BR122017012966B1 - SYSTEM, READIBLE MEDIUM, APPARATUS THAT IDENTIFIES OBJECTS AND METHOD FOR IDENTIFYING OBJECTS THAT FLOW IN A MIXTURE OF SAMPLE FLUID - Google Patents

Abstract

The present invention relates to an apparatus and a method for identifying objects that include: a microfluidic chip which are arranged in a plurality of channels, wherein the microfluidic chip includes: a main fluid channel in which a fluid mixture of sample of objects to be identified is introduced; a plurality of sheath fluid channels into which the sheath fluids are introduced wherein the sheath fluids orient objects in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel; an interrogation apparatus that detects and interrogates objects oriented in the main fluid channel and a focused energy apparatus that performs an action on the objects.

Description

Divided from BR112016009768-8, filed on 06.18.2014. CROSS REFERENCE TO RELATED ORDERS

[001] The present invention claims priority from Provisional Patent Application No. U.S. 61/897,743 filed on October 30, 2013, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION

[002] The present invention relates to a microfluidic system with an interrogation apparatus that detects and searches for objects in a mixture of sample fluid from a microfluidic chip and a focused energy apparatus that performs an action that affects the objects. In one modality, the research apparatus that performs a survey of objects to determine their identity and the focused energy apparatus is a device that acts on targeted objects. In one modality, the focused energy device is used to damage, kill, alter, disable or destroy the targeted objects.

2. DESCRIPTION OF RELATED TECHNIQUE

[003] In the separation of several particles or cellular materials - for example, the separation of sperm into viable sperm and with non-viable sperm motility and without motility or separation by gender - the process is, in general, a time-consuming task, with severe volume restrictions. Therefore, current separation techniques cannot, for example, produce the desired yield or process volumes of cellular materials in a timely manner.

[004] Photo damage laser systems utilize lasers to photodamage or kill unwanted cellular objects. However, the prior art required flow cytometers with the use of nozzles, to search and arrange the individual objects in droplet flow and to try to separate and photodamage the objects as they are covered in several containers - which were difficult to reach.

[005] Thus, there is a need for a method and an apparatus that identifies and discriminates between targeted objects that are continuous and have high yield, is effective in time and cost, and causes negligible or minimal damage to the various targeted objects. Furthermore, such apparatus and method should have additional applicability to other biological and medical areas, not only in discrimination of sperm, but in discrimination of blood and other cellular materials, including viral structures, cell and globular organelles, colloidal suspensions and other materials biological.

SUMMARY OF THE INVENTION

[006] The present invention relates to a microfluidic system with a search device that detects and searches for objects in a mixture of sample fluid from a microfluidic chip and a focused energy device that performs an action that affects the objects. In one modality, the research apparatus that performs a search of objects to determine their identity, and the focused energy apparatus is an apparatus that acts on the targeted objects. In one modality, the focused energy device is used to damage, kill, alter, disable or destroy the targeted objects.

[007] In one embodiment, an object identifying apparatus includes: a microfluidic chip into which a plurality of channels are disposed, including: a main fluid channel into which a fluid sample mixture of objects to be identified is introduced; a plurality of surrounding fluid channels into which the surrounding fluids are introduced, the surrounding fluids orienting objects in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel; a survey device that detects and surveys objects oriented in the main fluid channel and a focused energy device that performs an action on the objects.

[008] In one embodiment, the search apparatus detects and searches the objects to determine information about the objects.

[009] In one modality, the information about the objects determines if the objects are directed by the focused energy device.

[0010] In one modality, the action of the focused energy apparatus acts on the directed objects or a region that surrounds the directed objects.

[0011] In a modality, the action on the targeted objects is to damage, disable, alter, kill or destroy the targeted objects.

[0012] In one embodiment, the apparatus additionally includes at least one outlet channel leading from the main fluid channel, the at least one outlet channel that removes objects from the microfluidic chip.

[0013] In one embodiment, the at least one output channel removes both directed objects and undirected objects from the microfluidic chip.

[0014] In one embodiment, the apparatus further includes a plurality of side outlet channels leading from the main fluid channel, the plurality of side outlet channels disposed on either side of the at least one outlet channel, the plurality of lateral outlet channels that remove the fluids surrounding the microfluidic chip.

[0015] In one embodiment, the plurality of surrounding fluid channels includes: a first plurality of surrounding fluid channels that cross the main fluid channel at a first intersection such that the surrounding fluids compact the sample fluid mixture into at least two sides, so that the sample fluid mixture becomes a relatively smaller, lower transmission connected by the surrounding fluids while maintaining laminar flow in the main fluid channel.

[0016] In one embodiment, the plurality of surrounding fluid channels further includes: a second plurality of surrounding fluid channels that cross the main fluid channel at a second intersection downstream of the first intersection, so that the surrounding fluids of the second intersection The plurality of enveloping fluid channels compress the sample fluid mixture on one of the at least two sides, or on the two sides opposite the at least two sides, so that the sample fluid mixture is further compacted while still maintaining laminar flow in the main fluid channel.

[0017] In one embodiment, when the second set of surrounding fluid channels compresses the sample fluid mixture from at least two sides, the plurality of surrounding fluids further comprises: a third surrounding fluid channel disposed vertically to the main fluid channel at a third intersection and disposed downstream of the second intersection, the enveloping fluid of the third enveloping fluid channel further compresses the sample fluid while still maintaining laminar flow in the main fluid channel.

[0018] In one embodiment, the plurality of surrounding fluid channels hydrodynamically focus on objects such that objects are oriented in a predetermined direction and disposed in a constrained core volume as objects flow through the main fluid channel.

[0019] In one embodiment, the apparatus additionally includes an action chamber in which the search apparatus that performs a search for hydrodynamically focused objects in the sample fluid mixture, the action chamber disposed in the microfluidic chip downstream of the hair. minus one of the second intersection or the third intersection.

[0020] In one embodiment, the research apparatus includes: a light source that emits a beam of light into the action chamber to illuminate and excite objects in the sample fluid mixture.

[0021] In one embodiment, the light beam excites fluorescence in the objects so that the directed objects are distinguished from undirected objects.

[0022] In one embodiment, the light source is a laser.

[0023] In one embodiment, the apparatus further includes an optical signal detector that detects the light beam and converts it into an electronic signal; and a controller, which analyzes the electronic signal to determine whether objects should be steered or not steered.

[0024] In one embodiment, the focused energy apparatus is a laser.

[0025] In one embodiment, the microfluidic chip contains one or more layers or structural planes.

[0026] In one embodiment, the main fluid channel is disposed in a different structural layer or plane than the plurality of sheath channels.

[0027] In one embodiment, the at least one of the sample insertion channel and the plurality of sheath channels are disposed between the structural layers or planes of the microfluidic chip.

[0028] In one embodiment, the first plurality of sheath channels is disposed in a different structural layer or plane than the second plurality of sheath channels.

[0029] In one embodiment, the action chamber includes a first opening cut through at least one of the structural layers or planes on the microfluidic chip, wherein the first opening is configured to receive a first transparent cover.

[0030] In one embodiment, the action chamber includes a second opening cut through at least one of the structural layers or planes on an opposite side of the microfluidic chip from the first opening, wherein the second opening is configured to receive a second transparent cover.

[0031] In one embodiment, the microfluidic chip contains at least one functional layer that includes the plurality of surrounding fluid channels and the main fluid channel and a top layer that contains holes for accessing the at least one functional layer.

[0032] In one embodiment, a size of one of the second plurality of sheath fluid channels is different from the others of the second plurality of sheath channels.

[0033] In one embodiment, the size of the second plurality of sheath channels is different from a size of the first plurality of sheath channels.

[0034] In one embodiment, the apparatus additionally includes a first output disposed at one end of the at least one output channel.

[0035] In one embodiment, the apparatus additionally includes a plurality of outputs disposed at one end of each of the plurality of side output channels.

[0036] In one embodiment, the apparatus additionally includes at least one notch disposed on the microfluidic chip, the at least one notch provided between the outputs.

[0037] In one embodiment, a size of the plurality of side outlet channels increases from a size of the main fluid channel.

[0038] In one embodiment, the main fluid channel tapers into an entry point at the first intersection in the microfluidic chip.

[0039] In one embodiment, the main fluid channel tapers into the action chamber.

[0040] In one embodiment, the second plurality of sheath channels tapers prior to joining the main fluid channel.

[0041] In one embodiment, the second plurality of sheath channels includes at least a first vertical portion that joins the main fluid channel from approximately a right angle above the main fluid channel.

[0042] In one embodiment, the second plurality of sheath channels includes a second vertical portion that joins the main fluid channel from approximately a right angle below the main fluid channel.

[0043] In one embodiment, internal ramps are disposed in at least one of the main fluid channels before the first intersection.

[0044] In one embodiment, the inner ramps are disposed in the main fluid channel before the second intersection.

[0045] In one embodiment, the inner ramps are disposed in at least one of the second plurality of sheath channels.

[0046] In an embodiment, objects are cells.

[0047] In one embodiment, the cells to be actuated by the focused energy apparatus include at least one viable sperm or non-viable sperm motility and non-motility or gender discriminated sperm or other sex-discriminating variations.

[0048] In one embodiment, the cells to be actuated by the focused energy apparatus include: stem cells broken down from cells in a population; one or more sorted cells discriminated from unclassified cells; cells discriminated by desirable or undesirable characteristics; cells discriminated on the basis of surface markers; cells discriminated on the basis of membrane integrity or viability; cells that have genes that are broken down into nuclear DNA according to a specific trait; cells discriminated on the basis of potential or predicted reproductive status; cells discriminated on the basis of an ability to maintain integrity after freezing; cells broken down from contaminants or debris; healthy cells broken down from damaged cells; red blood cells broken down from white blood cells and platelets in a plasma mixture; or any cells discriminated from any other cell objects into corresponding fractions.

[0049] In one embodiment, the laser is one of a pulsed laser of 349 or 355 nm.

[0050] In one embodiment, the laser is a pulsed Q-switch laser capable of delivering 15 ns or smaller pulses of energy to objects at a rate of more than 1000 pulses per second.

[0051] In one embodiment, the laser is a 532 nm laser.

[0052] In one embodiment, the pulsed Q-switch laser preferably delivers 10 ns energy pulses to the objects at a rate of more than 200,000 pulses per second.

[0053] In one embodiment, the focused energy apparatus acts on the objects in a predetermined amount of time after searching the objects.

[0054] In one modality, the focused energy apparatus acts on the objects before searching the objects by the light source.

[0055] In one embodiment, the focused energy apparatus acts on objects when the objects exit at least one outlet before being collected in a container.

[0056] In one embodiment, the apparatus additionally includes a container that collects both targeted and untargeted objects.

[0057] In one embodiment, the apparatus further includes a pumping apparatus that pumps at least one of the sample fluid mixture or the plurality of fluids surrounding the microfluidic chip.

[0058] In one embodiment, the pumping apparatus pumps the at least one of the sample fluid mixture or the plurality of fluids surrounding the microfluidic chip with the use of external tubing.

[0059] In one embodiment, the apparatus further includes: at least one external reservoir holding at least one of the sample fluid mixture or the plurality of surrounding fluids.

[0060] In one embodiment, the apparatus further includes: a microfluidic chip retainer on which the microfluidic chip is mounted, wherein the microfluidic chip retainer includes openings through which the external tubing accesses the microfluidic chip of the at least one external reservoir .

[0061] In one embodiment, the apparatus further includes: a controller that controls the pumping of a one of the sample fluid mixture or the plurality of fluids surrounding the microfluidic chip.

[0062] In one embodiment, the apparatus further includes a plurality of microfluidic chips arranged in parallel, wherein the plurality of microfluidic chips contains a plurality of sample fluid mixtures; where a single research device is used for each of the plurality of microfluidic chips.

[0063] In one embodiment, a computer system identifies objects, including: at least one memory that contains at least one program that includes the steps of: controlling a flow of a sample fluid mixture that contains objects to be identified, through from a main fluid channel of a microfluidic chip; controlling an introduction of a plurality of surrounding fluid channels into the microfluidic chip, the plurality of surrounding fluids orienting objects in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel; and analyze a survey of objects oriented in the main fluid channel with the use of a survey apparatus; and controlling an action on objects with the use of a focused energy device; and a processor that runs the program.

[0064] In one embodiment, a non-transient computer readable medium contains instructions for identifying the objects, including: controlling a flow of a sample fluid mixture containing objects to be identified, through a main fluid channel of a chip microfluidic; controlling an introduction of a plurality of surrounding fluid channels into the microfluidic chip, wherein the plurality of surrounding fluids orients objects in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel; analyze a survey of objects oriented in the main fluid channel using a survey apparatus; and controlling an action on objects with the use of a focused energy device.

[0065] In one embodiment, an object identifying apparatus includes: a microfluidic chip into which a plurality of channels are disposed, including: a main fluid channel into which a fluid sample mixture of objects to be identified is introduced and a plurality of enveloping flow channels that perform at least a three-step hydrodynamic focusing process on objects such that objects are oriented in a predetermined direction as objects flow through the main fluid channel.

[0066] In one embodiment, the plurality of surrounding fluid channels includes: a first plurality of surrounding fluid channels that crosses the main fluid channel at a first intersection to perform a first step of the at least three steps of hydrodynamic focusing, of so that the surrounding fluids compress the sample fluid mixture on at least two sides so that the sample fluid mixture becomes a relatively smaller, lower transmission connected by the surrounding fluids, while maintaining laminar flow in the fluid channel. main.

[0067] In one embodiment, the plurality of surrounding fluid channels further includes: a second plurality of surrounding fluid channels crossing the main fluid channel at a second intersection downstream of the first intersection to perform a second step of the at least three hydrodynamic focusing steps such that the surrounding fluids of the second plurality of surrounding fluid channels further compact the sample fluid mixture on at least two sides so that the sample fluid mixture is further compacted while still maintaining laminar flow in the main fluid channel.

[0068] In one embodiment, the plurality of surrounding fluids further includes: a third surrounding fluid channel disposed vertical to the main fluid channel at a third intersection and disposed downstream of the second intersection to perform a third step of said at least three steps of hydrodynamic focusing, in which the enveloping fluid from the third enveloping fluid channel compresses the sample fluid while still maintaining laminar flow in the main fluid channel.

[0069] In one embodiment, the apparatus further includes: a search apparatus that detects and searches for objects oriented in the main fluid channel and a focused energy apparatus that performs an action on the objects.

[0070] In one embodiment, a method for identifying objects that flow in a sample fluid mixture, includes: flowing a sample fluid mixture that contains objects to be identified, through a main fluid channel of a microfluidic chip; introducing a plurality of surrounding fluid channels into the microfluidic chip, wherein the plurality of surrounding fluids orients objects in the main fluid channel in a predetermined direction while still maintaining laminar flow in the main fluid channel; surveying objects oriented in the main fluid channel with the use of a survey apparatus; and with the use of an object-focused energy device.

[0071] Thus, some features were highlighted in consistency with the present invention so that the detailed description of them below can be better understood and so that the present contribution to the technique can be better appreciated. There are, in fact, additional features consistent with the present invention which will be described below and which will form the subject matter of the claims appended hereto.

[0072] In this regard, before explaining at least one embodiment consistent with the present invention in detail, it should be understood that the invention is not limited in its application to the construction details and provisions of objects presented in the description below or illustrated in the drawings. The methods and apparatus consistent with the present invention are capable of other embodiments and of being practiced and performed in a variety of ways. Furthermore, it is to be understood that the phraseology and terminology employed in this document, as well as the abstract included below, are for the purpose of description and should not be construed as limiting.

[0073] Accordingly, those skilled in the art will appreciate that the design, upon which this disclosure is based, can readily be used as a basis for designing other structures, methods and/or systems to accomplish the various purposes of the present invention. Therefore, it is important that the claims be construed to include such equivalent constructions to the extent that they do not depart from the spirit and scope of the methods and apparatus consistent with the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The objects, features and advantages of the present invention will be more readily evident by reference to the following disclosure when considered in conjunction with the accompanying drawings, in which:

[0075] Figure 1A shows an exploded perspective view of an illustrative embodiment of a single-layer microfluidic chip with a top "raw block" layer, in accordance with an embodiment consistent with the present invention.

[0076] Figure 1B (a) shows an exploded perspective view of an illustrative embodiment of a two-layer microfluidic chip, with the functional layers on top of the bottom layer and on the underside of the top layer, according to further another embodiment consistent with the present invention.

[0077] Figure 1B (b) shows a top view of the bottom layer and the bottom face of the top layer of the modality shown in Figure 1B (a).

[0078] Figure 1C(a) shows an exploded perspective view of an illustrative embodiment of a three-layer microfluidic chip with three functional layers, the top and middle layers having the functional portions on the underside of the layers, according to with yet another embodiment consistent with the present invention.

[0079] Figure 1C (b) shows the illustrative embodiment of three layers of Figure 1C (a), from the opposite perspective, with the layers inverted, showing the underside, the functional layers of the top and middle layers.

[0080] Figure 1D shows an exploded perspective view of an illustrative embodiment of a four-layer microfluidic chip with the top layer being a "rough block" layer, according to yet another embodiment consistent with the present invention.

[0081] Figure 2 shows a top view of an illustrative embodiment of a microfluidic chip, according to an embodiment consistent with the present invention.

[0082] Figure 3A shows a perspective view of the sample and sheath or buffer channels in a two-layer (functional) microfluidic chip, in accordance with an embodiment consistent with the present invention.

[0083] Figure 3B shows a perspective view of the sample and sheath or buffer channels in a single-layer microfluidic chip, according to another embodiment consistent with the present invention.

[0084] Figure 4A shows a perspective view of the sample channel, with tapered and internal ramp, which enters the intersection of the first hydrodynamic focusing region according to an embodiment consistent with the present invention.

[0085] Figure 4B shows a perspective view of the main channel, with tapered and internal ramp, which enters the second region of hydrodynamic focusing, according to an embodiment consistent with the present invention.

[0086] Figure 4C shows a ramp feature in the sample channel before the action chamber, according to an embodiment consistent with the present invention.

[0087] Figure 5 shows an internal cross-sectional view of an illustrative search by a light source of objects flowing in a fluid mixture through the main channel of the microfluidic chip system of Figure 1A, according to a embodiment consistent with the present invention.

[0088] Figure 6A shows a schematic angled side view of object flow in the main channel of a microfluidic chip system, search for discrimination, with focused energy device activation after the search, according to a modality consistent with the present invention.

[0089] Figure 6B shows a schematic inclined side view of the flow of objects in the main channel of a microfluidic chip system, search for discrimination, with activation of the energy device focused on the output channel output, according to another modality consistent with the present invention.

[0090] Figure 6C shows a schematic angled side view of object flow in the main channel of a microfluidic chip system, search for discrimination, with focused energy device activation on a disconnected droplet between output channel output and collection , in accordance with another embodiment consistent with the present invention.

[0091] Figure 6D shows a schematic angled side view of object flow in the main channel of a microfluidic chip system, search for discrimination, with focused energy device activation before the search, according to another modality consistent with the present invention.

[0092] Figure 7 shows a perspective view of the shear force gradient through the main microfluidic channel, in accordance with an embodiment consistent with the present invention.

[0093] Figure 8A shows a cross-sectional view of the main channel of a microfluidic chip, after the first hydrodynamic focusing step, with the sample fluid compacted on the sides of the main channel and objects deviating from the central portion of the main channel.

[0094] Figure 8B shows a cross-sectional view of the main channel of a microfluidic chip, after the second step of hydrodynamic focusing, with the sample fluid compacted above and below the main channel, so that the objects are located substantially from central mode on the main channel.

[0095] Figure 9 shows a cross-section of the main channel of the microfluidic chip, with an object in the center of it, according to an embodiment consistent with the present invention.

[0096] Figure 10 is a diagram showing a flow velocity profile through the main channel of a microfluidic chip, according to an embodiment consistent with the present invention.

[0097] Figure 11 shows a cross-section of the main channel of a microfluidic chip, with an object offset from the center, according to an embodiment consistent with the present invention.

[0098] Figure 12A shows the second step of hydrodynamic focusing, in which the sheath or buffer channels are parallel to the sample channel of a microfluidic chip, above and below, and enter the sample channel from vertical directions, according to with an embodiment consistent with the present invention.

[0099] Figure 12B shows the second hydrodynamic focusing step of Figure 12A, with a sheath or buffer channel of a microfluidic chip that is smaller than the other channel, according to another embodiment consistent with the present invention.

[00100] Figure 13A shows a histogram of sperm cells in a center of the main channel of a microfluidic chip, in accordance with an embodiment consistent with the present invention.

[00101] Figure 13B shows a histogram of sperm cells shifted from a center of the main channel of a microfluidic chip, in accordance with an embodiment consistent with the present invention.

[00102] Figure 14 shows an internal, oblique, perspective view of objects flowing through the microfluidic chip and an illustrative two-stage hydrodynamic focusing operation, with single-sheath or plug fluids entering from an upper portion of the main channel, in accordance with an embodiment consistent with the present invention.

[00103] Figure 15 shows a perspective view of the microfluidic chip and an illustrative two-stage hydrodynamic focusing operation, with envelope or plug fluid channels entering vertically from either an upper or a lower portion of the channel of sample, in accordance with an embodiment consistent with the present invention.

[00104] Figure 16 shows a schematic, angled, side view of the microfluidic chip system with apparatus for research and discrimination and deployment of the beam optical elements from opposite sides of the sample channel, with the collection optical elements shared with the optical elements of focused energy device, in accordance with an embodiment consistent with the present invention.

[00105] Figure 17 shows a schematic, tilted side view of the microfluidic chip system with search and discrimination apparatus, and deployment of the beam optical elements on the same side of the sample channel, with the search and combined focused energy beams through them, or separate optical elements, in accordance with an embodiment consistent with the present invention.

[00106] Figure 18 shows a schematic view of multiple microfluidic chip systems arranged in parallel, with the use of a single research apparatus, according to an embodiment consistent with the present invention.

[00107] Figure 19 shows a schematic view of the flow control network of the microfluidic chip system, with individual external sample reservoirs and surrounding fluids or buffer, according to an embodiment consistent with the present invention.

[00108] Figure 20 shows a schematic view of the flow control network of the microfluidic chip system, with a single sheath or external buffer reservoir and sample reservoir, according to an embodiment consistent with the present invention.

[00109] Figure 21 shows a schematic view of a pressure-regulated network microfluidic chip system with a single sheath or buffer reservoir, in accordance with an embodiment consistent with the present invention.

[00110] Figures 22A and 22B show the front and rear, respectively, of a microfluidic chip retainer having three fluid ports for a functional multilayer microfluidic chip, in accordance with an embodiment consistent with the present invention.

[00111] Figures 23A and 23B show the front and rear, respectively, of a microfluidic chip retainer having four fluid ports for a single-layer microfluidic chip, in accordance with an embodiment consistent with the present invention. DESCRIPTION OF THE INVENTION

[00112] Before returning to the Figures, which illustrate the illustrative modalities in detail, it should be understood that the present disclosure is not limited to the details or methodology presented in the description or illustrated in the Figures. It should also be understood that the terminology is for descriptive purposes only and should not be construed as limiting. An effort has been made to use the same or similar reference numerals in all drawings to refer to the same or similar parts.

[00113] The present invention relates to a microfluidic chip with a search device that detects and searches for objects in a mixture of sample fluid and a focused energy device that performs an action on the objects or a region around the objects. In one embodiment, the survey apparatus that performs an object survey to identify the objects and to determine whether the objects should be targeted by the focused energy apparatus. In one modality, targeted objects are unwanted targeted objects.

[00114] In one modality, the focused energy apparatus is a discrimination apparatus that discriminates between directed and undirected objects through the damage, extermination, alteration, disabling or destruction of the directed objects. The present invention is conducted in a continuous fluid stream flowing within the microfluidic network, in which objects are subjected to hydrodynamic focusing, positioning and orientation, and undirected objects are allowed to flow through the unaltered microfluidic chip and targeted objects they can be actuated, including photodamaged, terminated, altered, disabled or destroyed, by a focused energy device.

APPLICATIONS

[00115] The various modalities of the present invention provide the selection of objects in a fluid mixture, for example: select viable sperm or sperm with non-viable motility and without motility; select sperm by gender, and other variations of selection by sex; select stem cells from cells in a population; select one or more sorted cells from unclassified cells that distinguish desirable/undesirable features; select cells for desirable characteristics; select genes in nuclear DNA in cells, according to a specific trait; select cells based on surface markers; select cells based on membrane integrity (viability), potential or predicted reproductive status (fertility), ability to maintain integrity after freezing, etc.; select cells from contaminants or debris; select healthy cells from damaged cells (ie, cancer cells) (with bone marrow extractions); RBCs from white blood cells and platelets in a plasma mixture and select any cells from any other cell objects in corresponding fractions; select damaged cells or contaminants or debris or any other biological materials that one wishes to undergo discrimination. Objects can be cells or microspheres treated or coated with binding molecules or embedded with a fluorescent or luminescent label molecule(s). Objects can have a variety of physical or chemical attributes such as size, shape, materials, texture, etc.

[00116] In one embodiment, a heterogeneous population of objects can be measured, with each object being scanned for different quantities or regimes in similar quantities (eg, multiplex measurements) or objects can be scanned and distinguished based on a marker (eg, fluorescent), image (due to size, shape, different absorption, scattering, fluorescence, luminescence characteristics, fluorescent or luminescence emission profiles, fluorescent or luminescent decreasing lifetime) and/or particle position, etc.

[00117] In addition, the subject matter of the present disclosure is also suitable for other medical applications. For example, the various laminar flows discussed below can be used as part of a kidney dialysis process, in which whole blood is cleaned from waste and returned to the patient. Additionally, the various modalities of the present disclosure may have additional applicability to other biological or medical areas, such as for selection of cells, viruses, bacteria, organelles or cell subparts, globular structures, colloidal suspensions, lipid and lipid globules, gels, immiscible particles , blastomeres, cell aggregations, microorganisms and other biological materials. For example, object selection in accordance with the present disclosure may include cell "washing", in which contaminants (such as bacteria) are removed from cell suspensions, which may be particularly useful in food and medical industry applications. Additionally, the present invention has applicability to select non-motile cellular objects from motile cellular objects.

[00118] The subject matter of the present disclosure can also be used to transfer a species from one solution to another solution, where separation by filtration or centrifugation is not practical or desirable. In addition to the applications discussed above, additional applications include selecting colloids of a certain size from colloids of other sizes (for research or commercial applications) and washing particles such as cells, eggs, etc. (effectively replacing the medium in which they are contained and removing contaminants) or washing particles, such as nanotubes of a salt solution and surfactants with a different salt concentration or without surfactants, for example.

[00119] The action of selecting species may depend on various physical properties of the objects or objects that include automotility, self-diffusion, free fall velocity or action under an external force such as an actuator, an electromagnetic field or a holographic optical container. Properties that can be selected include, for example, cell motility, cell viability, object size, object mass, object density, tendency of objects to attract or repel each other or other objects in the flow, object, object surface chemistry, and the tendency of certain other objects (ie molecules) to adhere to the object.

[00120] Although the discussion below focuses on the identification and selection of viable sperm or with non-viable and non-motile sperm motility, or selecting sperm by gender and other selection variations by sex, or selecting one or more sorted cells of cells unclassified that distinguish desirable/undesirable characteristics, etc., the apparatus, methods and systems of the present invention may extend to other types of particulate, biological or cellular matter, which are capable of being searched by fluorescence techniques within a fluid flow or capable of being manipulated between different fluid flows in one or more outlets.

SAMPLE PREPARATION:

[00121] In one embodiment, a concentration of objects 160, such as cells (ie, raw semen), is determined using a cell counting device such as a Nucleocounter. In one embodiment, the appropriate dye volume is obtained (ie using dye from a calculator spreadsheet) and the dye TALP volume and objects 160 (ie, pure semen) that need to be added for a concentration of predetermined cell (ie, sperm concentration of 200 x 106/ml) are calculated. For example, a quantity of 1 ml of stained sample is prepared when the concentration of pure semen = 1500 x 106/ml. Thus, 200 x 106/ml/1500 x 106/ml = 0.133ml pure semen, which is added to (in order) 0.012 ml of Hoechst 33342 (5 mg/ml raw material solution) and 0.855 of dye TALP (pH 7.4), to equalize dye volume from 1 ml total to 200 x 106/ml.

[00122] In one embodiment, dye TALP is prepared by filling a container (ie beaker) with Milli-Q water to 2/3 of the total desired volume. A stir bar and stir plate are used to mix the solution as chemicals are added. The chemicals, which are added in the order listed (up to Gentamicin, which is added later), include: TABLE DYE CHEMICAL COMPONENTS

[00123] In one modality, after proper mixing of the chemicals, the pH is adjusted to 7.4 with the use of NaOH. Additional Milli-Q water is used to bring the solution to a final volume in a container (ie, volumetric flask). A sterile filter (ie, 0.22 sterile filter) is used to filter/sterilize the volume. An antibiotic (ie Gentamicin solution) is added after filtration and the volume of dye TALP is stored at 5 °C and can be used for 7 to 10 days.

[00124] In this way, after the sample volume is dyed with dye TALP, in one modality, the 120 dyed samples are placed in containers (ie tubes) in a water bath set at 34 to 35 °C and incubated for a predetermined time (ie 45 minutes). In one embodiment, after incubation, stained samples were removed from the water bath and an equal volume of 4.0% egg yolk TALP with red food coloring that was heated in the water bath set to 34 to 35 °C , is added.

[00125] To obtain 4% TALP of egg yolk with red food coloring, the dye TALP as noted above is prepared and a desired volume of final solution is determined. The volume of dyeing TALP and egg yolk required to prepare a 4% egg yolk solution is calculated as follows:

[00126] The desired volume: (250 ml) x 0.04 = 10 ml of egg yolk required for 4% solution. 240 ml of dye TALP is added to a graduated container (ie cylinder) and 10 ml of egg yolk is added. FD&C #40 food coloring is added to the container (ie cylinder) to obtain: 0.261 ml/100 ml of solution. With a total desired volume of 250 ml, 0.261 ml x 250 ml / 100 = 0.653 ml of food color Red. The container (ie cylinder) is covered with parafilm and carefully inverted until the volume is completely mixed. The container volume is then allowed to rest overnight and be cooled in a cool environment. The bulking vessel is then carefully decanted into a sterile vessel, leaving any sediment at the bottom of the bulking vessel. The volume is then filtered/sterilized through a 0.22 µm bottle top filter and an appropriate amount of antibiotic solution is added (ie, 0.250 ml of Gentamicin/100 ml of egg yolk TALP) .

[00127] Thus, after adding an equal volume of 4% TALP egg yolk with red food coloring to the 120 dyed sample, the 120 dyed samples are filtered by pouring the 120 samples into a 20 micron filter (this is, CellTrics filter), which is extracted into another sterile 5 ml culture vessel. After a predetermined dyeing time (ie 45 minutes), an equal volume of 4% TALP egg yolk is added. The 120-stained sample (ie, cells) is passed through a filter (ie, Partec filter, 50 micron mesh) and the sample is placed in a 233 sample retainer or reservoir for introduction into the 100 microfluidic chip (see Figures 19 to 21).

[00128] In one modality, the final sperm concentration = 100 x 106/ml and the percentage of final egg yolk = 2%. A new 120 sample aliquot can be prepared and used every hour is desired.

MICROFLUIDIC CHIP SYSTEM

[00129] The various modalities of the microfluidic chip, as described below, use one or more flow channels, which have a plurality of substantially laminar flows, which allows one or more objects to be searched for identification by a search device and to be actuated by a focused energy device, with objects exiting the microfluidic chip at one or more outlets. In one modality, objects not directed by the focused energy apparatus are unaltered and the focused energy apparatus, photodamage, alters, disables, kills or destroys the directed objects.

[00130] The various embodiments of the present invention thus provide selection of objects on a continuous basis, such as within a continuous closed system without the potential damage and contamination of prior art methods, particularly as provided in sperm separation. The continuous process of the present invention also provides significant time savings when selecting and discriminating objects.

[00131] Although the present matter is discussed in detail in relation to a microfluidic chip 100 illustrated in Figures 1A to 2 and a microfluidic chip retainer 200 illustrated in Figures 20 to 21, it should be understood that this discussion applies equally to several others modalities discussed in this document or any variation thereof.

MICROFLUIDIC CHIP

[00132] Figure 1A is an illustrative embodiment of a microfluidic chip 100. The microfluidic chip 100 is manufactured from a suitable material such as glass or a thermoplastic (for example, low autofluorescence polymer etc.) or combination of materials, through of an etching process, soft photolithography or injection molding process, also known to a person of ordinary skill in the art and is of suitable size. Each layer may be of any suitable thickness, for example the thickness may be within a range of approximately 300 to 400 µm and more preferably the thickness may be approximately 400 µm.

[00133] The microfluidic chip 100 includes one or more structural layers in which microchannels are disposed that serve as sample insertion channel(s), buffer or envelope fluid channel(s), exit channel(s), etc. The microchannels are of adequate size to accommodate laminar flow streams that contain objects and can be arranged in any of the layers of the chip 100 in the appropriate length, as long as the object of the present invention is realized. In one embodiment, the dimensions of the microfluidic channels are in the range of 50 microns to 500 microns, with 100 to 300 microns being preferably used to prevent clogging.

[00134] The desired flow rate through the microfluidic chip 100 can be controlled by a predetermined inlet flow rate in the chip 100, which maintains the appropriate microchannel dimensions within the chip 100, by pumping mechanisms that pump external fluids into the chip. 100 and by providing narrowing or tapering of the microchannels at various locations and/or by providing obstacles, ramps or dividers within the microchannels (discussed further below).

[00135] More specifically, a plurality of inputs is provided on the microfluidic chip 100, where the inputs provide access to the microchannels/channels. In one embodiment, as shown in Figures 1A to 2, a sample inlet 106 is used to introduce a sample of particles or objects (i.e., cells) 160 into a mixture of sample fluid 120 in a main fluid channel 164 of the microfluidic chip 100 from at least one reservoir source (see Figure 19).

[00136] The microfluidic chip 100 also includes at least one sheath or buffer inlet for introducing surrounding fluids or buffer. In one embodiment, there are two sheath or buffer inlets on microfluidic chip 100, which include a sheath or buffer inlet 107 and sheath or buffer inlet 108, both disposed proximate to sample inlet 106 and both of which introduce fluid. envelopes or buffer 163 on the microfluidic chip 100 (see Figures 1A through 3B).

[00137] In one embodiment, there are three sheath or buffer entries 107, 108 and 172 (see Figure 1A and 3B) that introduce sheath or buffer fluids into channel 164 of microfluidic chip 100. The location of sheath or sheath entries of buffer 107, 108, 172 may vary and they may access channels in chip 100 that are in the same or different structural layers. In one modality, enclosing fluids or plug 163 are introduced into inlets 107, 108, 172 of a common reservoir (see Figures 20 through 21) or in another modality, of separate reservoirs (see Figure 19).

[00138] Surrounding or buffer fluids are well known in the microfluidic art and in one embodiment, may contain nutrients well known in the art to maintain the viability of objects 160 (i.e. sperm cells) in the fluid mixture. Commercially available Tris, as available from Chata Biosystems, is an example, and the buffer fluid or 163 can be formulated to include the following:

[00139] Water - 0.9712 L; Tris - 23.88 g; citric acid monohydrate - 11.63 g; D-fructose - 8.55 g. The pH is adjusted to 6.80 + 0.05 with hydrochloric acid and the osmolarity is adjusted, if necessary, to 270 to 276 mOsm with high fructose purity. The mixture is filtered using a 0.22 micron filter.

[00140] The microfluidic chip 100 may have one or more structural layers in which the microchannels are arranged. Channels can be arranged in one or more layers or between layers. The following modalities describe a bonding process, but an individual of ordinary skill in the art would know how to achieve the various features using an injection molding process. For example, in injection molding, instead of forming two layers, two molds could be produced and joined so that an injection is carried out in the cavity in order to obtain the chip of the present invention.

[00141] In one embodiment, as shown in Figure 1A, a structural layer 101 is included on the microfluidic chip, with a top "raw block" plastic layer 104 disposed thereon. The top "rough block" layer 104 connects to the functional layer 101 to form a closed microfluidic network and may have multiple holes to provide access to the lower layer(s) of the chip 100. For example, the top "rough block" layer 104 may have holes corresponding to inputs 106, 107, 108, 172, etc., or provide holes 145 for pins to secure layers 101, 102, 104, etc., of chip 100 together. In one embodiment, top layer 104 of microfluidic chip 100 includes a plurality of apertures configured to align with fits in a microfluidic chip retainer 200 (further described below).

[00142] In another embodiment, as shown in Figure 1B(a), two structural, functional plastic layers 101-102 are included in the microfluidic chip 100, with no top "raw block" layer. In this embodiment, the functional side of top layer 102 is disposed on the underside of layer 102 so that when the layers are placed together, channels 114, 115, 116, 117 are formed (see Figure 1B(b)).

[00143] In yet another embodiment, as shown in Figure 1C(a), three functional structural layers 101, 102, 103 are used in the microfluidic chip 100. As in Figure 1B(a), layers 102, 103 include functional sides at the lower portions of layers 102, 103, with layers 101 and 102 which form channels 116, 117 when placed together. Layer 103 has channels 114, 115 disposed on the underside of layer 103 (see Figure 1C(b)).

[00144] In yet another embodiment, as shown in Figure 1D, four structural plastic layers 101 to 103 and a top "rough block" layer 104 are used in microfluidic chip 100. In this embodiment, layer 102 includes 114, 115 and layers 101 and 103 each include one of channels 117, 116, respectively.

[00145] However, an individual of ordinary skill in the art would know that more or a few structural layers with functional sides and with or without "rough-block" layers, can be used and channels can be arranged in any of the structural layers, or in different structural layers and, in any arrangement, with access to those channels through a top "rough block" layer, provided that the object of the present invention is achieved.

[00146] In one embodiment, a sample fluid mixture 120 including objects 160, is introduced to the sample inlet 106 and the fluid mixture 120 flows through main channel 164 towards the action chamber 129 (see Figures 1A through two). Sheath or buffer fluids 163 are introduced to sheath or buffer inlets 107, 108 (see Figures 1A through 2) in most embodiments and to sheath or buffer inlets 107, 108, and 172 in another modality (see Figures 1A through 2) Figure 1A). Surrounding or plug fluids 163 flow through channels 114, 115 and 116, 117 in main channel 164 and towards action chamber 129 before flowing out through at least outlet channels 140 and 142 in laminar flow.

[00147] In one embodiment, the mixture of fluid 120 of main channel 164 joins with the surrounding fluids or buffer 163 of channels 114, 115 at intersection 161 of microfluidic chip 100. In one embodiment, the fluids of buffer 163 of channels 116, 117 join with the blend of combined fluid 120 and surrounding fluids or buffer 163 from the first intersection 161, downstream at the second intersection 162 (see Figures 1A through 2). In one embodiment, surrounding or plug fluids 163 are inserted through inlet 172 into main fluid channel 164, downstream of second intersection 162 (see Figures 1A and 3B).

[00148] In one embodiment, channels 114, 115 have substantially the same dimensions as channels 116, 117, as long as the desired flow rate(s) is achieved to accomplish the objective of the present invention, but an individual of ordinary skill in the technique would know that dimensions can be different as long as they achieve the desired results (discussed further below).

[00149] In one embodiment, channels 114 to 117 and 140 to 142 may have substantially the same dimensions, however, an individual of ordinary skill in the art would know that the size of any or all of the channels on the microfluidic chip 100 may vary. in dimension (for example, between 50 and 500 microns), as long as the desired flow rate(s) is achieved to accomplish the object of the present invention.

[00150] In an exemplary embodiment, channels 114, 115 or 116, 117 are arranged in the same layer or structural plane of microfluidic chip 100 as the layer or plane in which channel 164 is disposed (see Figure 1A, for example) or it can be arranged on a different layer or structural plane (see Figure 1B, for example). In another embodiment, input channel 164 and sheath channels 114, 115 or 116, 117 may be disposed between structural layers or planes of chip 100. In that way, one of ordinary skill in the art would know that channels 114 through 117, 164 and 140 to 142, etc. can be arranged in any layer or between any two layers. Additionally, although channels 114 to 117, 164 and 140 to 142, etc. are described in exemplary embodiments, as shown in the Figures, one of ordinary skill in the art would know that the particular arrangement or layout of channels on chip 100 can be of any desired arrangement as long as they achieve the described features of the present invention.

In one embodiment, channels 116, 117 are cut through layer 101 (see Figures 1A and 2) and join the fluid mixture 120 in channel 164 in the same plane through holes cut through the layers. In one embodiment, channels 116, 117 are substantially parallel to inlet channel 164 and to each junction intersection 161 at an angle to channel 164 (see Figures 2 to 3). The surrounding or plugging fluids of channels 116, 117 compact the fluid mixture 120 flowing horizontally from the sides, or laterally, so that the objects 160 in the fluid mixture 120 are flattened and/or oriented in a selected or desired direction, while still maintaining laminar flow in channel 164 (ie, the first in two steps of hydrodynamic focusing, as further described below).

[00152] Additionally, in one modality, channels 114, 115 join the fluid mixture 120 in channel 164 at intersection 162, with each channel 114, 115 at an angle of channel 164 (see Figure 1A). The enclosing or plugging fluids of channels 114, 115 compress the fluid mixture flow 120 relative to the channel 164 (see Figure 14), so that objects 160 in the fluid mixture 120 are further flattened and/or oriented in the selected or desired direction, while still maintaining laminar flow in channel 164 (ie, the second in two steps of hydrodynamic focusing, as further described below).

[00153] In addition to this modality, a third surrounding fluid or plug inlet 172 is disposed downstream of the intersection 162 (see Figure 3B), which allows a third hydrodynamic focusing step to occur, in which the fluid mixture from sample 120 is compacted from above channel 164 by the surrounding fluids or buffer 163 introduced therein.

[00154] In an alternative embodiment, after the first hydrodynamic focusing step described above, channels 114, 115 join the fluid mixture 120 in channel 164 at intersection 162 from an angle from above (see Figure 3A) - and may be above or below (see Figure 12A) - channel 164, to compact the fluid mixture 120 from a vertical direction to further flatten and/or orient objects 160 in channel 164 (i.e., the second step of hydrodynamic focusing).

[00155] However, an individual of ordinary skill in the art would verify the depicted configurations, angles and structural arrangements of the 100 microfluidic chip sheath or plug inlets, sample inlet and sample insertion channel, and sheath or sheath channels. buffer, as well as the hydrodynamic focusing steps, can be different as long as they achieve the desired features of the present invention.

[00156] In one embodiment, as shown in Figure 2, channels 114, 115 and 116, 117 are depicted as partially coaxial to each other with a center point defined by sample input 106. Thus, in one embodiment, the channels 114, 115 and 116, 117 are arranged in a substantially parallel arrangement, with channels 114, 115 and 116, 117 being equidistant from main channel 164. However, one of ordinary skill in the art would recognize that the depicted configuration may be different as long as it achieves the desired features of the present invention.

[00157] In one embodiment, the holes and pins/stops 145 are arranged in several convenient positions in layers 101, 102, 103, 104 etc. to secure and align the multiple layers during chip 100 fabrication.

[00158] In one embodiment, a gasket 105 of any desired shape, or O-rings, can be provided to maintain a hermetic seal between the microfluidic chip 100 and the microfluidic chip retainer 200 (see Figures 1D and 21 to 22, by example). In the case of a gasket 105, it can be a single sheet or a plurality of objects, in any configuration, or material (i.e., rubber, silicone, etc.), as desired. In one embodiment, as shown in Figure 1D, a first gasket 105 is disposed at one end of the microfluidic chip 100 and interfaces with or is bonded to layer 104. A plurality of holes 144 are provided in the first gasket 105 and are configured to align. with sample inlet 106, sheath/buffer inlet 107 and sheath/buffer inlet 108.

[00159] In one embodiment, a second gasket 143 may be disposed at the other end of the microfluidic chip 100 opposite the first gasket 105 (see Figure 1D, for example) and interfaces or is bonded to the (using epoxy) layer top structural 104 (see Figures 1D and 21 through 22).

[00160] In one embodiment, O-rings are used in place of gaskets to aid in sealing as well as to stabilize the microfluidic chip 100 in the chip retainer 200.

[00161] However, an individual of ordinary skill in the art would know that one or more gaskets or O-rings can be applied to the outer layers of chip 100 in order to protect chip 100 in a chip retainer 200 during chip operation. .

[00162] In one embodiment, channels 114 to 117 and 140 to 142 of the microfluidic chip 100 may not only vary in size, but may have tapered shapes at entry points to other channels on the chip 100 in order to control the flow of fluid through the channels. For example, the main channel 164 can taper at the entry point at intersection 161 (see Figure 4A, taper 166A) or at the entry point at intersection 162 (see Figure 4B, taper 166A) to control and accelerate the flow of sample 120 at intersection 161 and allowing the surrounding or buffer fluids 163 of channels 116, 117 or 114, 115, respectively, to compress the sample fluid mixture 120 in a first direction (i.e., horizontally or laterally) into at least two sides, if not, on all sides (depending on where fluid channel 164 enters intersection 161) and in a second direction (ie, vertically) (see Figures 3A to 3B, 4A to 4B and taper 166A).

[00163] In another embodiment, the ramps may be arranged in channel 164 or channels 114 to 117 to achieve the effect of controlling and accelerating the sample flow through the channels. Ramps can be in addition to, or instead of, bottlenecks.

[00164] For example, a ramp 166B may be disposed on channel 164 before the sample stream approaches intersections 161 and 162 respectively, or before entering action chamber 129 (see Figures 4A and 4B).

[00165] In this way, the sample fluid mixture 120 becomes a relatively minor transmission, inferior, bound or enveloped by surrounding fluids or plug 163, while maintaining laminar flow in channel 164. However, an individual of common skill in the The technique would know that the main channel 164, or the buffer channels 114 to 117 can be of any physical arrangement, such as a rectangular or circular shaped channel, with tapers, ramps or other internal features, provided that the purpose of the present invention is achieved. .

[00166] In one embodiment, a plurality of output channels coming from the main channel 164 (see Figure 2) are provided for removing fluid flowing through the microfluidic chip 100, including any directed or undirected objects 160 and/or fluids envelopes or plug 163. In one embodiment, as shown in Figures 1A to 2, there are three output channels 140 to 142 that include a left-hand output channel 140, a center output channel 141, and a left-hand output channel 141. right side 142. Left side output channel 140 ends in a first output 111, center output channel 141 ends in a second output 112, and right side output channel 142 ends in a third output 113. it is possible to have only one output channel 141 and output 112.

[00167] In one embodiment, output channels 140 to 142 separate from channel 164 within chamber 129 to outputs 111 to 113. In one embodiment, the cross-section and length of output channels 140 to 142 must be maintained at a predetermined volume ratio (ie 2: 1:2 or 1:2: 1 etc.) to obtain the desired hydraulic resistance of output channels 140 to 142.

[00168] In one embodiment, the output channels 140-142 increase in size from the channel 164, leaving the chamber 129, so that the output ratio for objects 160 is increased through the relevant channel 141.

[00169] In one embodiment, instead of a straight edge, where necessary, a plurality of notches or recesses 146 may be disposed on a bottom edge of the microfluidic chip 100 to separate the outputs (i.e., outputs 111 to 113) and for attaching containers and external piping (to recycle surrounding fluids or plug 163 - see Figures 19 to 21) etc. First output 111, second output 112, and third output 113 are reached via output channels 140 to 142 that originate from action chamber 129 (see Figure 2).

[00170] In one embodiment, a container 188 collects the objects 160 from the second outlet 112, although other containers may collect the output from the first outlet 111 and the third outlet 113 (see Figures 6A to 6D). In one embodiment, portions of the first, second, and third outputs 111 to 113 may be electronically characterized to detect object concentrations 160, pH measurement, cell count 160, electrolyte concentration, etc.

[00171] In one embodiment, the targeted objects 160 are actuated by the focused energy apparatus 157 and these objects 160, as well as non-targeted objects 160, can be collected as product 165 of the second output 112.

[00172] In one embodiment, the product 165 of targeted and untargeted objects 160 may continue to be processed for storage, for further separation, or for processing such as cryopreservation (discussed further below).

[00173] In one embodiment, the microfluidic chip 100 is provided in a sterile state and can be prepared with one or more solutions (i.e., buffer fluids or 163), or purged of any fluids or materials, or by draining the chip. microfluidic 100 or by flowing surrounding fluids or buffer 153 or other solutions through the microfluidic chip 100, in accordance with known methods.

ACTION CHAMBER

[00174] In one embodiment, downstream of intersection 162, objects 160 in fluid mixture 120 flow through channel 164 into an action chamber 129, in which objects 160 are searched and actuated. In one embodiment, channel 164 tapers into chamber 129 (see Figure 4B), which accelerates the flow of the fluid mixture through chamber 129. However, one of ordinary skill in the art would know that channel 164 would not need to. taper and could be of any dimension and size as long as the present invention is carried out in accordance with the desired requirements.

[00175] In one embodiment, a search apparatus 147 is used to search and identify objects 160 in the fluid mixture in channel 164 passing through chamber 129. Additionally, in one embodiment, focused energy device 157 also acts on objects 160 passing through chamber 129.

[00176] In one embodiment, chamber 129 includes a relatively small diameter aperture or window 150 (see Figure 5) cut through the microfluidic chip 100 and layers 101 to 102, through which objects 160 can be viewed as they pass through channel 164.

[00177] Additionally, a shallow and relatively larger diameter opening is cut in layer 104 as a top window, and in layer 101 as a bottom window. In one embodiment, the top window is configured to receive a first transparent cover 133 and the bottom window 152 is configured to receive a second transparent cover 132. Covers 133, 132 can be produced from any material with the requirements of transmission desired such as plastic, glass or can even be a lens. In another embodiment, instead of a window with coverings, a continuous plastic sheet can be used. Note that although the relative diameters of covers 132, 133 and opening are shown in Figure 5, they may vary based on design or manufacturing considerations.

[00178] In one embodiment, the above-mentioned first and second covers 133, 132 are configured to close the chamber 129. The windows and covers 133, 132 (see Figure 5), allow objects 160 to flow in the mixture of fluid 120 in channel 164 through chamber 129, are viewed through aperture 150 and actuated by a suitable light source 147 and focused energy apparatus 167 (discussed later).

[00179] In one embodiment, windows and/or openings are not required due to the chip layer structure (i.e., glass) and/or its configuration, or the focused energy device 157 and the light source wavelengths 147 and power levels are such that no damage to chip 100 will occur.

RESEARCH DEVICE

[00180] The research apparatus of the present invention includes a light source 147 that is configured to emit a high intensity beam 148 with any wavelength that corresponds to excitable objects in the fluid mixture 120 (see Figure 5). While a laser 147 is preferred, any other suitable light sources 147 can be used, such as a light emitting diode (LED) or arc lamp, etc. to emit a beam that excites the objects.

[00181] In one embodiment, such a high intensity laser beam 148 of a suitable laser 147 of a pre-selected wavelength - for example, a continuous wave of 349 nm or 355 nm (CW), or pulsed laser of near 147 CW - is required to excite objects 160 in fluid mixture (ie sperm cells). In another modality, a 532 nm 147 green laser is used.

[00182] In one embodiment, laser 147 (see Figure 5) emits a laser beam 148 through cover 133 on an uppermost portion of chip 100, through aperture 150, to illuminate objects 160 flowing through the channel. 164 into chamber 129 of chip 100 and then through cover 132 into layer 101 of chip 100.

[00183] In one embodiment, the light beam 148 can be distributed to the objects 160 by an optical fiber that is embedded in the microfluidic chip 100 at the aperture 150.

[00184] The high intensity beam 148 interacts with the objects 160 (see detailed explanation below) and passes through the first covers 133, to exit the cover 132 in the bottom window, so that the emitted light 151, which is induced by the beam 148, is received by an objective lens 153 or other optical collection elements. The objective lens 153 or other optical collection elements can be arranged in any suitable position relative to the microfluidic chip 100 - for example, parallel to the main channel with the optical axis perpendicular to the sample fluid flow 120. Due to the fact that the chamber 129 is sealed by first and second covers 133, 132, high intensity beam 148 does not impinge on microfluidic chip 100 and damage layers 101,104 (see Figure 1A). In this way, the first and second covers 133, 132 help to prevent damage to the microfluidic chip 100 from the high intensity beam 148 and photonic noise induced from the microfluidic chip material 100 (ie, plastic).

[00185] In one embodiment, the light beam 148 passes through the chip 100 and the emitted light 151 received by the objective lens 153 or other optical collection elements, is detected by the detector 154 and is converted into an electronic signal by an optical sensor 154, such as a photomultiplier tube (PMT) or photodiode, etc. The electronic signal can be digitized or processed by an analog to digital converter (ADC) 155 and sent to a controller 156 based digital signal processor (DSP) or computer. The electronic controller 156 can be any electronic processor with adequate processing power, such as a DSP, a Microcontroller Unit (MCU), a Field Programmable Port Array (FPGA), or even a Central Processing Unit (CPU).

[00186] In one embodiment, the DSP-based controller 156 monitors the electronic signal and, based on predetermined criteria, the focused energy apparatus 157 can be employed when a directed object 160 is detected.

[00187] However, in another embodiment, the survey apparatus simply searches the objects 160 in the sample fluid flow 120 for identity and is not connected to employing the focused energy apparatus 157 (see Figure 6C).

FOCUSED ENERGY APPLIANCE

[00188] In one embodiment, in order to deliver a desired energy level to the objects 160, a focused energy apparatus 157 is used to supply focused energy pulses to the objects 160. The focused energy apparatus 157 may be a device thermal, electrical, optical, or electromagnetic 157, which would have a desired wavelength and deliver high peak power with a very high repetition rate (or pulse frequency) to the targeted objects 160.

[00189] In one embodiment, the focused energy apparatus 157 is activated at a predetermined time (i.e., milliseconds) after activation by the controller 156 (where the timing is defined based on the object trajectory speed 160 through the channel 164, and is discussed further below), and emits a pulse to the selected object or target 160 (ie, unwanted).

[00190] Examples of pulsed 157 lasers include locked mode, Q switch, as well as those lasers using both lock mode and Q switch techniques. For example, a 157 focused energy device such as an Avia 3555-100 (available from Coherent, Inc., Santa Clara, CA) or the Q Explorer XP switch laser lasers, available from Spectra-Physics Inc., are capable of operating in a pulse-on-demand mode and can deliver pulses of power from 15 ns or less, at a rate of more than 1,000 pulses per second, for the 160 targeted objects.

[00191] In one embodiment, pulse energy levels from 0.5 to 8.0 µJ are used and in a preferred mode, a Q 157 switch laser in pulse-on-demand mode is used to deliver an average pulse energy 1.8 μJ with a range for individual pulses from 1.3 μJ to 2.3 μJ. In one embodiment, the pulse width ranges from 3 nanoseconds to 1 microsecond, and preferably is in a range of 5 to 9 nanoseconds. However, a person of ordinary skill in the art would know that any high power laser currently available or later developed, with the appropriate high energy pulses and pulse frequency, would be suitable for the present invention in order to achieve accuracy and/or desired target effect.

[00192] In one embodiment, the need for a hermetic action region (i.e., chamber 129, or space between chip 100 and container 188) in order to distribute pulsed energy from focused energy apparatus 157 to targeted objects 160 or a region around them, it is important to minimize the potential impact of distributing energy outside the targeted objects 160 or region, to otherwise untargeted or unselected objects 160. For example, an apparatus Focused energy 157 like Q Explorer XP 355-1 laser switch, has the ability to deliver <4 rms, provide pulse stability for high pulse when fired at even, regular intervals.

[00193] However, for flow cytometric analysis and action systems where objects 160 or cells enter the action region (ie chamber 129, or space between chip 100 and container 188 - see Figures 6A to D) at non-uniform intervals, additional measurements are employed to distribute uniform pulse energy 158 to impinge only on the 160 targeted objects or cells, or the region around them. Such measurements include matching laser performance parameters 157, such as pulse length and peak power levels to enable the system of the present invention to achieve targeted target accuracy (i.e., in one modality, photodamage or kill rate of 95% or higher impact rate on targeted objects 160).

[00194] In addition, additional tuning with pulse-on-demand operation and laser performance 157 to deliver extremely high pulse stability to pulse when fired at non-uniform intervals greatly reduces the spatial variability of the area impacted by pulse 158. , by reducing pulse-to-pulse variability in the focused energy device 157, unintended action, damage or destruction to untargeted objects 160 or cells, is greatly reduced, achieving, for example, a viability rate of 85% or more for 160 untargeted objects or active cells.

[00195] In one modality, the focused energy apparatus 157 is used in an action region 129, such as the camera 129, prior to the search by the research apparatus 157 (see Figure 6D), and in another modality, the focused energy 157 is used in the action region (ie chamber 129) after the search by searcher 157 (see Figure 6A). In yet another embodiment, the focused energy apparatus 157 acts on the sample fluid with objects 160 after it exits the chip 100 and enters a container 188 - either outlet 112, or in the form of a disconnected droplet 187 before entering the container 188 (see Figures 6B to C).

[00196] In the modality in which the focused energy apparatus 157 acts on the objects 160 after they are searched by the research apparatus 147 in the action region 129 (i.e., chamber 129), by determining that the objects 160 must be directed, the focused energy apparatus 157 emits a focused energy beam 158 to act on objects 160 flowing through channel 164 (see Figures 5 and 6A to 6C, for example).

[00197] In the modality, the focused energy apparatus 157 acts on the objects 160 after searching the action region 129 and after the objects 160 flow through the outlet channel 141, but before the sample fluid 120 is collected by the container 188. In this embodiment, focused energy apparatus 157 is used as mentioned above, but is positioned to beam the beam between chip 100 and container 188. In one embodiment, sample fluid 120 simply falls from outlet 112 through the air in the container. 188 in drop form 187 and, in another embodiment, there may be a transparent casing between the chip 100 and the container 188.

[00198] The focused energy apparatus 157 can be adjusted to damage, alter, disable, kill or destroy the targeted or unwanted object 160 in the sample fluid 120 with a pulse or to activate one of several mechanisms in the object 160 or cell, so that cell damage or death results.

[00199] However, depending on the desired arrangement (see additionally below for various modalities), the targeted or selected objects 160 may be desired objects 160, in which the focused energy apparatus 157 is not activated or triggered, or the targeted objects or selected 160 may be unwanted objects 160, wherein the focused energy apparatus 167 is actuated on the objects 160, such as to damage, alter, disable, kill or destroy the selected, unwanted objects 160. However, these are not the only modalities and the various modalities are discussed further below.

[00200] In one embodiment, when the selected object 160 is damaged, altered, disabled, killed or destroyed by the focused energy device 157, the object 160 continues to flow through the main channel 164 to the central output channel 141 and to the second outlet 112 and into container 188, along with any undirected objects 160. Enclosing or plugging fluids 163 proceeded in laminar flow through outlet channels 140, 142 to outlets 111, 112, respectively.

[00201] However, in one embodiment as noted above, objects 160 on channel 164 may flow out of chip 100 through output channel 141 and single output 112.

[00202] Accordingly, in one embodiment, the present methods and apparatus are capable of producing a discriminated product 165 (see Figures 6A to 6D) of objects 160 in container 188, including a high feasibility of undirected or desired objects 160 or cells and a high percentage of targeted objects photodamaged, altered, disabled, destroyed or killed 160.

CONFORMATION AND OPTICAL BEAM ELEMENTS

[00203] In order to achieve satisfactory signal repetition and effective damage, alteration, disabling, killing or destruction of objects 160, it is advantageous to use beam shaping optical elements for the search beam 148 as for the focused energy beam 158 ( see Figures 16 to 17). As used herein, the term "beam location" refers to a cross-section of either beam 148 or beam 158.

[00204] In one embodiment, the focused energy apparatus 157 is disposed downstream of the light source 147 and on the same side as the light source 147 (see Figure 17), but the focused energy apparatus 157 may also be arranged to downstream and on an opposite side of the light source 147 (see Figure 16 for example).

[00205] In the embodiment of Figure 16, an optical beam shaping element for the search beam 148 is disposed on one side of the chip 100. The search beam 148 from the light source 147 passes through the action region (i.e. , camera 129) and is received by objective lens 153.

[00206] In one embodiment, beam 148 is expanded by optical beam-shaping element 181, which may include a plurality of lenses, which arrangement would be well known to a person of ordinary skill in the art. For example, beam-shaping optical element 181 may include a pair of prisms and a pair of cylindrical lenses with appropriate focal lengths, or it may include other lenses, with or without prism layers, that would be available to an individual of ordinary skill in the field. technique. Beam expansion enables the final location size at the focal point in search region 129. In one embodiment, circular beam location 148 is expanded using a beam expander 180. Beam expansion also reduces the influence on an optical element downstream, limiting damage and extending service life.

[00207] However, in one modality, no beam expander is used. Alternatively, if the source beam has a very large diameter, the optical element would be used to reduce that diameter to a suitable size.

[00208] In one embodiment, the beam-shaping optical element 181 includes two perpendicular cylindrical lenses to change the beam shape 148 into an ellipse perpendicular to the sample fluid flow direction 120 and along the fluid flow direction of sample 120, when focused on the center of them. Elliptical beam location 148 serves to excite objects 160 passing through channel 164 of microfluidic chip 100 and provides maximum uniform illumination in a central area of beam location 148 to compensate for minor fluctuations in the flow of objects 160 through the channel. 164. Additionally, in one embodiment, the beam-shape ellipse that has a broader dimension perpendicular to the sample fluid flow 120, assists in reducing variation in the fluorescence signal coming from objects 160 (i.e., sperm cells ) that are not perfectly centered within the sample fluid flow stream 120. The narrow dimension keeps the beam 148 at a high enough intensity to properly excite the fluorescent ink for the search of objects 160 (ie, cells of sperm). While an elliptical beam location 148 is preferred, in other embodiments of the present invention, a differently shaped beam may be used. The power of the search beam can be adjusted, as well as aiding in the search and limiting the impact on searched objects.

[00209] In one embodiment, the focused energy beam 158 is also shaped by the optical beam shaping element 180. The shape of the focused beam location 158 of the focused energy device 157 influences the targeted target accuracy and potential to impact the undirected objects 160 on channel 164 and can have varying beam shapes as the application requires. In a flow-based system, the beamwidth along the flow direction of the sample fluid 120 must be adjusted to be narrow enough so that only the targeted object 160 is affected and the sufficient beam intensity concentration is achieved. The length of the beam location 158 through fluid channel 164 can be intentionally adjusted to compensate for any instability and slight variability in the focused flow of the sample fluid 120. The desired beam shaping can easily be achieved by a person skilled in the art. technique.

[00210] In one embodiment, as shown in Figure 16, the optical beam shaping element 180 for the focused energy beam 158 is used to focus the beam 158 down to a much smaller size to increase a laser flux in the region. action 129. In one embodiment, beam 158 is expanded by beam expander optical element 180 (see Figure 16) which may include a plurality of lenses or prisms with appropriate focal lengths that would be readily known to a skilled individual. common in the technique.

[00211] In one embodiment, the beam 158 passes through a pair of anamorphic prisms, for example, to shape the beam 158. The beam 158 is further focused and compressed in the vertical and horizontal directions by an optical object due to a property of Gaussian beam. In one embodiment, the optical object can be, for example, a detector optical element 153 such as a microscope object or a focusing lens with a short focal length. The beam spot provides a combination of energy concentration for effective action (ie, kill, etc.) and sufficient width to compensate for minor fluctuations in a flow of objects 160 through microfluidic channel 164. In one modality, a pair of Cylindrical lens is used to expand the beam in the vertical dimension, before a spherical focusing lens or objective lens is used to focus the beam to an elliptical beam location of a diameter smaller than 2 µm and a diameter larger than 20 µm.

[00212] In alternative embodiments, a different shape and/or dimension(s) may be used for the beam 158. Verify that other larger or smaller diameters can be obtained by a person skilled in the art and can be applied to the same process.

[00213] In another embodiment, the focused energy device 157 is deployed on the opposite side of the search beam 148, as shown in Figure 17. The configuration proves advantageous due to the fact that it is easily deployed and used effectively free space on one side of system photo detector.

[00214] In one embodiment, dichroic mirrors are used to split specific wavelengths or to integrate specific wavelengths into the optical path. Furthermore, although a dichroic mirror and mirror/beamsplitter may be used, the person of ordinary skill in the art will know that more than one mirror and/or dichroic mirror/beamsplitter may be used in the present system.

[00215] In one embodiment, the optical collection elements 153, which include a microscope objective, collect the fluorescence emission from the objects 160 on the chip 100 and a dichroic mirror passes the fluorescence emission from the optical collection elements 153 into direction to the optical signal detector 154. In one embodiment, the focused energy device 157 emits a beam 158 which passes through the beam shaping optical elements 180 (as described above) and which is directed by a mirror and also reflected by the dichroic mirror through the optical collection elements 153 on the chip 100. Specifically, in one embodiment, the objective lens of the optical collection elements 153 focuses the focused energy beam 158, which enters the rear aperture of the objective lens, at a restricted location on the objects. 160 just slightly downstream of the research/excitement point in the action region 129. However, the individual of ordinary skill in the technique will know that the energy beam the focused 158 can be disposed below the output 112 of the chip 100 or slightly upstream of the search/excitement region 129.

[00216] In one embodiment, a distance between the search beam beam location 148 and the focused energy beam beam location 158 is adjustable.

[00217] In one embodiment, a dichroic mirror, or any beam splitting device, can split a small piece of light to a camera 182 (see Figure 5), which allows the user to visually examine the alignment.

[00218] In one embodiment, camera 182 provides a visual image of the microfluidic flow environment for general alignment purposes. The camera can be used to determine the location and timing for triggering the focused energy device 157.

[00219] In another embodiment, the focused energy beam 158 is deployed from the same side as the search beam 148 (see Figure 17). With this approach, the focusing lens for the focused energy apparatus 157 is not shared with the sensing side, so it is more flexible for beam shaping and it eliminates the need for a microscope objective that is rated for high power at the action wavelength (ie, photodamage, death), which can reduce the cost of the system.

[00220] In this embodiment, the focused energy device 157 emits a beam 158 from the same side as the survey apparatus 147, and the beam 158 is shaped by optical beam shaping elements 180 (described above) to be directed and aligned by a mirror and a dichroic mirror, to be focused on objects 160 in channel 164 of chip 100. In one embodiment, beam focusing optical elements 181 (as described above) are disposed between a dichroic mirror and chip 100 to focus beam 158.

[00221] As stated above, in the modality, beam 158 is focused on a restricted point on objects 160 just slightly downstream of the research/excitement point in action region 129. However, the individual of ordinary skill in the technique will know that focused energy beam 158 may be disposed below output 112 of chip 100 or slightly upstream from search/excitation region 129 (see Figures 6A through D).

[00222] Furthermore, as stated above, in the modality, a distance between the beam location of the search beam 148 and the beam location of the focused energy beam 158 is adjustable.

OBJECT FOCUSING AND ORIENTATION

[00223] In conventional flow cytometry systems, since objects or cells, especially with asymmetric shapes, tend to orient themselves as they flow close to a solid surface, the function and improvement of object or cell orientation depends complex nozzle designs such as guide deflectors or a deflection structure within the nozzle. To avoid the complex design of nozzle-based flow cytometry systems and their high manufacturing cost, the microfluidic chip 100 design of the present invention focuses, positions, and orients the objects 160 in order to improve their analytical capability. Thus, in one embodiment, objects 160 (ie, cells) with non-spherical shapes are aligned in a restricted core volume in channel 164 and held in a similar and desired orientation as they pass through the search/detection beam. 148. As a result, more uniform scattering and detection signals will be obtained in this way, which helps to increase the sensitivity and stability of the system 100.

[00224] In one embodiment, object orientation 160 can be accomplished by positioning the sample core stream 120 off-set with respect to the center of the center plane of the channel cross section 164 using hydrodynamic focusing.

TWO-STEP HYDRODYNAMIC FOCUSING

[00225] The following describes the two-step hydrodynamic focusing that occurs during fluid flow in one embodiment of the microfluidic chip 100 (see Figures 1B to 1D).

[00226] In one embodiment, the first hydrodynamic focusing step of the present invention is performed by inserting a fluid sample 120 containing objects 160, which includes biological samples such as sperm cells 160 etc., through sample entry 106, and inserting surrounding fluids or plug 163 via sheath or plug insertion 107, 108. In one embodiment, objects 160 are pre-dyed with ink in accordance with known methods (e.g., Hoechst ink), in order to allow fluorescence and imaging of them.

[00227] In one embodiment, objects 160 in the sample fluid mixture 120 flow through the main channel 164, are enveloped and shaped by the fluid flow, and have a random orientation and position (see Figures 3A and 6A). At crossover 161, the sample mixture 120 flowing in main channel 164 is enveloped and shaped by the surrounding or buffer fluids 163 from channels 116, 117 and compacted in a first direction (i.e., at least horizontally, in at least on both sides of the flow, if not on all sides, depending on where the main channel 164 enters the intersection 161), when the surrounding or buffer fluids 163 meet the sample mixture 120. This compaction is called hydrodynamic focusing ( three-dimensional (3-D)) and is used to align objects 160 in channel 164 in a constrained core volume that can approximate a single file formation. Hydrodynamic focusing takes advantage of a significantly large sheath or plug flux in channel 164 to accelerate the vector velocity of travel of objects 160 through flat microfluidic channel 164. In one embodiment, sample core current 120 may also be bypassed from central plane by a ramp 166B or taper structure 166A in channel 164, which is prior to junction 161 of channel 164 and first stage sheath or plug channels 116, 117.

[00228] As a result, objects 160 are accelerated and the spacing between objects 160 in microfluidic channel 164 can also be lengthened. The vector velocity of the objects 160 is dependent on the sample flow rate 120 and the ratio of the value to the envelope or buffer flow rate 163. This function is useful to avoid object clogging and crowding problems 160 with highly concentrated object samples 120.

[00229] However, as shown in Figure 8A, at this stage, the resulting sample core stream 120 through main channel 164 still shows overlapping objects 160 or cells along the depth direction or vertical axis of channel 164. In particular, objects 160 are focused around the center of channel 164 and can be compacted into a thin strip across the depth of channel 164. Thus, at intersection 161, as sample fluid 120 is being compacted by the surrounding or flowing fluids. plug 163 from channels 114, 115, towards the center of channel 164, objects 160 (i.e. sperm cells) move in the direction of the width of the center of channel 164.

[00230] In one embodiment, the present invention includes a second focusing step, wherein the sample mixture 120 containing objects 160 is further compacted by surrounding fluids or buffer 163 from a second direction (i.e., the direction vertical, from top and bottom) entering from channels 114, 115 at intersection 162 (see Figure 14). The intersection 162 leading to channel 164B is the second focusing region. Note that although entries at junction 162 from channels 114, 115 are shown as rectangular, the person of ordinary skill in the art will understand that any suitable configuration (ie, tapered, circular) can be used.

[00231] In one embodiment, the enclosing or plug fluids 163 in channels 114, 114 enter from the same plane (see Figures 3A and 6A) or from different planes in channel 164 (see Figure 15, where channels 114, 115 are arranged above and below main channel 164, which enter channel 164 vertically) to align objects 160 at the center of channel 164B both by width and depth (i.e., horizontally and vertically) therewith flow along channel 164B. Then, the resulting flow in the main channel 164 is subsequently compacted and repositioned by the second stage enveloping flow through microfluidic channels 114, 115.

[00232] Thus, in the second focusing step of the present invention, the sample mixture 120 is again compacted by the envelope or vertical buffer fluids 163 entering channels 114, 115 and the sample stream 120 is focused at the center depth of channel 164, as illustrated in Figure 8B, and the flow of objects 160 along the center of channel 164 in a restricted core volume that can approximate a single file formation in a particular orientation.

[00233] Consequently, after these two subsequent steps of hydrodynamic focusing, a restricted core volume of 160 objects or cells is obtained and the position of the current can also be adjusted to a desired location along the vertical geometric axis (see Figure 8B ). In this way, objects 160 introduced into sample inlet 106 are subjected to two-step hydrodynamic focusing, which allows objects 160 to move through channel 164B in a restricted core volume that can approximate a single file formation, in a more uniform orientation (depending on the type of 160 objects), which allows an easier search of the 160 objects.

THREE-STEP HYDRODYNAMIC FOCUSING

[00234] In one embodiment, three-step hydrodynamic focusing is performed on objects 160 on chip 100. In this embodiment, as shown in Figures 1A and 3B, the first two steps of hydrodynamic focusing are performed by horizontally compacting the fluid current of sample 120 at intersections 161 and 162 and then, in a third step, the sample fluid stream 120 is vertically compressed in channel 164. Sheath or plug channels 114, 115, and 116, 117 enter channel 164a from a horizontal direction, at an angle of 45 degrees or less for each channel.

[00235] More specifically, in the first hydrodynamic focusing step, the sample stream 120 between the first intersection 161 and the surrounding or buffer fluids 163 from channels 116, 117 surround the sample stream 120 and immediately compact it into a slender sample stream 120 in channel 164. In one embodiment, sample fluid channel 164 is tapered with an internal ramp before crossing 161, wherein the enclosing fluid channel or plug 116, 117 enters channel 164 ( see Figure 3B). Meanwhile, since the channel 164 is shallower (i.e., smaller in dimension) than the first sheath channels 116, 117, the sample stream 120 is lifted by the surrounding fluid or buffer 163 from the channels 116, 117 to flow to the top of the main channel 164.

[00236] In the second hydrodynamic focusing step, the surrounding or plug fluids 163 are introduced from channels 114, 115 into channel 164 - channel 114, 115 which is disposed near the top of main channel 164 (see Figure 3B ). In one embodiment, as shown in Figure 3B, sheath or plug channels 114, 115 join channel 164 horizontally and may have a smaller dimension than that of sheath or plug channels 116, 117. whereas the depth of the channels 114, 115 is shallower than the main channel 164, the surrounding or buffer fluids 163 further compress the sample stream 120 along the width of the channel 164 to restrict the width of the sample stream 120. That flow of envelope fluid or buffer 163 significantly improves signal measurement sensitivity.

[00237] Without a third hydrodynamic focusing step, lack of vertical compaction at intersections 161 and 162 can result in multiple objects 160 or cells entering simultaneously into sensing region 129, thereby reducing detection sensitivity or causing sensing errors. measurement, especially for a high throughput flow cytometry application.

[00238] However, three-dimensional hydrodynamic focusing is an effective way to align objects 160 in a constrained core volume that can approximate a single file formation in channel 164. Also, consistent positioning of object 160 in channel 164 results in a minimal variability in vector velocity from object to object in the parabolic flow.

[00239] In microfluidic-based flow cytometry, the velocity vector profile of the flow is parabolic along the rectangular cross-section of the microchannel. Figure 7 illustrates the cross-sectional shear force profile of microfluidic channel 164. The shear force gradient shown in Figure 7 has a minimum shear force at the tapered tip and the larger region at the rear has the maximum shear value. In this way, the hydrodynamic shear force gradient is formed across the cross section of the channel 164. The large shear force near the wall of the channel 164 helps to orient asymmetric objects 160.

[00240] In the present embodiment, after the two steps of hydrodynamic focusing, the sample stream 120 shrinks in a very thin current in both the horizontal and vertical directions. The ratio of independently controlled surrounding fluids or buffer 163 from channels 116, 117 and 114, 115 each determines the size of the resulting sample stream 120 in channel 164. After compaction caused by the surrounding fluid or buffer 163 from channels 114, 115, objects 160 may propagate somewhat along the depth of channel 164, but objects 160 still follow sample stream 120, which is very close to the top ceiling of main channel 164 ( note: with the chip described as horizontally arranged for ease of reference). Therefore, it is necessary to have a third flow of enveloping fluid or plug 163 from channel 172 to further compact the sample stream 120 along the vertical direction and tightly confine the objects 160 within the thin sample stream 120.

[00241] Additionally, since the surrounding fluid or buffer flow 163 is introduced perpendicularly into the pre-confined sample stream 120 from the sheath or buffer channel 172, it helps to position the sample stream 120 at a location along the cross-section of channel 164 (ie, achieves a final result as shown in Figure 8B). By precisely regulating the flow rate of the surrounding fluid or plug 163 from the channel 172, the positions of the objects 160 can be precisely controlled as they pass through the sensing region 129.

[00242] In one embodiment, envelope fluid or buffer 163 is introduced through channel 172 from outer sheath tubing (see Figure 20) rather than through microchannels that pass through microfluidic chip 100. Thus, a controller External flow is required to provide a constant and stable flow rate through input channel 172 (see Figures 19 through 21).

[00243] The design of the present invention allows the core sample stream 120 to orient flat-shaped objects, position the objects 160 in channel 164 in a physical arrangement that approximates uniformity, all of which improve precision action to downstream of the focused energy apparatus 157.

[00244] Although the three steps of hydrodynamic focusing are disclosed above, the person of ordinary skill in the art will know that the configuration and number of the sheath or plug channels can change as long as they achieve the desired features of the present. invention, with respect to the orientation and focusing of objects in the sample fluid 120.

FLOW CONTROL METHODS

[00245] To perform the exemplary three-dimensional hydrodynamic focusing methods described above, it is required that both the sample fluid 120 and the surrounding or buffer fluids 163 be delivered precisely so that a flow can be transmitted through the microfluidic chip 100 After being compacted by the enveloping fluid or buffer flows 163, the objects 160 or cells were accelerated and the average spacing between the objects 160 or cells in the sample core stream 120 was also significantly elongated therefrom. The ratio of the envelope fluid or total buffer 163 flow rate and the 120 sample flow rate can be adjusted between 100: 1 and 1000: 1. Preferably, the ratio of 200 to 400: 1 is used in the microfluidic chip 100 of the present invention. The overall fluid flow rate on the microfluidic chip 100 is about 2 to 4 ml/min. The enveloped fluid or buffer flows 163 introduced must be constant and pulse-free to ensure a stable path scalar velocity of the objects 160 during the search and signal detection and between the detection/search position and the action position of the focused energy apparatus 157 (see Figure 6A). This facilitates an accurate signal reading and action on the object 160 directed by the focused energy apparatus 157. With the precise control of fluid flow through the main channel 164, the overall flow rate variation is less than 1% of the flow rate. set flux and the scalar velocity of path of directed objects 160 for potential action by the focused energy apparatus 157 varies less than 1% from the position where the search and detection of objects 160 occurs to the position where the focused energy 157 acts on objects 160 (see Figure 6A).

OBJECT ORIENTATION

[00246] One of the challenging problems in detecting flat 160 shaped objects (ie sperm cells) is to constrain the 160 objects to a uniform orientation as they pass through search beam 148. objects 160 and a corresponding object orientation 160 on channel 164 help to increase the sensitivity of the system. With the aforementioned hydrodynamic focusing strategy, the position of objects 160 along channel 164 can be manipulated in a controlled manner. In this way, by adjusting the ratio of the surrounding fluid or buffer flows 163 from channels 116, 117, and 114, 115, and 172, the position of the focused sample current deviation 120 from the center of the channel 164 of about 5 to 20 microns (based on a cross section of channel 164 with a width of 150 microns and heights of 100 microns, for example) is preferred for detecting flat-shaped objects 160. In general, an adjustment of 0 to 100 microns of oblique position of objects 160 can be achieved.

[00247] Specifically, in order to align objects 160 in channel 164 to improve its orientation, the high aspect ratio of microfluidic channel 164 takes advantage of inducing shear force to rotate the flat surface of object 160 (ie, cell of sperm) which faces the wall of the channel 164. In addition, the enveloping fluid or plug 163 flow can be actively employed to compact and position the objects 160 in the channel 164. These methods are described in more detail below.

i) PASSIVE METHOD

[00248] In one embodiment, an asymmetric geometry structure can be used to position focused objects 160 in channel 164, through one of: a) placing an asymmetric ramp 166B in main sample channel 164 to raise the sample stream 120 ( see the explanation above in relation to Figure 4A); and b) placing an asymmetric ramp 166B in the main channel 164 before the action chamber 129 to raise the focused sample current 120 (see explanation above in relation to Figure 4B). The asymmetric features above can be used individually or in an appropriate combination of two or more. However, the person of ordinary skill in the art will know that these features are not necessary to achieve the position of objects 160 in channel 164.

[00249] In one modality, as noted above, ramps can be used on channels 114 to 117 to raise the sample flow 120, although these are not required. The placement of ramps in the channels is dependent on the direction in which the objects 160 of the sample core stream 120 are required to be offset from the center to improve the orientation of the object 160 or the cell. However, the passive method above has less flexibility to vary the position of object 160 on main channel 164.

ii) ACTIVE METHOD

[00250] In an alternative method to bypass the sample core 120 current in channel 164, an asymmetrical envelope flow 163 is introduced to adjust the positions of objects 160 or cells in channel 164. There are several methods for realizing envelope fluid flow or of asymmetric plug 163, the two modalities of which are described below.

[00251] One embodiment is to introduce enveloping or single plug fluid flow 163 that forms a 90 degree angle with the wall of the main channel 164, as shown in Figure 3A (in a two-step hydrodynamic focusing method) or in the Figure 3B (in a three-step hydrodynamic focusing method). In the two-stage hydrodynamic focusing modality, the enveloping fluid or plug flow 163 introduced at the second stage hydrodynamic focusing intersection 162 further compacts the sample core stream 120 subsequent to the first stage hydrodynamic focusing at the intersection 161.

[00252] In the alternative three-stage hydrodynamic focusing mode, this compaction of the sample core stream 120 occurs at the third stage hydrodynamic focusing intersection, where channel 172 joins main channel 164. final hydrodynamic focusing positions 160 objects or cells to a desired location along the vertical geometric axis. By controlling the ratio of the hydrodynamic focusing flow rates, a desired object position 160 can be obtained to achieve optimal orientation.

[00253] In a second embodiment, using the two-stage hydrodynamic focusing method, two second-stage sheath or plug channels 114, 115 merge at an angle to the wall of the main channel 164, and the main channel 164 parallel from above and below, as shown in Figures 12A, -12B. The angle of the hydrodynamic focusing channels of the second stage 114 115 and the main channel 164 can vary and is dependent on the manufacturing methods. Preferably, an angle of 90 degrees is selected (see Figures 12A to 12B). Flow rates of different envelope or plug 164 fluids can flow through the two channels 114, 115, which have the ability to reposition sample core current 120 in the main channel 164. After the objects 160 are offset from the center plane of the channel 164, the orientation of objects 160 is improved. In a specific embodiment, such as sperm cells 160, sperm cells 160 tend to rotate their flat sides towards the wall of channel 164 on the geometric vertical axis.

[00254] As can be seen from the embodiment of Figure 12B, the dimensions of the channels 114, 115 are not necessarily identical (as shown in Figure 12A), in order to obtain different hydraulic resistances. In this way, the same flow rate of the envelope or plug 164 fluid flows of the second stage in channels 114, 115 will also generate oblique fluid flow.

[00255] To summarize, both passive and active methods above can help to position objects 160 optimally and improve their orientation on channel 164.

[00256] In one embodiment, pancake-shaped sperm cells 160 are taken as an example of objects 160. Due to their pancake-shaped or flat teardrop-shaped heads, sperm cells 160 will reorient themselves in a predetermined direction as they undergo the second or third (depending on the modality) step of focusing - that is, with their flat surfaces perpendicular to the light beam direction 148 (see Figure 6). In this way, sperm cells 160 develop a preference in their body orientation as they go through the hydrodynamic focusing process. Specifically, sperm 160 cells tend to be more stable with their flat bodies perpendicular to the direction of compaction. Thus, with control of the surrounding fluids or buffer 163, sperm cells 160 that start out with random orientation now reach a uniform orientation. In this way, sperm cells 160 are not only arranged in a restricted core volume in the center of channel 164B, but they also achieve a uniform orientation with their flat surface normal to the compaction direction in the last step of hydrodynamic focusing.

[00257] The above methods improve the orientation of sperm 160 cells and the ability to differentiate the DNA content of X and Y sperm chromosomes (and thereby distinguish between X and Y sperm). The histograms of Figures 13A and 13B show sperm cells 160 at the center of channel 164 and the offset from the center of channel 164, respectively. The circles on the left and right of the histogram in Figure 13A show the misoriented sperm cell populations 160. The left bulge of the main population weakens the ability to differentiate X and Y sperm populations and contributes to the asymmetric distribution of sperm populations X and Y.

MICROFLUIDIC CHIP SYSTEM OPERATION OBJECT RESEARCH

[00258] In one embodiment, the search light source 147 is an excitation laser 147 (see Figure 16) that has a power of 350 mW, a wavelength of 355nm, and a pulse width of 12 ps.

[00259] In one embodiment, additionally downstream of the hydrodynamic focusing steps, in channel 164, objects 160 are detected in action chamber 129 at aperture 150 through cover 133, with the use of light source 147. light 147 emits a beam of light 148 (which may be through an optical fiber) which is focused in the center of channel 164 at aperture 150.

[00260] In one embodiment, the objects 160 are sperm cells 160, which are guided by the hydrodynamic focusing steps such that the flat surfaces of the sperm cells 160 are facing towards the light beam 148. all objects 160 or sperm cells 160 are moved in a restricted core volume that can approximate a single file formation by the hydrodynamic focusing steps as they pass under the light beam 148. As objects 160 pass under the light source 147 and light beam 148 act on them, objects 160 emit fluorescence which indicates the identity of the desired objects 160.

[00261] The light source 147 provides the fluorescence excitation energy for detecting objects 160 in the action region 129. In an exemplary embodiment, with respect to objects 160 that are sperm cells 160, the X chromosome cells fluoresce in a different intensity than Y chromosome cells (based on DNA content, as is well known in the art) (note: 355nm is selected for the Hoescht 33342 ink used on DNA). Also, in other embodiments, objects 160 that are cells carrying a trace may fluoresce at a different intensity or wavelength than cells carrying a different set of traces. In addition, objects 160 can be seen by indicators of shape, size or any other indicator of distinction.

[00262] Thus, in the 160 sperm cell modality, the illumination for the flat surface and edge of the 160 cells is quite different with 160 sperm cells as compared to other cells. The fluorescence signal derived from the edge of sperm cells 160 is significantly stronger than that from the flat surface, which makes it more difficult for the digital processor 156 to deal with the stronger edge signal and the lower normal signal from the flat surface of X and Y of cells 160. In this way, rotating the flat surface of sperm cell 160 to face the laser illumination (ie, light beam 148) helps to reduce orientation variability and increase capacity for the system differentiate 160X or Y sperm cells.

[00263] In the beam-induced fluorescence mode, the emitted light beam 151 (in Figure 5) is then collected by the objective lens 153 and subsequently converted to an electronic signal by the optical sensor 154. The electronic signal is, then digitized by an analog to digital converter (ADC) 155 and sent to an electronic controller 156 for signal processing.

[00264] As noted above, in one embodiment, the DSP-based controller 156 monitors the electronic signal, and when a particular signal is observed, a focused energy apparatus 157 can be employed to act on a targeted object 160 (see Figures 6A-6C). However, in an alternative modality, the search apparatus that performs a search of the objects 160 after the focused energy device 157 acts on them (see Figure 6D).

[00265] In one embodiment, the search of sample 120 that contains 160 objects (ie biological material) is performed by other methods. In this way, portions of, or outlets, microfluidic chip 100 can be inspected optically or visually. In general, methods for research may include direct visual imaging, such as with a camera, and may use direct intense light imaging or fluorescent imaging; or more sophisticated techniques can be used, such as spectroscopy, transmission spectroscopy, spectral imaging or dispersion, such as dynamic light scattering or diffuse wave spectroscopy.

[00266] In some cases, optical search region 129 can be used in conjunction with additives, such as chemicals, which bind to or affect the objects 160 of the sample mixture 120, or with granules, which are functionalized to bind and/or fluoresce in the presence of certain materials or diseases. These techniques can be used to measure cell concentrations, to detect disease, or to detect other parameters that characterize objects 160.

[00267] However, in another embodiment, if fluorescence is not used, then polarized light backscatter methods can also be used. Using spectroscopy methods, objects 160 are searched as described above. The spectrum of those objects 160 that tested positive and fluoresced (ie, those objects 160 that reacted with a tag) are identified by selection by the focused energy apparatus 157.

[00268] In one embodiment, objects 160 can be searched and identified based on the reaction or binding of objects 160 with additives or surrounding fluids or buffer 163, or using the natural fluorescence of the objects 160 or the fluorescence of a substance associated with the object 160, such as an identity tag or a background tag, or a corresponding size, dimension, or surface feature, etc. selected.

[00269] In one modality, upon completion of an analysis, the selection can be made, through a computer 182 (which monitors the electronic signal and employs the focused energy device 157) and/or an operator, of which 160 objects to discard and which to collect.

APPLICATIONS FOR FOCUSED ENERGY APPLIANCE

[00270] The focused energy apparatus 157 of the present invention can perform various actions on objects 160 in channel 164 or between a chip 100 and a container 188.

[00271] In one embodiment, the focused energy device 157 acts to photodamage or destroy the objects 160 in various ways.

[00272] Specifically, the focused energy apparatus 157 acts to extinguish objects 160 (ie, cells). For example, the targeted objects 160 can be unwanted cells 160 and, upon action by the focused energy apparatus 167, cell death can be caused by overheating the intracellular environment, which can promote, but without limitation, denaturation or reduction of protein in enzyme activity.

[00273] In another method, the energy dosing action 158 from the focused energy apparatus 157 is strong enough to cause disruption of the plasma membrane and leakage of cell contents outside the cell 160 and into the surrounding environment (i.e., surrounding fluid or buffer 163).

[00274] In another method, the death of the object 160 or the cell may be caused by the formation of radical oxygen species (ROS) due to the absorption of energy from the focused energy pulses 158 from the focused energy apparatus 157, which will cause, among other things, DNA and protein damage.

[00275] In another embodiment, the focused energy apparatus 157 may temporarily or permanently disable the targeted objects 160, such as cells 160, with the use of focused energy pulses 158 from the focused energy apparatus 157.

[00276] For example, exposing 160 sperm cells to focused energy pulses 158, such as those produced by a laser or LED 157, generates a photoactivation within the 160 cells and results in temporary or permanent disabling of cellular mechanisms responsible for motility of sperm 160. After disabling targeted sperm cells 160, the resulting sample 120 contains sperm with motility 160 and sperm with immotility (targeted) 160, where sperm with immotility does not have the ability to fertilize oocytes naturally.

[00277] In another embodiment, it may also be desirable to use focused energy pulses 158 to render sperm 160 infertile by dimerizing nucleotides in DNA. Dimerization occurs when 160 cells, such as 160 sperm cells, are exposed to UV light, which causes bonds between pyrimidine bases and results in a type of "transversality" which, if not repaired, inhibits replication and the transcript. Thus, although the targeted sperm cells 160 are still alive, as evidenced by their motility, the fertility of the targeted sperm cells 160 is highly reduced.

[00278] In addition to sperm cells 160, the individual can use powered focused energy sources 157, such as LEDs or lasers 157, to photobleach the fluorescence in objects 160, such as cells or colloids 160, which expresses a predetermined level of fluorescence. For example, in many self-assembly object formulations, a wide size range of 160 objects is formed, which are difficult to separate from each other. To produce an improved sample 120 of objects 160 that have a desired size, the subject can fluorescently label all objects 160 using methods known in the art, and the subject can use optical research to determine a size of the object 160, and photobleach the objects. 160 objects that have the predetermined level of fluorescence.

[00279] In a specific example, a 120 semen sample may contain contaminants, such as bacterial or viral cells, which are the target of photobleaching. Another example might include sperm cells 160 that contain a given cell 160 or DNA trace and that are labeled with the fluorophore for quantitative and/or qualitative measurement. Sperm cells 160 that contain the trait can be targeted by the focused energy apparatus 157. In another embodiment, sperm cells 160 that do not contain the trait can be targeted for photobleaching. Specific 160 cells in other cell mixtures, such as blood, are also candidates for photobleaching treatments to reduce viability. In yet another embodiment, photobleached cells/objects 160 may be undetectable downstream and therefore not subjected to subsequent processing steps (i.e., may bypass subsequent processing steps).

[00280] In another embodiment, in contrast to disabling objects 160, focused energy pulses 158 can be used to activate materials, such as caged molecules or compounds, within objects 160.

[00281] In one application, caged compounds represent, but are not limited to, fluorescent tagged or cell responsive molecules. In these applications, focused energy pulses 158 are used to cause photoactivation of the caged molecules or compounds that alter cell signaling kinetics 160 for ex vivo therapies.

[00282] In another embodiment, the focused energy pulses 158 can activate light curing events that disable cells or colloids 160 by altering the internal properties of the object 160.

[00283] In another embodiment, with respect to intracellular signaling pathways, focused energy pulses 158 are used to activate hot shock proteins or induce biogenesis or mitochondrial activation within objects 160 or cells, which includes germ cells, to improve cell viability and functionality. Additional intracellular pathways can also be activated to repair damage done either by the search/detection device 147 or by several other factors that are too numerous to list (ie, environmental, thermal, chemical, etc.)

[00284] In one embodiment, the individual skilled in the art can generate photopolymerization through focused energy pulses 158 to temporarily encapsulate or permanently contain 160 targeted cells, colloids or other objects, with the use of PEG-acrylate/PEG-vinyl/etc . of multiple branches, which promotes encapsulation of the 160 targeted objects or cells. Sperm 160 cells can be encapsulated to improve preservation of viability and fertility through commercial storage and distribution processes.

[00285] In another embodiment, focused energy pulses 158 are used to cause light curing events on the surface of cells or targeted objects 160, which increase the size or density of the encapsulating material in order to change the size or object density 160 or improve the properties and performance of the encapsulating material.

[00286] In another embodiment, a photopolymerizable sequence in the hydrophobic portion of the vesicle can be used to permanently seal the desired molecule or object 160 for encapsulation therein.

[00287] In another method, the focused energy apparatus 157 can be used to act externally or change the environment around the targeted objects 160.

[00288] In one method, focused energy pulses 158 are used to heat the local environment around the targeted cells or objects 160 so that a thermal enhancement is sufficient to cause toxicity to the targeted cells 160.

[00289] In another embodiment, focused energy pulses 158 are used to promote disruption of analyte containing delivery vehicles (such as vesicles) that are in close proximity to targeted cells 160. Delivery vehicles carry molecules such as sodium fluoride (NaF), which cause temporary immobility of sperm cells 160, or heparin, which promotes capacitation of sperm 160. When the concentration of analytes or activating agents is increased locally, targeted sperm cells 160 or other objects respond to local signals without activating similar responses in untargeted cells 160.

[00290] In another embodiment, when objects 160 or cells are fixed to a surface, the enveloping environment is modified with focused energy pulses 158 to vary the surface cell's modulus of elasticity or chemical-responsive release from surface of 160 objects or enveloping cells.

[00291] In one embodiment, the production of heat through the absorption of light/waves EM 158 causes a temperature change that kills objects 160 or cells.

[00292] In another embodiment, focused energy pulses 158 are used to form predetermined chemical bonds or break chemical bonds in the fixation material, thereby directing the differentiation of objects 160, such as stem cells 160, into cell lines differentiated.

[00293] In one embodiment, for some applications, it is desirable to use focused energy pulses 158 to promote cell uptake or adhesion on targeted objects 160.

[00294] In one embodiment, cellular uptake or antibodies, cell probes or DNA are enhanced by local heating.

[00295] In one embodiment, with said local heating, when the temperature rise is also optimized to maintain object 160 or cell viability, internalization of objects 160 into targeted viable cells 160 is selectively promoted.

[00296] In one embodiment, the object 160 to be delivered is fixed to an object, which, when directed with a light source 147, can cause a slight microbubble. For example, an oligonucleotide can be conjugated to a gold nanoparticle. When the other is heated with a light source 147 at an optimized wavelength, a microbubble is lightly formed. By cavitation of the microbubble, the gold nanoparticles are broken up and the parts of the nanoparticle and the object attached to them are permeabilized through a cell membrane.

[00297] In another modality, the temperature increase of the object 160 directed is not enough to cause the formation of a microbubble. The localized temperature rise is optimized to maintain cell viability and selectivity that promote the internalization of objects 160 into targeted viable cells.

[00298] Similarly, in another embodiment, by using focused energy pulses 158 to adhere materials into colloids or objects, the geometry of the object 160 or the properties of the object 160 are altered, thereby allowing for additional separation techniques , such as magnetic or electric fields, to separate materials that are not normally susceptible to such forces.

FOCUSED ENERGY APPLIANCE OPERATION

[00299] In general, flow cytometric analysis and action systems that use an electromagnetic radiation source, such as a focused energy device 157 or laser, to act on selected objects 160, typically wish to deliver a level of controlled energy to individual objects 160. In one embodiment, such systems can extinguish, alter, damage or destroy objects 160 or targeted cells using the focused energy apparatus 157. In other embodiments, such systems can, among other methods, activate targets in objects 160 or selected cells or in the fluid, medium or matrix surrounding the selected objects 160, as described above.

[00300] In the above methods, which use focused energy pulses 158, radiation can be applied by both continuous shooting and continuous elimination methods, so that the desired objects 160 are not affected and the unwanted objects 160 are altered, dead, destroyed, or damaged are discriminated from sample 120. Similar considerations, as noted above, are given when selecting a laser wavelength and laser power for continuous shooting and continuous shooting modes.

The. DIRECTED INFLAMMATION MODE:

[00301] More specifically, in continuous shooting, the focused energy apparatus 157 is employed for aimed objects 160. Specifically, an object 160 in a mixture of sample fluid 120 can pass through a search/detection area in chamber 129, for example, where specified characteristics of object 160 are evaluated using one or more of the above methods.

[00302] Thus, in a flow-based system, for example, the action area 129 of the focused energy apparatus 157 is downstream from the optical research area using the light source 147 for research. Alternatively, the focused energy apparatus 157 is used prior to the search further downstream. In one embodiment, the focused energy apparatus 157 acts on objects in the action chamber 129. The distance between the optical search region and the action region can be adjusted to accommodate different timings.

[00303] Based on predetermined criteria, a decision is made whether to keep, discard or act on the selected object 160. Objects 160 marked for action are hit by a triggered energy pulse 158 from the focused energy apparatus 157 (see Figures 6A to 6C, for example).

[00304] When the object 160 or the cell is not targeted, it remains unaffected and flows through chamber 129, through channel 164, to channel 141 and output container 188, which collect objects 160 targeted and not targeted as an itemized product 165.

[00305] In one embodiment, the laser pulse 158 is of short duration and can selectively target 160 objects or individual cells while not intentionally impacting 160 objects or untargeted cells that may be nearby, thereby preventing "excessive sprinkling" on 160 objects or untargeted cells. Pulse energy is selected to impart the desired effect while avoiding unwanted disturbances to the surrounding environment or, for example, in flow systems, not causing unwanted cavitation or bubble formation. A variety of laser wavelengths can be used; however, the flow requirement may be different depending on the characteristics of the object 160, the targeted ink and environment.

[00306] Laser units 157 are power limited, especially compact models that operate at high pulse frequency (>100 kHz typically) and it may be preferable to choose a laser wavelength that minimizes the flux required. For example, matching the laser wavelength 157 to the absorption of inks, other targets, or objects 160 (ie molecules) used in the action process greatly improves effectiveness and efficiency. Additionally, pulse energy 158 is selected to impart the desired effect while avoiding unwanted disturbances to the surrounding environment or, for example, in flow systems, which does not cause unintended cavitation or bubble formation.

[00307] In a specific example related to sperm cells 160, such as objects 160, a laser 157 of 355 nm was used to take advantage of the ink (ie Hoechst ink 33342) used for the cell staining process. In a similar example, a 349 nm 157 laser can be used. In such examples, when unwanted sperm 160 cells are photodamaged, destroyed or killed, pulse energy levels of 0.5 to 8.0 µJ are used.

B. CONTINUOUS SHOOTING:

[00308] In continuous shooting, as in a flow-based system, the focused energy pulse 158 is constantly employed and only interrupted for the passage of undirected objects 160 (i.e., desired objects 160 that should not be damaged, destroyed, altered or dead) or debris or contaminants that do not require action on them.

[00309] As stated above, based on predetermined criteria, a decision is made about either keeping or disposing of an object 160, or acting on it, which includes photodamaging, extinguishing, altering, disabling or destroying an object 160. focused energy 157, such as a continuous wave (CW) or fast pulse laser or LED, delivers a continuous current of focused energy 158 to objects 160 and is used to act on (i.e., including, photodamage, alter, disable, destroy or extinguish) each object 160 that passes through a particular location in the flow stream of the sample fluid stream 120 in an exemplary flow system. When untargeted objects 160 are found in the action region, laser beam 158 is turned off, deflected, or otherwise stopped for a short period of time to allow untargeted objects 160 (in some cases, items discarded) pass unaffected. Objects 160 - directed or undirected - flow through channel 141 to container 188.

[00310] Methods for interrupting or diffusing beam 158 include mechanical (shutters, cutters, galvanometer mirror), optical (acoustic optical deflector, acoustic optical modulator, spatial light modulator, digital micromirror device, polarization modification, crystal display liquid), electronic (pulse conditioning, dropping or altering a Q-switch laser), or acoustic. Any other known or suitable future methods or techniques can be used to interrupt the focused energy beam 158.

[00311] With both directed and continuous methods, the energy pulse 158 has a short duration and the focused energy apparatus 157 can selectively direct only a single object 160 and not impact other objects 160 that are close to the sample fluid flow 120 (ie, may limit "collateral damage"). Energy pulse 158 is selected to be sufficient to achieve the desired action on object 160 (ie, damage, alter, extinguish, or destroy object 160) per user requirements. The pulsed energy 158 from the focused energy apparatus 157 must be in a range where it will not cause a disturbance in the sample fluid 120 due to cavitation, bubble formation or energy absorption method.

[00312] Other technologies to reduce unwanted action, damage, and destruction to untargeted objects 160 include those that absorb a significant portion of pulse energy 158, alter beam direction 158, or discharge excess energy by igniting pulses 158 in the flow current 120. Specifically, these may include mechanically moving a mirror or lenses in order to defocus or deflect excess laser pulses 158 in an energy absorbing device, electronically altered lenses in order to change the angle of laser 157 propagation and sophisticated drive technologies that coordinate pulse energy data coordinated from laser 157 with object-to-object timing data of objects 160 in the immediate flux current.

ç. PULSE TIME

[00313] The timing between actions by the focused energy device 157 on objects 160 is not uniform and follows a Poisson distribution in which many short and extremely long intervals occur. Because a laser-based 157 action system includes inherent limiting factors, it is preferable to include a short “recharge time” between laser pulses 158. Latency time (inherent in focused energy apparatus 157), in addition to the "charge/recharge" time, it is the minimum time that the focused energy apparatus 157 can react (deliver a pulse) and still deliver the energy level required for the targeted object 160.

[00314] In a mode, the charging time must be in the range of .1 to 1 second and, preferably, it must be from 0.1 μs to 4 ms. Pulse-to-pulse variability in energy levels affects the rate of producing the desired effect on 160 targeted objects or cells and the potential to impact 160 untargeted objects or cells. When ignited at non-uniform intervals, pulse-to-pulse stability should be high. In one example, a 157 Q-switch laser in pulse-on-demand mode was used to deliver an average pulse energy of 1.8 µJ with a range for individual pulses of 1.3 µJ to 2.3 µJ.

[00315] In a flow-based system, the action region 129 may be located downstream from the optical search region in chamber 129, or upstream thereof, and the distance between the optical search region and the region action can be adjusted to accommodate different timings. To accommodate a sufficient charge time for a pulsed laser 157, the minimum delay between searching 160 objects or cells, and acting on selected 160 objects or cells, should not be less than 1 µs.

[00316] The focused energy apparatus 157 successfully operates, for substantial periods, at action rates of up to 5600 objects per second, with accuracy rates that can be selected and are in the range of, for example, 75 to 95%. In systems where the spacing between objects 160 in the flux stream is controlled, system 157 can operate at action rates up to the repetition rate of laser 157.

d. SELECTION OF OBJECTS

[00317] In one embodiment, the focused energy apparatus 157 is employed prior to searching the object 160. However, in another embodiment, in order to determine which objects 160 are selected for action by the focused energy apparatus 157, as noted above, a histogram, or any graphical representation of the measured/calculated characteristics of the objects 160 after the search, can be used in order to make the decision for its action on the population of objects 160. In one modality, after the search is carried out, the plot the range (i.e., transit time through the search region of chamber 129) may reflect the relative size of the sample core stream 120 under different flow conditions, the object 160 or cell distribution across the main channel 164, and the object vector velocity 160 as well as the object vector velocity variation 160 within a particular chip 100 design.

[00318] As noted above, the high aspect ratio of the main channel 164 is important for the hydrodynamic focusing, migration and orientation of the object 160 or cell. In one embodiment, less than one to the high aspect ratio for microfluidic channel 164 is used in the present invention. Preferably, the high aspect ratio of 2/3 is used for the main channel 164. The span value itself approximately indicates the vector velocity of the object 160. The large span value indicates that the objects 160 pass through the light beam of 148 search slowly. The restricted span size indicates that the sample core stream 120 is closer to the center plane of channel 164 and there is less object vector velocity variation 160.

[00319] In one embodiment, to act precisely on selected objects 160 as they flow through the chip 100 as they move away from the output 112, it allows less object vector velocity variation 160 to ensure that the focused energy apparatus 157 can precisely target the 160 objects or selected cells. In this way, based on the positioning and orientation methods described above (i.e., active and passive methods), objects 160 are positioned near the center of the cross section of channel 164 to reduce the vector velocity variation of the objects 160.

[00320] In one embodiment, for object 160 or flat-shaped cells such as live sperm cells 160, both the orientation and the variation in vector velocity need to be taken into account. Thus, sperm cells 160 offset from the center plane of channel 164 along the vertical geometric axis (see Figure 9B) tend to have better resolution (for example, the differentiation of sperm cells 160 X and Y is greater than 50% separated in the histogram obtained after the search) and a 160 less misguided cell population. In this way, the resolution and targeting efficiency (i.e., photodamage, killing) is balanced with, in an exemplary embodiment, the sample core stream 120 which is preferably shaped to be about 10 microns wide and 5 microns wide. at 10 microns in height of the main channel 164, through the cross section of the main channel 164, in the search/detection region of the chamber 129 and offset by about 2 to 10 microns from the central plane of the main channel 164, controlling, of preferably, the hydrodynamic focusing steps.

[00321] In an embodiment where the objects 160 are sperm cells, a targeted sperm cell 160 may be a male sperm-bearing sperm cell (ie, a Y-chromosome-bearing sperm cell) and a sperm cell untargeted 160 may be a female sperm-carrying sperm cell (ie, an X-chromosome-bearing sperm cell). In another embodiment, a targeted sperm cell 160 may be a female sperm-bearing sperm cell (i.e., an X-chromosome-bearing sperm cell) and an untargeted sperm cell 160 may be a sperm-bearing sperm cell male (ie, a Y-chromosome-bearing sperm cell).

[00322] In one embodiment, objects 160 are acted upon prior to the survey using localized heat shock to incorporate molecules, such as DNA or other probes, through protective outer layers and into objects - that is, through protective membranes and in the cells (see Figure 6D). Traditional methods for incorporating molecules using high voltages require successful electroporation to permeabilize membranes which may be desirable, as evidenced by high cell mortality rates. Localized heat shock represents a milder procedure and, therefore, may be more desirable to maintain cell viability. The focused energy apparatus 157 is used to generate a localized rise in temperature, thereby achieving thermal shock. Localized thermal shock results in permeabilizing objects 160, thereby facilitating the incorporation of the desired molecules. A searcher 147 is hereinafter used to detect and search the objects 160 from which the searcher 147 determines the number or proportion of objects 160 for which incorporation of molecules has been achieved. Such a method may be particularly desirable in biodetection, cell engineering, targeted therapeutics and drug/gene delivery.

and. ACTION ZONE

[00323] In one modality, after the search is performed and after an acceptable histogram is obtained (that is, with acceptable resolution and relatively small coverage distribution), then a decision is made to employ the focused energy apparatus 157 to act on 160 objects or selected cells. One of the most important parameters is the setting of timing for the pulse from the focused energy apparatus 157 (i.e. delay or time interval for the object 160 between the search/detection beam 148 and the focused energy beam 158 ).

[00324] The action zone of the focused energy apparatus 157 is the area in the cross-section of the main channel 164 in which the objects 160 or selected cells can be acted on effectively (i.e., photodamaged, altered, disabled, killed, destroyed , etc.), as shown in Figure 16. Based on a predetermined energy level and a beam shape of the focused energy apparatus 157, and the design of the microfluidic channel 164 and flow conditions, the action zone can be estimated by a percentage of action (for example, <97%). The energy level of the focused energy apparatus 157 is dependent on the current and charge time of the focused energy apparatus 157. The overall higher flow rate in the main channel 164 means that objects 160 are traveling faster. In this way, the transit time of objects 160 through the focused energy beam 158 is shorter. For example, for a focused energy apparatus 157 energy level of 2.3 µJ with a certain beam shape (eg 2.5 microns x 15 microns) and object travel velocity vector 160 of about 7, 5 m/s, the action zone of the focused energy apparatus 157 is estimated to be about 20 microns in the Y axis direction and about 16 microns in the vertical direction.

[00325] Within the action zone, the percentage of targeted objects 160 affected (ie, damaged, altered, or dead) also depends on the shape and position of the sample core stream 120. Adjusting the flow rates of the streams Of hydrodynamic sheath or cap focusing, the size and position of the sample core stream 120 can be adapted to accommodate the zone of action in the horizontal and vertical directions. Eventually, at the desired energy level of the focused energy apparatus 157, the flow conditions for the present microfluidic chip 100 can be determined. Thus, the shape of the sample stream 120 can preferably be restricted to about 10 microns in width and 5 to 10 microns in height by way of example.

[00326] In other embodiments, the action zone, as substantially described above, is arranged prior to the survey or after the sample fluid 120 leaves the chip 100 into the container 188 (see Figures 6B to D).

f. OPERATION IN SPERM CELLS

[00327] As discussed above, in an exemplary embodiment using sperm cells 160 as the objects 160, live sperm cells 160 (i.e., bovine sperm cells, with approximately 50-50 X chromosome and de Y chromosome) are introduced into the sample input 106 and go through the hydrodynamic focusing steps to reach the search region 129. In the search region 129, the ink, i.e. Hoechst ink 33342) is excited by a search beam 148a from a light source 147, such as a laser 147, which generates fluorescence in cells 160, which is captured by an optical signal detector 154 after passing through objective lenses 153.

[00328] Based on the characteristics of the fluorescence signal, eg a difference in reflective properties, the controller 156 can individually identify and distinguish targeted sperm cells 160 from untargeted sperm cells 160. If sperm cell 160 is a desired sperm cell (i.e., an X- or Y-chromosome sperm cell), a determination is made to allow the desired or untargeted sperm cell 160 to pass through microfluidic channel 164 to collection apparatus 188 in an untargeted manner. affected.

[00329] However, if the sperm cell is an unwanted sperm cell (ie the other sperm cell of X or Y chromosome), the focused energy apparatus 157 will be employed to act on the targeted sperm cell 160, after a predetermined delay time, to allow the unwanted/targeted sperm cell to reach the action zone (which may be in chamber 129 or between outlet 112 and collection apparatus 188.

[00330] The present invention allows for flotation in the system of the present invention, in order to have the most efficient operation of the focused energy device 157 in the action zone, after the predetermined delay time. Depending on the vector speed of travel of the targeted sperm cell 160, the targeted sperm cell 160 will be in the action zone for about 2 µs and the focused energy beam 148 is most effective when aimed at the center of the targeted sperm cell 160. Therefore, it is preferable to limit the fluctuation to within 1 µs or less.

[00331] As noted above, targeted sperm cells 160 can be altered, photodamaged, killed, altered, disabled or destroyed by: 1) targeted mode or "pulse on demand" or 2) a "continuous firing" mode of the device of focused energy 157. As noted above, it is preferred to choose a laser wavelength for the focused energy device 157 that minimizes the required flux. For example, matching the laser wavelength to the absorption of the dye used in sperm cell staining can improve efficiency and effectiveness. For example, if Hoechst ink 33342 is used for the dyeing process, a laser wavelength of 355 nm for the focused energy apparatus 157 is ideal.

[00332] In one embodiment, targeted sperm cells are killed or destroyed. In another embodiment, targeted sperm cells 160 are sufficiently disabled so that they no longer have the capacity to perform a defined function. For example, a targeted sperm cell tail 160 can be disabled so that it no longer exhibits progressive motility. In this way, the targeted and disabled sperm cell 160 will be prevented from fertilizing an egg.

[00333] In one embodiment, sophisticated software for the controller 156 can be designed for a high power laser 157 that meets the requirements described above that allow single pulses of laser 158 to be ignited. In this way, the continuous firing or "pulse on demand" mode delivers consistent laser pulses 158 whenever required by the focused energy apparatus 157. The continuous firing mode is preferably used for samples containing 120 to a greater extent. part, untargeted sperm cells 160 and only a relatively small number of targeted sperm cells 160 that need to be eliminated. However, with commercially available scalar high-speed pulse-on-demand systems, continuous firing mode can be implemented for samples that have other non-targeted to targeted sperm cell ratios.

[00334] In an alternative embodiment, when targeted sperm cells 160 largely outnumber untargeted sperm cells 160, the focused energy apparatus 157 may not have the ability to generate laser pulses 158 fast enough to act on (ie, extinguish or disable) all targeted sperm cells 160. In this way, the continuous firing mode becomes less effective. In this case, the "continuous shooting" mode becomes more favorable.

[00335] In continuous shooting mode, as discussed above, the focused energy apparatus 157 is a continuous wave (CW) or quasi-CW, or fast pulsed laser 157, used to act on (i.e. extinguish or disable) every sperm cell 160 that passes through a certain location in the microfluidic channel 164 without discriminating between targeted and untargeted sperm 160 cells. The focused energy beam 158 is turned off, deflected, or otherwise interrupted for a short period of time to allow untargeted sperm cells (ie, one of the X-chromosome or Y-chromosome cells) to pass. unaffected mode when a group of untargeted sperm cells 160 is identified. The group can be any predetermined number of untargeted sperm cells 160. After the untargeted sperm cells 160 pass the action zone, the continuous firing mode resumes.

[00336] In both directed and continuous firing modes of operation, sperm cells 160 that are too close together or that overlap in the sample fluid stream 120 are either killed or disabled, even if one of the 160 sperm cell is an untargeted 160 sperm cell instead of a 160 targeted sperm cell. As used herein, the term "very close" refers to the presence of two or more sperm cells within the range of the focused energy beam 158 such that both objects 160 are sufficiently acted upon by the focused energy beam 158 that the desired action takes place in both cells. In addition, sperm cells 160 that are unable to be effectively identified, either as an untargeted or targeted sperm cell 160, are killed or disabled by the focused energy beam 158 to ensure an overall sample purity for the discriminated product. 165. The reasons for the discrimination system of the present invention not having the ability to effectively identify an untargeted sperm cell 160 may be due to flow issues within the microfluidic channel 164, staining problems, ducts, etc. Due to the fact that the system of the present invention errs by killing or disabling targeted sperm cells 160, or any sperm cells 160 that cannot be effectively identified as either targeted or untargeted, more pulses 158 can be used than the total number of real targeted sperm cells 160 present in the sample 129.

[00337] In one embodiment, as noted above, it is preferable to include a short "recharge/charge time" between the laser pulses 158 of the focused energy apparatus 157. First, the spacing between consecutive sperm cells 160 is not uniform , but instead follows a Poisson distribution. In sample fluid flow 120, there is a percentage of sperm cells 160 that are relatively close together. To employ the focused energy apparatus 157 on events that are more closely spaced. close (ie, shorter elapsed time), a smaller “recharge/charge time” of the focused energy beam 158 is allowed. Also, if two sperm cells 160 are too close together, so that their fluorescence signals interfere with each other, and there is no clear identification of whether sperm cell 160 is a targeted or untargeted cell, focused energy apparatus 157 will have to fire multiple laser pulses 158 in a short period of time to quench or disable all 160 unidentifiable sperm cells in order to maximize sample purity. Thus, to achieve higher throughput, in general, a shorter average elapsed time between pulses 158 is required.

[00338] As noted above, the discriminated 120 sample, after being acted upon by the focused energy apparatus 157, is collected in a collection device 188. Thus, in one modality, the collected product 165 contains both sperm cells 160 untargeted as targeted sperm cells 160 (ie, killed, altered, destroyed, disabled), still at the same gender ratio as the original 120 sample. Collection in a single container 188 does not affect an overall 120 sample quality. Thus, in one embodiment, the end product 165 that is used for eventual fertilization includes both untargeted and targeted sperm cells 160. Alternatively, subsequent product 165 separation techniques (i.e., flow cytometry, electrostatic plaques, holographic optical trapping, etc.) can be used to, for example, separate sperm cells 160 from product 165 into sperm cells 160 living or dead/disabled, or centrifugation can be used to remove unwanted debris such as the remnants of dead or disabled targeted sperm cells 160.

[00339] In one embodiment, the microfluidic chip system of the present invention is used in conjunction with a separation or isolation mechanism, such as the piezoelectric actuator mounting device described in pending Patent Application No. US 13/943,322, filed in July 16, 2013, or an optical trapping system as described in US Patents 7,241,988, 7,402,131, 7,482,577, 7,545,491, 7,699,767, 8,158,927 and 8,653,442, for example, the contents of which are incorporated herein by reference in their entirety.

[00340] In one embodiment, as shown in Figures, 6B to C, the main channel 164 is shortened after the action chamber 129, so that the discriminated objects 160 leave the output channel 141 and the output 112 in drop shape 187, before falling towards a collection apparatus 188. The operation of the focused energy apparatus 157 is the same, except that the objects 160 are acted upon as they leave the output of output channel 112 (see Figure 6B) or as the disconnected drops 187 fall (see Figure 6C), and before they enter the collection apparatus 188.

POST- COLLECTION OF THE DISCRIMINATED PRODUCT

[00341] In one embodiment, the discriminated objects 160 are collected in a container 188, which has 20% Tris (note: commercially available, as sold by Chata Biosystems), which is disposed below the output 112 of the chip 100. In one embodiment, the contents of container 188 are circulated at predetermined intervals to ensure mixing of product 165 therein. In one embodiment, when container 188 reaches a predetermined volume (i.e., 18 ml), container 188 may be replaced with a new container 188.

[00342] In one embodiment, antibiotics are added to a filled container 188 (i.e., 0.5 ml of CSS antibiotics to 30 ml of product 165), with the time recorded, and a plurality of containers 188 with the product 165 are collected in a large container and cooled. In one example, three containers 188 with antibiotic-containing product 165 at a time are placed in a 400 ml plastic container and placed in a cold environment.

[00343] In one embodiment, after a predetermined amount of time in cooling (i.e., two hours), a Tris B extender (14% Tris glycerol) is added in two aliquots, separated by a predetermined time (i.e., 15 minutes) for each tube. After the last 120 sample has been cooled for a predetermined time (ie, two hours) and extender B is added, the 120 samples are centrifuged for 20 minutes at 850 xG in a centrifuge refrigerated at 5°C. The supernatant is aspirated from each tube, leaving -0.2 ml with the granule. All beads are combined in a single pre-weighed container (ie tube) and a combined bead concentration using commercially available counting protocols (ie Sperm View) is determined and the final concentration is calculated to 11.35 x 106 cells/ml. The final volume is adjusted with a complete Tris A+B+CSS antibiotic extender (ie, 50:50 ratio of 20% egg yolk Tris + 14% Tris glycerol).

[00344] In the exemplary embodiment of sperm cells 160, the imprinted semen ducts are filled and sealed and the ducts are frozen using liquid nitrogen vapor.

[00345] In one embodiment, post-freeze quality control measures include bacterial contamination steps, in which a frozen duct is thawed in a water bath, and the thawed duct is cleaned with alcohol to disinfect it. The duct contents are transferred to plates on a blood agar plate (5% sheep blood) and incubated at 37°C for 24 hours to determine the bacterial content.

[00346] In one embodiment, quality control measurements for progressive motility include thawing a frozen duct in a water bath and expelling the duct contents into a small tube placed in the tube heating stage. Commercial methods, such as Motility Analysis or Sperm Vision, are used to determine the number of cells with motility per pipeline.

[00347] In one embodiment, quality control measurements for sample purity include thawing a frozen duct in a water bath and isolating live cells by processing them through glass wool. Then, FISH analysis is performed on the live cell population to determine the sex chromosome ratio.

MULTIPLE SYSTEMS

[00348] In one embodiment, a plurality of 100 microfluidic chips, optical research apparatus, and focused energy apparatus are mounted in parallel at increasing throughput.

[00349] Referring now to Figure 18, in one embodiment, a multiple system layout 400 with, for example, "n" systems 401, 402, etc., each equipped with a focused energy apparatus 157, is illustrated . In one embodiment, the multi-system layout 400 includes a single search apparatus 147 and a single search beam 148 that is configured to provide a fluorescence excitation energy for detecting objects 160 - e.g., sperm DNA content .

[00350] In one embodiment, the multi-system layout 400 further includes beamshaping optical elements 181, with a series of beamsplitters 189, wherein each beamsplitter provides a 50/50 split of the incoming power. These results in nearly equal power beams 158 for each of multiple systems 401, 402, etc.

EXTERNAL DEVICES SEPARATION APPLIANCE

[00351] In one embodiment, after the focused energy apparatus 157 acts on objects 160 flowing through the microfluidic chip 100, rather than flowing in the output channel 141, a separation mechanism (i.e., actuator mounting device) piezoelectric (external or internal), an optical trapping assembly, electrostatic plates, or other separation mechanism well known to the person of ordinary skill in the art, can separate directed and undirected objects. (See Patent Application No. 13/ 943,322, filed July 16, 2013 or US Patents 7,241,988, 7,402,131, 7,482,577, 7,545,491, 7,699,767,8,158,927 and 8,653,442, for example, the contents of which are incorporated herein document for reference in its entirety).

[00352] In this way, in an exemplary modality, the 160 directed objects that have been damaged, killed, disabled, destroyed or altered by the focused energy apparatus 157, can be separated from undirected objects 160, in which the separation mechanism that separates objects directed on one of output channels 140 to 142 and objects 160 not directed on another of output channels 140 to 142.

[00353] In another embodiment, the directed objects are separated with the use of a separation mechanism after leaving the output 112 with the use of electrostatic plates - well known in the art - for example.

[00354] In yet another embodiment, the focused energy apparatus 157 acts on the objects 160 as they exit or fall from the outlet 112, and the separation mechanism also separates the directed objects 160 from the undirected objects 160 hereafter or the product 165 .

PUMPING MECHANISMS

[00355] As shown in Figures 19 to 21, in one embodiment, a pumping mechanism includes a system having a pressurized gas 235 that provides pressure to pump the sample fluid mixture 120 from reservoir 233 (or sample tube 233), through tubing 242, to the sample inlet 106 of the microfluidic chip 100.

[00356] A central reservoir 240 (see Figures 20 to 21) or individual reservoirs 241 (see Figure 19) contain surrounding fluids or buffer 163 and are connected to each sheath or buffer insert 107, 108, 172 of the microfluidic chip 100 through tubing, in order to introduce surrounding fluids or plug 163 therein. In one embodiment, the reservoirs are collapsible containers 237 (see Figures 20 through 21), which are disposed in pressurized vessels 240 and pressurized gas 235 pushes envelope or buffer fluids 163 into the microfluidic chip 100. In one embodiment, the pressurized vessel 240 pushes capping or enclosing fluids 163 into a manifold 238 (see Figure 21) which has a plurality of different outlets such that enclosing or buffer fluids 163 are distributed through tubing 231a, 231b, or 231c to the sheath or plug entries 107, 108, or 172, respectively, of the chip 100. Although a three-way plug or envelope fluid arrangement is shown (ie, Figure 1D), the person of ordinary skill in the art will know that less or more surrounding fluids or tubing distribution buffer for the microfluidic chip 100 is possible. Also, although the tubing is shown as entering microfluidic chip 100 in Figures 18 through 21, the arrangement of tubing relative to the entries in chip 100 is not shown in the exact order. In addition, the person of ordinary skill in the art will know that tubing enters chip 100 through chip retainer 200 (discussed below).

[00357] In one embodiment, each individual reservoir 241 or central reservoir 240 includes a pressure regulator 234 that regulates the pressure of gas 235 within reservoir 241, 240 (see Figures 19 to 21). In one embodiment, a pressure regulator 239 regulates the pressure of gas 235 within sample vessel 233. Flow meters 243 and flow valves 244 control the envelope or buffer fluids 163 pumped through tubing 231a, 231b, or 231c , respectively, at sheath or plug entries 107, 108 or 172, respectively (via retainer 200). In this way, tubing 230, 231a, 231b, 231c is used in the initial loading of the surrounding fluids or buffer 120 into chip 100 and can be used throughout the chiplOO operation to load sample fluid 120 into sample inlet 106 or inlets. of sheath or plug 107, 108 (and 172).

[00358] In one embodiment, flow meters 243 are used to provide feedback to flow valves 244 (see Figures 19 to 20) placed in the surrounding flow paths to achieve a steady flow with a constant flow rate in the channels microfluidics. With precise flow control, the overall flow rate variation is less than 1% of the set flow rate and the travel scalar velocity for 160 targeted objects varies less than 1% during detection and between search site/ detection and the place of action.

[00359] In one embodiment, the surrounding fluid or buffer 163 is pumped through a vacuum chamber (not shown) that is disposed between the pressure box and the manifold in order to remove dissolved gas in the surrounding fluid or plug. Inside the vacuum chamber, a segment of gas permeable tubing is disposed between the inlet port and the outlet port. Although the enclosing or buffer fluid travels inside the vacuum chamber through the gas permeable tubing, the dissolved gas passes through the tubing wall while the liquid remains inside the tubing. The applied vacuum helps the gas to permeate through the pipe wall. In one embodiment, gas permeable piping is produced from a porous hydrophobic material, eg expanded poly(tetrafluoroethylene) (EPTFE), which has a high water-to-liquid inlet pressure but is permeable to gas.

COMPUTER CONTROL

[00360] In one embodiment, the computer system user interface 156 includes a computer screen that displays objects 160 in a field of view acquired by a CCD camera 182 on the microfluidic chip 100.

[00361] In one embodiment, the computer 156 or a controller 156 controls any external devices, such as pumps (i.e., pump mechanisms of Figures 19 to 20), if used, to pump any sample fluids 120, surrounding fluids, or of buffer 163 on microfluidic chip 100, and also control any heating devices that set the temperature of fluids 120, 163 that are inserted on microfluidic chip 100.

[00362] According to an illustrative modality, any of the operations, steps, control options, etc., can be implemented by instructions that are stored in a computer-readable medium, such as a computer memory, database , etc. By executing the instructions stored on the computer-readable medium, the instructions can cause a computing device 156 to perform any of the operations, steps, control options, etc., described herein.

[00363] The operations described in this specification may be implemented as operations performed by a data processing apparatus or a processing circuit on data stored in one or more computer-readable storage devices or received from other sources. A computer program (also known as a program, software, software application, writing or code) can be written in any form of programming language, which includes compiled or interpreted languages, declarative or process languages, and it can be implemented in any form, included as a standalone program or as a module, component, subroutine, component, or other unit suitable for use in a computing environment. A computer program can, but need not, match a file on a file system. A program can be stored in a portion of a file that holds other programs or data (for example, one or more or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple files coordinates (for example, files that store one or more modules, subprograms or pieces of code). A computer program can be deployed to run on one computer or on multiple computers that are located at one site or distributed at multiple sites and interconnected through a communication network. Processing circuits suitable for executing a computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any type of digital computer.

MICROFLUIDIC CHIP SUPPORT

[00364] In one embodiment, the microfluidic chip 100 is loaded into a chip holder 200 (see Figures 22A to 23B). Chip detent 200 is mounted in a translation stage (not shown) to allow positioning of detent 200 relative to the research apparatus and the focused energy apparatus. The microfluidic chip retainer 200 is configured to retain the microfluidic chip 100 in one position so that the light beam 148 from the search apparatus can intercept the objects 160 in the manner described above, at the opening 150, and the scanning apparatus. focused energy 157 can act on objects 160.

[00365] The mechanisms for attaching the chip 100 to the retainer 200 are described below in conjunction with its method of operation, but the person of ordinary skill in the art will know that these devices can have any configuration to accommodate the microfluidic chip 100, provided that the objectives of the present invention are met.

[00366] As illustrated in Figures 22A to 23B, in one embodiment, a microfluidic chip retainer 200 is produced from a suitable material, such as aluminum alloy, or any other suitable metallic/polymer material, and includes a main plate 201 and a snap-in board 202. The main board 201 and snap-in board 202 can be of any suitable shape, but their configuration depends on the layout of the chip 100 and the requirements for accessing them.

[00367] In one embodiment, the main board 201 has a substantial L-shape, but the shape of the retainer 200 depends on the layout of the chip 100 (see Figures 22A to 23B). An L-shaped main plate leg 201 includes a plurality of slots that are configured to receive mounting screws 219, which are used to mount retainer 200 in the translation stage. Any number of slots of any suitable shape and size can be included on main board 201. In one embodiment, four screws 219 are used to mount retainer 200 in the translation stage and adjust the position of retainer 200. However, the skill individual Anyone in the art will know that any number of mounting slots and screws of any size can be used.

[00368] In one embodiment, a switching valve 220 is secured to the main board 201 above mounting screws 219 on the leg of the L-shaped main board 201 (see Figures 22A to 23B). In addition, on the other leg of the L-shaped main board 201, a pair of linear actuators, such as pneumatic cylinder actuators 207A and 207B, are disposed on the same side of main board 201 as switching valve 220 (see Figures 22A to 23B).

[00369] In one embodiment, the snap plate 202 is secured to the main plate 201, on the other side and along the other leg of the L-shaped main plate 201 (see Figures 22A to 23B). The socket plate 202 includes a plurality of openings that accommodate a plurality of sockets 204 to 206 (see Figures 22A to 22B, for example) configured to receive and engage external piping (see Figures 19 to 23B) to communicate fluids/samples to the microfluidic chip 100. In one embodiment, the socket plate 202 includes three sockets 204 to 206, which are used to align with the sheath or buffer inlets 107, 108 and with the sample inlet 106, respectively (see Figures IB and 22A to 22B). In another embodiment, four grooves 204-206 and 216 are used to align with the sheath or plug entries 107, 108, with the sample entry 106 and with the sheath or plug inserts 172, respectively (see Figures 1A and 23A to 23B). However, the person of ordinary skill in the art will know that the sockets are arranged anyhow by number and position in order to align with the number of entries in the microfluidic chip 100 and allow the transmission of fluids from one or more reservoirs through the piping (see Figures 19 to 21).

[00370] In one embodiment, a pair of slit wipers 203A, 203B affix snap plate 202 and pneumatic cylinder actuators 207A, 207B to main plate 201 (see Figures 22A through 23B). In this way, the snap plate 202 is easily removed from the main plate 201 without disassembling the switching valve 220 and pneumatic cylinder actuators 207A, 207B from the main plate 202.

[00371] In one embodiment, pneumatic cylinder actuators 207A, 207B each include a piston (not shown) having a piston rod coupled thereto and extensible and retractable relative to the cylindrical body portion. In one embodiment, air is supplied through port 209 to switching valve 208 and exits through ports 210, 211 to ports 212, 213, respectively, in pneumatic cylinder actuator 207A. Air is additionally supplied from ports 214A, 214B on pneumatic cylinder actuator 207A to ports 215A, 215B, respectively, on pneumatic cylinder actuator 208A.

The toggle switch 220 of the switch valve 208 opens and closes to allow or prevent, respectively, air from the air supply from entering through the port 209 and to the pneumatic cylinder actuators 207A, 207B. When air is supplied to pneumatic cylinder actuators 207A, 207B, the piston rods of pneumatic cylinder actuators 207A, 207B are extended outward to an open position (not shown) and the snap plate 202 is pushed out of the microfluidic chip 100 to an open position to allow a user to charge (or unload) microfluidic chip 100 (see Figures 22A–23B). When the air supply is closed, the piston rods of the pneumatic cylinder actuators 207A, 207B are retracted inward to a closed position (as illustrated in Figures 22A to 23B) and the snap plate 202 is drawn towards the chip. 100 and pressed against chip 100, which forms a liquid seal between chip 100 and the tubing connections for the surrounding or buffer and sample fluids.

[00373] In one embodiment, when the microfluidic chip 100 is in the closed position, the O-rings, which are disposed on the surface of the snap plate 202, between the snap plate 202 and the chip 110, form a seal substantially free of leakage to protect the microfluidic chip 100 from damage. However, the individual of ordinary skill in the art will know that any number and configuration of O-rings or gaskets can be used.

[00374] In one embodiment, the retainer 200 is positioned such that the chip 100 is at a height sufficient to accommodate at least one collection vessel 188 disposed under the chip 100. In another embodiment, the collection vessels are arranged between each output 111 to 113.

[00375] The construction and arrangements of the microfluidic chip system with the focused energy device, as shown in the various illustrated embodiments, are illustrative only. It should be noted that the orientation of various elements may differ according to other illustrative embodiments, and that such variations are intended to be covered by the present disclosure. Although only a few modalities have been described in detail in this disclosure, many modifications are possible (eg variations in sizes, dimensions, structures, shapes and proportions of the various elements, parameter values, mounting arrangements, use of materials, colors, orientations , etc.) without departing materially from the teachings and innovative advantages of the subject described in this document. Some elements shown as integrally formed may be constructed from multiple parts or elements, the position of elements may be reversed or otherwise varied, and the nature or number of distinct elements or positions may be changed or varied. The order or sequence of any process, logical algorithm or method steps can be varied or resequenced according to alternative modalities. Other substitutions, modifications, alterations and omissions may also be made in the design, conditions and operating arrangement of the various illustrative modalities without departing from the scope of the present disclosure. All such modifications and variations are intended to be included herein within the scope of the invention and protected by the following claims.

Claims (45)

1. Computer system identifying objects (160) characterized in that it comprises: at least one memory containing at least one program comprising the steps of: controlling a flow of a sample fluid mixture containing objects (160) to be identified, through a main fluid channel (164) of a microfluidic chip (100); controlling an introduction of a plurality of sheath fluids into said microfluidic chip (100), said plurality of sheath fluids orienting said objects (160) in said main fluid channel (164) in a predetermined direction while still maintaining flow laminar in said main fluid channel (164); wherein said plurality of sheath fluids is in a plurality of sheath fluid channels comprising: a first plurality of sheath fluid channels (116, 117) intersecting said main fluid channel (164) at a first intersection (161) to perform a first hydrodynamic focusing step such that sheath fluids compress said sample fluid mixture on at least two sides so that said sample fluid mixture becomes a relatively smaller flow. , narrower, limited by said sheath fluids while maintaining laminar flow in said main fluid channel (164); and a second plurality of sheath fluid channels (114, 115) intersecting said main fluid channel (164) at a second intersection (162) downstream of said first intersection (161) to perform a second hydrodynamic focusing step. , such that said sheath fluids from said second plurality of sheath fluid channels (114, 115) compress said sample fluid mixture on said at least two sides, or on two opposite sides of said at least two sides. , so that said mixture of sample fluid is further compressed while still maintaining laminar flow in said main fluid; wherein said second plurality of sheath fluid channels (114, 115) includes at least a first portion disposed vertically above said main fluid channel (164) so that said first portion joins said main fluid channel ( 164) from a substantially right angle to and above said main fluid channel (164); wherein said plurality of sheath fluid channels hydrodynamically focuses said objects (160) such that said objects (160) are oriented in a predetermined direction and disposed in a restricted core volume as said objects (160) flow through said main fluid channel (164); and wherein a ratio of a sheath fluid flow rate of said first and second plurality of sheath fluid channels to a flow rate of said sample fluid mixture is 200-400:1 such that said mixture of sample fluid is displaced from a position having a maximum flow velocity in a cross section of said main fluid channel (164); analyzing, in an action chamber (129), a search of said oriented objects (160) in said main fluid channel (164) with the use of a search apparatus, wherein said action chamber (129) is arranged on said microfluidic chip (100) downstream of said second intersection (162), wherein said search apparatus comprises a first laser (147) which detects and searches said oriented objects (160) in said main fluid channel (164 ), wherein said first laser (147) excites fluorescence in said oriented objects (160) so that target objects are differentiated from non-target objects; and controlling an action on said objects (160) with the use of a second laser (157), which supplies electromagnetic energy to said oriented objects (160), said second laser (157) is disposed downstream of said search apparatus. ; where the action comprises damaging, disabling, altering, extinguishing or destroying said targeted objects; and a processor (156) that executes the program. 2. Non-transient computer readable medium containing instructions for identifying objects (160) characterized in that it comprises: controlling a flow of a sample fluid mixture containing objects (160) to be identified, through a main fluid channel (164) of a microfluidic chip (100); controlling an introduction of a plurality of sheath fluid channels into said microfluidic chip (100), wherein said plurality of sheath fluids orients said objects (160) in said main fluid channel (164) in a predetermined direction while still maintaining laminar flow in said main fluid channel (164); wherein said plurality of sheath fluids is in a plurality of sheath fluid channels comprising: a first plurality of sheath fluid channels (116, 117) intersecting said main fluid channel (164) at a first intersection (161) to perform a first hydrodynamic focusing step such that sheath fluids compress said sample fluid mixture on at least two sides so that said sample fluid mixture becomes a relatively smaller flow. , narrower, limited by said sheath fluids while maintaining laminar flow in said main fluid channel (164); and a second plurality of sheath fluid channels (114, 115) intersecting said main fluid channel (164) at a second intersection (162) downstream of said first intersection (161) to perform a second hydrodynamic focusing step. , such that said sheath fluids from said second plurality of sheath fluid channels (114, 115) further compress said sample fluid mixture on said at least two sides, or on two opposite sides of said at least two sides, such that said mixture of sample fluid is further compressed while still maintaining laminar flow in said main fluid; wherein said second plurality of sheath fluid channels (114, 115) includes at least a first portion disposed vertically above said main fluid channel (164) so that said first portion joins said main fluid channel. (164) from a substantially right angle to and above said main fluid channel (164); wherein said plurality of sheath fluid channels hydrodynamically focuses said objects (160) such that said objects (160) are oriented in a predetermined direction and disposed in a restricted core volume as said objects (160) flow through said main fluid channel (164); and wherein a ratio of a sheath fluid flow rate of said first and second plurality of sheath fluid channels to a flow rate of said sample fluid mixture is 200-400:1 such that said mixture of sample fluid is displaced from a position having a maximum flow velocity in a cross section of said main fluid channel; analyzing, in an action chamber (129), a search of said oriented objects (160) in said main fluid channel (164) with the use of a search apparatus, wherein said action chamber (129) is arranged on said microfluidic chip (100) downstream of said second intersection (162), wherein said search apparatus comprises a first laser (147) which detects and searches said oriented objects (160) in said main fluid channel (164 ), wherein said first laser (147) excites fluorescence in said oriented objects (160) so that target objects are differentiated from non-target objects; controlling an action on said objects (160) with the use of a second laser (157), which supplies electromagnetic energy to said oriented objects (160), said second laser (157) being disposed downstream of said research apparatus; and wherein said action comprises damaging, disabling, altering, extinguishing or destroying said targeted objects. 3. Method for identifying objects (160) flowing in a sample fluid mixture, characterized in that it comprises: flowing a sample fluid mixture containing objects (160) to be identified through a main fluid channel (164 ) of a microfluidic chip (100); introducing a plurality of sheath fluids into said microfluidic chip (100), said plurality of sheath fluids orients said objects (160) in said main fluid channel (164) in a predetermined direction while still maintaining laminar flow in said channel. of main fluid (164); wherein said plurality of sheath fluids is in a plurality of sheath fluid channels comprising: a first plurality of sheath fluid channels (116, 117) intersecting said main fluid channel (164) at a first intersection (161) to perform a first hydrodynamic focusing step such that sheath fluids compress said sample fluid mixture on at least two sides so that said sample fluid mixture becomes a relatively smaller flow. , narrower, limited by said sheath fluids while maintaining laminar flow in said main fluid channel (164); and a second plurality of sheath fluid channels (114, 115) intersecting said main fluid channel (164) at a second intersection (162) downstream of said first intersection (161) to perform a second hydrodynamic focusing step. , such that said sheath fluids from said second plurality of sheath fluid channels (114, 115) further compress said sample fluid mixture on said at least two sides, or on two opposite sides of said at least two sides such that said sample fluid mixture is further compressed while still maintaining laminar flow in said main fluid channel (164); wherein said second plurality of sheath fluid channels (114, 115) includes at least a first portion disposed vertically above said main fluid channel (164) so that said first portion joins said main fluid channel. (164) from a substantially right angle to and above said main fluid channel (164); and wherein said plurality of sheath fluid channels hydrodynamically focus said objects (160) such that said objects (160) are oriented in a predetermined direction and disposed in a restricted core volume conforming to said objects (160) flow through said main fluid channel (164); and wherein a ratio of a sheath fluid flow rate of said first and second plurality of sheath fluid channels to a flow rate of said sample fluid mixture is 200-400:1 such that said mixture of sample fluid is displaced from a position having a maximum flow velocity in a cross section of said main fluid channel (164); searching, in an action chamber (129), said objects oriented (160) in said main fluid channel (164) with the use of a search apparatus, wherein said action chamber (129) is disposed in said microfluidic chip (100) downstream of said second intersection (162), wherein said search apparatus comprises a first laser (147) which detects and searches said oriented objects (160) in said main fluid channel (164) wherein said first laser (147) excites fluorescence in said oriented objects (160) so that target objects are differentiated from non-target objects; and using a second laser (157) to act on said objects (160), which supplies electromagnetic energy to said oriented objects (160), said second laser (157) being disposed downstream of said search apparatus; and in which the action comprises damaging, disabling, altering, extinguishing or destroying said targeted objects. 4. Apparatus identifying objects (160) characterized in that it comprises: a microfluidic chip (100) in which a plurality of channels are arranged, including: a main fluid channel (164) in which a mixture of sample fluid of objects (160) to be identified is introduced; and a plurality of sheath fluid channels into which sheath fluids are introduced, said sheath fluids orienting said objects (160) in said main fluid channel (164) in a predetermined direction while still maintaining laminar flow. in said main fluid channel (164); wherein said plurality of sheath fluid channels comprises: a first plurality of sheath fluid channels (116, 117) that intersect said main fluid channel (164) at a first intersection (161) such that the said sheath fluids compress said sample fluid mixture on at least two sides so that said fluid mixture becomes a relatively smaller, narrower flow limited by said sheath fluids, while maintaining laminar flow in the said main fluid channel (164); and a second plurality of sheath fluid channels (114, 115) intersecting said main fluid channel (164) at a second intersection (162) downstream of said first intersection (161) so that said fluids from sheath of said second plurality of sheath fluid channels (114, 115) compress said mixture of sample fluid on one of said at least two sides, or on two opposite sides of said at least two sides, so that said sample fluid mixture is further compressed while still maintaining laminar flow in said main fluid channel (164); wherein said second plurality of sheath fluid channels (114, 115) includes at least a first portion disposed vertically above said main fluid channel (164) so that said first portion joins said main fluid channel. (164) from a substantially right angle to and above said main fluid channel (164); and wherein a ratio of a sheath fluid flow rate of said first and second plurality of sheath fluid channels to a flow rate of said sample fluid mixture is 200-400:1 such that said mixture of sample fluid is displaced from a position having a maximum flow velocity in a cross section of said main fluid channel (164); a search apparatus comprising a first laser (147) that detects and searches said oriented objects (160) in said main fluid channel (164); and a second laser (157) providing electromagnetic energy to said objects (160), said second laser disposed downstream of said research apparatus. 5. Apparatus according to claim 4, characterized in that said search apparatus detects and searches said objects (160) to determine information about said objects (160). 6. Apparatus according to claim 5, characterized in that said information about said objects (160) determines whether said objects (160) are to be directed by said second laser (157). 7. Apparatus according to claim 6, characterized in that said action of said second laser (157) acts on said targeted objects or a region surrounding said targeted objects. 8. Apparatus according to claim 7, characterized in that said action on said targeted objects is to damage, disable, alter, extinguish or destroy said targeted objects. The apparatus of claim 6, further comprising: at least one outlet channel (112) leading from said main fluid channel (164), said at least one outlet channel (112 ) removes said objects (160) from said microfluidic chip (100). 10. Apparatus according to claim 9, characterized in that said at least one output channel (112) removes directed and non-directed objects from said microfluidic chip (100). 11. Apparatus according to claim 9, characterized in that it further comprises: a plurality of side outlet channels (140, 142) leading from said main fluid channel (164), said plurality of outlet channels side (140, 142) disposed on each side of said at least one outlet channel (112), said plurality of side outlet channels (140, 142) removes said sheath fluids from said microfluidic chip (100). 12. Apparatus according to claim 4, characterized in that said second set of sheath fluid channels (114, 115) compresses said sample fluid mixture from said at least two sides. 13. Apparatus according to claim 4, characterized in that said plurality of sheath fluid channels hydrodynamically focuses said objects (160) so that said objects (160) are oriented in a predetermined direction and disposed in a restricted core volume as said objects flow through said main fluid channel (164). 14. Apparatus according to claim 13, further comprising: an action chamber (129) in which said first laser (147) of said search apparatus searches for said hydrodynamically focused objects (160) in the said sample fluid mixture, said action chamber (129) disposed on said microfluidic chip (100) downstream of said second intersection (162) and said first laser (147) disposed adjacent to said main fluid channel (164 ). 15. Apparatus according to claim 14, characterized in that said first laser (147) emits a beam of light to said action chamber (129) to illuminate and excite said objects (160) in said mixture of sample fluid. 16. Apparatus according to claim 15, characterized in that said light beam excites fluorescence in said objects (160), so that said directed objects are distinct from said undirected objects. 17. Apparatus according to claim 15, characterized in that it further comprises: an optical signal detector (154) that detects said light beam and converts it into an electronic signal; and a controller (156) which analyzes said electronic signal to determine whether said objects (160) should be targeted. 18. Apparatus according to claim 14, characterized in that said microfluidic chip (100) contains one or more structural or plane layers. 19. Apparatus according to claim 18, characterized in that said main fluid channel (164) is disposed in a layer or structural plane different from said plurality of sheath fluid channels. 20. Apparatus according to claim 18, characterized in that at least one of said sample inlet channel and said plurality of sheath fluid channels are disposed between said structural layers or said planes of said chip. microfluidic (100). 21. Apparatus according to claim 18, characterized in that said first plurality of sheath fluid channels (116, 117) is disposed in a different structural layer or plane than said second plurality of sheath fluid channels (114, 115). 22. Apparatus according to claim 18, characterized in that a width of one of said second plurality of sheath fluid channels (114) is different from another of said second plurality of sheath fluid channels (115) . 23. The apparatus of claim 18, wherein a width of said second plurality of sheath fluid channels (114, 115) is different from a width of said first plurality of sheath fluid channels (116 , 117). 24. Apparatus according to claim 11, further comprising: a plurality of outlets disposed at one end of each of said plurality of side outlet channels (140, 142). 25. The apparatus of claim 24, further comprising: at least one notch (146) comprising a recessed portion disposed on a lower edge of said microfluidic chip (100) between each outlet of said plurality of outlets . 26. Apparatus according to claim 24, characterized in that a size of said plurality of side outlet channels (140, 142) is different from a size of said main fluid channel (164). 27. Apparatus according to claim 13, characterized in that said main fluid channel (164) comprises an angled taper at an entry point at said first intersection (161) on said microfluidic chip (100) of so that the width of said main fluid channel (164) gradually decreases before said inlet point. 28. Apparatus according to claim 14, characterized in that said main fluid channel (164) comprises an angled taper in said action chamber (129) so that the width of said main fluid channel ( 164) gradually narrows before crossing said action chamber (129) 29. Apparatus according to claim 13, characterized in that said second plurality of sheath fluid channels (114, 115) comprise an angular taper at an entry point in said main fluid channel (164) of so that the width of said second plurality of sheath fluid channels (114, 115) gradually decreases prior to said entry point. 30. Apparatus according to claim 4, characterized in that said second plurality of sheath fluid channels (114, 115) includes a second vertical portion that joins said main fluid channel (164) of substantially a right angle below said main fluid channel (164). 31. Apparatus according to claim 13, characterized in that said main fluid channel (164) comprises an internal ramp disposed before said first intersection (161) so that the width of said main fluid channel ( 164) narrows before said first intersection (161). 32. Apparatus according to claim 13, characterized in that said main fluid channel (164) comprises an internal ramp disposed before said second intersection (162) so that the width of said main fluid channel ( 164) narrows before said second intersection (162). 33. Apparatus according to claim 13, characterized in that said second plurality of sheath fluid channels (114, 115) comprises an internal ramp disposed before said second intersection (162) so that the width of the said second plurality of sheath fluid channels (114, 115) narrows before said second intersection (162). 34. Apparatus according to claim 4, characterized in that said objects (160) are cells. 35. Apparatus according to claim 34, characterized in that said cells to be actuated by said second laser (157) include at least one of viable or mobile sperm from non-viable or non-motile sperm, and sperm discriminated by gender or other variations of sex discrimination. 36. Apparatus according to claim 4, characterized in that said first laser (147) is a pulsed Q-switch laser preferably capable of delivering 10 ns energy pulses to said objects (160) at a rate of more than 200,000 pulses per second. 37. Apparatus according to claim 4, characterized in that said second laser (157) acts on said objects (160) for a predetermined period of time after said search of said objects (160). 38. Apparatus according to claim 23, characterized in that said second laser (157) acts on said objects (160) when said objects (160) leave said at least one outlet before being collected in a container (188). 39. Apparatus according to claim 37, characterized in that it further comprises: a container (188) that collects targeted objects and non-targeted objects. 40. The apparatus of claim 18, further comprising: a pumping apparatus that pumps at least one of said sample fluid mixture or said sheath fluids to said microfluidic chip (100). 41. Apparatus according to claim 40, characterized in that said pumping apparatus pumps said at least one of said mixture of sample fluid and said sheath fluids to said microfluidic chip (100) using tubing external. 42. The apparatus of claim 41, further comprising: at least one external reservoir containing at least one of said mixture of sample fluid and said sheath fluids. 43. The apparatus of claim 42, further comprising: a microfluidic chip retainer (200) on which said microfluidic chip (100) is mounted, said microfluidic chip retainer (200) including openings through which said external tubing accesses said microfluidic chip (100) from said at least one external reservoir. 44. The apparatus of claim 43, further comprising: a controller (156) that controls said pumping of one of said mixture of sample fluid or said sheath fluids into said chip microfluidic. 45. The apparatus of claim 44, further comprising: a plurality of microfluidic chips (100) arranged in parallel, said plurality of microfluidic chips (100) containing a plurality of sample fluid mixtures; wherein a single research apparatus is used for each of the plurality of microfluidic chips (100).

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