NA of 0.42 and imaged onto a digital CCD camera. The camera recorded images of RBCs at a frame rate of approximately 80 fps with a reduced field of view of approximately 35 μm × approximately 35 μm.

[0161] Use a tube to connect the inlet of the microfluidic channel to a syringe pump. Control the flow rate in the channel by controlling the rate of the pump. Use another tube to connect the outlet of the channel to a container to collect waste.

[0162] Cell sample preparation

[0163] About 5 mL of rabbit blood samples were obtained from the Animal and Plant Care Laboratory (APCF) of the Hong Kong University of Science and Technology. About 10% heparin was added to the blood samples for anticoagulation. The blood was diluted in about 0.6x phosphate buffered saline (PBS) buffer solution to osmotically swell the RBCs, and the cell concentration was estimated to be about 10 8cells / ml. Prior to the experiment, the red blood cells were incubated in a buffer solution for about 10 minutes. For chemical treatment of the cells, the RBCs were incubated in about 0.6x PBS buffer solution with a glutaraldehyde concentration of about 0.002 v / v%. Other embodiments based on this protocol include studying the dependence of cell deformation and elasticity on the concentration of chemical treatment, extracting the sensitivity of cell stretching.

[0164] Image Processing

[0165] Using the Image Processing Toolbox and Computer Vision System Toolbox in Matlab, a program was compiled to process all the frame images in the video recorded by the CCD camera. Image processing mainly consists of three steps, including (i) edge detection, (ii) object segmentation and (iii) feature extraction.

[0166] In edge detection, a gradient image is calculated from the recorded top view images of the cells and an adaptive threshold is applied to create a binary mask containing the cell edge contours. In object segmentation, the cell edge contours are obtained from the binary mask and irrelevant edges are removed. In feature extraction, based on the extracted cell contours, the cell centroid position, velocity, and size are measured.

[0167] Using the extracted position of the cell in each frame, the cell trajectory can be tracked and the cell velocity can be measured as it passes through the ~35μm × ~35μm field of view. The dx, dy, and dx / dy ratios are extracted and plotted as the x position of the centroid position to show the change in cell deformation as the cell passes through the field of view.

[0168] Cell capture by optical tweezers in microfluidics

[0169] Figure 3 Figure (a) shows the extracted trajectories of multiple osmotically swollen rabbit RBCs flowing through the field of view at a flow rate of 2.5 μl / hr from a video of about 78 s. The xy position of the optical tweezers is marked with a white circle. The optical tweezers are located at x = about 17 μm and y = about 18 μm, and the estimated optical power is about 45 mW. Given the beam waist diameter of about 1.1 μm, the estimated optical intensity is about 10 6 W / cm 2 Other implementations based on this scheme include using lower optical power and reduced flow rate.

[0170] A region of interest was defined with dimensions of approximately 6 μm (y) × approximately 25 μm (x), indicated by a black box in the figure, where the incoming cells overlapped with the optical tweezers as they passed through the field of view. A total of N ≈ 190 cells passed through the region of interest. Of these, N' ≈ 90 cells were attracted and temporarily captured on the optical tweezers and subsequently escaped along the line in the direction of flow. This phenomenon is attributed to the fact that when the laser is turned on (approximately 250 milliseconds), the cells are attracted and captured by the optical gradient force, while when the laser is blocked (approximately 250 milliseconds), the cells pass through the region of interest without being captured.

[0171] For comparison, cells outside the region of interest (with no spatial overlap with the optical tweezers) were also observed to keep moving along the flow without being affected by the optical tweezers.

[0172] The velocity of cells moving through the field of view was extracted from the video. Figure 3 Figure (b) shows a scatter plot of cell velocity as a function of x position within the region of interest. The curve shows the average of the cell velocity. The gray background bar marks the x position of the optical tweezers. The total number of cells N in the region of interest is labeled, and the number of captured cells N' is labeled as (N, N').

[0173] The average entry velocity of the cells was observed to be about 120 μm / s. The optical tweezers attracted and captured the cells at x = about 19 μm, where the average cell velocity dropped to almost zero.

[0174] When x> about 19 μm, the stretched cells are released and accelerated by the flow, with the cell velocity increasing from almost zero to about 150 μm / s. It should be noted that the average velocity of the released cells after acceleration is higher than the entry velocity of about 120 μm / s. This phenomenon may be attributed to the fact that the cells are slightly lifted and captured by the optical tweezers at a plane higher than their entry plane (closer to the substrate due to gravity). When the cells are released, their flow velocity is higher in laminar flow distribution (maximum flow velocity near the center of the channel) than the flow velocity close to the substrate.

[0175] Figure 3 The middle (c) panel shows the cell velocity as a function of the area outside the region of interest (e.g. Figure 3 Figure 3. Scatter plot of the x-position of the cells in the image (a). The cell velocities are uniformly distributed with an average value of about 120 μm / s, indicating that the cell velocities outside the region of interest are not affected by the optical tweezers.

[0176] Stretching captured cells using microfluidic flows

[0177] Figure 4 Figures (a)-(e) show optical micrographs of representative cells across the region of interest. Figure 4As shown in Figure (a), before capture, the cell presents an almost symmetrical shape due to osmotic swelling. Figure 4 As shown in Figures (b) and (c), during the stretching process, the cell stretches along the flow direction (x direction) due to the fluid drag force. Figure 4 As shown in Figures (d)-(e), after the cell is released from the optical tweezers, it gradually recovers to an almost symmetrical shape. Figure 4 In the figure (a), the lengths of the cells along the flow direction (x direction) and transverse to the flow direction (y direction), namely dx and dy, are marked.

[0178] Extract as Figure 3 The dx, dy, and dx / dy ratio of cells in the region of interest indicated by the black box in (a). Figure 4 Figures (f)-(h) show scatter plots of the extracted dx, dy and dx / dy ratio as a function of the cell's x position. In each case, an average value was obtained, as shown by the curve in each figure. A gray background bar is used on the three figures to indicate the position of the optical tweezers.

[0179] Before the incoming cell reaches the optical tweezers at x = about 17 μm, the average dx and dy values ​​of the cell are both about 6.7 μm (for example, for x < 13 μm), as Figure 4 Correspondingly, the average dx / dy ratio of the input cells is about 1.0 (for x < 13 μm), as shown in Figures (f) and (g). Figure 4 These results indicate that the cell shape before the cell interacts with the optical tweezers is almost spherical (osmotic swelling) before being trapped.

[0180] The cell captured at x = about 19 μm is stretched and deformed by the microfluidic flow. The average dx increases and the average dy decreases, as shown in Figure 4 As shown in (f) and (g), the cell is stretched along the x direction and compressed along the y direction. When x = about 20 μm, dx reaches a maximum value of about 7.5 μm (Δdx is about 0.8 μm), and dy reaches a minimum value of about 6.1 μm (Δdy is about -0.6 μm). Accordingly, the dx / dy ratio reaches a maximum of about 1.2 at the maximum deformation.

[0181] After the cell was released from the optical tweezers, within a displacement of about 2 μm, the average dx recovered to about 6.8 μm and the average dy recovered to about 6.6 μm. Correspondingly, the dx / dy ratio recovered to about 1.0, indicating that the cell shape recovered to a nearly spherical shape after the optical tweezers and fluid dragging process.

[0182] The fluid force F exerted on the trapped cell was estimated to be in the pN level using a first-order approximation according to Stokes' law: F = 6πηRv, where η (~10 -3 Nm-2 ·s) is the dynamic viscosity of the PBS solution, R (about 3.4 μm) is the cell radius, and v (about 150 μm / s) is the relative velocity between the cell and the fluid. Assuming a linear spring model, considering that the change in cell dx is about 1 μm, the spring constant of RBC is estimated to be in the order of μN / m.

[0183] During the entire approximately 78 s video, N' approximately 90 cells were captured and dragged, corresponding to an average cell extension flux of approximately 1.2 cells / s.

[0184] For comparison, we extracted the regions outside the region of interest (e.g. Figure 3 dx, dy and dx / dy ratio of the cell (shown in the white box in (a)). Figure 4 Panels (i)-(k) show the dx, dy, and dx / dy ratio (N is about 170) of the extracted cells as a function of the cell's x position. Figure 4 Figures (i)-(j) show that dx and dy are consistent along the x position, with an average value of about 6.7 μm. Figure 4 The middle panel (k) shows that the dx / dy ratio is consistent along the x position, with an average value of about 1.0, indicating that there is no obvious deformation of the cell that does not interact with the optical tweezers.

[0185] Differentiate between healthy (untreated) and unhealthy (chemically treated) cells

[0186] To further demonstrate the applicability of optical tweezers and fluid-towed cell stretching, osmotic swelling of red blood cells was measured with and without chemical treatments that altered cell stiffness. For chemical treatment of cells, red blood cells were treated with glutaraldehyde at a concentration of approximately 0.002% v / v. Glutaraldehyde is known to crosslink cell proteins, thereby increasing cell stiffness.

[0187] Erythrocytes treated with and without glutaraldehyde were characterized at various flow rates from about 3.0 μl / hour to about 1.0 μl / hour. For each condition tested, the dx / dy ratio of cells in the region of interest was extracted. Figure 5 Figures (a)-(j) show a comparison of the dx / dy ratios of untreated and chemically treated RBCs at different flow rates. As shown in the black curve, the average value of the dx / dy ratio is calculated from multiple cells. The standard error is represented by the gray shading around the black curve. For each figure, the total number of cells N and the number of cells captured N' are marked as (N, N').

[0188] The average dx / dy ratio entering the cell was approximately 1.0 under all conditions tested, indicating that flow rate or chemical treatment had no significant effect on cell shape before reaching the optical tweezers. Figure 5Except for the middle panel (j), the average dx / dy ratio increases when the cell stretches. After the cell is released from the optical tweezers, the average dx / dy ratio recovers to about 1.0 in all conditions tested, indicating that the cell shape recovers to a nearly spherical shape.

[0189] Figure 6 The maximum dx / dy ratios for extracted untreated and chemically treated RBCs are shown. The maximum dx / dy ratio increases with flow rate for both untreated and chemically treated cells. This phenomenon can be attributed to the fact that the captured cells are subject to greater stretching forces when flowing at high speeds. At all flow rates, the chemically treated cells showed less deformation than the untreated cells, which is consistent with cell stiffening due to the chemical treatment. Other embodiments based on the embodiments include testing the dependence of cell deformation on chemical treatment concentration to establish the sensitivity of stretching to chemical treatment.

[0190] Other embodiments of the present application are described below.

[0191] Figure 7 Another embodiment of the present application is schematically shown, which uses two optical tweezers for two parallel optical tweezers and fluid dragging processes. The two optical tweezers are arranged at a sufficiently large interval in a direction transverse to the flow direction so that they can independently and simultaneously perform optical tweezers on two cells or particles, and realize two independent optical tweezers and fluid dragging processes in the same microfluidic flow. Compared with the above embodiment, this embodiment can achieve 2 times the characterization throughput.

[0192] Figure 8 Schematic illustration of a method to generate two optical tweezers in a microfluidic channel, using two separate laser sources whose output powers or wavelengths are not necessarily the same. After simultaneously focusing the two laser beams using the same high NA microscope objective, two optical tweezers can be generated within the microfluidic channel.

[0193] Fig. 9 Another method to generate two optical tweezers in a microfluidic channel is schematically shown, where a single laser source is employed. Using an interferometer setup including two beam splitters and two mirrors, the input laser beam is split into two parallel propagating displaced laser beams. After simultaneously focusing the two laser beams using the same high NA microscope objective, two optical tweezers can also be generated within a microfluidic channel.

[0194] Fig.10Figures (a) and (b) in the figure schematically show another embodiment of the present application, which uses one-dimensional and two-dimensional optical tweezers arrays for multiple parallel optical tweezers and fluid dragging processes. The optical tweezers array includes multiple optical tweezers, which can be arranged into a one-dimensional 1×N grating, a two-dimensional N×M rectangular grating, a two-dimensional hexagonal grating, etc. When multiple cells or particles enter the array area, the arrayed optical tweezers in the microfluidics can simultaneously and independently implement the optical tweezers and fluid dragging processes, provided that the spacing between the optical tweezers should be large enough. Compared with the above embodiment, this embodiment can significantly improve the characterization throughput, depending on the number of optical tweezers in the array.

[0195] Fig.11 Schematic illustration of a method for generating a one-dimensional optical tweezers array in a microfluidic channel, where a single laser source is employed. An interferometer setup comprising two beam splitters and two mirrors is used to split the input laser beam into two spatially overlapping, parallel propagating laser beams. After simultaneously focusing the two laser beams using the same high-NA microscope objective and interfering inside the microfluidic channel, a one-dimensional optical tweezers array can be generated inside the microfluidic channel. By controlling the phase difference and spatial overlap between the two beams, the optical interference pattern can be fine-tuned.

[0196] Fig.12 A method for generating one-dimensional and two-dimensional optical tweezers arrays in a microfluidic channel is schematically shown, where a single laser source is employed. Using a passive multimode interference (MMI) waveguide, the input Gaussian beam is shaped into an output multi-beam array based on the multimode interference inside the waveguide. A variety of optical lattices can be generated using MMI waveguides by adopting different cross-sectional shapes as well as the size and length of the waveguide. The inset schematically shows a 3×3 optical lattice generated from the waveguide output end face as an example. A microscope objective is used for input and output coupling with the MMI waveguide. By controlling the input coupling conditions, the optical lattice pattern can be fine-tuned. The resulting optical lattice beam is focused by a high NA microscope objective into the microfluidic channel to form an optical tweezers array.

[0197] Fig.13 Schematic illustration of another approach to generate one-dimensional and two-dimensional optical tweezers arrays in microfluidic channels, where a single laser source is employed. A computer-controlled spatial light modulator (SLM) is used to shape the expanded single beam into an optical tweezers lattice (called holographic optical tweezers). The lattice can be one-dimensional or two-dimensional and can be actively reconfigured. The beam is focused by a high-NA microscope objective into a microfluidic channel to form an optical tweezers array.

[0198] Finally, it should be understood that, although the various aspects of this specification describe specific embodiments, it is easy for those skilled in the art to understand that these disclosed embodiments are only illustrations of the principles of the subject matter disclosed herein. Therefore, it should be understood that, unless explicitly stated, the disclosed subject matter is not limited to the specific combinations, methods and / or formulations described herein, etc. In addition, it will be appreciated by those of ordinary skill in the art that, without departing from the spirit of this specification, certain changes, modifications, substitutions, alterations, increases, decreases and sub-combinations can be made according to the teachings of this article. Therefore, the claims attached to this application are intended to be interpreted as including all these changes, modifications, substitutions, alterations, increases, decreases and sub-combinations within their true spirit and scope.

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Claims

1. A method for determining the mechanical properties of biological cells or biological cell-like particles, comprising the following steps: Passing a fluid containing the biological cells or biological cell-like particles through a microfluidic channel in a first direction; During the process of the fluid passing through the microfluidic channel, at least a portion of the biological cells or biological cell-like particles are captured by optical tweezers in a second direction transverse to the first direction, and the captured cells or particles are stretched and deformed by fluid force; Acquiring an image of the biological cell or biological cell-like particle passing through the microfluidic channel in a third direction transverse to the first direction; as well as determining the mechanical properties of the biological cells or biological cell-like particles based on the deformation amount of the biological cells or biological cell-like particles captured by the optical tweezers in the image, The optical refractive index of the medium of the fluid is lower than that of the biological cells or biological cell-like particles.

2. The method of claim 1, wherein the cross-section of the microfluidic channel is large enough so that the biological cells or biological cell-like particles do not physically contact the wall of the microfluidic channel to generate deformation force when passing through the microfluidic channel.

3. The method of claim 1, wherein the optical tweezers is a highly focused laser beam with a Gaussian distribution.

4. The method of claim 1, wherein the spectral window of the optical tweezers is transparent to one or more of: The biological cell or biological cell-like particle, the fluid medium, a wall of the microfluidic channel.

5. The method of claim 1, wherein the optical tweezers are periodically modulated to periodically capture and release the biological cells or biological cell-like particles. The method according to claim 5 , wherein the periodic modulation is to periodically activate and deactivate the optical tweezers. The method of claim 5 , wherein the modulation is performed by a modulator. The method of claim 5 , wherein the frequency of the modulation is at a Hertz level.

9. The method of claim 8, wherein the frequency of the modulation is 1-5 Hz.

10. The method of any one of claims 1 to 9, wherein a plurality of optical tweezers are used.

11. The method of claim 10, wherein the plurality of optical tweezers are generated by an interferometer configured with a single laser source.

12. The method of claim 10, wherein the plurality of optical tweezers are generated by a plurality of laser sources. The method of claim 10 , wherein the plurality of optical tweezers are arranged into an optical tweezers array.

14. The method of claim 13, wherein the optical tweezers array is a one-dimensional optical tweezers array or a two-dimensional optical tweezers array.

15. The method of claim 13, wherein the optical tweezers array is generated using a multi-mode interference waveguide with a single laser source.

16. The method of claim 13, wherein the optical tweezers array is generated using a spatial light modulator with a single laser source.

17. The method of any one of claims 1-9, wherein the focus of the optical tweezers is set so that the cells captured by the optical tweezers are away from the wall of the microfluidic channel.

18. The method of any one of claims 1-9, wherein the image is acquired using a microscope objective and a digital camera.

19. The method of any one of claims 1 to 9, wherein the image is acquired using a long working distance microscope objective and a CCD digital camera.

20. The method according to any one of claims 1 to 9, wherein images of the biological cell or biological cell-like particle are collected before, during and after being captured by the optical tweezers.

21. The method according to any one of claims 1 to 9, wherein the dimensions of the biological cells or biological cell-like particles captured by the optical tweezers in the image along the first direction and perpendicular to the first direction are measured, and the ratio of the two is used to characterize the deformation amount.

22. The method of claim 21, wherein the dimension is path length.

23. A system for determining mechanical properties of biological cells or biological cell-like particles, comprising: A microfluidic device comprising a microfluidic channel for passing a fluid containing the biological cells or biological cell-like particles in a first direction; An optical tweezers providing device is used to capture at least a portion of the biological cells or biological cell-like particles in a second direction transverse to the first direction by using optical tweezers during the process of the fluid passing through the microfluidic channel, and to stretch and deform the captured cells or particles by using fluid force; An image acquisition device, used to acquire an image of the biological cell or biological cell-like particle passing through the microfluidic channel in a third direction transverse to the first direction; as well as an analysis device for determining the mechanical properties of the biological cell or biological cell-like particle based on the deformation amount of the biological cell or biological cell-like particle captured by the optical tweezers in the image, The optical refractive index of the medium of the fluid is lower than that of the biological cells or biological cell-like particles.

24. The system of claim 23, wherein the cross-section of the microfluidic channel is large enough so that the biological cells or biological cell-like particles do not physically contact the wall of the microfluidic channel to generate deformation force when passing through the microfluidic channel.

25. The system of claim 23, wherein the optical tweezers is a highly focused laser beam having a Gaussian distribution.

26. The system of claim 23, wherein the spectral window of the optical tweezers is transparent to one or more of: The biological cell or biological cell-like particle, the fluid medium, a wall of the microfluidic channel.

27. The system of claim 23, wherein the optical tweezers providing device further comprises a component for periodically modulating the optical tweezers so as to periodically capture and release the biological cells or biological cell-like particles.

28. The system of claim 27, wherein the periodically modulated component is used to periodically activate and deactivate the optical tweezers.

29. The system of claim 27, wherein the component that periodically modulates the optical tweezers is a modulator.

30. The system of claim 29, wherein the frequency of the modulation is at the Hertz level.

31. The system of claim 30, wherein the frequency of the modulation is 1-5 Hz.

32. The system of any one of claims 23-31, wherein the optical tweezers providing device is capable of using a plurality of optical tweezers.

33. The system of claim 32, wherein the plurality of optical tweezers are generated by an interferometer configured with a single laser source.

34. The system of claim 32, wherein the plurality of optical tweezers are generated by a plurality of laser sources.

35. The system of claim 32, wherein the plurality of optical tweezers are arranged into an optical tweezers array.

36. The system of claim 35, wherein the optical tweezers array is a one-dimensional optical tweezers array or a two-dimensional optical tweezers array.

37. The system of claim 35, wherein the optical tweezers array is generated using a multimode interference waveguide with a single laser source.

38. The system of claim 35, wherein the optical tweezers array is generated using a spatial light modulator with a single laser source.

39. The system of any one of claims 23-31, wherein the focus of the optical tweezers is set so that the cells captured by the optical tweezers are away from the wall of the microfluidic channel.

40. The system of any one of claims 23-31, wherein the image acquisition device comprises a microscope objective and a digital camera.

41. The system of any one of claims 23-31, wherein the image acquisition device comprises a long working distance microscope objective and a CCD digital camera.

42. The system of any one of claims 23-31, wherein the image acquisition is configured to acquire images of the biological cell or biological cell-like particle before, during, and after being captured by the optical tweezers.

43. A system as described in any one of claims 23-31, wherein the analysis device is configured to measure the dimensions of the biological cells or biological cell-like particles captured by the optical tweezers in the image along the first direction and perpendicular to the first direction, and use the ratio of the two to characterize the deformation amount.

44. The system of claim 43, wherein the dimension is path length.

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