System and method for high-throughput single-stream particle focusing that is insensitive to particle size

Abstract

A tunable inertial-sheathing (TIS) system and method for particle-size-insensitive high-throughput single-stream focusing of particles suspended in a particle-carrying fluid is provided. The TIS regulates the local distribution of particles within one of the compartments of the inertial force field, followed by inertial focusing to migrate them to a single focal point. For particle positioning, the TIS system generates and accumulates a sheath fluid from the particle-carrying fluid by a combination of inertial focusing, channel bifurcation, and channel convergence, thereby introducing an arbitrary form of peripheral sheath. A variety of forms of TIS systems are also provided, each including a main channel and at least one bypass channel. The main channel includes and cascades at least three segments, at least one bifurcation junction, and at least one convergence junction.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 266543, filed January 7, 2022, the entire contents of which, including any forms, figures or drawings, are incorporated herein by reference. Background Technology

[0003] Focusing particles in a high-speed fluid flow into a single stream provides high precision, efficiency, and throughput for particle analysis, classification, counting, and filtration. High-throughput single-stream focusing is crucial for a wide range of applications, including flow cytometry and single-cell sorting. Traditional methods in this field employ external force fields, such as electromagnetic or acoustic fields, or sheath fluid / particle-free fluids used for hydrodynamic focusing, to actively confine the position of particles.

[0004] However, due to the need for precise control of the force field and fluid flow rate, these conventional systems designed to achieve optimal focusing performance are typically very expensive to manufacture. Furthermore, when using hydrodynamic focusing mechanisms, the large amount of sheath fluid significantly dilutes the particle concentration, making it difficult to scale up throughput for further downstream particle analysis.

[0005] Inertial focusing is a proven focusing method that bypasses expensive force field control equipment and sheath fluid. It aligns particles in a way that relies solely on high-speed fluid flow in microchannels to induce a converging force field (called an inertial force field), promising for reduced manufacturing and operating costs. While passive, high-throughput, and highly precise, its drawback is the lack of naturally formed single-flow. Naturally partitioned inertial force fields (one focus per partition) arrange uniformly distributed particles into multiple particle streams.

[0006] Therefore, achieving single-flow inertial focusing requires reducing the number of particle streams. State-of-the-art methods can be categorized into single-field (Category 1) and multi-field (Category 2), with the former further subdivided into single-stage (Category 11) and multi-stage (Category 12). Single-stage single-field and multi-field methods employ a single-focus force field to align particles into a single flow at once. Conversely, multi-stage single-field methods employ a series of multi-focus inertial force fields to gradually align particles from multiple flows into a single flow. In other words, these methods rely on the use of multiple force fields, but with different implementation methods: parallel (Categories 12 and 2) and sequential (Category 12). Notable methods within each category include: (Category 1) straight tubes with non-rectangular cross-sections, (Category 12) straight tubes with variable cross-sections, (Category 2) bent tubes with rectangular cross-sections, (Category 2) straight rectangular tubes with periodically varying aspect ratios, and (Category 2) straight rectangular tubes for non-Newtonian fluids.

[0007] These different methods all share a key drawback: a trade-off between throughput (e.g., flow rate) and particle size tolerance. This is understandable from the limited coverage of effective flow rates and particle sizes for each force field. The use of multiple force fields leads to a narrowing of effective coverage. For example, high-throughput methods effectively focus only monodisperse particles (e.g., size variations less than 10 μm), while high-tolerance methods typically have a maximum throughput of less than 10,000 particles per second. These are more than 3 times and 10 times lower, respectively, than commercially available hydrodynamic focusing quantities. This is insufficient for real-world scenarios involving large-scale polydisperse particles (i.e., particles with large size variations), such as flow cytometry, where typical biological cell sizes span 5 to 30 micrometers or larger, with concentrations >1e5 per mL of sample. Furthermore, achieving high practicality for existing single-flow inertial focusing methods is challenging due to design trade-offs between various factors such as accuracy, flow rate tolerance, and fabrication friendliness. Summary of the Invention

[0008] There is still a need in this field for improved designs and techniques for passive, cost-effective, and high-performance single-stream particle focusing systems and methods for particle analysis and filtration at the microscale.

[0009] Embodiments of the present invention relate to particle focusing systems and methods for high-throughput single-flow inertial focusing that are insensitive to particle size, such as arranging polydisperse particles into a single flow without requiring multiple internal force fields or any external force fields or external sheath fluid. The method of the present invention is based on positioning the particle distribution across the channel cross-section to accommodate the compartmentalized nature of the inertial force field. In the absence of an external force field, a particle distribution positioned within only one compartment of the inertial force field results in the formation of a single flow. This method avoids perturbing the inertial force field, thereby maximizing the effective coverage of fluid velocity and particle size to meet practical needs. Particle positioning is achieved in an adjustable inertial sheath (TIS) system, which can utilize inertial forces to generate an arbitrary pattern of peripheral sheath. In other words, TIS, like conventional fluid dynamics focusing, condenses particles into a narrower flow with arbitrary shape and position across the channel cross-section while using inertial forces instead of a sheath fluid. Uniquely, it accumulates inertial wall effects, transforming a physically defined sheath into an arbitrary sheath. This accumulation involves two repetitive processes: (1) inducing an inertial wall effect to the outer sheath particles, and (2) physically separating the periphery of the channel to isolate the sheath fluid from the particles, while simultaneously inducing a further wall effect to the particles. In physics terms, this repetition continuously converts the work done by the abstract wall effect into potential energy in the actual form of sheath fluid. After sufficient accumulation, removing all septa releases the accumulated wall effect (sheath fluid), which instantaneously positions the particle distribution across the channel cross-section.

[0010] According to an embodiment of the present invention, a TIS system for particle localization is provided, comprising a main channel and at least one bypass channel for accumulating and consuming inertial wall effects (equivalent to sheath fluid in the case of TIS). All bypass channels transport sheath fluid and each includes at least one inlet and at least one outlet. The main channel transports the fluid carrying the particles and includes at least three straight sections; at least one bifurcation contact; and at least one confluence contact. The straight sections are connected by bifurcation and confluence contacts disposed therebetween, respectively connecting to the inlet and outlet of the bypass channel. From the sheath perspective, the TIS system can be decomposed into four types of functional blocks. In the initial long straight section, it initiates inertial focusing on the particles in the outer sheath, serving as the initiation unit (block A). The end is attached to the bifurcation contact of the long straight section, wherein the bifurcation contact diverts some of the previously generated sheath fluid to the bypass channel and has a sufficiently long straight section to recover the sheath fluid, thereby constituting an accumulation unit (block B). A short, straight confluence junction is attached at the end, where some sheath fluid is returned to the main channel to temporarily position the particles within the straight section, forming a consumption unit (block C). A bypass channel in which only sheath fluid flows serves as a storage unit (block D).

[0011] The main channel of a TIS system includes a block A at the beginning, at least one block C downstream, and at least one block B in between to achieve particle localization. The tunability of the TIS system can be enhanced by using a larger number of blocks B and C to form a complex structure, which can be characterized by arranging blocks B and C into three categories: staggered (i.e., BCBC…BCBC), blocky (i.e., BB…BBCC…CC), and partial (i.e., a mixture of staggered and blocky). Note that the above structure only allows TIS in one direction (unidirectional TIS). A TIS system capable of achieving arbitrary particle localization across the channel cross-section would include at least four unidirectional TIS systems to provide sufficient degrees of freedom in two dimensions.

[0012] In some embodiments of the invention, the unidirectional TIS system includes a microchannel comprising a main channel having a high aspect ratio rectangular cross-section for single-stream focusing in a planar channel design. The main channel, i.e., a high aspect ratio rectangular tube, is formed for continuous inertial focusing, and its inertial force field is configured to integrate the cross-section of the channel into two horizontally parallel compartments. The unidirectional TIS system first focuses uniformly distributed particles into the focal point of the two compartments by inertial force, where the focal point is the equilibrium point of the inertial force field, located away from the long wall of the channel and at its center, to form two particle streams. It then gradually increases the inertial sheath on one side, placing the two streams into the same compartment, which allows them to be merged into a single stream by separate inertial focusing.

[0013] In a particular embodiment of the invention, a unidirectional TIS system tailored to simplify the channel design process includes an interleaved configuration comprising block A and four pairs of blocks B and C, each pair connected to block D with gradually decreasing hydraulic resistance. In another embodiment of the invention, a unidirectional TIS system tailored for biological applications includes a segmented configuration comprising block A, six blocks B and C, all connected to the same block D. In these embodiments, a bifurcation junction is first formed, configured to divert sheath fluid from the main channel to a bypass channel. It physically separates the channel cross-section such that a thin sheath fluid slip between one of the two particle streams and its nearest channel wall is precisely isolated from the main channel. In particular, this fluid slip, generated by the inertial wall effect, is non-adjustable, and its thickness depends on the Reynolds number, particle size, and channel geometry. This separation results in sheath fluid in the branch; the main channel has two particle streams, one of which moves relative to the wall. A segment of the main channel follows and is configured with a similar inertial force field to refocus the two particle streams to a focal point, thereby generating additional particle-free slip. In the staggered embodiment, a confluence point is then formed and configured to sheath the particle-carrying fluid on one side, temporarily positioning the two particle streams in a smaller compartment biased to one side. This process is then repeated more than three times, gradually increasing the amount of sheath fluid extracted. Eventually, the amount of sheath fluid in the bypass channel equals or greater than the amount of particle-carrying fluid in the main channel (equivalent to occupying one compartment of the inertial field), positioning the two particle streams in one compartment of the inertial field. In the segmented embodiment, the extraction process is repeated more than five times to complete sheath accumulation before the sheath fluid returns to the main channel. Furthermore, all bifurcation points are associated with orifices at the bifurcation points to avoid direct contact with the particles, thereby reducing the impact on the particles and improving their survivability. After positioning, a long straight channel, identical or similar to the previous straight section, is formed to continue inertial focusing, merging the two positioned particle streams into a single stream. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of a microfluidic chip according to an embodiment of the present invention, wherein the annotations define the geometric terms used herein.

[0015] Figure 2A This is a schematic diagram illustrating the compartmentalized nature of an inertial force field. An inertial force field consists of many repulsive regions connected by lines. These lines prevent particles from crossing and traverse the entire field, acting as imaginary dividers that segment the force field and create compartmentalized structures. Due to this property, particles migrate only to the focus corresponding to the compartment where they initially resided.

[0016] Figure 2BThis is a schematic diagram illustrating the reduction of the particle flow achieved by a localized particle distribution at the start of inertial focusing. According to an embodiment of the invention, single-flow focusing can be achieved by inertial focusing, which has a localized particle distribution confined within a compartment of an inertial force field. It differs from traditional inertial focusing, which has a uniform particle distribution in the quantity of the synthesized particle flow.

[0017] Figure 3A A schematic diagram is shown of a passive sheath with a square-shaped channel cross-section, caused by the lift generated by the wall during inertial focusing.

[0018] Figure 3B A schematic diagram of the peripheral sheath through inertial focusing is shown in cross-sections of channels of different shapes. The peripheral sheath is given for all cross-sections of the channel, regardless of the exact mode of its inertial force field.

[0019] Figure 3C This is a cross-sectional diagram illustrating the principle of the Adjustable Inertial Sheath (TIS) process. The TIS focuses the particle distribution at the channel boundary, and the lift generated by the inertial focusing, particularly its wall effect, repels the particles from this boundary. This repulsion is equivalent to passively enveloping the particle-carrying fluid. The channel then branches to form a physical separation, diverting this particle-free fluid to a bypass channel. It simultaneously stores the wall effect as sheath fluid and resets the particle distribution in the original channel. This is followed by another inertial focusing process to generate additional wall effects on the particles in the original channel, thus creating another sheath. Finally, the channels merge, connecting the two separate channels to form a thickened sheath, resulting in a more localized particle distribution away from the boundary. The repetition of these steps achieves a passive and adjustable sheath.

[0020] Figure 4A This is a schematic diagram illustrating the principle of arbitrary particle positioning through the TIS process. A complete TIS system comprises at least four unidirectional TIS systems, each of which compresses the particle distribution in one direction by adjusting the peripheral inertial sheath to provide sufficient degrees of freedom to position the particles to a distribution of arbitrary size and position on the channel cross-section. In each unidirectional TIS, it includes at least one of the following tandemly connected sequences of bifurcation (bi), inertial focusing (In), and confluence (Con) to form an interlaced structure for accumulating sufficient sheath fluid.

[0021] Figure 4B This is a schematic diagram of an alternative design for a unidirectional TIS system, including a series of bifurcations (bi) and inertial focusing (In) upstream, and a confluence (Con) downstream, to form a block-like structure.

[0022] Figure 5This is a schematic diagram of single-stream focusing via TIS in rectangular cross-sections with different aspect ratios. The extreme aspect ratios reduce the complexity of partitioning the inertial force field from 2D to 1D, thereby reducing the complexity of the TIS system used for single-stream focusing.

[0023] Figure 6A This is a schematic diagram of microfluidic pattern design for a TIS system according to an embodiment of the present invention. The image titled "TIS System" shows a 3D view and a top view of the TIS system, which includes a main channel and a bypass channel. The main channel, with a high aspect ratio rectangular cross-section, includes a bifurcation joint and a confluence joint connected by three straight segments sandwiched in the middle. The bypass channel is composed of segments of arbitrary shapes. The system can be decomposed into four building blocks (i.e., blocks A, B, C, and D) for different sheath controls, as detailed on the right side of the top view.

[0024] Figure 6B This is a top view illustrating the particle trajectory in the pressure-driven flow within the TIS system. Cross-sectional views at six representative locations show the corresponding cross-sectional migration. According to an embodiment of the invention, particles initially migrate into two flows and are then deflected by the TIS into one of the two compartments. Under this localized particle distribution, subsequent inertial focusing results in a single flow.

[0025] Figure 6C A schematic representation of the equivalent circuit model of a TIS system in its basic form is shown, and the changes in velocity flow distribution before and after development and their relationship with width and volumetric flow velocities of fluids containing particles and those without particles are illustrated.

[0026] Figure 7A A schematic diagram is shown of the inertial focusing effect of particles of two different sizes in a channel with a high aspect ratio rectangular cross section in an embodiment of the present invention, and its influence on single-flow formation using different TIS methods.

[0027] Figures 7B-7C These are schematic diagrams of different embodiments of the TIS system according to embodiments of the present invention, wherein... Figure 7B This is a schematic diagram of a TIS system with an interleaved configuration. Figure 7C This is a schematic diagram of a TIS system with a modular form. For each embodiment, its geometry, exploded view showing the component blocks, and equivalent circuit model of the bifurcation structure for analysis are given according to embodiments of the present invention.

[0028] Figure 8A simulation of the extraction process through the peripheral sheath of the TIS system is shown. The results are illustrated by a series of streamline diagrams, where the fluid portion designed to be diverted to the bypass is shown in orange. By varying the width of the bypass channel, the volume of fluid diverted to the bypass is shown as being controlled by the hydraulic resistance ratio between the bypass and the bridge. Furthermore, according to an embodiment of the invention, the bifurcation only receives peripheral fluid from the bypass side – providing a clear separation effect for the channel cross-section.

[0029] Figure 9A A simulation of particle flow trajectories in a TIS system with a basic structure is shown. According to an embodiment of the invention, the bottom particle flow shows an instantaneous offset after channel convergence, which supports the particle localization provided by the TIS system.

[0030] Figure 9B A scaled schematic diagram of a TIS system with a basic structure according to an embodiment of the present invention is shown, wherein a magnified window shows the shapes of the branching and merging points of the TIS system, indicated by gray boxes.

[0031] Figure 9C Two pairs of fluorescent stripe images according to an embodiment of the present invention are shown, respectively illustrating the flow trajectories of solutions carrying fluorescent polystyrene beads with a diameter of 8 μm and solutions carrying fluorescent polystyrene beads with a diameter of 20 μm through the bifurcation and confluence junctions of a TIS system having a basic form, showing the clear flow movement after the confluence junction.

[0032] Figure 9D Ultrafast laser scanning images of a rapidly flowing biological cell stream according to an embodiment of the present invention are shown (1) before reaching the bifurcation junction, (2) after passing through the bifurcation junction, and (3) after passing through the confluence junction, wherein red arrows indicate the gaps between the biological cell stream and the adjacent channel walls, and yellow dashed lines indicate the center of the main channel.

[0033] Figure 10A The image shows fluorescent stripe patterns illustrating differences in particle flow trajectories within a TIS system comprising bifurcation junctions with and without expansion wells. The images demonstrate that the expansion wells effectively prevent particle collisions, which do not affect post-merging particle localization, but may cause cell deformation or damage. Figure 10B The illustration shows cell images captured by ultrafast laser scanning microscopy before and after particle collision according to an embodiment of the invention, wherein triangular cells (distinctly deformed cells indicated by red arrows) are only found after particle collision.

[0034] Figure 11AA simulation of particle flow trajectories in a TIS system with an interleaved configuration is shown. According to an embodiment of the invention, the bottom particle flow exhibits a time offset with an increasing magnitude after each channel convergence, which supports the asymptotic accumulation of an inertial sheath that positions the particles into one of the inertial focusing compartments and results in single-flow focusing.

[0035] Figure 11B This is a scaled schematic diagram of a microfluidic pattern design for a TIS system with an interlaced form for particle positioning according to an embodiment of the present invention, wherein enlarged windows show the bifurcation and merging points indicated by gray boxes. Figure 11C Seven pairs of fluorescent stripe images are shown, illustrating the flow trajectories of 8 μm and 20 μm fluorescent polystyrene beads at all junctions.

[0036] Figure 12A The diagram illustrates the co-current flow of water and ink in a TIS system with a block structure. According to an embodiment of the invention, by increasing the water-to-ink ratio, the flow pattern shows the bypass channel gradually drawing in peripheral fluid from the main channel and returning all stored fluid to the main channel.

[0037] Figure 12B This is a scaled schematic diagram of a microfluidic pattern design for a TIS system with a segmented form for particle positioning according to an embodiment of the present invention. Figure 12C Four fluorescent stripe images are shown, illustrating the flow trajectory of 6 μm fluorescent polystyrene beads through the entire TIS system and the subsequent long straight section.

[0038] Figure 13 The illustration shows laser scan images of a test evaluating the performance of TIS-assisted inertial focusing using a TIS system when microparticles and cultured cells flow through a microfluidic channel at three different flow rates, according to an embodiment of the present invention. The laser scan images are captured before (equivalent to inertial focusing alone) and after the TIS system to evaluate particle size-insensitive single-flow focusing performance.

[0039] Figure 14A This illustration shows a compressed image containing 10,000 cells captured in one second, according to an embodiment of the present invention. Figure 14B It shows Figure 14A Images of the first 120 cells were used to demonstrate the performance of TIS-assisted inertial focusing on heterogeneous cell samples at high throughput. Detailed Implementation

[0040] Embodiments of the present invention relate to a high-throughput single-flow inertial sheath (TIS) system and method for particle size insensitive inertial focusing of particles suspended in a fluid carrying particles.

[0041] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. As used herein, the singular forms “a,” “an,” and “the” are intended to include both the plural and singular forms unless the context clearly indicates otherwise. It will also be understood that when the terms “comprising” and / or “including” are used in this specification, they specify the presence of the stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof.

[0042] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms, such as those defined in common dictionaries, shall be interpreted as having meanings consistent with their meanings in the relevant field and in the context of this disclosure, and shall not be interpreted as having idealized or overly formal meanings unless expressly defined herein.

[0043] When the term “about” is used in conjunction with a numerical value in this document, it should be understood that the value can be in the range of 90% to 110% of that value, that is, the value can be + / - 10% of the stated value. For example, “about 1 kg” means from 0.90 kg to 1.1 kg.

[0044] In describing this invention, it should be understood that numerous techniques and steps are disclosed. Each of these techniques has its own advantages, and each can be used in combination with one or more other disclosed techniques, or in certain cases, with all other disclosed techniques. Therefore, for clarity, this description will avoid unnecessarily repeating every possible combination of the individual steps. However, the specification and claims should be understood to mean that such combinations are fully within the scope of this invention and the claims.

[0045] Working principle of particle positioning for size-insensitive single-stream inertial focusing

[0046] The various embodiments of the TIS systems and methods described below are based on the concept of microfluidic channel design, which enables the focusing of particles with a wide size distribution into a high-speed single flow, such as a Newtonian microfluidic flow. The TIS systems and methods enable high-precision, high-efficiency, and high-throughput particle detection, analysis, and filtration at the microscale. Therefore, the systems and methods of this invention are applicable to various fields of microfluidics, analytical chemistry, cell biology, clinical diagnostics, healthcare, marine, and life science research.

[0047] The TIS system and method of this invention are designed as inertial focusing to assist single-flow particle focusing. Note that the term "particle focusing" as used herein refers to confining the position of particles in the channel cross-section by means of lateral streamline migration of the particles. Terms describing microfluidic patterns, cross-sections, and other geometric terms are as follows: Figure 1 As shown. In the inertial focusing technology solution, particle focusing is introduced by two reaction-driven inertial lift forces: lift caused by the shear gradient and lift caused by the wall, which act on and away from the channel wall, respectively. Reference Figure 2A This force creates an inertial force field across the cross-section of the channel, naturally forming multiple compartments, each with a repulsive boundary and a focus. The repulsive boundary prevents particles from passing through, while the net force at the focus is zero. When the Reynolds number of the flow is sufficiently high, the travel distance is sufficiently long, and the particle size is similar to the hydraulic diameter of the channel cross-section, the particle automatically migrates to the focus corresponding to the compartment on the cross-section during its downward flow. Reynolds number (Re) and hydraulic diameter (D) h () is defined by the following formula:

[0048] and

[0049] in,

[0050] ρ = liquid density

[0051] U m =Maximum flow rate

[0052] μ = dynamic viscosity of the fluid

[0053] D h =Channel hydraulic diameter=4A / P

[0054] A = Area of ​​the channel cross-section

[0055] P = Circumference of the channel cross-section

[0056] In some embodiments of the invention, Re is between 1 and 2000, and D... h Between 100nm and 100mm, it is used to achieve inertial focusing. (Reference) Figure 2B Due to the separating nature of the inertial force field, the number of particle streams formed is also controlled by the particle distribution in the channel cross-section. Under normal conditions with uniform particle distribution, single inertial focusing will form multiple particle streams. Conversely, a single stream can be obtained entirely through inertial focusing, where the particle distribution is located within a single compartment—this is the central idea of ​​the present invention.

[0057] The advantage of single-stream focusing via particle localization is the expansion of the effective particle size, which meets the needs of applications involving particles with a wide size distribution, such as particle focusing for flow cytometry and particle filtration. Compared to existing single-stream inertial focusing methods, this method can be performed without significant force field modification to bypass its associated size dispersion effects. A significant example is the use of Dean flow, which introduces an additional Dean force (F). D This force has a second-order correlation with particle size, such as the force ratio (R). f As shown in the figure.

[0058]

[0059] in,

[0060] F L =Inertial lift

[0061] F D =Deanli

[0062] R = radius of curvature

[0063] a = Particle size

[0064] D h =Hydraulic diameter

[0065] This force naturally distributes particles of different sizes across the channel cross-section, generating multiple particle streams, each formed by particles of a specific size. In other words, the definition of single-stream focusing in existing methods only applies to certain particles of a specific size. This phenomenon inevitably limits traditional size-based particle separation methods and deviates them from single-stream particle focusing. Therefore, avoiding perturbed inertial force field particle localization is key to expanding the effective coverage of fluid velocity and particle size in single-stream inertial focusing.

[0066] Systems and methods for adjustable inertial sheaths (TIS) for particle positioning

[0067] The TIS system and method of this invention achieve arbitrary particle positioning on the channel cross-section by designing an outer sheath caused by the inertial wall effect, without any change in the force field, which effectively forms an envelope for the particle distribution. (Reference) Figure 3A One characteristic particle migration caused by inertial focusing is wall-free motion, which is equivalent to a wall-induced biomechanically induced outer sheath. (Reference) Figure 3B The fact that all foci of the inertial force field must lie on the equilibrium line means that the concept of an outer sheath applies to any cross-sectional shape of the channel, regardless of the exact form of the inertial force. Note that the thickness of this outer sheath is not arbitrary, but is defined by Re, the flow profile, and the size of the focused particles. (Reference) Figure 3CThe TIS system designs thickness through sheath accumulation via at least one cycle of channel bifurcation, inertial focusing, and channel merging. Specifically, channel bifurcation creates a physical separation to divert some sheath fluid to a bypass channel. This action simultaneously stores a wall effect in the form of sheath fluid and resets the particle distribution in the original channel. Subsequent inertial focusing again induces a wall effect to repel particles from the original channel, thus creating additional encapsulation. Channel merging then connects the two separate channels together to form a thickened sheath. (Reference) Figure 4A The unidirectional TIS system of this invention can repeat this sheath accumulation cycle, and this flexibility allows for adjustment of the sheath thickness in one direction. Therefore, at least four unidirectional TIS systems can deliver arbitrary particle distributions. (Reference) Figure 4B A unidirectional TIS system can alternatively be formed by at least one cycle of channel bifurcation and inertial focusing, followed by a final channel convergence. Generally, the fewer the number of compartments in the inertial field, the fewer unidirectional TIS systems are required for single-compartment positioning, making it more practical. For example, reference... Figure 5 At least three unidirectional TIS systems are required to position particles into a single compartment of inertial force within a channel with a square cross-section. In practice, channels with rectangular cross-sections of high aspect ratio are used to create only two compartments in the inertial force field, which simplifies the design and fabrication of the TIS system, where the aspect ratio (AR) is defined as:

[0068]

[0069] in,

[0070] H = Channel height

[0071] W = Channel width

[0072] In some embodiments of the invention, the height H and width W are each between 1 micrometer and 10 millimeters, and the aspect ratio AR is between 0 and 0.75 or greater than 1.33, for dividing the inertial force field into two compartments. Reference Figure 6AThe TIS system of the present invention can be implemented through a suitably designed and constructed pattern comprising a main channel and a bypass channel. The main channel has a high aspect ratio rectangular cross-section and includes at least one bifurcation joint, at least one merging joint, and at least three straight segments, while the bypass channel comprises arbitrary segments. The shape of each segment can be configured based on different design criteria. Typically, each straight segment has an extended cube for continuous inertial focusing and may have structures other than cubes for other design purposes, such as channel expansion or contraction for interparticle spacing control and slight bending for flexible design, provided that no significant secondary flow is introduced into the straight segment. The bifurcation joint has one inlet and two outlets, connecting to the two straight segments and the bypass channel, respectively. The merging joint has two inlets and one outlet, connecting to the two straight segments and one bypass channel, respectively. Both types of joints may have expansion wells attached downstream. At each joint, the angle between the two straight segments can vary between -90 degrees and 90 degrees, and the angle between the straight segment and the bypass channel at the inlet can vary between 0 degrees and 180 degrees.

[0073] The TIS system can be decomposed into four building blocks, each responsible for a different function. The first straight section of the main channel is block A, which is used to initialize inertial focusing; the bifurcation point of the main channel with a long straight section attached to one end is block B, which is used to accumulate sheath fluid in the bypass channel; the confluence point of the main channel with a straight section attached to one end is block C, which is used to deplete the sheath fluid from the bypass channel; and any section of the bypass channel is block D, which is used to store sheath fluid.

[0074] In embodiments of the present invention, the fluid channels of the TIS include silicones, such as, but not limited to, polydimethylsiloxane.

[0075] In an alternative embodiment of the invention, the fluid channels of the CPC include cyclic olefin copolymers (COC), polymethyl methacrylate (PMMA), or polycarbonate (PC).

[0076] exist Figure 6A In one embodiment shown, the simplest functional form of the TIS system, i.e. the basic form, includes a main channel assisted by a unified structure.

[0077] refer to Figure 6BIn a TIS system of basic form, the first straight section provides inertial focusing to continuously drag particles to one of two foci on the intermediate plane surrounded by the outer sheath fluid. The bifurcation junction diverts a defined portion of the sheath fluid to a bypass channel on one side, leaving a pair of deflected particle streams in the main channel. Before reaching the confluence junction, the particle streams are repositioned to the focal point by inertial focusing to reform the sheath. At the confluence junction, the sheath fluid previously stored in the bypass merges with the particle-carrying fluid in the main channel to further envelop the particles away from the dividing side, resulting in temporary particle positioning. Given sufficient downstream propagation, if the two streams are located in the same compartment, the inertial force field merges the particle streams into a single stream.

[0078] To form and maintain the particle flow on the intermediate plane through inertial focusing, the average cross-sectional area of ​​each straight segment of the TIS system is configured to have a high aspect ratio. Therefore, the width W of block A... A1 The width W of block B B1 Width W B2 and width W B3 And the width W of block C C1 Width W C2 and width W C3 Configured to meet the following conditions:

[0079]

[0080] in,

[0081] n = A1, B1, B2, B3, C1, C2 and C3

[0082] W n =Channel width of segment n

[0083] H = Channel height

[0084] AR = Aspect Ratio ≥ 1.33

[0085] In some embodiments of the invention, the height H and width W are each between 1 micrometer and 10 millimeters, and the aspect ratio AR is between 1.33 and 4, for dividing the cross-section of the channel into two horizontally parallel compartments, thereby forming a pair of flows on the intermediate plane.

[0086] In one embodiment of the invention, the height H is approximately 80 micrometers, the width W is between 20 and 60 millimeters, and the aspect ratio AR is between 1.33 and 4.

[0087] Furthermore, the theoretical range of focusable dimensions can be determined by the minimum channel width that satisfies the following condition:

[0088] 0.07H≤D p <min(W n )

[0089] in,

[0090] n = A1, B1, B2, B3, C1, C2 and C3

[0091] D p =Particle size

[0092] W n =Channel width of segment n

[0093] H = Channel height

[0094] In some embodiments of the present invention, D p Between 0.1 micrometers and 10 millimeters.

[0095] In one embodiment of the present invention, D p Between 5.6 and 30 micrometers.

[0096] In addition, the length L of block A A1 And the length L of block B B2 Configure it to meet the following conditions in order to achieve inertial focusing:

[0097]

[0098] in,

[0099] n = A1 and B2

[0100] ρ = liquid density

[0101] μ = dynamic viscosity of the fluid

[0102] D n = hydraulic diameter of segment n

[0103] U m =Maximum flow rate

[0104] a = minimum particle size

[0105] H = Channel height

[0106] W = the channel width of segment n

[0107]

[0108]

[0109] Note that the particles migrate from random positions to the focal point along the vertical (y-axis) and horizontal (x-axis) axes of the straight segment of block A, while once focused (e.g., in block B), the particles migrate primarily along the horizontal (x-axis). Therefore, the length L of block A... A1 Configured to be equal to or greater than the length L of block B B2 .

[0110] In one embodiment, the length L of block A A1 The configuration is on a scale of tens of millimeters, while the length L of block B is... B2 It is configured on a scale of a few millimeters.

[0111] On the other hand, it is desirable to minimize particle migration at the bifurcation point between blocks B and C. Therefore, the length L of block B... B1 And the length L of block C C1 and L C2 Configured to be longer than the length L of block A A1 Or the length L of block B B2 Much shorter.

[0112] In one embodiment, the length L of block B B1 And the length L of block C C1 and L C2 Each is configured to operate on a scale of hundreds of micrometers.

[0113] refer to Figure 6C The bifurcation point is configured to perform fluid bifurcation, diverting the sheath fluid to the bypass channel. At the inlet, the relationship between fluid flow rate and flow width is shown in the following equation:

[0114] Q A =W M ·H·v

[0115] Q B =W′ FB ·H·v

[0116] Q D =W FD ′·H·v

[0117] in,

[0118] Q n = The volumetric flow rate of block n, where n = A, B, and D

[0119] W n =Channel width of segment n

[0120] H = Channel height

[0121] v = linear fluid velocity

[0122] W FB = Inlet flow width of block B

[0123] W FD ′ = Inlet flow width of block D

[0124] To ensure the fluid in the bypass channel is particle-free, the width of the fully developed flow toward the branch cannot exceed the width of the particle-free fluid slice in the straight section. This is entirely determined by the fluid's Reynolds number (Re), particle size, and channel geometry. (See again...) Figure 6C Considering the development of the velocity distribution, the following conditions are obtained:

[0125]

[0126] in,

[0127] W P = Distance from particle to the nearest channel wall

[0128] W FD = Fully developed flow width of block D

[0129] W′ FD =Inlet flow width of block D

[0130] f para =Mapping function between the entry point and the fully developed flow

[0131] In some embodiments of the invention, the width W of the fully developed flow reaching block D is... FD It is configured to be between 0 and 60 micrometers.

[0132] In pressure-driven microfluidic flow, the volumetric flow rate is controlled by the following equation:

[0133]

[0134] in,

[0135] Q = Volumetric flow rate

[0136] ΔP = Pressure difference between inlet and outlet

[0137] R = hydraulic resistance

[0138] Therefore, controlling the hydraulic resistance is crucial for controlling bifurcation. For cubic channels with a high aspect ratio, the hydraulic resistance of the channel is roughly determined by the following conditions:

[0139]

[0140] in,

[0141] R H = Hydraulic

[0142] L = Channel length

[0143] W = Channel width

[0144] As a result, as shown in the equation below, by appropriately configuring the ratio of hydraulic resistance, thereby configuring the ratio of width to length between blocks B and D, sheath extraction can be achieved:

[0145]

[0146] in,

[0147] W FB = Width of the fully developed flow of block B

[0148] W FD = Width of the fully developed flow of block D

[0149] W F ′ B = Width of undeveloped flow at the exit section

[0150] W F ′ D = Width of the undeveloped flow of the branch

[0151] f para =Mapping function between undeveloped flow and fully developed flow

[0152] W n =Channel width of segment n

[0153] L n = Length of segment n

[0154] To facilitate the analysis of complex hydraulic resistance in pressure-driven microfluidic patterns of a basic TIS system, methods such as... Figure 6C Analysis of equivalent circuit models similar to the basic form of the TIS system shown.

[0155] Note that if the thickness of the extracted sheath is close to the gap between the particle and the channel wall, bifurcation causes the particle to collide with the bifurcation point. An expansion well attached to the end of the bifurcation point will avoid this strong impact on the particle, thereby improving particle viability, which is essential in applications involving living biological cells. Similarly, a well attached to the end of the confluence point also suppresses particle collisions under strong inertial influence. Furthermore, the transition between the expansion well and the subsequent straight section should not be too blunt to avoid generating strong secondary flows. In some embodiments of the invention, the width W... B2 and width W C2 Each is between 1 micrometer and 10 millimeters, with an angle θ B2 and angle θ C2 Each is between 120 degrees and 180 degrees. In one embodiment of the invention, the width W B2 and width W C2 Each is between 40 micrometers and 100 millimeters, at an angle θ. B2 and angle θC2 Each is between 120 degrees and 170 degrees.

[0156] Assuming the final amount of sheath fluid is the sum of all sheath fluid produced in the TIS system, the sufficient condition for positioning all particles within a single compartment for single-stream focusing is defined as:

[0157]

[0158] in,

[0159] W m =Channel width of block A

[0160] W m = Distance from particle to the nearest channel wall

[0161] Although the sheath fluid is adjustable in a TIS system of basic form, after one sheath accumulation cycle in a TIS, the volume of sheath fluid is typically insufficient to deflect all particles into a single compartment of the original channel cross-sectional area. (Reference) Figure 7A Two different sized particles are now inertially focused in a channel with a rectangular cross-section having a high aspect ratio. In addition to all particles concentrating on the mid-plane, size-dependent particles are enveloped by inertial wall effects along the horizontal direction. Specifically, larger particles are farther from the channel wall. To satisfy the single-flow particle positioning condition in a single-sheath aggregation cycle, the branched fluid must be large enough to inherently cover small particles, causing sheath extraction failure and resulting in size-dependent single-flow focusing. On the other hand, if small volumes are extracted, it is generally insufficient to satisfy the single-compartment positioning condition. As a result, a pair of symmetrical focal points still exist.

[0162] To achieve size-insensitive single-stream focusing and equivalent wide effective particle size coverage, it is necessary to resort to methods such as Figure 7A The multi-cycle TIS shown is designed to accumulate a sufficient volume of sheath fluid without separating small particles. It can be implemented in complex forms by cascading multiple building blocks, including but not limited to interlaced, modular, or hybrid forms by combining one or more of these aforementioned building blocks.

[0163] In one embodiment, such as Figure 7B As shown, the TIS system has an interleaved configuration, comprising multiple interconnected blocks B and C to form a periodic cascade with branching and merging loops, with each pair of blocks B and C connected to block D. The drag ratio between blocks B and D gradually decreases downstream to monotonically increase the amount of sheath stored in the bypass channel.

[0164] In another embodiment, such as Figure 7CAs shown, the TIS system has a modular form, comprising multiple blocks B and C integrated into a single structure, ultimately forming only a single block D with multiple inlets. Compared to the staggered form, the modular design offers advantages in terms of smaller size and lower overall resistance, but requires more complex channel analysis.

[0165] Different forms of TIS systems can be combined in various ways to create more complex architectures for TIS. For example, in one embodiment, a hybrid form comprising random combinations of building blocks can be constructed for microfluidic networks in laminar flow.

[0166] The same set of design rules in the TIS system can be applied to these complex forms. The sufficient condition for positioning all particles within a single compartment for single-flow focusing now becomes:

[0167]

[0168] in,

[0169] W m =Channel width of block A

[0170] W P = Distance from particle to the nearest channel wall

[0171] n = the number of blocks B

[0172] refer to Figure 8 A computational fluid dynamics (CFD) simulation of an embodiment of a basic TIS system shows a linear relationship between the amount of fluid diverted to the bypass channel and the ratio of hydraulic resistance between the bypass channel (block D) and the bridge (block B), demonstrating that precise sheath extraction can be achieved by controlling the hydraulic resistance of the channel.

[0173] refer to Figure 9A Another CFD simulation of an embodiment of a TIS system with basic form illustrates the trajectory of the particle flow within. The temporary upward movement of the bottom flow indicates that the TIS successfully positioned the particle. Now refer to... Figure 9B In one embodiment, the TIS system in its basic form is shown in enlarged form as block B. In this embodiment, the hydraulic resistance ratio of the bridge to the bypass is set to 1:7 to produce sheath fluid with a thickness of one-eighth the channel width. The bypass channel is formed with a serpentine pattern to illustrate that only the channel resistance affects the amount of sheath fluid in the branch.

[0174] To detect particle flow movement, two solutions, each containing fluorescent polystyrene beads of a specific size, were injected into the TIS system. For example, one solution might contain fluorescent polystyrene beads with a diameter of 8 μm, while the other might contain fluorescent polystyrene beads with a diameter of 20 μm. The trajectories of the moving fluorescent polystyrene beads were then recorded.

[0175] First, the particles are focused into a single stream in the upstream focusing unit for better observation, and their individual trajectories can be observed. Figure 9C As seen in the striped image, during the flow across the bridge, the flow immediately maintains close contact with the main channel wall after passing the bifurcation point, while moving away from the bridge wall, indicating that inertial focusing resets the position of the particles.

[0176] However, after passing the confluence point, the flow did not approach the wall of the main channel, indicating successful sheath fluid accumulation. The same effect was observed when the bead size changed, suggesting that sheath fluid accumulation may be insensitive to particle size. To better verify this, ultrafast laser scanning microscopy was used to image rapidly flowing biological cells at three different locations, cells with more non-uniform size distributions, such as… Figure 9C As shown.

[0177] refer to Figure 9D Cells of different sizes are focused to form a single stream near the main channel wall, similar to the microbeads at the scanning point. Due to light diffraction, the main channel wall, which forms the longitudinal side of the channel cross-section after imaging, spans the focal plane of the microscope. Therefore, in Figure 9D In this process, a portion of the cell wall becomes out of focus, resulting in reduced contrast in the expanded wall. Furthermore, no gaps are formed between the cell and the main channel wall, such as... Figure 9D The red arrow at scan point one indicates this. Furthermore, it has been shown that no gap forms at scan point two. However, a 7.5 μm thick gap forms at scan point three, which is approximately one-eighth the width of the 60 μm channel. Therefore, the results demonstrate that precise cross-sectional separation can be achieved through the design of a bifurcation and uniform channel structure for particle flow.

[0178] In addition to single-stream inertial focusing, TIS systems and methods can also be used in the field of deformable cytometry to evaluate the mechanical properties of particles.

[0179] refer to Figure 10A In one embodiment, the TIS system is configured without expansion wells, which set the bifurcation boundary on the particle stream, forcing the particles to collide with the channel at the bifurcation junction. This impact introduces significant forces, deforming the elastic particles into a triangular shape. With good control, the elasticity of the cells can be quantified using various imaging techniques. On the other hand, the expansion wells effectively suppress collisions without affecting the sheath accumulation performance shown at the confluence junction.

[0180] Figure 10BImages of cells captured by ultrafast laser scanning microscopy before and after particle collision are shown. The triangular-like cells indicated by the red arrows are clearly deformed cells, only visible after particle collision. Therefore, the application of the TIS system and method is not limited to single-stream focusing, but can include a wide range of microfluidic maneuvers requiring precise single-particle localization.

[0181] refer to Figure 11A A CFD simulation of an embodiment of a TIS system with an interleaved configuration illustrates the trajectory of particles flowing within the system. The bottom flow exhibits a temporal upward movement at the confluence point, with gradually increasing amplitude, and eventually merges with the top flow, demonstrating successful particle localization via the TIS system with an interleaved configuration.

[0182] refer to Figure 11B The microfluidic channel comprises an interlaced TIS system with long, high aspect ratio cubic channels attached downstream, as illustrated in a scaled schematic diagram.

[0183] By fixing the width of the bypass channels, the resistance of each bypass channel is proportional only to the length of the corresponding branch. As a result, the stepped structure of the TIS system achieves gradually decreasing resistance in the bypass channels (equivalent to gradually increasing sheath fluid volume).

[0184] Refer again Figure 11B A magnified window shows all the bifurcation and merging points where fluorescent stripe images were captured. Deionized water containing fluorescent polystyrene beads with diameters of 8 μm and 20 μm was injected into the microfluidic chip, respectively. Images of the formation and changes in the flow were then captured and shown in [the image]. Figure 11C In the middle. For example Figure 11C As shown in (1), due to inertial focusing, two parallel fluorescence streams are formed after passing through the extended high aspect ratio cubic channel. Next, as... Figure 11C As shown in the transition region between the basic forms in (3)-11C(5), the flow deflection increases as it travels downhill after each basic form of the TIS system, and finally deflects towards the upper half after passing through the four basic forms, as... Figure 11C (6) is shown. Then, after passing through the second extended high aspect ratio cubic channel, the two streams (one with 8μm beads and the other with 20μm beads) merge into one stream, as shown. Figure 11C As shown in (7).

[0185] refer to Figure 12A In one embodiment of a TIS system with an interleaved configuration, a series of images showing the co-current flow of water and ink illustrates fluid intake in the bypass channel at different water-to-ink ratios. The results demonstrate how the bypass channel continuously draws in up to 45% of the total peripheral fluid from the main channel and then returns it to the main channel.

[0186] refer to Figure 12B The diagram illustrates, to scale, a microfluidic channel comprising a modular TIS system with a long, high aspect ratio cubic channel attached downstream.

[0187] refer to Figure 12C Deionized water containing fluorescent polystyrene beads with a diameter of 6 μm was injected into the microfluidic chip. Then, in Figure 12C Images of flow formation and changes throughout the main channel were captured and shown. As shown, due to inertial focusing, two parallel fluorescence streams are formed after passing through block A, which has a high aspect ratio cube. The streams then deflect and recover as they travel downhill after each block B of the TIS system, ultimately deflecting upwards after passing through the confluence junction. The two streams, each with 6μm beads, then merge into a single stream after passing through the long, high aspect ratio cube channel, completing the single-stream focusing.

[0188] Now for reference Figure 13 Laser scanning images were captured to examine the single-flow focusing performance of TIS-assisted inertial focusing as microparticles and biological cells flowed through the microfluidic chip at different flow rates. At all flow rates, laser scanning images captured before the TIS system showed two distinct particle sequences, while images captured after the TIS system showed only one particle sequence in the upper half. Furthermore, these particles were all “focal-aligned,” meaning they were all located within a narrow range of vertical positions within the channel cross-sectional area, indicating that the TIS system and method achieved accurate 3D focusing. Moreover, the wide range of applicable flow rates suggests that the single-flow inertial focusing provided by the TIS system and method has a large tolerance for flow rate variations.

[0189] like Figure 14A and 14B As shown, the 3D focusing performance of the TIS system and method was also tested in high-throughput operations.

[0190] refer to Figure 14A This shows a compressed image of 10,000 cells obtained within 1 second at the outlet of a microfluidic channel, corresponding to a high throughput of 10,000 particles per second. Figure 14A The magnified window shows magnified images of 45 cells. These cells are heterogeneous in size and shape, but all are clearly focused at high throughput, demonstrating the advantageous capability of the CPC system and method for size-insensitive high-throughput 3D focusing. Images of the first 120 cells are shown below. Figure 14B As shown, there was no pleasant picking in the test to verify the focusing performance. Therefore, a direct application of the present invention is the particle focusing operation of imaging / non-imaging flow cytometers for high-throughput particle analysis.

[0191] The advantages of the TIS systems and methods of the present invention are that they do not require any external sheath fluid or external force field, thereby suppressing particle dilution to facilitate downstream particle analysis, reducing hydraulic pressure to improve system robustness, and reducing manufacturing and operating costs.

[0192] Furthermore, because TIS systems and methods achieve single-flow focusing based on inertial focusing and particle distribution constraints, this contrasts with traditional systems and methods that require external force fields such as secondary forces / Dean forces. Therefore, TIS systems and methods can be developed without the need for a delicate balance between inertial lift and secondary forces, which are complexly dependent on the flow velocity, the square of the particle size, and the cube of the channel cross-sectional area. Consequently, TIS systems and methods offer greater tolerance in fluid velocity control and scalable flux, cover a wide range of focusable particle sizes, have greater tolerance in manufacturing precision such as lithographic resolution, and simplify the channel design process.

[0193] The advantage of the TIS system and method of this invention lies in the fact that all particles are focused on the same horizontal plane in coplanar focusing, regardless of particle size differences. This is a necessary feature for acquiring high-quality data in techniques sensitive to changes in particle position in the vertical direction (e.g., optical analysis techniques). Specifically, this system, through integration with high-speed imaging systems and camera technologies (e.g., scientific CMOS), enables high-throughput imaging flow cytometry with a throughput of >10,000 cells, which is used to capture images of rapidly flowing cells after the TIS system.

[0194] Because the TIS system and method of this invention do not require the use of non-Newtonian fluids, including water and biological fluids (such as phosphate-buffered saline and blood), for routine samples, the cumbersome fluid exchange process in sample preparation is eliminated. Therefore, not only is operation simplified and accelerated, but the disturbances introduced into the sample are minimized, enabling the analysis of live samples for life science applications.

[0195] The TIS system and method of this invention can be widely applied to various flow cytometry applications and optical interrogation of particles. Further analysis and more advanced applications can be achieved when a particle classifier is integrated downstream of the TIS system. By utilizing force fields and corresponding particle migration simulations, focusing effects can be optimized and enhanced, minimizing the size of microfluidic chips designed based on the TIS system and method.

[0196] All patents, patent applications, provisional applications, and publications mentioned or cited herein, including all figures and tables, are incorporated herein by reference in their entirety, without contradicting the express teachings of this specification.

[0197] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or variations based on these examples and embodiments will occur to those skilled in the art, and such modifications or variations will be included within the spirit and scope of this application and the appended claims. Furthermore, any element or limitation of any invention or embodiment disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment thereof, and all such combinations are considered to be within the scope of the invention, but not limited thereto.

Claims

1. A microfluidic system for focusing particles suspended in a particle-carrying fluid, comprising: Microfluidic channels, including: The first part includes an accumulation unit, a consumption unit, and a storage unit. The accumulation unit includes a bifurcated joint with a long straight section attached to its end, and the consumption unit includes a confluence joint with a short straight section attached to its end. The first part is configured to perform an adjustable inertial sheath (TIS). The second part includes a startup unit with a straight segment, configured to perform inertial focusing. The TIS internally confines multiple particles to have a spatial distribution within one of multiple compartments of the inertial force field in the cross-sectional area of ​​the microfluidic channel. The microfluidic channel further includes a main channel and a bypass channel serving as a storage unit. The main channel includes an initiation unit at the beginning, at least one consumption unit downstream, and at least one accumulation unit in between. The bypass channel is in which only sheath fluid flows.

2. The microfluidic system according to claim 1, wherein, The microfluidic channel is formed with a cubic structure.

3. The microfluidic system according to claim 1, wherein, Within the cross-sectional area of ​​the microfluidic channel, there exists only one inertial focal point within one of the multiple compartments of the inertial force field.

4. An adjustable inertial sheath (TIS) system for particle positioning of multiple particles, the TIS system comprising: Fluid channels including main channels and bypass channels; The main channel includes: At least three straight segments; At least one branching point; and At least one junction point; and The bypass channel includes: At least one entrance; and At least one export, Each of the forking and merging points has a Y-shaped intersection structure, which includes: The three ends include: Connected to both ends of the straight segment; and One end of the bypass channel is connected; and Expansion well.

5. The TIS system according to claim 4, wherein, The fluid channel is made of cyclic olefin copolymer (COC), polymethyl methacrylate (PMMA), or polycarbonate (PC).

6. The TIS system according to claim 4, wherein, The fluid channel is formed with a rectangular channel cross-section with a high aspect ratio to confine particles to a pair of focal points in the intermediate plane through inertial focusing.

7. The TIS system according to claim 4, wherein, The fluid channel has an aspect ratio greater than 1.

8. The TIS system according to claim 4, wherein, The fluid channel is made of silicone containing polydimethylsiloxane.

9. The TIS system according to claim 4, wherein, The fluid channel is configured to have a basic form, including: Two straight sections are used for inertial focusing to generate a peripheral particle-free fluid. A bifurcation point for separating the particle-free fluid in the bypass channel from the particle-carrying fluid, and concentrating the particle-carrying fluid in the main channel; and A confluence point is used to encapsulate a particle-carrying fluid through a particle-free fluid in a bypass channel and temporarily position the particle distribution within a smaller area of ​​the cross-sectional area of ​​the fluid channel.

10. The TIS system according to claim 9, wherein, A particle-free fluid slides in the bifurcation junction as a self-generated sheath fluid without affecting the inertial focusing.

11. The TIS system according to claim 10, wherein, The thickness of the particle-free fluid is determined by the Reynolds number of the fluid carrying particles, the size of the particles, and the geometry of the channel.

12. The TIS system according to claim 9, wherein, The volumetric flow rate of the particle-free fluid is equal to or greater than the volumetric flow rate of the particle-carrying fluid in the final straight section connected to the final confluence point.

13. The TIS system according to claim 4, wherein, The TIS system is configured to obtain different sheath extraction conditions, including: Small volume extraction; Large volume extraction; or Multiple small-volume extractions.

14. The TIS system according to claim 13, wherein, The small-volume extraction configuration is configured to allow fluid to slide with a thickness less than the distance between the center of the smallest particle to be focused and the nearest wall of the channel.

15. The TIS system according to claim 13, wherein, The large-volume extraction is configured with a sliding particle-free fluid, the thickness of which is less than the distance between the center of the largest particle to be focused and the nearest wall, and greater than the distance between the center of the smallest particle to be focused and the nearest wall.

16. The TIS system according to claim 13, wherein, The multiple small-volume extractions are configured to have multiple stages, including small-volume extraction, large-volume extraction, or a combination of both.

17. The TIS system according to claim 13, wherein, The fluid channel is configured to have a pattern including the following: Interlacing form; Block format; or A hybrid form, which has a combination of interlaced or block-like forms.

18. The TIS system according to claim 4, wherein, The fluid channel is configured with multiple branching and converging points to form an overall asymmetric structure to achieve the conditions of claim 14 for focusing size-insensitive single-stream particles.

19. The TIS system according to claim 4, wherein, The fluid channels are configured to have varying forms to introduce particle collisions at the bifurcation points used for cell deformation.

20. The TIS system according to claim 11, wherein, The Reynolds number of the fluid carrying the particles is between 1 and 2000.

21. The TIS system according to claim 4, wherein, The fluid channel is formed with a rectangular channel cross-section having a height H and a width W, each between 1 micrometer and 10 millimeters, and an aspect ratio AR between 0 and 0.75 or greater than 1.33, for dividing the inertial force field into two compartments.

22. The TIS system according to claim 21, wherein, The height H is 80 micrometers, the width W is between 20 and 60 millimeters, and the aspect ratio AR is between 1.33 and 4.

23. The TIS system according to claim 4, wherein, The particle size is between 0.1 micrometers and 10 millimeters.

24. The TIS system according to claim 4, wherein, The particle size is between 5.6 and 30 micrometers.

25. The TIS system according to claim 4, wherein, The fluid channel comprises four blocks, including block A, which is initially a straight section to initiate inertial focusing on the peripheral sheath particles; Block B is a bifurcation junction that diverts some of the previously generated sheath fluid to the bypass channel and has a straight section of sufficient length to restore the sheath. Block C is a confluence point that returns some sheath fluid to the main channel to temporarily position the particles in the straight section; And block D, which is a bypass channel through which only sheath fluid flows.

26. The TIS system according to claim 25, wherein, The width W of the fully developed flow to block D FD It is configured to be between 0 and 60 micrometers.

27. The TIS system according to claim 25, wherein, The width W of block B B2 and width W C2 Each is between 1 micrometer and 10 millimeters, and the angle θ of block B. B2 and the angle θ of the block C C2 Each is between 120 degrees and 180 degrees.

28. The TIS system according to claim 25, wherein, The width W of block B B2 and the width W of the block C C2 Each is between 40 micrometers and 100 millimeters, and the angle θ of block B. B2 and the angle θ of the block C C2 Each is between 120 degrees and 170 degrees.

29. A system for imaging and analyzing multiple biological cells, comprising: The microfluidic system according to claim 1 is used to focus biological cells into a single stream; A microfluidic channel includes a third portion having the accumulation unit, the consumption unit, and the storage unit, and the third portion is configured to perform a second TIS for high-quality optical probing of focused biological cells; as well as A real-time image acquisition system for optically focused biological cell imaging.

30. The system according to claim 29, wherein, The optically focused biological cells are located within the depth of field of the real-time image acquisition system.

31. The system according to claim 29, wherein, The image acquisition system records the contrast of focused biological cell images, including: Bright field contrast ratio; Quantitative phase contrast; and Fluorescence contrast.

Images