AU2023286012B2 - Microfluidic chip device for optical force measurements and cell imaging using microfluidic chip configuration and dynamics - Google Patents

Abstract

#$%^&*AU2023286012B220250925.pdf##### ABSTRACT A microfluidic chip configuration wherein injection occurs in an upwards vertical direction, and fluid vessels are located below the chip in order to minimize particle settling before and at the analysis portion of the chip's channels. The input and fluid flow up through the bottom of the chip, in one aspect using a manifold, which avoids orthogonal re-orientation of fluid dynamics. The contents of the vial are located below the chip and pumped upwards and vertically directly into the first channel of the chip. A long channel extends from the bottom of the chip to near the top of the chip where it takes a short horizontal turn. The fluid is pumped up to a horizontal analysis portion that is the highest channel/fluidic point in the chip and thus close to the top of the chip, which results in clearer imaging. A laser may also suspend cells or particles in this channel during analysis. ABSTRACT A microfluidic chip configuration wherein injection occurs in an upwards vertical direction, and fluid vessels are located below the chip in order to minimize particle settling before and at the analysis portion of the chip's channels. The input and fluid flow up through the bottom of the chip, in one aspect using a manifold, which avoids orthogonal re-orientation of fluid dynamics. The contents of the vial are located below the chip and pumped upwards and vertically directly into the first channel of the chip. A long channel extends from the bottom of the chip to near the top of the chip where it takes a short horizontal turn. The fluid is pumped up to a horizontal analysis portion that is the highest channel/fluidio point in the chip and thus close to the top of the chip, which results in clearer imaging. A laser may also suspend cells or particles in this channel during analysis. 20 23 28 60 12 27 D ec 2 02 3 A B S T R A C T 2 0 2 3 2 8 6 0 1 2 2 7 D e c 2 0 2 3 A m i c r o f l u i d i c c h i p c o n f i g u r a t i o n w h e r e i n i n j e c t i o n o c c u r s i n a n u p w a r d s v e r t i c a l d i r e c t i o n , a n d f l u i d v e s s e l s a r e l o c a t e d b e l o w t h e c h i p i n o r d e r t o m i n i m i z e p a r t i c l e s e t t l i n g b e f o r e a n d a t t h e a n a l y s i s p o r t i o n o f t h e c h i p ' s c h a n n e l s . T h e i n p u t a n d f l u i d f l o w u p t h r o u g h t h e b o t t o m o f t h e c h i p , i n o n e a s p e c t u s i n g a m a n i f o l d , w h i c h a v o i d s o r t h o g o n a l r e - o r i e n t a t i o n o f f l u i d d y n a m i c s . T h e c o n t e n t s o f t h e v i a l a r e l o c a t e d b e l o w t h e c h i p a n d p u m p e d u p w a r d s a n d v e r t i c a l l y d i r e c t l y i n t o t h e f i r s t c h a n n e l o f t h e c h i p . A l o n g c h a n n e l e x t e n d s f r o m t h e b o t t o m o f t h e c h i p t o n e a r t h e t o p o f t h e c h i p w h e r e i t t a k e s a s h o r t h o r i z o n t a l t u r n . T h e f l u i d i s p u m p e d u p t o a h o r i z o n t a l a n a l y s i s p o r t i o n t h a t i s t h e h i g h e s t c h a n n e l / f l u i d i c p o i n t i n t h e c h i p a n d t h u s c l o s e t o t h e t o p o f t h e c h i p , w h i c h r e s u l t s i n c l e a r e r i m a g i n g . A l a s e r m a y a l s o s u s p e n d c e l l s o r p a r t i c l e s i n t h i s c h a n n e l d u r i n g a n a l y s i s .

Description

MARKED-UP COPY MICROFLUIDIC CHIP DEVICE FOR OPTICAL FORCE MEASUREMENTS AND CELL IMAGING USING MICROFLUIDIC CHIP CONFIGURATION AND DYNAMICS

CROSS-REFERENCE TO RELATED APPLICATIONS 2023286012

[0001] This is a divisional application of Australian Patent Application No. 2017443695, which is the Australian National Phase of International Patent Application PCT/US2017/068373, the disclosure of which is incorporated herein by reference in its entirety. BACKGROUND Field of the Invention:

[0001A] This invention relates in general to a device and method for particle analysis and imaging for particles or cells in fluids, and in particular to a device and method for particle imaging for fluids using pressure, fluid dynamics, electrokinetic, and optical forces.

[0002] The present invention is directed to a microfluidic chip wherein injection occurs in an upwards vertical direction, and fluid vials are located below the chip in order to minimize particle settling before and at the analysis portion of the chip’s channels.

[0003] Changes had to be made to existing microfludic chip designs to implement the invention herein. For example, to keep the vials vertically in-line with the chip, a different interface with the chip had to be established as compared to the prior art. Specifically, instead of the input tube interfacing with the chip via a port attached to the largest face of the chip (as is typically done in microfluidic lab-on-a-chip systems) in an orthogonal manner and then pumping fluid first across and then up the chip, the current invention has the input and fluid coming up through the bottom of the chip, in one aspect using a manifold, which avoids horizontal re-orientation of fluid/fluid dynamics.

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[0004] According to the invention herein, the contents of the vial are located below the chip and pumped upwards and vertically directly into the first channel of the chip. A long channel extends from the bottom of the chip to near the top of the chip. Then the 2023286012

channel takes a short horizontal turn but the new channel is so short as to almost negate

1A

any influence of cell settling due to gravity and zero flow velocity at the walls. Then, any influence of cell settling due to gravity and zero flow velocity at the walls. Then,

contrary to the prior art, the fluid is pumped up to the analysis portion. The horizontal contrary to the prior art, the fluid is pumped up to the analysis portion. The horizontal

analysis portionisistherefore analysis portion therefore thethe highest highest channel/fluidic channel/fluidic point point in the in theand chip chip and thus thus close to close to

the top of the chip, which results in less chip material (e.g., glass) between the the top of the chip, which results in less chip material (e.g., glass) between the

microscope/camera microscope/camera and and thethe sample sample than than thethe prior prior artand art andtherefore thereforeclearer clearer imaging. imaging.The The laser also laser also suspends suspends cells cellsin inthis channel this channelduring duringanalysis analysiswhich whichprevents preventsthem them from from 2023286012

settling. settling.

Description of the Related Art: Description of the Related Art:

[0005] According to the prior art, microfluidic chip vials containing cells or particles to

[0005] According to the prior art, microfluidic chip vials containing cells or particles to

be separated be separated and/or and/or analyzed analyzedare are located located to to the the side side and and horizontally horizontally pumped into the pumped into the channels in the microfluidic chip. First the contents of the vials (e.g., particles or cells) channels in the microfluidic chip. First the contents of the vials (e.g., particles or cells)

were pumped were pumpedin in theupwards the upwards verticaldirection, vertical direction,then thenmake makea au-turn u-turntototravel travel down, down,and and are then pumped horizontally into the chip. (See, e.g., U.S. Patent No. 9,594,071.) are then pumped horizontally into the chip. (See, e.g., U.S. Patent No. (9,594,071.)

[0006] The

[0006] Theconnection connectiontotothe thechip chipisis horizontal, horizontal, which, which, combined withthe combined with thedead deadvolume volume (empty space in fluidic connections that is to some extent unavoidable) in the connection, (empty space in fluidic connections that is to some extent unavoidable) in the connection,

led to significant additional settling due to gravity. Such a configuration also necessitates led to significant additional settling due to gravity. Such a configuration also necessitates

a relatively large diameter channel required by the connection, which, in addition to the a relatively large diameter channel required by the connection, which, in addition to the

dead volume, creates an area of relatively low velocity, further increasing the problem of dead volume, creates an area of relatively low velocity, further increasing the problem of

particle settling. The current chip according to the present invention eliminates the need particle settling. The current chip according to the present invention eliminates the need

for a large horizontal input channel and a rather abrupt change from a large horizontal for a large horizontal input channel and a rather abrupt change from a large horizontal

input channel to the relatively thin upward flow in the first vertical chip channel. Such a input channel to the relatively thin upward flow in the first vertical chip channel. Such a

configuration eliminates configuration eliminates the the horizontal horizontal settling settling and anand an unnecessary unnecessary change in change in direction direction

which causes settling. Having the cells enter the bottom edge of the chip also solves the which causes settling. Having the cells enter the bottom edge of the chip also solves the

issue of settling in the dead volume by orienting it vertically with respect to gravity so issue of settling in the dead volume by orienting it vertically with respect to gravity SO

that the cells or particles cannot settle in the bottom of a horizontal channel, but rather that the cells or particles cannot settle in the bottom of a horizontal channel, but rather

they are they are constantly constantly guided upwardsbybythe guided upwards theflow. flow.This Thisisisnot notintuitive intuitive and and required required much much

experimentationtoto realize experimentation realize the the problem before designing problem before designingthe the current current embodied embodiedsolution. solution. Currently available Currently available microfluidic microfluidic devices devices incorporate incorporate custom orcommercially custom or commercially available available

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connections on the polished surface and larger area of the glass, in contrast to the present connections on the polished surface and larger area of the glass, in contrast to the present

invention, which generally forces any particles, such as cells, contained within the sample invention, which generally forces any particles, such as cells, contained within the sample

stream to take an immediate tum and travel horizontally upon entering the chip. stream to take an immediate turn and travel horizontally upon entering the chip.

[0007] Also in the prior art, the cells or particles have several horizontal runs on a

[0007] Also in the prior art, the cells or particles have several horizontal runs on a

microfluidic chip before reaching the analysis channel, which leads to settling. At the microfluidic chip before reaching the analysis channel, which leads to settling. At the 2023286012

point the vial contents enter the chip and the channels in the chip, the contents are point the vial contents enter the chip and the channels in the chip, the contents are

pumpedhorizontally pumped horizontallycompared comparedto to thethe verticalin-chip vertical in-chipchannel. channel.The The channel channel then then flows flows

upwards and takes a long horizontal tum at which point the cells tend to settle at the upwards and takes a long horizontal turn at which point the cells tend to settle at the

bottom of the channel due to gravity, as well as experience lower velocity at the wall due bottom of the channel due to gravity, as well as experience lower velocity at the wall due

to laminar flow conditions. In essence, due to parabolic velocity profile, the flow is to laminar flow conditions. In essence, due to parabolic velocity profile, the flow is

highest in the middle of a channel and decreases to zero at or near the channel wall. highest in the middle of a channel and decreases to zero at or near the channel wall.

After the first horizontal in-chip channel, the fluid takes a downward tum before the After the first horizontal in-chip channel, the fluid takes a downward turn before the

analysis channel where the particles are imaged or separated. Due to this configuration, analysis channel where the particles are imaged or separated. Due to this configuration,

there exists a relatively large distance between the microscope/camera and the analysis there exists a relatively large distance between the microscope/camera and the analysis

channel. In this typical prior art configuration, the particles are forced downward and channel. In this typical prior art configuration, the particles are forced downward and

ultimately exit the bottom of the chip. ultimately exit the bottom of the chip.

[0008] Moreover, due to constraints in the prior art, multiple horizontal runs are required,

[0008] Moreover, due to constraints in the prior art, multiple horizontal runs are required,

causing cells to settle in multiple places in the channels. This, in tum, causes a decrease causing cells to settle in multiple places in the channels. This, in turn, causes a decrease

in image in quality because image quality of the because of the need to image need to throughadditional image through additionalmaterial material at at the the edge edge of of

the chip. The prior art channels in the chip had to be pumped in the upwards vertical the chip. The prior art channels in the chip had to be pumped in the upwards vertical

direction, then horizontal, then in a zigzag nature for adequate cell or particle suspension, direction, then horizontal, then in a zigzag nature for adequate cell or particle suspension,

then down then downand andout outthe thechip. chip. Zigzag Zigzagchannels channels areobviated are obviated byby thecurrent the currentinvention. invention.

[0009] Prior art also exists regarding rendering 3D images of cells or particles in fluid.

[0009] Prior art also exists regarding rendering 3D images of cells or particles in fluid.

For example, For example,M.M.Habaza, Habaza,M. M. Kirschbaum, Kirschbaum, C. Guemth-Marschner, C. Guernth-Marschner, G. Dardikman, G. Dardikman, I. I. Bamea, R. Barnea, R. Korenstein, Korenstein, C. C. Duschl, Duschl,N. N.T.T.Shaked, Shaked,Adv. Adv.Sci. Sci.2017, 2017,4,4,1600205, 1600205, teaches teaches

trapping a cell, rotating it at high speeds, and using interferometry to measure refractive trapping a cell, rotating it at high speeds, and using interferometry to measure refractive

index distribution within a cell. Interferometry has also been used to analyze cells in index distribution within a cell. Interferometry has also been used to analyze cells in

microfluidic channels (see e.g., Y. Sung et al., Phys. Rev. Appl. 2014 Feb. 27; 1: microfluidic channels (see e.g., Y. Sung et al., Phys. Rev. Appl. 2014 Feb. 27; 1:

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014002). The current invention, however, claims taking multiple images of a cell or

particle as it travels in the fluid flow and passes through the focal planes of the imaging

device(s), thereby negating any need to trap the cell in order to render a 3D image. Other

techniques have been taught, such as moving a cell or particle using a mechanical

translation stage (e.g., N. Lue et al., Opt. Express 2008 Sep. 29; 16(20): 16240-6), none

of which use bright field imaging as described herein and do not utilize fluid flow to 2023286012

provide cell positioning relative to the image focal plane.

[0010] All prior art references cited herein are incorporated by reference in their entirety.

SUMMARY

[0011] The present invention is directed to a microfluidic chip wherein injection occurs

and sample vials are located below the chip in order to minimize particle settling.

Therefore, the contents of the vial are located below the chip and pumped upwards and

vertically directly into the channel of the chip. A long channel extends from the bottom

of the chip to near the top of the chip. Then the channel takes a short horizontal turn but

the new channel is sufficiently short as to be insignificant with respect to any influence of

cell settling due to zero flow velocity at the walls of the channel. Then, contrary to the

prior art, the sample is pumped up to the analysis portion. The horizontal analysis

portion is therefore the highest channel/fluidic point in the chip and thus close to the top

of the chip, which results in less glass between microscope/camera than the prior art and

therefore clearer imaging. Distance of the analysis channel from the top of the chip may

be from 100 microns to 2 mm, but as great as 100 mm such as from 100 microns to 200

microns, from 200 microns to 300 microns, from 300 microns to 400 microns, and SO

on. In one embodiment, after the analysis portion of the chip, the sample, e.g., fluid,

cells, and/or particles, are pumped downwards to the bottom of the chip and forced

outwards.

[0012] The present invention is further directed to a microfluidic chip wherein horizontal

runs are minimized, especially at the point fluid enters in the chip channels. Prior art

chips contain about 13 mm in horizontal channels (non-analysis portions), around 2 mm

of which is of the much larger diameter injection port, exacerbating settling due to the

low velocity. The chip described herein has, in a preferred embodiment, about 0.2 to 3.0

mm in horizontal channels (non-analysis), although the horizontal channels may range in

length from 0.01 to 100.0 mm, such as from 0.01 mm to 0.02 mm, from 0.02 mm to 0.03

mm, from 0.03 mm to 0.04 mm, and SO on. This is a result of the invented channel 2023286012

system and is an order of magnitude different from the prior art, which improves flow

and elimination of cell/particle settling.

[0013] Another aspect of the invention is directed to a microfluidic chip wherein imaging

occurs and analysis is based at or near the corner of the chip, whereby imaging from

multiple viewpoints is improved because less glass and distance exists between the

camera and analysis channel. This also allows a higher numerical objective lens to be

used to improve detailed imaging by increasing magnification. Such a design

improvement decreases the distortion of the image caused by glass (or other substance

comprising the chip, such as plastic or any transparent or semi-transparent material) and

distance (e.g., due to imperfections in glass). The distance between the imaging device

and analysis channel may be from 100 microns to 2 mm, but as great as 100 mm, such as

from 100 microns to 200 microns, from 200 microns to 300 microns, from 300 microns

to 400 microns, and SO on.

[0014] Further, the present invention is directed to a microfluidic sorting chip with

separation downstream from an analysis channel allowing for using both pressure and/or

a laser (or other optical force) simultaneously or sequentially separately to activate a

sorting function. In one aspect, flow would continue from the analysis channel for the

sorting function. For example, the particles would be directed into a vertical channel and

then to a horizontal sorting channel. In one embodiment, an optical force and/or pressure

would be applied in the direction of the flow with the sorting channel to push particles

through the channel. Particles not directly acted upon by the optical force would divert to

an alternate channel due to, for example, gravity, electrokinetic forces, magnetic forces,

laminar flow lines, stream lines, decreased flow rate, an orthogonal optical force, or a

vacuum being applied to suck the particles into the alternate channel. In another aspect

related to the sorting post-analysis channel, the present invention allows for directing an

optical force from a back side of the chip (the laser or optical force being oriented in the

same direction of flow) and, in some aspects, splitting this primary laser. In

embodiments, the optical force and/or pressure may be applied in the opposite direction

of the movement of the substance through the channel, e.g., against the flow, or may be 2023286012

applied in the same direction as the movement of the substance in the channel, e.g., with

the flow. Cell or particle sorting could occur on a single device or a separate chip. For

example, in FIG. 1B, the fifth channel prior to the outlet tubing 145, contains one or more

bifurcations to enable single or multiple sorting regions.

[0015] In another aspect of the current invention, a manifold is connected to a vial in a

way such that tubing goes through the manifold and connects to a vial or other vessel on

the other side making contact with the substance (e.g., fluid) in the vial. The manifold

allows for vials to be connected to the microfluidic chip but stored under the chip, and/or

the manifold allows for contents of the vials to be injected from the bottom of the chip,

alleviating several problems experienced by the prior art, such as cell or particle

settlement where the vial or tubing from the vial connects to or communicates with the

microfluidic chip.

[0016] In another aspect, the present invention is directed to a microfluidic chip holder,

this chip holder comprising a structure to guide a light source including an integrated

prism cavity, which when fitted with a prism causes light to exit at an angle relative to the

chip and is a preferred method for illuminating constrained geometry. In embodiments,

the light source includes but is not limited to fiber optics or a collimated or focused light

source. This light source is precisely directed, or oriented, or directed into the analysis

channel, in particular.

[0017] In another aspect of the current invention, the device includes a second imaging

device oriented orthogonally to the first camera and channel view. Reasons for the

second camera vary. In one aspect, the reason for a second imaging device is to aid in

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visual alignment of the laser or optical force in the analysis channel. In another aspect, using the method described herein, data from the first camera can be recorded. With the second camera, data can be combined with data from the first camera, resulting in additional data that can be used to more accurately extrapolate cell position, size, shape, volume, etc. This additional information about the same cell (or particle) 2023286012

increases the accuracy and range of measurements and analysis. In a further aspect, the second camera, combined with the first camera, allows for a 3D reconstruction of a cell or particle, or group of cells or particles, imaged by the orthogonal camera and a camera in the flow or a camera located towards the side of the chip. Using the algorithm described herein or others, including reversing or slowing the flow and taking one or more images of a particular cell or particle, or group of cells or particles, the invention allows for multiple images to be analyzed and processed thereby allowing for determination of characteristics/attributes/quantitative measurements, such as the cell volume, cell shape, nucleus location, nucleus volume, organelle or inclusion body location, etc. In another aspect of the current invention a camera is oriented to image in the axis that is in the direction of flow.

[0018] In one aspect, the current invention does not require a serpentine or zigzag channel, as preferred according to the prior art, to keep particles properly suspended. Because of the vertical nature of fluid and particles or cells being injected in the chip, the zigzag channel is obviated by the vertically integrated pumped particles that flow directly up through the channel to the first horizontal channel (referred to herein as the second channel).

[0018A] In a further aspect, the present invention provides a method for assessing biological particles for use in a cell-based therapy, comprising: the use of a device wherein the device comprises: a substrate comprising a plurality of channels configured to transport one or more biological particles, wherein the plurality of channels comprises: a first channel disposed vertically within the substrate, a second channel in operable communication with the first channel and disposed horizontally within the substrate, a third channel in operable communication with the second channel and

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disposed vertically within the substrate, and a fourth channel in operable communication with the third channel and disposed horizontally within the substrate; wherein the first, second, third and fourth channels are disposed in such a manner as to provide a path for movement of the one or more biological particles through the substrate from the first channel to the second channel to the third channel to the fourth 2023286012

channel; and injecting the biological particles into the first channel disposed vertically within the substrate through a bottom horizontal planar surface having an opening to the first channel, to maintain directional and volumetric continuity with the first channel, wherein the biological particles comprise CAR T cells, CAR-M cells, TILs, NK cells, other engineered or effector cells, or stem cells; and assessing the biological particles in the fourth channel using optical force-based measurements.

[0018B] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0018C] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings illustrate certain aspects of some of the embodiments of the present invention, and should not be used to limit or define the invention. Together with the written description the drawings serve to explain certain principles of the invention.

7A

[0020] Figure 1A is a diagram depicting a global view of the device configuration,

including the chip, manifold, and vials. Figure 1B is a diagram which shows certain

depictions of the chip channel orientations and locations.

[0021] Figures 2A and B contain diagrams depicting the microfluidic chip holder

according to the present invention. 2023286012

[0022] Figures 3A and 3B are a diagram depicting angles and aspects of the chip holder

according to the present invention.

[0023] Figures 4A and 4B are diagrams showing examples of the cell path and imaging

configuration related to the chip. Figure 4C is a diagram which shows an alternative cell

path.

[0024] Figure 5 is a diagram showing on chip multi-plane imaging and how it may be

used to render a 3D images and information.

[0025] Figures 6A and 6B are diagrams showing on chip multi-plane imaging in fluid

flow and how it may be used to render a 3D image.

[0026] Figure 7 is a diagram showing how a cell may be trapped and/or balanced within

the analysis portion of the chip and imaged from multiple angles.

[0027] Figure 8 is a diagram showing how a camera and illumination source can be

placed in line with the laser and direction of flow, such that the particles move away from

the camera.

[0028] Figure 9 is a diagram showing how a camera and illumination source can be

placed in line with the laser and direction of flow, such that the particles move toward the

camera.

DETAILED DESCRIPTION DETAILED DESCRIPTION

[0029] The

[0029] Thepresent presentinvention inventionhas hasbeen beendescribed describedwith withreference referencetotoparticular particular embodiments having embodiments having various various features. features. It will It be will be apparent apparent to those to those skilled in skilled the art in the art that that

various modifications and variations can be made in the practice of the present invention various modifications and variations can be made in the practice of the present invention

without departing from the scope or spirit of the invention. One skilled in the art will without departing from the scope or spirit of the invention. One skilled in the art will 2023286012

recognize that recognize that these these features featuresmay may be be used singularly or used singularly or in inany any combination basedononthe combination based the requirementsand requirements andspecifications specifications of of aa given given application application or or design. design. Embodiments Embodiments

comprising various features may also consist of or consist essentially of those various comprising various features may also consist of or consist essentially of those various

features. Other embodiments of the invention will be apparent to those skilled in the art features. Other embodiments of the invention will be apparent to those skilled in the art

from consideration of the specification and practice of the invention. The description of from consideration of the specification and practice of the invention. The description of

the invention the invention provided is merely provided is exemplaryororexplanatory merely exemplary explanatoryininnature natureand, and,thus, thus, variations variations that do not depart from the essence of the invention are intended to be within the scope of that do not depart from the essence of the invention are intended to be within the scope of

the invention. the invention.

[0030] Before explaining at least one embodiment of the invention in detail, it is to be

[0030] Before explaining at least one embodiment of the invention in detail, it is to be

understood that the invention is not limited in its application to the details of construction understood that the invention is not limited in its application to the details of construction

and the and the arrangement arrangementofofthe the components components setforth set forthininthe the following followingdescription description or or illustrated ininthe illustrated drawings. the drawings. The The invention invention is iscapable capable of ofother otherembodiments or of embodiments or of being being practiced or carried out in various ways. Also, it is to be understood that the phraseology practiced or carried out in various ways. Also, it is to be understood that the phraseology

and terminology and terminologyemployed employed herein herein is is forthe for thepurpose purposeofofdescription descriptionand andshould shouldnot notbebe regarded as limiting. regarded as limiting.

[0031] Turning

[0031] Turningnow nowto to thefigures, the figures, FIG. FIG.1AIAshows shows a global a global view view of of thethe device device taught taught

herein. The herein. Thesample samplevials vials130 arelocated 130are locatedbelow belowthe themicrofluidic microfluidicchip chip100 (which 100(which may may

also be referred to generically herein as a substrate) and are not limited in terms of also be referred to generically herein as a substrate) and are not limited in terms of

number of vials or sizes. The vials are configured to hold any type of sample that can be number of vials or sizes. The vials are configured to hold any type of sample that can be

movedthrough moved throughthethedevice, device,such suchasasone oneorormore more substance,including substance, including butnotnotlimited but limitedtoto one or more of fluids, liquids, gas, plasma, serum, blood, cells, platelets, particles, etc. or one or more of fluids, liquids, gas, plasma, serum, blood, cells, platelets, particles, etc. or

combinations thereof. In the context of this specification, the term fluid or sample may combinations thereof. In the context of this specification, the term fluid or sample may

be used be used generically generically to to refer referto tosuch suchone oneor ormore more substances. The vials substances. The vials are are connected connected

9

either directly or indirectly, such as indirectly using air tubing 110 in operable either directly or indirectly, such as indirectly using air tubing 110 in operable

communication with communication with thethe underside underside of of thechip. the chip.They They maymay alsoalso be connected be connected by of by way waya of a manifold120 manifold 120asasfurther further shown shownininFIG. FIG.1A. IAFIG. FIG. 1B IB shows shows a preferred a preferred embodiment embodiment of of the microfluidic chip 100 whereby the substance, fluid, particles, and/or cells is/are the microfluidic chip 100 whereby the substance, fluid, particles, and/or cells is/are

injected at one exterior surface, such as an edge, of the microfluidic chip (e.g., the XZ injected at one exterior surface, such as an edge, of the microfluidic chip (e.g., the XZ

plane in plane in FIG. IB). Injection FIG. 1B). Injection is is shown in FIG. shown in FIG. 1B IBalong alongone oneofofthe thefaces faces shared sharedby bythe the 2023286012

smallest distance, smallest distance, such such as as the theXZ XZ face face or or the theXY face. In XY face. In this this particular particularembodiment, the embodiment, the

length of side X 160 is less than the length of side Y 170 and the length of side Z180. length of side X 160 is less than the length of side Y 170 and the length of side Z 180.

Suchaa configuration Such configuration allows allowsaa sample sampletotohave haveminimal minimal deviationupon deviation upon entering entering channels channels

on the chip (e.g., no right tum is necessary as is generally the case in current state of the on the chip (e.g., no right turn is necessary as is generally the case in current state of the

art). art).

[0032] In

[0032] In FIG. FIG. 1A, IA,aa manifold manifold120 120isisshown shown connecting connecting thethe vialororvials vial vials130 130totothe the microfluidic chip 100 so that the substance, fluid, particles, and/or cells may be injected microfluidic chip 100 SO that the substance, fluid, particles, and/or cells may be injected

or pumped or intothe pumped into themicrofluidic microfluidicchip chipin in the the vertical verticaland and upwards direction. The upwards direction. The manifold manifold allows injection of substances from the bottom of the chip, while also positioning the allows injection of substances from the bottom of the chip, while also positioning the

electronics, flow sensors, and tubing (both liquid and air) in such a way as to minimize electronics, flow sensors, and tubing (both liquid and air) in such a way as to minimize

cell settling, cell settling,which whichoptimizes optimizesthroughput. throughput. Air Air tubing tubing provides pressure or provides pressure or vacuum, vacuum,

which, when which, whensealed, sealed,provides providesfor foraa closed closed system systemwithin withinthe theconfines confinesofofthe the manifold manifold apparatus. In apparatus. In one one embodiment embodiment there there is is a adistance distancebetween between thevial(s) the vial(s)and andthe themanifold manifold indicating that there is no seal and the pressure of the internal volume is atmospheric. indicating that there is no seal and the pressure of the internal volume is atmospheric.

This allows This allows for for pumping substancesfrom pumping substances from or or totoa acontainer containeropen opentotothe theatmosphere atmosphere (vacuumisisrequired (vacuum requiredto to pump pumpfrom from anan open open container).TheThe container). manifold manifold positions positions the the

electronics, flow sensors, and tubing (both liquid and air) in such a way as to minimize electronics, flow sensors, and tubing (both liquid and air) in such a way as to minimize

cell cell settling settling which optimizes which optimizes throughput. throughput.

[0033] By

[0033] Byinjecting injecting the the contents contents from the bottom from the bottomofofthe the chip, chip, the the invention invention minimizes minimizes

horizontal movement horizontal movement inin inlettubing inlet tubing140 140and andoutlet outlettubing tubing145, 145, which whichcauses causessuch such problems as settlement of the particles or cells in the channel(s) 150. The outlet tubing is problems as settlement of the particles or cells in the channel(s) 150. The outlet tubing is

offset from the inlet tubing, in this example, in the Zand X dimensions (FIG. IB). In offset from the inlet tubing, in this example, in the Z and X dimensions (FIG. 1B). In

embodiments, the outlet tubing is offset, not offset, straight, in-line, or angled in relation embodiments, the outlet tubing is offset, not offset, straight, in-line, or angled in relation

to the inlet tubing. Such a confi guration obviates the need for serpentine or zigzag to the inlet tubing. Such a configuration obviates the need for serpentine or zigzag

vertical channels vertical channels because the current because the current configuration configuration resolves resolves problems with settlement problems with settlement that that occur in the prior art, such as in the case in which fluid combined with cells and/or occur in the prior art, such as in the case in which fluid combined with cells and/or

particles is injected or pumped into the chip from the side in a horizontal direction that particles is injected or pumped into the chip from the side in a horizontal direction that

then must then must change changedirection directionand andfluid fluid dynamics dynamicstotobebepushed pushedupwards. upwards. The The manifold manifold and and injection from injection from the the bottom of the bottom of the chip chip also also allow allow for foradditional additionalelements, elements,components, components, 2023286012

machinery,oror hardware machinery, hardwaretotobebeplaced placedbelow below thechip. the chip.(See (SeeFIG. FIG. IA) 1A.)

[0034] The

[0034] Themanifold manifold120120 works works by by adjusting adjusting thethe airair pressureabove pressure above thecontents the contentsininthe the vial and vial and providing correct geometry providing correct for flow geometry for flow sensors sensors and andelectronics. electronics. Tubing, Tubing,such suchasas fluid or air tubing, passes through the manifold and connects to a vial on the other side. fluid or air tubing, passes through the manifold and connects to a vial on the other side.

Pressurized air passes through one side of manifold and creates a closed pressurized Pressurized air passes through one side of manifold and creates a closed pressurized

systemwithin system withinthe the manifold. manifold. ByByadjusting adjustingpressure pressureininthe theenclosed enclosedregion, region,the the system system allows for allows for changes to parameters changes to suchasas flow parameters such flowspeed speedand andfluid fluiddynamics. dynamics.TheThe pressurized pressurized

region is in both the vial(s) 130 and the manifold 120. In another aspect, no air region is in both the vial(s) 130 and the manifold 120. In another aspect, no air

connection is needed for the vial, because it is open to the ambient atmosphere. This connection is needed for the vial, because it is open to the ambient atmosphere. This

enables sampling enables samplingofofaa larger larger variety variety of of containers containersand and sources. sources. In In such such an an embodiment, embodiment, a a

vacuum is applied to the other one or more vials such that a pressure differential is vacuum is applied to the other one or more vials such that a pressure differential is

created to drive fluid flow from the open container. created to drive fluid flow from the open container.

[0035] In

[0035] In one one embodiment, embodiment, pressure-based pressure-based sample sample injection injection is is used.TheThe used. vials vials areare filled filled

with a sample in a fluid and sealed either with a lid or tubing connected to the chip. Prior with a sample in a fluid and sealed either with a lid or tubing connected to the chip. Prior

to attachment to the lid, the vial can be either open to the air or sealed with a septum or to attachment to the lid, the vial can be either open to the air or sealed with a septum or

other gas-tight device. In one aspect, the lid may contain two connections, one for a fluid other gas-tight device. In one aspect, the lid may contain two connections, one for a fluid

such as such as aa gas gas and and one for aa liquid. one for liquid. Optionally, Optionally, the themethod method embodiment furtherincludes embodiment further includes providing providing aa sample sampleinlet inlet line line tip tipcommunicating withaasample communicating with sampleinlet inlet line, line, which which

communicates communicates with with thethe first channel. first channel.

[0036] In

[0036] In another another embodiment, embodiment, a vacuum a vacuum based based sample sample injection injection is used. is used. The The vials vials are are

filled with a sample in a fluid and sealed either with a lid or tubing connected to the chip. filled with a sample in a fluid and sealed either with a lid or tubing connected to the chip.

Prior to attachment to the lid, the vial can be either open to the air or sealed with a Prior to attachment to the lid, the vial can be either open to the air or sealed with a

11

septum. In one aspect, the lid may contain two connections, one for a fluid such as a gas septum. In one aspect, the lid may contain two connections, one for a fluid such as a gas

and one for a liquid. Optionally, fluid can be aspirated from a vial open to the and one for a liquid. Optionally, fluid can be aspirated from a vial open to the

atmospherebybyapplying atmosphere applyingvacuum vacuum pressure pressure to one to one or or more more of the of the other other vials.Optionally, vials. Optionally, the method the embodiment method embodiment further further includes includes providing providing a sample a sample inlet inlet linetip line tipcommunicating communicating with aa sample with inlet line, sample inlet line,which which communicates withthe communicates with thefirst first channel. channel. 2023286012

[0037] In

[0037] In FIGS. FIGS.2A-B 2A-B and and 3A-B, 3A-B, a chip a chip holder holder 200,300 200, is shown. 300 is shown. The The chip chip holder holder

comprises a structure comprises a structure to to guide guide a light a light source source 210,assuch 210, such as aoptic a fiber fiberlight opticsource, light source, light light emitting diode, or laser, and an integrated prism cavity 220,320 that can be fitted with a emitting diode, or laser, and an integrated prism cavity 220, 320 that can be fitted with a

prism. The light source is guided or aligned by an integrated structure or channel 240 prism. The light source is guided or aligned by an integrated structure or channel 240

within the chip holder to the desired location. The built-in space for the fiber optic light within the chip holder to the desired location. The built-in space for the fiber optic light

source allows for illumination, such as a cone of illumination 250, even in constrained source allows for illumination, such as a cone of illumination 250, even in constrained

geometricenvironments, geometric environments,such suchasasonona amicrofluidic microfluidicchip chip230, 330orora achannel 230,330 channelininaa microfluidic chip. This light source is precisely directed, or oriented, or focused on the microfluidic chip. This light source is precisely directed, or oriented, or focused on the

analysis channel 260 in particular, in a preferred aspect. In a preferred embodiment, the analysis channel 260 in particular, in a preferred aspect. In a preferred embodiment, the

chip holder includes a built-in space for a prism 220,320 and fiber optic light source 210 chip holder includes a built-in space for a prism 220, 320 and fiber optic light source 210

allowing for allowing for illumination illumination with with constrained constrained geometry. Thechip geometry. The chipfurther furtherincludes includesholes holesoror openings in the bottom of the holder 350 to precisely align fluidic tubing as described openings in the bottom of the holder 350 to precisely align fluidic tubing as described

herein. In herein. In one embodiment,adjustable one embodiment, adjustablescrews screws areintegrated are integratedinto intothreaded threadedholes holes360 360onon one or one or more faces for more faces for proper proper alignment. alignment.

[0038] FIG.

[0038] FIG.4A 4Aisisaa preferred preferred embodiment embodiment of of thethemicrofluidic microfluidicchip chip400 400 described described herein. herein.

As shown, fluid travels first upwards in a vertical direction through a first channel 410 As shown, fluid travels first upwards in a vertical direction through a first channel 410

then communicates then communicates with with a second a second horizontal horizontal channel channel 420. 420. Another Another vertical vertical channel channel 430 430 takes the fluid even closer to the top of the chip at which point a fourth channel 440 is takes the fluid even closer to the top of the chip at which point a fourth channel 440 is

horizontal and, horizontal and, as as show in the show in the FIG. FIG. 4, 4, comprises the analysis comprises the analysis channel. Thechannels channel. The channelsare are in operable in operable communication with communication with one one another another to to allow allow forfor a a sample sample to to bebe moved moved through through

the system the fromone system from onechannel channeltotoanother. another.InInembodiments embodimentsthe the sample sample can can flowflow fromfrom the the first channel to the second channel to the third channel to the fourth channel, or in the first channel to the second channel to the third channel to the fourth channel, or in the

reverse, or reverse, or combinations thereof. AApump combinations thereof. pump and/or and/or vacuum vacuum apparatus apparatus can can be provided be provided to to provide positive and/or negative pressure at either or both the opening and the exit of the provide positive and/or negative pressure at either or both the opening and the exit of the

12

channels to channels to enable movement enable movement of of thesubstances the substances through through thethe channels. channels. TheThe analysis analysis

channel is close to one or more exterior surface of the substrate, such as the faces, edges, channel is close to one or more exterior surface of the substrate, such as the faces, edges,

or sides of the chip. For example, the analysis channel, according to this confi guration, is or sides of the chip. For example, the analysis channel, according to this configuration, is

close to close to the thetop topand and side sideof ofthe chip, the thereby chip, improving thereby improvingimaging imaging and and analysis analysis through through the the

substance of substance of the the microfluidic microfluidic chip. chip. In In preferred preferred embodiments, theanalysis embodiments, the analysis channel channelis is from around from around1 1mmmm to to around around 2 mm 2 mm fromfrom the and the top top and side side of the of the chip. chip. However, However, the the 2023286012

distance of distance of the the analysis analysischannel channel from from the the top top of ofthe thechip chipmay may be be from from 0.1 0.1 mm mm toto100 100mm, mm, such as such as from 0.1 mm from 0.1 mmtoto0.2 0.2mm, mm, from from 0.20.2 mm mm to 0.3 to 0.3 mm, mm, from from 0.3tomm 0.3 mm 0.4to 0.4and mm, mm,SO and so on. Expressed on. Expressedanother anotherway, way,thetheanalysis analysischannel channelcan canbebedisposed disposed within within thetop the top50%, 50%, 33%,25%, 33%, 25%,10%, 10%, or or 5% 5% of the of the substrate.TheThe substrate. length length of of thethe horizontalanalysis horizontal analysischannel channel accordingthe according the present present invention invention may maybebefrom fromaround around 250 250 microns microns to around to around 10 mm. 10 mm.

However,the However, thelength lengthofofthe the analysis analysis channel channel may maybebefrom from100100 microns microns to to 100100 mm,mm, suchsuch

as from as 0.1 mm from 0.1 mmtoto0.2 0.2mm, mm,from from 0.20.2 mm mm to 0.3 to 0.3 mm,mm, from from 0.3tomm 0.3 mm 0.4tomm, 0.4and mm,SO and on. so on. Expressedanother Expressed anotherway, way,the thelength lengthofofthe the analysis analysis channel channel can can be be about about75% 75%ororless lessofofthe the height, width or length of the substrate/chip, such as 50% or less, 33% or less, 25% of height, width or length of the substrate/chip, such as 50% or less, 33% or less, 25% of

less, 10%, or 5% or less of the height, width or length of the substrate/chip. less, 10%, or 5% or less of the height, width or length of the substrate/chip.

[0039] In

[0039] In FIG. FIG. 4B, 4B, two twoimaging imagingdevices devices 450,such 450, such as as machine machine vision vision cameras, cameras, areare shown. shown.

In one In one embodiment, embodiment, a acamera cameramaymay be located be located above above the the chipchip and and be oriented be oriented

orthogonally to orthogonally to the the analysis analysis channel. channel. In In another another embodiment, embodiment, a acamera camera may may be be located located to to the side of the chip and be oriented orthogonal to the direction of the flow or diagonal to the side of the chip and be oriented orthogonal to the direction of the flow or diagonal to

the direction of the flow (such as above the channel, below the channel, or at angles to the direction of the flow (such as above the channel, below the channel, or at angles to

the side the side of of the thechannel). channel). In Inembodiments, the camera embodiments, the cameracan canbebepositioned positionedsuch suchthat thatthe the imagingisis performed imaging performedatatany anyangle anglerelative relative to to the the flow flow of of the theone oneor ormore more substance, substance, such such

as orthogonal or 90 degrees relative to the flow of the one or more substance, or such as as orthogonal or 90 degrees relative to the flow of the one or more substance, or such as

from0Oto from 90 degrees, to 90 degrees, or or from 10 to from 10 to 80 80 degrees, degrees, or or from 30 to from 30 to 60 60 degrees and SO degrees and so on. on. In In another embodiment, another embodiment,twotwo or or more more cameras cameras may may be used be used to image to image cellscells or particles or particles in in thethe

analysis channel. analysis For example, channel. For example,a acamera cameramay may be be above above the the chip chip andand be be oriented oriented

orthogonally to orthogonally to the the analysis analysis channel. channel. A secondcamera A second cameramay may be be located located to to theside the sideofofthe the chip and be oriented orthogonally to the direction of the flow or diagonal to the direction chip and be oriented orthogonally to the direction of the flow or diagonal to the direction

of the flow (such as above the channel, below the channel, or angles to the side of the of the flow (such as above the channel, below the channel, or angles to the side of the

13

channel). As channel). Aspictured picturedin in FIG. FIG.4B, 4B,one oneorormore morelight lightsources sources460 may 460may be be used used to to

illuminate the illuminate the analysis analysis channel 440, and channel 440, and such light sources such light sources may be located may be located under under the the chip chip and shining up, to the side of the chip and shining in the flow direction or opposite the and shining up, to the side of the chip and shining in the flow direction or opposite the

flow direction, above the chip and shining down, or diagonally to the analysis channel. flow direction, above the chip and shining down, or diagonally to the analysis channel.

[0040] Alternatively, a dichroic mirror 840 or other appropriate optical element can be

[0040] Alternatively, a dichroic mirror 840 or other appropriate optical element can be 2023286012

used to selectively divert a specific wavelength range of light while allowing others to used to selectively divert a specific wavelength range of light while allowing others to

pass, such pass, such as as shown in FIG. shown in FIG.8. 8. This Thiswould wouldallow allowforforcameras cameras to to 810 810 be be placed placed in in line line

with the with the analysis analysis channel. Aspictured channel. As pictured in in FIGS. FIGS. 88 and and9,9, several several embodiments embodiments ofof this this

occur, including placing the optical force laser 830 and camera 810 at the same or occur, including placing the optical force laser 830 and camera 810 at the same or

opposite ends of opposite ends of the the analysis analysis region. region. An illumination source An illumination source 860 for the 860for the camera might camera might

also be also be required required and and can be oriented can be oriented in in several several ways, ways, such such as as what what is is shown in FIGS. shown in FIGS. 88

and 9. and 9. The Thelight light source source could could be be aa broad broad spectrum spectrumsource, source,such suchasasone oneorormore moreLEDs LEDs or or a a narrowsource narrow sourcesuch suchasasaalaser. laser. The Thecamera cameracould couldbebeused used asas a asingle singlecamera cameraororasaspart partof of a multi-camera a systeminincombination multi-camera system combination with with otherviewpoints other viewpoints as as described described herein. herein.

[0041] Also pictured in FIG. 4A, a light source 480, such as a laser, may be used to affect

[0041] Also pictured in FIG. 4A, a light source 480, such as a laser, may be used to affect

cell flow. The laser may be placed in line with the cell flow, or opposing the cell flow. cell flow. The laser may be placed in line with the cell flow, or opposing the cell flow.

The laser may also be placed and/or oriented orthogonally or diagonally to the cell flow. The laser may also be placed and/or oriented orthogonally or diagonally to the cell flow.

[0042] An

[0042] Anembodiment embodiment of the of the invention invention includes includes a device a device forfor particleanalysis. particle analysis.(See, (See, e.g., FIGS. e.g., FIGS. 4-9.) 4-9.) The embodiment The embodiment of of theinvention the inventionincludes includesatatleast least one one camera camera450 for 450for capturing images of particles or cells in the microfluidic channels (e.g., 440). In one capturing images of particles or cells in the microfluidic channels (e.g., 440). In one

embodiment, a laser or other optical force 480, such as a collimated light source operable embodiment, a laser or other optical force 480, such as a collimated light source operable

to generate at least one collimated light source beam, is included. The at least one to generate at least one collimated light source beam, is included. The at least one

collimated light collimated light source source beam includesat beam includes at least least one one beam cross-section. The beam cross-section. Theembodiment embodiment of the invention includes a substrate with a first channel 410 extending in a vertical of the invention includes a substrate with a first channel 410 extending in a vertical

direction in the substrate such that a first plane traverses first channel 410 substantially direction in the substrate such that a first plane traverses first channel 410 substantially

along its length and whereby the fluid sample is injected into the substrate/chip from the along its length and whereby the fluid sample is injected into the substrate/chip from the

bottomofofthe bottom the chip chip and and is is forced forced by by positive positive or ornegative negative pressure pressure upwards. The upwards. The

embodiment embodiment of of theinvention the inventionincludes includesa asecond secondchannel channel 420420 orthogonal orthogonal to the to the first first

14

channel and thus disposed horizontally in the substrate such that a second plane traverses channel and thus disposed horizontally in the substrate such that a second plane traverses

secondchannel second channel420 420substantially substantiallyalong alongits its length length and the second and the plane is second plane is disposed disposed

orthogonal to the first plane. This second channel is in the horizontal direction of the orthogonal to the first plane. This second channel is in the horizontal direction of the

chip. The chip. Thesecond secondchannel channelcommunicates communicates directly directly or indirectly or indirectly with with thefirst the first channel. channel. The The secondchannel second channelcommunicates communicates directly directly or or indirectlywith indirectly witha ashort shortupward upwardvertical verticalthird third channel 430 that takes the channel network closer to the top of the chip. The third channel 430 that takes the channel network closer to the top of the chip. The third 2023286012

channel communicates directly or indirectly with a fourth horizontal channel 440 that is channel communicates directly or indirectly with a fourth horizontal channel 440 that is

located near the top and/or corner of the chip. In a preferred embodiment, the fourth located near the top and/or corner of the chip. In a preferred embodiment, the fourth

channel is the channel closest to the top of the chip. In an embodiment, the fourth channel is the channel closest to the top of the chip. In an embodiment, the fourth

channel is an analysis channel. In one aspect, a camera 450 is oriented orthogonally to channel is an analysis channel. In one aspect, a camera 450 is oriented orthogonally to

the flow the flow direction direction in in the thefourth fourthchannel. channel. The The embodiment embodiment ofof theinvention the inventionincludes includesa a focused particle stream nozzle operably connected to the first channel. In another aspect focused particle stream nozzle operably connected to the first channel. In another aspect

of the of the current current invention, invention,the thesecond second channel channel undergoes undergoes aa size size change andpasses change and passesthrough through a nozzle a nozzle before before communicating with communicating with thethe thirdchannel. third channel.

[0043] Figure

[0043] Figure4C 4Cshows shows another another embodiment embodiment of sample of the the sample path path of microfluidic of the the microfluidic chip. chip.

As shown, fluid travels in the chip first upwards in a vertical direction through a first As shown, fluid travels in the chip first upwards in a vertical direction through a first

channel 410, channel 410, which whichchannel channelthen thencommunicates communicates directly directly or or indirectlywith indirectly with a a second second

horizontal channel 420 at, in this example, the top of the chip and comprises the analysis horizontal channel 420 at, in this example, the top of the chip and comprises the analysis

channel in channel in this this embodiment. The embodiment. The analysischannel, analysis channel,according according to to thisconfiguration, this configuration,isis close to close to the the top topof ofthe thechip, chip,thereby improving thereby improvingimaging imaging and analysis through and analysis the through the

substance of substance of the the microfluidic microfluidic chip. chip. In In preferred preferred embodiments, theanalysis embodiments, the analysis channel channelisis fromaround from around1 1mmmm to to around around 2 mm 2 mm from from the and the top top side and side of the of the chip. chip. However, However, the the distance of distance of the the analysis analysischannel channel from from the the top top of of the thechip chipmay may be be from 0.1 mm from 0.1 mm toto100 100mm, mm, such as such as from 0.1 mm from 0.1 mmtoto0.2 0.2mm, mm, from from 0.20.2 mm mm to 0.3 to 0.3 mm, mm, from from 0.3tomm 0.3 mm 0.4 to 0.4and mm, mm,SO and so on. Expressed on. Expressedanother anotherway, way,thetheanalysis analysischannel channelcan canbebedisposed disposed within within thethe top50%, top 50%, 33%,25%, 33%, 25%,10%, 10%, or or 5% 5% of the of the substrate.TheThe substrate. length length of of thethe horizontal horizontal analysischannel analysis channel accordingthe according the present present invention invention may maybebefrom fromaround around 250250 microns microns to around to around 10 10 mm. mm. However,the However, thelength lengthofofthe the analysis analysis channel channelmay maybebefrom from 100 100 microns microns to 100 to 100 mm, mm, such such

as from as 0.1 mm from 0.1 mmtoto0.2 0.2mm, mm, from from 0.20.2 mm mm to 0.3 to 0.3 mm, mm, from from 0.3tomm 0.3 mm 0.4 to 0.4and mm, mm,SO and on. so on. Expressed anotherway, Expressed another way,the thelength lengthofofthe the analysis analysis channel channel can canbe beabout about75% 75%oror lessofofthe less the

15

height, width or length of the substrate/chip, such as 50% or less, 33% or less, 25% of height, width or length of the substrate/chip, such as 50% or less, 33% or less, 25% of

less, 10%, or 5% or less of the height, width or length of the substrate/chip. In less, 10%, or 5% or less of the height, width or length of the substrate/chip. In

embodiments,thethesubstrate embodiments, substratecan cancomprise compriseoneone or or more more analysis analysis channel, channel, such such as as 1, 1, 2,2,3,3,4, 4, 5, 5, 6, 6, 7, 7, 8, 8, 9, 9, or or 10 10 analysis channels. analysis channels.

[0044] Also pictured in FIG. 4C, a light source 480, such as a laser, may be used to affect

[0044] Also pictured in FIG. 4C, a light source 480, such as a laser, may be used to affect 2023286012

substance flow, such as cell flow. The laser may be placed in line with the cell flow, or substance flow, such as cell flow. The laser may be placed in line with the cell flow, or

opposingthe opposing the cell cell flow. Thelaser flow. The laser may mayalso alsobe beplaced placedand/or and/ororiented orientedorthogonally orthogonallyoror diagonally relative to the cell flow. diagonally relative to the cell flow.

[0045] In FIGS. 1 and 4, a fluid flow containing cells or particles is directed through a

[0045] In FIGS. 1 and 4, a fluid flow containing cells or particles is directed through a

first channel vertically. The one or more substance (fluid, cells, and/or particles) enter first channel vertically. The one or more substance (fluid, cells, and/or particles) enter

through the bottom of the chip at an opening of the first channel and enter the substrate in through the bottom of the chip at an opening of the first channel and enter the substrate in

the vertical the verticaldirection. direction.The The first firstvertical channel vertical is between channel 100 is between microns 100 micronsand and100 100 mm in mm in

length, such length, such as as from 0.1 mm from 0.1 mm toto0.2 0.2mm, mm,from from 0.20.2 mmmm to 0.3 to 0.3 mm,mm, andon. and SO so The on. first The first channel is channel is followed by aa second followed by secondorthogonal/horizontal orthogonal/horizontalchannel, channel,which, which,ininaapreferred preferred embodiment,isisshorter embodiment, shorterthan thanthe the first first channel. channel. The secondchannel The second channelmay maybebe 250 250 microns microns to to 100 mmininlength, 100 mm length,such suchasasfrom from0.25 0.25mmmm to to 0.50.5 mm, mm, fromfrom 0.5 0.5 mm mm to to 0.75 0.75 mm,0.75 mm, from from 0.75 mm to 1.0 mm, and so on. A third channel runs vertically and parallel to the first mm to 1.0 mm, and SO on. A third channel runs vertically and parallel to the first

channel. The channel. Thethird thirdchannel channelmay maybebe 5050 microns microns to to 100100 mm mm in length, in length, suchsuch as from as from 0.050.05

mmtoto0.1 mm 0.1mm, mm, from from 0.10.1 mm mm to 0.15 to 0.15 mm, mm, from from 0.15 0.15 mm tomm 0.2 to 0.2 mm, mm, and and The SO on. so on. The channels are disposed in operable communication either directly or indirectly in such a channels are disposed in operable communication either directly or indirectly in such a

mannerasastoto allow manner allowone oneorormore moresubstance substancetotomove move through through multiple multiple channels. channels. The The typical direction of the fluid flow is given by the flow arrows in FIG. 4A and 4C, but can typical direction of the fluid flow is given by the flow arrows in FIG. 4A and 4C, but can

be reversed. be reversed.

[0046] In

[0046] In preferred preferred embodiments, embodiments,thethefourth fourthchannel channelcomprises comprises thethe analysischannel, analysis channel, whichisis aa channel which from250 channel from 250microns micronstoto100 100mmmm in length, in length, such such as as from from 0.25 0.25 mm mm to to 0.5 0.5 mm,from mm, from0.5 0.5mmmm to to 0.75 0.75 mm,mm, fromfrom 0.75 0.75 mm tomm 1.0tomm, 1.0and mm,SO and on. so In on. thisInembodiment, this embodiment, the fourth channel is the channel closest to the top of the chip. The distance of the fourth the fourth channel is the channel closest to the top of the chip. The distance of the fourth

channel from channel fromthe thetop top of of the the chip, chip, measured vertically, may measured vertically, be from may be from100 100microns micronstoto2 2mm, mm,

16

but as but as great great as as 100 100 mm suchasasfrom mm such from100 100microns microns to to 200 200 microns, microns, from from 200 200 microns microns to to 300 microns, 300 microns, from from300 300microns microns to to 400 400 microns, microns, andand SO so on.on. Expressed Expressed another another way,way, the the fourth channel fourth can be channel can be disposed disposedwithin withinthe the top top 50%, 50%,33%, 33%,25%, 25%, 10%, 10%, or of or 5% 5%the of the chip. chip.

The imaging The imagingdevice, device,such suchasasaacamera, camera,can canbebeoriented orientedorthogonal orthogonaltotothe thefourth fourthchannel channel and around and around100 100microns micronstoto2 2mmmm from from the the fourth fourth channel, channel, butbut as as great great asas 100 100 mmmm fromfrom

the fourth the fourth channel, channel, such such as as from 100 microns from 100 micronstoto200 200microns, microns,from from200200 microns microns to to 2023286012

300 microns, 300 microns, from from300 300microns microns to to 400 400 microns, microns, andand SO so on.on.

[0047] In one embodiment, a laser or other optical source is present with a focusing lens

[0047] In one embodiment, a laser or other optical source is present with a focusing lens

element. FIG. 4A depicts the invention with the laser 480 operating, emitting a laser element. FIG. 4A depicts the invention with the laser 480 operating, emitting a laser

beam,directing beam, directing the the beam througha afocusing beam through focusinglens lenselement elementinto intothe thefourth fourth flow flowchannel channel 440. The 440. Theparticles particles are are aligned aligned within within the the laser laserbeam due to beam due to the the gradient gradient force force which which

draws particles toward the region of highest laser intensity. The laser scatter force draws particles toward the region of highest laser intensity. The laser scatter force

propels particles in the direction of laser beam propagation (e.g., left to right in FIG. 4A). propels particles in the direction of laser beam propagation (e.g., left to right in FIG. 4A).

[0048] In

[0048] In another another embodiment, embodiment,a asecond second camera camera or image or image capturing capturing device device (see(see 450), 450), in in addition to addition to the the camera camera or or image capturing device image capturing deviceoriented oriented orthogonally orthogonallytoto the the fourth fourth channel, is oriented to the side of the analysis (or fourth) channel here and directed channel, is oriented to the side of the analysis (or fourth) channel here and directed

orthogonally or at any angle relative to the fourth channel; for example, as pictured in orthogonally or at any angle relative to the fourth channel; for example, as pictured in

FIG. 4B. Taking FIG. 4B. Takingone one image image of of each each cellatatorthogonal cell orthogonalviews views allows allows forfor multiple multiple cell cell

properties, for example size and shape, to be calculated in two dimensions, increasing the properties, for example size and shape, to be calculated in two dimensions, increasing the

amountofofinformation amount informationthat thatcan canbe becaptured capturedfor for each eachcell. cell. This This would wouldalso alsoenable enablethe the calculation of volumetric properties of the cells, including total volume, shape and give calculation of volumetric properties of the cells, including total volume, shape and give

insight into cells that are not symmetric about the axis parallel to the direction of flow insight into cells that are not symmetric about the axis parallel to the direction of flow

(the Z-axis (the Z-axis as as drawn in Fi drawn in gure 5). Figure 5). Using twooror more Using two morecameras, cameras,a abasic basic3D3Dmodel model of of thethe

cell 550 cell 550 can can be be constructed constructed by by combining theorthogonal combining the orthogonalimages images using,forforexample, using, example, existing 3D existing reconstruction algorithms, 3D reconstruction algorithms, such such as as diffraction-theory diffraction-theory method or illumination method or illumination rotation-method. A A3D3D rotation-method. model model and and analysis analysis of3D of a a 3D model model willwill allow allow for for a more a more accurate accurate

analysis of the cell, such as cell size, shape, orientation, and other quantitative and analysis of the cell, such as cell size, shape, orientation, and other quantitative and

qualitative measurements regarding the particle(s) or cell(s) in the fourth channel of the qualitative measurements regarding the particle(s) or cell(s) in the fourth channel of the

microfluidic chip. microfluidic chip.

17

[0049] In

[0049] In FIG. FIG. 5, 5, aa portion portion of of the theanalysis analysischannel 540 is channel540 isshown shown from twodifferent from two different planes, such as planes that might be imaged from an imaging device (see, e.g., FIG. 4). planes, such as planes that might be imaged from an imaging device (see, e.g., FIG. 4).

In the first plane 520, one part of the cell or particle 510 is imaged in a certain In the first plane 520, one part of the cell or particle 510 is imaged in a certain

orientation. In the second plane 530, another part of the same cell or particle is imaged orientation. In the second plane 530, another part of the same cell or particle is imaged

from aa different from different perspective perspective from another camera. from another camera.This Thisallows allowsfor forthe thecalculation calculation of of 2023286012

multiple cell properties, creating a matrix of data per cell per camera and a 3D rendition multiple cell properties, creating a matrix of data per cell per camera and a 3D rendition

of the cell or particle 550. of the cell or particle 550.

[0050] As different portions of the cell pass through the focal planes (e.g., 630 (the XZ

[0050] As different portions of the cell pass through the focal planes (e.g., 630 (the XZ

focal plane) and 640 (the YZ focal plane)) of the imaging device(s), different slices of the focal plane) and 640 (the YZ focal plane)) of the imaging device(s), different slices of the

cell can be imaged as the cell is moved by, for example, the fluid flow 620. This is cell can be imaged as the cell is moved by, for example, the fluid flow 620. This is

shown in Figure 6A. In Figure 6A, parts of the cell or particle are imaged at multiple shown in Figure 6A. In Figure 6A, parts of the cell or particle are imaged at multiple

points in time and/or space in different orientations and from different perspectives. In points in time and/or space in different orientations and from different perspectives. In

this example, as the cell moves and rotates through the focal plane, it appears as a this example, as the cell moves and rotates through the focal plane, it appears as a

different size different sizeininthe successive the successiveimages images(such (suchas asthe thefour fourexample example images images per per plane plane from from

two different two different cameras as shown cameras as shownininFigure Figure6A). 6A).Using Using 3D 3D reconstruction reconstruction algorithms, algorithms, this this

allows for allows for aa more complex3D3D more complex rendering rendering of of 650650 thethe particleororcell. particle cell. From Fromsuch sucha a rendering, certain attributes of the cell or particle may be extrapolated, such as cell size, rendering, certain attributes of the cell or particle may be extrapolated, such as cell size,

volume,location volume, location and andsize size of of the the nucleus nucleus as as well well as as other otherorganelles, organelles,and andmeasurement or measurement or

outlining of cellular topography. Additionally, cell rotation can be measured as a outlining of cellular topography. Additionally, cell rotation can be measured as a

function of function of optical optical force forcebased based torque torque due due to to changes changes in in biophysical biophysical or or biochemical biochemical

properties, including but not limited to, refractive index, birefringence, or cell shape or properties, including but not limited to, refractive index, birefringence, or cell shape or

morphology. morphology.

[0051] FIG.

[0051] FIG.6B6Bdepicts depictsononchip chipmulti-plane multi-planeimaging imaging with with multiple multiple images images of of thethe same same

cell being taken over time, as the cell moves through the focal plan, for example. In such cell being taken over time, as the cell moves through the focal plan, for example. In such

cases, a view of the cell is referred to in the art as a "slice" or "image slice." An image cases, a view of the cell is referred to in the art as a "slice" or "image slice." An image

slice is effectively the thickness of the optical plane being imaged. The thickness of the slice is effectively the thickness of the optical plane being imaged. The thickness of the

image plane, or slice, is dictated, among other things, by the optical magnification of the image plane, or slice, is dictated, among other things, by the optical magnification of the

imagingsystem. imaging system.AtAthigher highermagnifications magnifications thethe working working distance distance of of thethe objective objective lens lens

18

decreases resulting in the need to have the lens closer to the cell or particle to be imaged. decreases resulting in the need to have the lens closer to the cell or particle to be imaged.

In one In one embodiment, embodiment, a alaser laseroror other other optical optical force 670 may force 670 beused may be usedtoto affect affect the the flow flow of of

the cell in the analysis channel. In a preferred embodiment, cells or particles may be the cell in the analysis channel. In a preferred embodiment, cells or particles may be

purposefully induced into or out of the focal plane of the channel for imaging by either purposefully induced into or out of the focal plane of the channel for imaging by either

movingthe moving thelaser laser and/or and/or camera, camera,oror adjusting adjusting flow(s), flow(s), or or position position using using hydrodynamic hydrodynamic

focusing. For focusing. Forexample, example,the thelaser laser source sourcecould couldbe bemoved movedby by a distance660660 a distance using,forfor using, 2023286012

example, a piezo electric actuator or linear electric optomechanical stage. This could be example, a piezo electric actuator or linear electric optomechanical stage. This could be

performedonona aper performed percell cell or or per per population population basis. basis. The hydrodynamic The hydrodynamic focusing focusing of of cells cells

could be changed to affect the initial position and trajectory of the cells. For example, could be changed to affect the initial position and trajectory of the cells. For example,

particles may be aligned or oriented in the focal plane within a laser beam due to the particles may be aligned or oriented in the focal plane within a laser beam due to the

gradient force which draws particles toward the region of highest laser intensity. The gradient force which draws particles toward the region of highest laser intensity. The

laser scatter force propels particles in the direction of laser beam propagation. See FIG. laser scatter force propels particles in the direction of laser beam propagation. See FIG.

6B. Moving the laser, in this case in the X axis, allows for imaging of features in 6B. Moving the laser, in this case in the X axis, allows for imaging of features in

different parts of the cell, illustrated by the disc 680. This could represent, for example, different parts of the cell, illustrated by the disc 680. This could represent, for example,

the nucleus, organelles, inclusion bodies, or other features of the cell or particle. The the nucleus, organelles, inclusion bodies, or other features of the cell or particle. The

laser pulls the cell to its center as a result of the gradient force. It is further contemplated laser pulls the cell to its center as a result of the gradient force. It is further contemplated

that two that two or or more camerasmay more cameras may increasedetail increase detailand andaccuracy. accuracy.

[0052] An

[0052] Anembodiment embodiment of the of the invention invention shown shown in FIG. in FIG. 7 is7 aisstatic a staticmode mode where where the the

particle or cell 710 is stopped at a specified differential retention location by balancing particle or cell 710 is stopped at a specified differential retention location by balancing

the optical the 730 and optical 730 and fluidic fluidicforces 735. The forces735. The optical optical force forcemay may be be applied applied by, by, for for example, example,

a laser a laser or orcollimated collimatedlight lightsource. source.Images Images can can be be taken taken in in multiple multiple planes planes such such as asshown shown

and described and described for for Figures 5 and Figures 5 and 6A-B. 6A-B.A A flow flow sensor sensor is is usedtotomeasure used measurethethe flow flow rateatat rate

which each particle stops in the flow for a given laser power. Because the optical and which each particle stops in the flow for a given laser power. Because the optical and

fluidic forces are balanced the fluidic drag force (i.e., from flow rate and channel fluidic forces are balanced the fluidic drag force (i.e., from flow rate and channel

dimension) is equal to the optical force. The properties of each cell can be measured dimension) is equal to the optical force. The properties of each cell can be measured

sequentially in sequentially in this thismanner. manner. Although notaa high Although not highthroughput throughputmeasurement measurement system, system, thisthis

embodiment embodiment of of theinvention the inventionallows allowsclose closeobservation observationandand imaging imaging of of thethe trapped trapped cell cell

and also and also dynamic changesininoptical dynamic changes opticalforce force resulting resulting from biochemicalororbiological from biochemical biological changesin changes in aa cell. cell. Reagent streams containing Reagent streams containingchemicals, chemicals,biochemicals, biochemicals,cells, cells, or or other other

standard biological agents can be introduced into the flow channels to interact with the standard biological agents can be introduced into the flow channels to interact with the

19

trapped cell(s). trapped cell(s). These These dynamic processescan dynamic processes canbebequantitatively quantitativelymonitored monitoredbybymeasuring measuring changes in optical force during experiments on a single cell or cells. changes in optical force during experiments on a single cell or cells.

[0053] In

[0053] In one one embodiment, embodiment, a camera a camera or or other other imaging imaging device device is oriented is oriented and/or and/or focused focused

in flow or opposite the flow of the analysis channel, such that it is in line and parallel to in flow or opposite the flow of the analysis channel, such that it is in line and parallel to

the flow. the (See, e.g., flow. (See, e.g.,FIGS. FIGS. 88 and and 9.) 9.) Figure Figure 88 shows shows aa camera camera810 810ininline line with with the the 2023286012

analysis channel analysis andlaser 820 and channel 820 laser or or collimated collimated light light source 830. A source 830. dichroic mirror A dichroic mirror or or similar device similar device 840 reflects the 840 reflects thelaser laserlight away 835835 light awayfrom from the thecamera camera to to prevent prevent damage damage

but passes but passes light 865 produced light 865 by the produced by the illumination illumination source source 860 to allow 860to allow for for imaging. The imaging. The

camera is oriented parallel to the fluid flow 870 such that the cells or particles 880 are, in camera is oriented parallel to the fluid flow 870 such that the cells or particles 880 are, in

one embodiment, one embodiment,moving moving awayaway from from the camera. the camera. The illumination The illumination sourcesource is oriented is oriented

orthogonal to the channel and laser. A second dichroic 845 that passes laser light and orthogonal to the channel and laser. A second dichroic 845 that passes laser light and

reflects illuminating light is used to direct both the illuminating light and laser light reflects illuminating light is used to direct both the illuminating light and laser light

through the through the channel. channel. AnAnalternative alternativeembodiment embodimentof of thisconfiguration this configurationswitches switches the the

locations of the laser and illumination source such that the laser is orthogonal to the locations of the laser and illumination source such that the laser is orthogonal to the

channel and channel andthe the illumination illumination source source is is parallel paralleltotothe channel. the channel.The The second second dichroic dichroic would would

still direct both the laser light and visible light through the channel. still direct both the laser light and visible light through the channel.

[0054] An

[0054] Analternative alternative embodiment embodiment is is shown shown in in FIG. FIG. 9. 9. In In thiscase, this case,the thecamera cameraoror imaging device 910 is oriented such that the cells or particles 980 are traveling toward the imaging device 910 is oriented such that the cells or particles 980 are traveling toward the

camerainin the camera the fluid fluid flow flow 970. Thus, the 970. Thus, the camera cameraand andlaser laser930 areon 930are onthe the same sameside sideofofthe the channel, while channel, while the the illumination illumination source source 960 is on 960 is on the the opposite opposite end end of of the the channel. channel. Two Two

dichroics 940 and 945 are used to direct laser light 935 and illumination light 965 into the dichroics 940 and 945 are used to direct laser light 935 and illumination light 965 into the

channel, direct the illumination light to the camera, and divert the laser light away from channel, direct the illumination light to the camera, and divert the laser light away from

the illumination the illumination source. Analternative source. An alternative embodiment embodiment ofof thisconfiguration this configurationswitches switchesthe the locations of the laser and camera such that the laser is orthogonal to the channel and the locations of the laser and camera such that the laser is orthogonal to the channel and the

illumination source illumination is parallel source is paralleltoto thethechannel. channel.The Thesecond second dichroic 945 would dichroic 945 thendirect would then direct the laser light through the channel and illuminating light to the camera. the laser light through the channel and illuminating light to the camera.

[0055] Optionally, the embodiment of the invention further includes at least one optical

[0055] Optionally, the embodiment of the invention further includes at least one optical

elementbetween element betweena asource sourceofofoptical optical force force and and said said fourth fourth channel, channel, and operable to and operable to

20

produceaa standard produce standard TEM00 TEMoo mode mode beam, beam, a standard a standard TEM01 TEM01 modeabeam, mode beam, a standard standard TEM10 TEM10 modebeam, mode beam,a astandard standardHermite-Gaussian Hermite-Gaussian beambeam mode,mode, a standard a standard Laguerre-Gaussian Laguerre-Gaussian

beammode, beam mode,Bessel Bessel beam, beam, or or a standard a standard multimodal multimodal beam. beam. Optionally, Optionally, theleast the at at least oneone

optical element optical element includes includes a standard a standard cylindrical cylindrical lens, lens, a standard a standard axicon, axicon, a standard a standard

concave mirror, concave mirror, a standard a standard toroidal toroidal mirror, mirror, a standard a standard spatialspatial light modulator, light modulator, a standarda standard

acousto-optic modulator, a standard piezoelectric mirror array, a diffractive optical acousto-optic modulator, a standard piezoelectric mirror array, a diffractive optical 2023286012

element, aa standard element, standard quarter-wave plate, and/or quarter-wave plate, and/or a a standard standard half-wave plate. Optionally, half-wave plate. Optionally, the source of optical force may include a standard circularly polarized beam, a standard the source of optical force may include a standard circularly polarized beam, a standard

linearly polarized beam, or a standard elliptically polarized beam. linearly polarized beam, or a standard elliptically polarized beam.

[0056] Optionally,

[0056] Optionally, aa device device is is embodied comprising embodied comprising a a microfluidicchannel, microfluidic channel,a asource sourceofof laser light focused by an optic into the microfluidic channel, and a source of electrical laser light focused by an optic into the microfluidic channel, and a source of electrical

field operationally connected to the microfluidic channel via electrodes; flowing particles field operationally connected to the microfluidic channel via electrodes; flowing particles

in a liquid through the microfluidic channel; and manipulating the laser light and the in a liquid through the microfluidic channel; and manipulating the laser light and the

electrical field to act jointly on the particles in the microfluidic channel, thereby electrical field to act jointly on the particles in the microfluidic channel, thereby

separating the particles based on size, shape, refractive index, electrical charge, electrical separating the particles based on size, shape, refractive index, electrical charge, electrical

charge distribution, charge mobility, permittivity, and/ or deformability. In yet another charge distribution, charge mobility, permittivity, and/ or deformability. In yet another

embodiment, embodiment, a a devicecomprises device comprises a microfluidic a microfluidic channel channel configured configured to supply to supply a a

dielectrophoretic (DEP) field to an interior of the channel via an (1) electrode system or dielectrophoretic (DEP) field to an interior of the channel via an (1) electrode system or

(2) insulator DEP system, and a source of laser light focused by an optic into the (2) insulator DEP system, and a source of laser light focused by an optic into the

microfluidic channel; flowing a plurality of particles in a liquid into the microfluidic microfluidic channel; flowing a plurality of particles in a liquid into the microfluidic

channel; and operating the laser light and field jointly on particles in the microfluidic channel; and operating the laser light and field jointly on particles in the microfluidic

channel to trap the particles or modify their velocity, wherein said DEP field is linear or channel to trap the particles or modify their velocity, wherein said DEP field is linear or

non-linear. Another non-linear. Anotherpossible possibleembodiment embodimentof of a device a device includes includes a microfluidic a microfluidic channel channel

comprising an inlet and a plurality of exits, and a source of laser light focused by an optic comprising an inlet and a plurality of exits, and a source of laser light focused by an optic

to cross the microfluidic channel at a critical angle matched to velocity of flow in the to cross the microfluidic channel at a critical angle matched to velocity of flow in the

microfluidic channel microfluidic channel SO so as as to to produce an optical produce an optical force force on on the the particles particleswhile whilemaximizing maximizing

residence time in the laser light of selected particles, thus separating the particles into the residence time in the laser light of selected particles, thus separating the particles into the

plurality of exits, wherein the laser light is operable to apply forces to particles flowing plurality of exits, wherein the laser light is operable to apply forces to particles flowing

through the microfluidic channel, thereby separating the particles into the plurality of through the microfluidic channel, thereby separating the particles into the plurality of

exits. exits.

21

[0057] Optionally, the embodiment of the invention further includes at least one particle

[0057] Optionally, the embodiment of the invention further includes at least one particle

interrogation unit interrogation unit communicating withone communicating with oneorormore moreofof thechannels, the channels,such suchasasthe theanalysis analysis channel(s) and channel(s) and in in particular particular thethe fourth fourth channel. channel. The particle The particle interrogation interrogation unit aincludes a unit includes

standard illuminator, standard optics, and a standard sensor. Optionally, the at least one standard illuminator, standard optics, and a standard sensor. Optionally, the at least one

particle interrogation unit includes a standard bright field imager, a standard light scatter particle interrogation unit includes a standard bright field imager, a standard light scatter 2023286012

detector, a standard single wavelength fluorescent detector, a standard spectroscopic detector, a standard single wavelength fluorescent detector, a standard spectroscopic

fluorescent detector, fluorescent detector, aastandard standardCCD camera,a astandard CCD camera, standardCMOS CMOS camera, camera, a standard a standard

photodiode, aa standard photodiode, standard photomultiplier photomultipliertube, tube, aa standard standard photodiode array, aa standard photodiode array, standard

chemiluminescent detector,aastandard chemiluminescent detector, standardbioluminescent bioluminescentdetector, detector,and/or and/oraastandard standardRaman Raman spectroscopy detector. spectroscopy detector.

[0058] The

[0058] Theatat least least one one particle particleinterrogation interrogationunit unitcommunicating with the communicating with the fourth fourth channel channel

comprises a laser-force-based apparatus or device that facilitates cell disease comprises a laser-force-based apparatus or device that facilitates cell disease

identification, selection, and sorting. In one aspect, the unit utilizes inherent differences identification, selection, and sorting. In one aspect, the unit utilizes inherent differences

in optical pressure, which arise from variations in particle size, shape, refractive index, or in optical pressure, which arise from variations in particle size, shape, refractive index, or

morphology,asasa ameans morphology, meansof of separatingand separating and characterizingparticles. characterizing particles. InInone oneaspect, aspect, aa near- near­ infrared laser beam exerts a physical force on the cells, which is then measured. Optical infrared laser beam exerts a physical force on the cells, which is then measured. Optical

force via radiation pressure, when balanced against the fluidic drag on the particles, force via radiation pressure, when balanced against the fluidic drag on the particles,

results in changes in particle velocity that can be used to identify differing particles or results in changes in particle velocity that can be used to identify differing particles or

changes with populations of particles based on intrinsic differences. The fluidic and changes with populations of particles based on intrinsic differences. The fluidic and

optical forcebalance optical force balancecancan alsoalso be used be used to change to change the relative the relative position position of particles of particles to one to one another based upon their intrinsic properties thus resulting in physical separations. another based upon their intrinsic properties thus resulting in physical separations.

Another embodiment Another embodiment of theof the interrogation interrogation unit includes unit includes a device a device for foranalysis particle particle analysis and/or separation, such as at least one collimated light source operable to generate at least and/or separation, such as at least one collimated light source operable to generate at least

one collimated one collimated light light source source beam. Theatatleast beam. The least one one collimated collimatedlight light source beamincludes source beam includes at least one beam cross-section. at least one beam cross-section.

[0059] An

[0059] Anembodiment embodiment of the of the instant instant invention invention involves involves thecombination the combination of of several several of of

the above-mentioned the designelements above-mentioned design elements discussed discussed above above in ainunitary a unitary device.Embodiments device. Embodiments also include also include methods of using methods of usingsuch suchdevices. devices. AnAnexample example of of such such a unitary a unitary device device is is

22

illustrated in FIG. 1. The illustrated embodiment of the invention is a 5-layer structure illustrated in FIG. 1. The illustrated embodiment of the invention is a 5-layer structure

with all 5 layers bonded to each other to yield a solid microfluidic chip, although the chip with all 5 layers bonded to each other to yield a solid microfluidic chip, although the chip

maybebeone may onestructure structureas as opposed opposedtotobonded bondedlayers. layers.The Thechip chipcould couldbebeconstructed constructedusing usinga a number of standard materials including, but not limited to, fused silica, crown glass, number of standard materials including, but not limited to, fused silica, crown glass,

borosilicate glass, soda lime glass, sapphire glass, cyclic olefin polymer (COP), borosilicate glass, soda lime glass, sapphire glass, cyclic olefin polymer (COP),

poly(dimethyl)siloxance(PDMS), poly(dimethyl)siloxance (PDMSOSTE, ), OS polystyrene, TE, polystyrene, poly(methyl)methacrylate, poly(methyl)methacrylate, 2023286012

polycarbonate, other polycarbonate, other plastics plastics or orpolymers. This chip polymers. This chip allows allows for for sample input, sample input,

hydrodynamic hydrodynamic focusing,optical focusing, opticalinterrogation, interrogation, imaging, imaging,analysis, analysis, sample sampleexit exit and andclear clear optical access for the laser light to enter and exit the regions. The chip in embodiments optical access for the laser light to enter and exit the regions. The chip in embodiments

can also can also be be 3D printed, molded, 3D printed, or otherwise molded, or otherwise shaped. shaped.

[0060] Optionally, the at least one particle type includes a plurality of particle types.

[0060] Optionally, the at least one particle type includes a plurality of particle types.

Each particle type of the plurality of particle types includes respective intrinsic properties Each particle type of the plurality of particle types includes respective intrinsic properties

and respective induced properties. Optionally, the intrinsic properties include size, shape, and respective induced properties. Optionally, the intrinsic properties include size, shape,

refractive index, morphology, intrinsic fluorescence, and/or aspect ratio. Optionally, the refractive index, morphology, intrinsic fluorescence, and/or aspect ratio. Optionally, the

induced properties include deformation, angular orientation, rotation, rotation rate, induced properties include deformation, angular orientation, rotation, rotation rate,

antibody label antibody label fluorescence, fluorescence, aptamer label fluorescence, aptamer label fluorescence, DNA labelfluorescence, DNA label fluorescence,stain stain label fluorescence, a differential retention metric, and/or a gradient force metric. This label fluorescence, a differential retention metric, and/or a gradient force metric. This

methodembodiment method embodiment further further includes includes identifying identifying andand separating separating thethe pluralityofofparticles plurality particles according to the respective particle types based on at least one of the intrinsic properties according to the respective particle types based on at least one of the intrinsic properties

and the and the induced properties. Optionally, induced properties. Optionally, this this method embodiment method embodiment further further includes includes

interrogating or interrogating or manipulating manipulating the the sample flow. Optionally, sample flow. Optionally, interrogating interrogating the the sample flow sample flow

includes determining at least one of the intrinsic properties so and the induced properties includes determining at least one of the intrinsic properties SO and the induced properties

of the particle types, and measuring particle velocity of the plurality of particles. of the particle types, and measuring particle velocity of the plurality of particles.

Measurement of at least one of the intrinsic properties can be used for a range of Measurement of at least one of the intrinsic properties can be used for a range of

applications, including but not limiting to: determining the viral infectivity of a cell applications, including but not limiting to: determining the viral infectivity of a cell

sample (thenumber sample (the number of functionally of functionally infectious infectious virus particles virus particles present present in a particular in a particular cell cell population, similar to a plaque assay or end point dilution assay) for the purposes of viral population, similar to a plaque assay or end point dilution assay) for the purposes of viral

quantification, quantification,process process development andmonitoring, development and monitoring,sample sample releaseassays, release assays,adventitious adventitious agent testing, clinical diagnostics, biomarker discovery, determining the productivity of a agent testing, clinical diagnostics, biomarker discovery, determining the productivity of a

cell ininterms cell termsof ofantibody antibody or orprotein proteinfor process for development process development and and monitoring, determining monitoring, determining

23

the efficacy, quality, or activation state of cells produced as a cell-based therapy, the efficacy, quality, or activation state of cells produced as a cell-based therapy,

including CAR including CART and T and other other oncology oncology applications applications andand stem stem cells, cells, determining determining thethe effect effect

of a chemical, bacteria, virus, antimicrobial or antiviral on a specific cell population, and of a chemical, bacteria, virus, antimicrobial or antiviral on a specific cell population, and

determining determining thethe disease disease state state or potential or potential of a of a research research or clinical or clinical cell sample. cell sample.

Optionally, a source of optical force includes at least one beam axis, and the sample flow Optionally, a source of optical force includes at least one beam axis, and the sample flow

includes a sample flow axis. The step of determining at least one of the intrinsic includes a sample flow axis. The step of determining at least one of the intrinsic 2023286012

properties and the induced properties of the particle types, and the step of measuring properties and the induced properties of the particle types, and the step of measuring

particle velocity of the plurality of particles together comprise offsetting the beam axis particle velocity of the plurality of particles together comprise offsetting the beam axis

from the sample flow axis. Optionally, the step of determining at least one of the from the sample flow axis. Optionally, the step of determining at least one of the

intrinsic properties and the induced properties of the particle types, and the step of intrinsic properties and the induced properties of the particle types, and the step of

measuring particle velocity of the plurality of particles together comprise calculating a measuring particle velocity of the plurality of particles together comprise calculating a

slope and a trajectory of a particle of the plurality of particles deviating from a sample slope and a trajectory of a particle of the plurality of particles deviating from a sample

flow axis toward at least one beam axis. flow axis toward at least one beam axis.

[0061] One skilled in the art will recognize that the disclosed features may be used

[0061] One skilled in the art will recognize that the disclosed features may be used

singularly, ininany singularly, any combination, combination, or or omitted omitted based on the based on the requirements andspecifications requirements and specifications of aa given of given application application or or design. design. When anembodiment When an embodiment refers refers to to "comprising" "comprising" certain certain

features, it is to be understood that the embodiments can alternatively "consist of' or features, it is to be understood that the embodiments can alternatively "consist of" or

"consist "consist essentially essentiallyof' of"any anyone one or ormore more of of the thefeatures. features.Other Other embodiments embodiments ofofthe the invention will be apparent to those skilled in the art from consideration of the invention will be apparent to those skilled in the art from consideration of the

specification andpractice specification and practice of of thethe invention. invention.

[0062] It is noted in particular that where a range of values is provided in this

[0062] It is noted in particular that where a range of values is provided in this

specification, each value between the upper and lower limits of that range is also specification, each value between the upper and lower limits of that range is also

specifically disclosed. specifically disclosed. The The upper and lower upper and lowerlimits limits of of these these smaller smaller ranges ranges may may

independentlybebeincluded independently includedororexcluded excludedininthe therange rangeasas well. well. The Thesingular singularforms forms"a," "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is "an," and "the" include plural referents unless the context clearly dictates otherwise. It is

intended that intended that the the specification specificationand andexamples be considered examples be considered as as exemplary exemplaryororexplanatory explanatory in nature and that variations that do not depart from the essence of the invention fall in nature and that variations that do not depart from the essence of the invention fall

within the scope of the invention. Further, all of the references cited in this disclosure are within the scope of the invention. Further, all of the references cited in this disclosure are

each individually incorporated by reference herein in their entireties and as such are each individually incorporated by reference herein in their entireties and as such are

24

intended to provide an efficient way of supplementing the enabling disclosure of this intended to provide an efficient way of supplementing the enabling disclosure of this

invention as well as provide background detailing the level of ordinary skill in the art. invention as well as provide background detailing the level of ordinary skill in the art. 2023286012

25

Claims (18)

MARKED-UP COPY The claims defining the invention are as follows:

1. A method for assessing biological particles for use in a cell-based therapy, comprising: the use of a device wherein the device comprises: a substrate comprising a plurality of channels configured to transport one or more biological particles, wherein the plurality of channels comprises: 2023286012

a first channel disposed vertically within the substrate, a second channel in operable communication with the first channel and disposed horizontally within the substrate, a third channel in operable communication with the second channel and disposed vertically within the substrate, and a fourth channel in operable communication with the third channel and disposed horizontally within the substrate; wherein the first, second, third and fourth channels are disposed in such a manner as to provide a path for movement of the one or more biological particles through the substrate from the first channel to the second channel to the third channel to the fourth channel; and injecting the biological particles into the first channel disposed vertically within the substrate through a bottom horizontal planar surface having an opening to the first channel, to maintain directional and volumetric continuity with the first channel, wherein the biological particles comprise CAR T cells, CAR-M cells, TILs, NK cells, other engineered or effector cells, or stem cells; and assessing the biological particles in the fourth channel using optical force-based measurements.

2. The method of claim 1, wherein the optical force-based measurements are used to yield information pertaining to morphology, motility, binding affinities, binding profiles, effect on other biological particles, effect on target cells, susceptibility to external forces such as biological, biochemical, chemical, physical, temperature influences, or a combination thereof, of the biological particles, and/or wherein the optical force-based measurements are used to yield information pertaining to size, shape, refractive index, internal cellular structural changes, cell aggregation, or a combination thereof of the biological particles.

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3. The method of claim 1 or 2, further comprising using the optical force-based measurements: i) to determine the total number and the number of functionally infectious virus particles present in the biological particles, ii) for viral quantification, iii) for process development 2023286012

iv) for process monitoring, v) for a sample release assay, vi) for adventitious agent testing, vii) for sterility testing viii) for clinical diagnostics, ix) for biomarker discovery, x) to determine the cell killing capacity of the CAR T cells, xi) to measure transfection efficiency, xii) to measure viral infectivity, xiii) to measure differentiation of the stem cells, xiv) to measure differentiation capability of the stem cells, xv) to measure the viral transduction efficiency, xvi) to determine productivity of a cell from among the biological particles in terms of antibody, protein, or cellular phenotype for process development and monitoring, xvii) to determine the efficacy, quality, or activation state of the biological particles, xviii) to determine the effect of a chemical, bacteria, virus, antimicrobial or antiviral on the biological particles, xix) to determine a disease state of the biological particles, xx) to measure cell death, viability, apoptosis, cytotoxicity, or another cell health related parameter, or xxi) a combination thereof.

4. The method of any one of claims 1 to 3, further comprising separating the biological particles based on size, shape, refractive index, morphology, electrical charge, electrical charge distribution, charge mobility, permittivity, deformability, or a combination thereof.

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5. The method of any one of claims 1 to 4, wherein the assessing comprises imaging the biological particles.

6. The method of any one of claims 1 to 5, further comprising using the imaging to determine volume, shape, nucleus location, nucleus volume, organelle location, inclusion body location, or a combination thereof of the biological particles. 2023286012

7. The method of claim 5 or 6, wherein the imaging: i) comprises capturing images of the biological particles in multiple focal planes, ii) comprises capturing a 3-dimensional image of the biological particles, iii) comprises capturing images of the biological particles during movement of the biological particles, iv) comprises capturing images of the biological particles from multiple angles and/or orientations, v) comprises the use of a plurality of imaging devices, vi) comprises analysis of multiple image slices of the biological particles as focal planes move relative to the biological particles, vii) comprises moving an imaging device to change a focal plane being imaged in the fourth channel, viii) comprises analysis of multiple image slices of the biological particles as the biological particles move through a focal plane, ix) comprises analysis of multiple image slices of a suspended or static biological particle from among the biological particles, x) directing light away from an imaging device while collecting images in the fourth channel, or xi) a combination thereof.

8. The method of any one of claims 5 to 7, further comprising moving the biological particles in the fourth channel with an optical force, a fluidic force, or a combination thereof, wherein a region in which the biological particles are imaged within the fourth channel changes during movement of the biological particles.

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9. The method of any one of claims 5 to 8, wherein the imaging comprises the use of a bright field imager, a light scatter detector, a single wavelength fluorescent detector, a spectroscopic fluorescent detector, a CCD camera, a CMOS camera, a photodiode, a photodiode array (PDA), a spectrometer, a photomultiplier tube or tube array, a photodiode array, a chemiluminescent detector, a bioluminescent detector, a standard Raman spectroscopy detection system, a surface enhanced Raman spectrometer (SERS), a coherent antistokes 2023286012

Raman spectrometer (CARS), a coherent stokes Raman spectrometer (CSRS), or a combination thereof.

10. The method of any one of claims 1 to 9, further comprising moving the biological particles in one or more of the plurality of channels with an electrical force, an optical force, a fluidic force, an electrophoretic force, a dielectrophoretic force, or a combination thereof.

11. The method of any one of claims 1 to 9, wherein the bottom horizontal planar surface has at least one shorter length from one edge to another edge compared to at least one length from one edge to another edge on a vertical planar surface of the substrate.

12. The method of any one of claims 1 to 9, wherein the device further comprises a collimated or focused light source oriented to interact with the biological particles in the fourth channel.

13. The method of any one of claims 1 to 12, wherein: i) the fourth channel is located closer to the top of the substrate than either the first channel, the second channel, or the third channel, ii) the fourth channel is located from 100 microns to 100 millimeters from the top of the substrate, iii) the first channel ranges from 0.1 millimeters to 100.0 millimeters in length, iv) the second channel ranges from 0.1 millimeters to 100.0 millimeters in length, v) the third channel ranges from 0.05 millimeters to 100.0 millimeters in length, vi) fourth channel ranges from 0.1 millimeters to 100.0 millimeters in length, vii) the first channel is greater in length than the second channel, the third channel, or the fourth channel, or

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viii) a combination thereof.

14. The method of any one of claims 1 to 13, wherein the device further comprises a cell or particle interrogation unit, a cell or particle collection channel, or a combination thereof.

15. The method of any one of claims 1 to 13, wherein the second and fourth channels are 2023286012

shorter than the first channel.

16. The method of any one of claims 1 to 13, wherein the injecting comprises providing a sample inlet line tip communicating with a sample inlet line, which communicates with the first channel.

17. The method of any one of claims 1 to 16, wherein the fourth channel is located from 100 microns to 100 millimeters from the top of the substrate.

18. The method of any one of claims 1 to 16, wherein the combined length of the first, second, third and fourth channels is from 0.35 millimeters to 400.0 millimeters.