METHOD, DEVICE AND SYSTEM FOR HYDRODYNAMIC FLOW FOCUSING

Abstract

In a method for hydrodynamic focusing of a laminar and planar sample fluid flow, a system is provided for analysis and/or sorting of microscopic objects in the sample fluid comprising an optical objective for optical inspection of the microscopic objects. Microscopic objects are conveyed in the laminar flow of the sample fluid, and two laminar and planar flow of sheath fluids are provided. The flow of the sample fluid is hydrodynamically focused at an optical inspection zone of the system by the sheath fluids. Focusing of the flow of the sample fluid is controlled such that all of the microscopic objects in the sample fluid are caused to be conveyed in a common flow direction in one single plane at the inspection zone of the system, and the microscopic objects in the fluid are optically inspected through the optical objective.

Claims

1. A method for hydrodynamic focusing of a laminar and planar sample fluid flow in a system for analysis and/or sorting of microscopic objects in the sample fluid, wherein said system comprises an optical objective for optical inspection of the microscopic objects, the method comprising: conveying the microscopic objects in the laminar flow of the sample fluid; providing at least a first laminar and planar flow of a first sheath fluid and a second laminar and planar flow of a second sheath fluid; hydrodynamically focusing the flow of the sample fluid at an optical inspection zone of the system by causing each of the first and second sheath flows to make planar contact with the flow of the sample fluid at two opposed planar flow surfaces of the sample fluid flow; controlling the flow of the sample fluid and the first and second flows of the sheath fluids such that the sample fluid and the first and second sheath fluids flow in a common flow direction at the inspection zone of the system; controlling said step of focusing the flow of the sample fluid in such a way that all of the microscopic objects in said sample fluid are caused to be conveyed in said flow direction in one single plane at the inspection zone of the system; optically inspecting at least one of the microscopic objects in the fluid through said optical objective.

2. A method according to claim 1, wherein the optical objective defines a depth of focus and is arranged to provide a view onto the sample fluid at the inspection zone in a viewing direction which is perpendicular to the common flow direction, and wherein the planar flow of the sample fluid has a height in said viewing direction, which is smaller than or equal to the depth of focus of the optical objective.

3. A method according to claim 1, wherein flows of the first and second sheaths fluids form planar inlets to said inspection zone, each of said planar inlets being wider in a direction perpendicular to the common flow direction than the width of the inspection zone when seen in the plane of each respective planar inlet.

4. A method according to claim 1, wherein the sample fluid and the first and second sheath fluids are conveyed at a common flow velocity in said common flow direction at the inspection zone.

5. A method according to claim 1, wherein respective flow rates of the sample fluid flow and the first and second sheath fluid flows are controlled by applied pressure gradients in said flows.

6. A method according to claim 1, wherein said flows are three-dimensionally guided by at least three planar and mutually parallel substrate elements providing: respective inlets, including said planar inlets formed by the sheath fluids, for said flows upstream of said inspection zone, at least one waste outlet downstream of the inspection, and at least one further outlet for a selected flow downstream of the inspection zone.

7. A method according to claim 6, comprising the step of splitting a combined flow of the flows of sheath fluids and the sample fluid into the selected flow and a waste flow downstream of the inspection zone.

8. A method according to claim 6, wherein the step of splitting said combined flow is carried out as the combined flow flows across a flow-separating edge extending parallel to said substrate elements and normal to the common flow direction.

9. A method for selecting microscopic objects included in a laminar and planar sample fluid flow, said method comprising: hydrodynamically focusing said sample fluid flow by means of a method according to claim 1; microscopically inspecting and analysing the microscopic objects in the sample fluid at the optical inspection zone; selecting at least one microscopic object in the sample fluid on the basis of said microscopic analysis; ejecting the at least one selected microscopic object out of the sample fluid flow by means of light or an electromagnetic beam; subsequently splitting a combined flow of the flows of sheath fluids and the sample fluid into a selected flow including the at least one selected microscopic object, and a waste flow.

10. A hydrodynamic flow focusing device for optical analysis for analysis and/or sorting of microscopic objects in a sample fluid, the system comprising: an optical inspection zone for optically inspecting the microscopic objects in the sample fluid flow; an optical objective at the optical inspection zone; a sample flow controller for controlling a laminar and planar flow of the sample fluid; a sheath flow controller for controlling a laminar and planar flow of a first sheath fluid and a laminar and planar flow of a second sheath fluid; a flow structure configured to hydrodynamically cause each of the first and second sheath flows to make planar contact with the flow of the sample fluid at two opposed planar flow surfaces of the sample fluid flow, so as to focus the flow of the sample fluid at said optical inspection zone; wherein said flow structure is shaped and dimensioned such that the microscopic objects in said sample fluid are conveyed in one single plane at the inspection zone of the system during use of the system.

11. A hydrodynamic flow focusing device according to claim 10, wherein at least one dimension of a flow channel for said flows is constant throughout a length of the inspection zone, said at least one dimension being transverse to a flow direction of said flows and extending in a viewing direction of the optical objective.

12. A hydrodynamic flow focusing device according to claim 10, comprising at least three planar and mutually parallel substrate elements providing respective inlets for said flows upstream of said inspection zone, at least one waste outlet downstream of the inspection, and at least one selected outlet for a selected flow downstream of the inspection zone.

13. A hydrodynamic flow focusing device according to claim 10, comprising a flow separating means downstream of the inspection zone for splitting the combined flow of the sheath fluids and the sample fluid into a waste flow and the selected flow.

14. A hydrodynamic flow focusing device according to claim 13 wherein said flow separating means comprises a flow-separating edge extending parallel to said substrate elements and normal to a flow direction of said fluid flows.

15. A system for selecting microscopic objects included in a laminar and planar sample fluid flow, said system comprising: a hydrodynamic flow focusing device according to claim 10; means for microscopically inspecting and analysing the microscopic objects in the sample fluid at the optical inspection zone; means for ejecting at least one selected microscopic object out of the sample fluid flow by means of light or an electromagnetic beam; flow-separating means for splitting a combined flow of the flows of sheath fluids and the sample fluid into a selected flow including the at least one selected microscopic object and a waste flow.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Embodiments and features of the invention will now be described with reference to the accompanying drawings wherein:

[0094] FIG. 1 illustrates the optical sorting chamber with the sample sheath flow providing optical access with an optical objective;

[0095] FIG. 2 illustrates the cross sections of sample and sheath flow inlet;

[0096] FIG. 3 shows the hydrodynamic compression of the sample flow creating the thin sample flow;

[0097] FIG. 4 illustrates the flow of sample and sheath fluids in an embodiment of the present invention;

[0098] FIG. 5 illustrates shows the flow of the microparticles through the field of view of the optical objective;

[0099] FIG. 6 a cross section illustrating the horizontal flow focusing along a streamline using the sheath flow;

[0100] FIG. 7 a cross section of an analysis system illustrating the flow of microparticles;

[0101] FIG. 8 a cross section of a sorting system illustrating the flow of microparticles;

[0102] FIGS. 9-11 shows a positive microparticle being catapulted to another streamline;

[0103] FIG. 12 illustrates an embodiment of the microfluidic flow cell comprising four substrates;

[0104] FIG. 13 a micrograph of an embodiment of the microfluidic flow cell;

[0105] FIG. 14 illustrates the flow of fluids through a hydrodynamic flow focusing device according to the present invention;

[0106] FIG. 15 illustrates the cross sectional flow through the optical sorting chamber;

[0107] FIGS. 16 and 17 illustrate the lumped circuits of the present invention; and

[0108] FIGS. 18 shows the coefficient of variation obtained with an embodiment of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Terms

[0109] The term “microparticle” refers to small particles but not limited to the micrometer scale, <500 microns, and not limited to biological cells. The microparticle is preferably dielectric but can be metallic optically as well.

[0110] The term “substrate” as used herein refers to a piece of material with constant thickness preferably transparent and of optical quality but not limited to this. Preferably of glass, quartz, SU-8, Polycarbonate, Cyclic Olefin Copolymer (COC) polymers such as TOPAS®, polystyrene, Poly(methyl methacrylate) (PMMA). The substrate does not have to be hard, rigid sheet, but can be soft foil as well, such as Polydimethylsiloxane (PDMS) or other elastomeric material. The thickness of the substrate is typically 0.25 mm to 1 mm thick, but not limited to this thickness range.

[0111] The term “substrate plane” refers to a geometric plane which is parallel to the top or bottom of a substrate.

[0112] The term “substrate normal” refers to the direction of a vector having two 90 degrees angles to the substrate plane.

[0113] The term “pump” refers to an electronic controlled device capable of realizing a pressure driven fluidic flow in a tube of inside a structure

[0114] The term “flow controlled pump” refers to a pump where the output flow rate is the primary parameter, and the pressure may be a floating parameter. It is generally realized by displacing a piston or a peristaltic pump.

[0115] The term “pressure controlled pump” refers to a pump where the output pressure is the primary parameter, and the flow rate may be a floating parameter.

[0116] By ‘fluid channel’ is understood a pathway for fluid, such as tubing, such as a hollow channel in a solid element, such as a channel bounded by walls. The dimension of the fluid channel is typical of 100-1,000 μm wide, but not limited to this dimension. The depth of the fluid channel is typical 50-300 μm, but not limited to this dimension.

[0117] The term “microfluidic flow cell” as used herein refers to the flow cell providing the optical analysis or the optical sorting.

[0118] The term “optical sorting chamber” as used herein refers to a fluid channel in the microfluidic flow cell. The optical sorting chamber is connected to fluidic inlets and one or more outlets, as defined below. The optical sorting chamber is typically 600 μm wide, 350 μm high and 1 mm long, but not limited to these dimensions.

[0119] By ‘inlet’ is understood an entrance into the optical sorting chamber through which fluid may enter.

[0120] By ‘outlet’ is understood an exit from the optical sorting chamber, through which fluid may exit.

[0121] The term “sheath fluid” as used herein refers to a sheath of compatible liquid surrounding a microparticle for carrying one or more particles through a channel.

[0122] The term “top substrate” as used herein refers a plate of substrate located on the top part of the microfluidic flow cell and is the first substrate counting from top to bottom of the microfluidic flow cell.

[0123] The term “top middle substrate” as used herein refers a plate of substrate located just beneath the top substrate and is the second substrate counting from top to bottom of the microfluidic flow cell.

[0124] The term “bottom middle substrate” as used herein refers a plate of substrate located just beneath the top middle substrate and is the third substrate counting from top to bottom of the microfluidic flow cell.

[0125] The term “bottom substrate” as used herein refers a plate of substrate located just beneath the bottom middle substrate and is the fourth substrate counting from top to bottom of the microfluidic flow cell.

[0126] The term “microfluidic system”, as used herein refers to the microfluidic flow cell with the necessary auxiliary components for operating the flow through the chip will be referred to as the microfluidic system. The microfluidic system may include tubing, interconnects, tubing, valves, pumps, control electronics, sample injection loop, other microfluidic flowcells in connection with the chip, and additional on-chip functionality, known by the persons skilled in the art, such as filtering, PCR, on-chip staining by biomarkers.

[0127] The term “sample chamber” as used herein refers to a chamber of being typically, but not limited to, 2-5 μl in volume structured in the bottom middle substrate of the microfluidic flow cell. The chamber is connected to a sample inlet and externally to a pump.

[0128] The term “sample inlet” as used herein refers to an inlet into the optical sample chamber. The inlet introduces a fluid medium in which microparticles are suspended. The width at the exit of the sample inlet channel is typical of 125 μm, 250 μm, 500 μm, but not limited to these dimension. The depth at the exit of the sample inlet channel is typical of 70 μm, but not limited to this dimension.

[0129] The term “sheath flow inlet” as used herein refers to a channel to pinch the sample of microparticles into a thin layer of hydrodynamically focused sample flow. The width of the sheath flow inlet is typical of 500 μm, 750 μm, 1000 μm, but not limited to these dimensions. The height of the sheath flow inlet is typically 300 μm, but not limited to this dimension.

[0130] The term “waste flow channel” as used herein refers to one channel structured in the bottom substrate. The un-selected microparticles exit from the optical sorting chamber and enter into the waste flow channel.

[0131] The term “selected flow channel” as used herein refers to one channel structured in the top middle substrate. The selected microparticles exit from the optical sorting chamber and enter into the selected flow channel.

[0132] The term “microscope”, as used herein refers to any optical system compromising one or more optical objectives. The term is used in broader sense than to laboratory optical microscopes. Typically, microscopes may also compromise electronics devices for image acquisition, such as CCD and CMOS devices. They may also include lasers for excitation of fluorescence for imaging of cells.

[0133] The term “light emitting device” as used herein refers to a light source, in particular a laser in the visible or in the infrared domain, a laser diode, a fiber laser or a laser suitable for inducing fluorescence.

[0134] The invention presents an optical cell sorter relying on the usual sorting procedure:

[0135] 1. Hydrodynamically focusing the fluid with microparticles in a thin file (or sheath layer).

[0136] 2. Detection and analysis (Optical fluorescence, cell morphology etc.) for the basis of sorting.

[0137] 3. Deflect cells of interest with an optical laser in flowing liquid medium (selected cells).

[0138] 4. Two outlets which are asymmetrically biased such that each cell goes into the waste outlet if not deflected in step 3.

[0139] These steps are indicated in FIGS. 7-11.

Detailed Description of Hydrodynamic Focusing

[0140] The invention presents a new approach to hydrodynamically focus the sample to thin sheath (sorting procedure step 1) which passes the field of view allowing detection and analysis (sorting procedure step 2), deflect microparticles based on selection (sorting procedure step 3), and separate the deflected microparticles by a Y-branch with two outlets (sorting procedure step 4).

[0141] Hydrodynamic focusing is used to spatially focus a sample fluid to a thin layer. In FIG. 2 the principle is shown applied to a sample fluid in a sample inlet channel of height H.sub.sample by two opposing channels with sheath fluid of height H.sub.sheath. The flow rates of the sheath fluid, Q.sub.sheath, is higher than the sample flow rate, Q.sub.sample, causing the sample flow to be compressed to a smaller thickness, H.sub.sample<T.sub.sample according to the continuity equation of fluids. The flow is generally laminar with low Reynold's number (Re<1000) giving a non-turbulent, laminar flow. Microparticles suspended in the sheath fluid generally follow the streamlines of the flow, thus allowing the microparticles to be tightly focused. Obvious to the persons skilled in the art, the principle of hydrodynamic focusing can be extended to 3 dimensional structures to achieve 2 dimensional focusing, although such structures can be exceedingly difficult to fabricate due to their complexity. Also obvious is that due to the properties of laminar flow the orientation of the sheath flow inlets is of less importance since the flow is laminar. Thus the sheath flow inlets 31, 32 may equally well be oriented normal to the sample flow, and the hydrodynamic focusing will be similar.

[0142] FIG. 3 shows the optical sorting chamber 4 with the connections of inlets 31, 32, 33 and outlets 34, 35. The sheath inlets 31, 32 are positioned at one end with a sample inlet 33 in between two opposing sheath inlets. The three inlets 31, 32, 33 focus the sample fluid in the sample inlet to a thin sheet with a width that is close to the width of the sample inlet 43. The sheath inlets 31, 32 have a width 41, which is wider than the sample inlet 43 and this is important in the formation of the sample sheet 3. The spatially thin sample sheath 3 forms a plane through the remainder of the optical sorting chamber 4 orthogonal to the optical axis 101 as seen in FIG. 5 and FIG. 6.

[0143] An embodiment of the three inlets 31, 32, 33 can be seen in FIG. 4 in scale.

[0144] In one embodiment the CV was 1.9% in FIG. 18. The sheath flow rate was Q.sub.sheath=2.5 microL/min, the sample flow rate was Q.sub.sample=0.025 microL/min. The sample was a suspension of 10 micron diameter polystyrene beads in distilled water. The sample was focused to sheath of approximately 150 micron width 64 with thickness approximately 12 micron 65.

[0145] On the other end of the optical sorting chamber there are two outlets, a ‘selected’ outlet 35 for one type of species and a ‘waste’ outlet 34 for the second type of species. In spite of the name, the ‘waste’ outlet can also output purified suspensions of microparticles 1.

[0146] At the outlet side the focused sample sheath 3 exits at the ‘waste’ outlet 34. The microparticles 1 which are to be selected exits at the selected outlet 35. The microparticles in the sample sheath layer 3 follows per default a streamline 61 with terminal in the waste outlet.

[0147] Using computational simulation tools such as computational fluid dynamics (CFD) the flow profile in complex geometries can be accurately predicted. FIG. 15 (left) shows a section though the optical sorting chamber 4 demonstrating the resulting sample sheath. The width of the optical sorting chamber 45 is 600 micron and the height 46 is 350 micron. The width of the sample inlet 43 is 300 micron and its height is 70 micron 44. The resulting sample sheath 3 is 346 microns wide 64 and 13 microns high 65. The flow is oriented in a direction normal to the section. CFD simulations show that the sample flow experiences a broadening of about 20% dependent on geometrical design and flow rates.

Sheath Flow

[0148] In a preferred embodiment of the present disclosure, the device is configured such that two planar sheath flows are established parallel to each other within the flow chamber.

[0149] In another preferred embodiment of the present disclosure, the width of the planar sheath flows are less than 100 microns, less than 200 microns, less than 300 microns, less than 400 microns, less than 500 microns, less than 600 microns, less than 800 microns, less than 1000 microns.

[0150] According to the sample flow, and the relation with the sheath flow, the velocity profile of the planar sheath flows may be constant within 20%, within 15%, within 10% or within 5%.

[0151] In a preferred embodiment of the present disclosure, the thickness of each of the sheath flows is less than 500 micron, or less than 40 micron, or less than 30 micron, or less than 20 micron, or less than 15 micron, or less than 14 micron, or less than 13 micron, or less than 12 micron, or less than 11 micron, or less than 10 micron, or less than 9 micron, or less than 8 micron, or less than 7 micron, or less than 6 micron, or less than 5 micron, or less than 4 micron, or less than 3 micron, or less than 2 micron, or less than 1 micron.

[0152] In another preferred embodiment of the present disclosure, the microfluidic flow cell is configured such that a sample flow incident to the optical sorting chamber through the sample flow inlet 33 is hydrodynamically focused to one of the planar sheath flows by means of hydrodynamic flow compression. In this way there may be a natural flow of liquid, such that sorting may be configured for sorting microparticles 1 away from the natural flow following the streamline 61, such that the microparticles 1 may be guided into the other planar sheath flow following the streamline 62.

Sheath Flow and Sample Flow Inlets

[0153] In a preferred embodiment of the present disclosure, the sheath flow inlets 31, 32 and the sample inlet 33 are formed in separate substrate plates. In this way, there may at least be three separate substrate plates.

[0154] In another preferred embodiment of the present disclosure, the top and bottom sheath flow inlets 31, 32 are arranged normal to the substrate plates. In this way, it may be possible to obtain sheath flow which connects to the sample flow inlet 33 in an identical manner such that the sheath flow from the sheath inlets 31, 32 may be close to being identical and thereby optimally configured. Another advantage of this configuration may be related to the ease of manufacture of the individual substrates, which are subsequently precision bonded.

[0155] In some embodiments of the present disclosure, one of the top 31 and bottom 32 sheath flow inlets are arranged normal to the substrate plates.

[0156] In some embodiments of the present disclosure, one of the top 31 and bottom 32 sheath flow inlets are arranged with an angle less than 90 degrees to the substrate plates preferably inclining such that the flow through sheath inlets 31, 32 experiences a change of direction less than 90 degrees.

[0157] In a preferred embodiment of the present disclosure, the width and/or height of the sheath flow inlet channels are less than 50 microns, less than 100 microns, less than 200, or less than 300 microns. In another preferred embodiment of the present disclosure, the width of the sheath flow inlets are less than 100 microns, less than 200 microns, less than 300 microns, less than 400 microns, less than 500 microns, less than 600 microns, less than 800 microns, less than 1000 microns.

[0158] In one embodiment of the present disclosure, the cross sectional area of the sheath flow inlets 31, 32 are identical, such that for example the pressure gradient across the top sheath flow inlet 31 and the sheath flow outlet(s) and over the bottom sheath flow inlet 32 and the sheath flow outlet(s) may be able to establish identical sheath flow in the flow chamber. The cross sectional area of the channels may be any suitable shape, in particular rectangular, elliptical or circular.

[0159] In a preferred embodiment of the present disclosure, the width of the optical sorting chamber is identical to the width of any of the sheath flow inlets 31, 32.

Sheath Flow Outlets Channels and Sheath Flow Outlets

[0160] In another preferred embodiment of present disclosure, the sheath flow outlet channels are formed in separate substrate plates.

[0161] In a preferred embodiment of the present disclosure, the width and/or height of the sheath flow outlet channels are less than 50 microns, less than 100 microns, less than 200, or less than 300 microns. In another preferred embodiment of the present disclosure, the width of the sheath flow outlets are less than 100 microns, less than 200 microns, less than 300 microns, less than 400 microns, less than 500 microns, less than 600 microns, less than 800 microns, less than 1000 microns.

[0162] In a preferred embodiment of the present disclosure, the width of the optical sorting chamber is identical to the width of any of the sheath flow outlets 34, 35.

Flow Chamber

[0163] In a preferred embodiment of the present disclosure, the length of the optical sorting chamber 4 is less than 0.5mm, less than 1 mm, less than 1.5 mm or less than 2.0 mm. The length of the optical sorting chamber 4 may be 0.5 mm, 1 mm, 1.5 mm or 2.0 mm.

[0164] In another preferred embodiment of the present disclosure, the width of the optical sorting chamber 4 is less than 0.3 mm, less than 0.6 mm, less than 0.9 mm or less than 1.2 mm. The width of the optical sorting chamber 4 may be 0.3 mm, 0.6 mm, 0.9 mm or 1.2 mm.

[0165] In yet another preferred embodiment of the present disclosure the height of the optical sorting chamber 4 is less than 0.1 mm, less than 0.2 mm, less than 0.3 mm or less than 0.4 mm. The height of the optical sorting chamber 4 may be 0.1 mm, 0.2 mm, 0.3 mm or 0.4 mm.

[0166] In a preferred embodiment of the present disclosure, the thickness of the sample flow is less than 50 micron, or less than 40 micron, or less than 30 micron, or less than 20 micron, or less than 15 micron, or less than 14 micron, or less than 13 micron, or less than 12 micron, or less than 11 micron, less than 10 microns, less than 9 microns, less than 8 microns, less than 7 microns, less than 6 microns, less than 5 microns, less than 4 microns, less than 3 microns, less than 2 microns or less than 1 micron. The sample flow may be between two sheath flows, in particular inside a flow chamber. Accordingly, the optical sorting chamber 4 and the sheath flows may be configured for establishing the sample sheath layer 3 flow as described above.

Optics

[0167] One purpose of having the thickness 65 of the sample sheath layer 3 as described may be that microparticles 1 may then be in a well-defined plane, wherein optimal optical focus may be established, in particular from the imaging means of an optical objective 104 in connection with an optical detection system 201.

[0168] More preferably, the optical sorting chamber 4 may comprise optical access in the plane of the substrate plates. The optical access may be for optical analysis or optical analysis and optical sorting.

[0169] The microfluidic flow cell 4 has an aspect that is thin in the direction of the optical axis 101. This allows the use of objectives 104 with a short working length less than 1 mm, such as objectives with a magnification of 20×, 50×, and 100×. These objectives have a high NA for efficient light collection and high optical resolution and contrast. A high NA indicates that the optical objective 104 accepts a wide light cone from each point in the conveyor belt. Thus the aspect of the optical sorting chamber and the distance from the conveyor belt to the sides of the optical sorting chamber must be designed such that the light cone is not refracted from the sides giving a distorted image close to the sides of the channel.

[0170] In a preferred embodiment of the present disclosure, the distance from the sheath sample layer 3 to the side of the optical sorting chamber 4 is longer than half the height of the optical sorting chamber times the numerical aperture of the optical objective 23 divided by the refractive index of sheath buffer. The light cone of the objective may thus avoid interfering with the sides of the optical sorting chamber. The distance from the sheath sample layer 3 to the side of the optical sorting chamber 4 is obviously half the width optical sample chamber minus half the width of sample sheath layer.

[0171] In a preferred embodiment of the present disclosure, two objectives 104 may be used for light condenser and light collection. The condenser objective focuses the illuminating light onto the image plane, and the collection objective guides the light to the electronic imaging device 201 or human eye.

Optical Sorting with a Laser

[0172] Optical sorting may be of the microparticles 1 residing in the fluid that may flow in the microfluidic flow cell 2.

[0173] In a preferred embodiment, an optical laser beam 103 is configured to provide an optical force normal to the substrate plates as seen in FIG. 10 and FIG. 12. Accordingly, the optical force of the laser beam 103 may be in a direction normal to the substrate plates and adapted to displace a microscopic object suspended in a liquid medium in the flow chamber. The optical laser beam 103 may also be configured to yield optical forces in the plane with the substrate plates.

[0174] By displacing the microparticles 1 normal to the streamlines by the optical force, the microparticle may be to follow a streamline 62 with terminal in the selected outlet, thereby being optically and physically sorted.

[0175] According to the present disclosure, the sorting controller 202 may be configured to identify a plurality of predefined/pre-marked/specific microparticles in a liquid medium flowing in the optical sorting chamber 4. In this way, a selective sorting process may be obtained.

[0176] In a particular embodiment, an electronic imaging device is provided 201 as seen in FIG. 5. By image analysis the positions and preferably the velocity of the microparticle 1 can be found. A controller 202 can pass the detected position of microparticle to a system 203 that provides a laser beam 103 that coincides with position of the microparticle 1 such as to displace the microparticle by optical forces. The optical sorting is illustrated in FIGS. 8-11.

[0177] The optical force of the laser beam 103 in the plane with the substrate plates may be slowing down the microscopic objects. The decrease in microscopic object velocity may be an advantage in that it may allow for increasing the exposure time of the imaging means. A further advantage is that the exposure time to the manipulating laser beam 103 is increased. In this way, the decrease in microscopic object velocity may be used to increase the manipulation time of the force normal to the substrate plates.

[0178] In a particular embodiment, the microfluidic system provides optical access to the sample flow. A microscope consisting of least one optical objective 104 has a field of view and depth of focus which is in relation with the width 64 and the thickness 65 of the sample sheath layer. The microscope provides a light source for illuminating the sample sheath layer 3.

[0179] In a further embodiment the microscope has specifically one optical objective 104 that is used sample illumination and light collection.

[0180] In another embodiment the microscope has specifically two optical objectives 104, one optical objective 104 provides optical sample illumination and the other objective 104 provides light collection.

[0181] In a further embodiment the microfluidic system provides an outlet Y-branch and a pump connected to one of any outlet 34, 35 for separating the ‘selected’ microparticles and the ‘waste’ microparticles.

[0182] In a further embodiment the field of view 102 is divided into an analysis region 105 and a manipulation region 106 as seen in FIG. 6.

[0183] It is contemplated that the microscope can include one or more lasers for excitation of fluorescence and the necessary filters before the electronic imaging device.

[0184] It is contemplated that the microparticles may be used specific attachment of optically active labels, such as fluorescent labels of specific biomarkers known in the field of cytology.

Flow Management

[0185] The embodiment also provides means for applying a pressure gradient in order to drive the sheath and sample fluid.

[0186] In a preferred embodiment of the present disclosure, the microfluidic flow cell is configured such that a pressure gradient can be applied over the top sheath, bottom sheath, and sample inlets 31, 32, 33 and the selected and waste outlets 35, 34.

[0187] The fluidic experiences a pressure drop along the channel, and the total pressure drop is proportional to the flow rate by a constant known as hydraulic resistance: ΔP=R.sub.hydQ. This is analogous to Ohm's law for electrical resistance, and the same circuitry schematics can be applied. ΔP, the pressure difference across the ends of the channel, corresponds to an electric voltage, Q., the flow rate, corresponds to electrical current.

[0188] Q.sub.sheath=Q.sub.top+Q.sub.bottom is the sum of volumetric flow rate through the top, Q.sub.top, 31 and bottom sheath inlet Q.sub.bottom, 32, Q.sub.sample is the volumetric flow rate though the sample inlet 33, Q.sub.waste is the volumetric flow rate through the waste outlet 34, and Q.sub.selected is the volumetric flow rate through the selected outlet 35.

[0189] FIG. 16 shows the equivalent lumped circuit of the flow in the microfluidic flow cell for analysis and FIG. 17 shows the circuit for sorting. Using Kirchhoff's law on the lumped circuit we get the following equation for the flow

Q.sub.sample+Q.sub.top+Q.sub.bottom=Q.sub.wasteQ.sub.selected

[0190] In order to focus the sample fluid the sample flow should be much lower than the sheath flow. Typical values are: Q.sub.sheath=0.1 microL/min, Q.sub.sheath=1 microL/min, Q.sub.sheath=10 microL/min, Q.sub.sheath=100 microL/min, Q.sub.sheath=1mL/min, and Q.sub.sample<Q.sub.sheath/10, Q.sub.sample<Q.sub.sheath/20, and Q.sub.sample<Q.sub.sheath/30.

[0191] Accordingly, it may be very important that the pressure gradient is established as described and/or the inlets and/or outlets and/or channels are manufactured as described to allow for the herein described sample flow. Accordingly, the pressure gradient of the terminals of the channels connecting the inlets 31, 32, 33 and/or the outlets 34, 35 may be configured such that the velocity profile of the sample flow may be laminar and non-turbulent. In this way, the microparticles may be carried through the optical sorting chamber 4 with a velocity in relation to the flow and further move in a thin sample sheath layer 3. Typical velocity of microscopic objects may be 10 microns/s, 100 microns/s, 500 microns/s, 1000 microns/s, 2000 microns/s, or 5000 micron/s.

[0192] In an embodiment a flow bias can be configured to prevent microparticles 1 bound for the waste outlet 34 from entering into the selected outlet 35 given false positive or false negatives and vice versa for microparticles bound for the selected outlet 35 from entering the waste outlet 34. The flow rate at the waste outlet 34 is set to

Q.sub.waste=Q.sub.sample+Q.sub.bottom+Q.sub.bias

where Q.sub.bias is a small parameter, such as Q.sub.sheath/100, Q.sub.sheath/50, Q.sub.sheath/25, Q.sub.sheath/10, that creates a retention distance such that a thin layer of sheath fluid is on top of the sample sheath layer 3 in the Y-junction of the outlets. The purpose being to avoid false positive microparticles entering the selected outlet 35. Q.sub.waste is a flow rate that the waste pump draws liquid away from the microfluidic flow cell 2, as defined in FIG. 17. Since there is no pump connected to channel connecting the selected outlet 35, the flow rate through the selected outlet 35, Q.sub.selected is determined by

Q.sub.selected=Q.sub.top−Q.sub.bias

Q.sub.selected and thereby sorting purity are perturbed by any fluctuation in either Q.sub.sample, Q.sub.top, Q.sub.bottom, or Q.sub.waste. A fluctuation may cause the selected streamline 62 to enter the waste outlet 34, or the waste streamline 61 to enter the selected outlet 35. In order to control the fluid flow in the microfluidic flow cell, only four of the five total inlets and outlets 31, 32, 33, 34, 35 need to have the flow rate actively controlled. The sheath flow rates, Q.sub.top, Q.sub.bottom can be identical, Q.sub.sheath/2, for both two sheath inlets located on top and bottom 31, 32 in order to center the sample sheath layer 3 flow exactly in the optical sorting chamber. Only three flow rates are unique (Q.sub.waste, Q.sub.sample, Q.sub.sheath).

[0193] In an embodiment three pumps are used for sorting. One pump is used for controlling the flow rates Q.sub.waste, Q.sub.sample, and Q.sub.sheath.

[0194] In another embodiment, two pumps are connected to the sample inlet 33 and the sheath inlets 34, 35 used for optical analysis of the microparticles 1.

Detailed Description of Fabrication of Microfluidic Flow Cell

[0195] Method for fabrication of microfluidic flow cell described here is not limited to construct the chip in one kind of material. The material of the microfluidic flow cell is preferably to be optically transparent, such as polymer, glass and elastomeric polymer.

[0196] For the substrates in polymer, milling, injection molding, hot embossing or femtosecond laser machining may be used to create the structures in each individual substrate. Thermal or other bonding methods such as ultrasonic welding or femtosecond laser welding can be used to form an embodiment of the microfluidic flow cell for mass production of the microfluidic flow cell or other techniques known to those skilled in the art.

[0197] A typical method for structuring of the glass substrates is by wet etching in the hydrofluoric acid (HF) based solution, or dry etching by using deep reactive-ion etching (DRIE) technology. The glass substrates are typically bonded by fusion bonding technique to form the microfluidic flow cell or other techniques known to those skilled in the art.

[0198] In a preferred embodiment of the present disclosure, the thickness of the substrate plates are less than 0.05 mm, less than 0.1 mm, less than 0.2 mm, less than 0.4 mm, less than 0.6 mm, less than 0.8 mm, less than 1.0 mm, less than 1.2 mm, less than 1.4 mm, less than 1.6 mm, less than 1.8 mm or less than 2 mm.

[0199] In a preferred embodiment of the present disclosure, he two or more bonded substrates are parallel to each other. In this way, it may be possible to connect structures from one substrate plate to another, thereby forming one complete microfluidic flow cell. The substrates are fabricated individually with structures such as fluid channels and optical sorting chambers in the substrate plane, and through-holes normal to the substrate plane. Once the substrates are bonded the grooves and structures may be closed to form a network of channels and chambers. In these channels it is possible to transport a medium by inducing a flow pressure on the open channel terminals. FIG. 12 shows schematically a stack of four substrates that may be bonded to form a microfluidic flow cell.

[0200] In some embodiments, the hydrodynamic flow focusing device is comprising four bonded parallel substrate plates, 21, 22, 23, 24, wherein four substrate plates are single sided, or wherein two substrate plates are double sided and two substrate plates are lids, or wherein two substrate plates are single sided, one substrate plate is double sided and one plate is a lid.

[0201] An embodiment of the invention can be seen in a micrograph in FIG. 4. From the left there are three inlet channels 31, 32, 33. The sample inlet 33 has a broader section in the far left that acts as a sample chamber. This part tapers to a channel which has a meandering part to disperse the suspended microparticles. The meander is not necessary for operating the invention. FIG. 13 shows another embodiment of a microfluidic flow cell according to the present invention. FIG. 7 and FIG. 8 show a side view of the hydrodynamic flow focusing device, and FIG. 14 is a perspective view of the microfluidic flow cell wherein the sample flow is shown as going from the sample flow inlet 33 and to the waste flow outlet 34. Referring to

[0202] FIG. 7 and FIG. 8, it can be seen that there is a top and a bottom sheath flow inlet, 31 and 32, respectively, and a waste and a selected outlet, 34 and 35, respectively. The sample flow is then hydrodynamically compressed to a sample sheath layer 3 which runs through the optical sorting chamber 4 with a cross section shown in FIG. 15. The optical sorting chamber 4 allows for both detection, analysis and deflection by a laser beam 103 of individual microparticles 1 in the sample sheath 3. This is seen in FIG. 7. The flow is then separated into the ‘waste’ and ‘selected’ outlets, 34 and 35. The arrangement of the outlets is such that the selected outlet 35 is a continuation of the optical sorting chamber 4, i.e. whereas the ‘waste’ outlet 34 is oriented at a 90 degrees angle with respect to the flow direction through the optical sorting chamber 4. This aspect of the embodiment is seen in FIG. 8. This embodiment is more robust to sedimentation of microparticles 1 which improves sorting purity, and is superior to a design where the selected outlet 34 and the waste outlet 35 both forms an angle of 90 degrees with respect to the flow direction through the optical sorting chamber 4 and are oriented normal to the substrate plane.

[0203] Referring to FIG. 1 and FIG. 8, show a possible schematic configuration of a system for sorting microparticles comprising a microfluidic flow cell wherein the optical sorting chamber comprises optical access in the plane of the substrate plates, imaging means having an optical axis 101 normal to the optical access and configured to image a first part of the optical sorting chamber 4, a laser beam 103 having incidence normal to the optical access and configured to target a second part of the optical sorting chamber 4.