TWI901456B - Microfluidic device for optical force measurements, cell imaging, and assessing biological particles and the method using the same - Google Patents

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. Then the channel takes a short horizontal turn that nearly negates any influence of cell settling due to gravity and zero flow velocity at the walls. 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 which prevents them from settling.

Description

Microfluidic device for optical force measurement, cell imaging, and evaluation of biological particles and method of using the same

The present invention generally relates to devices and methods for particle analysis and imaging of particles or particles in cells, and more specifically to devices and methods for particle imaging of fluids using pressure, fluid dynamics, electrokinetics, and optical forces.

The present invention relates to a microfluidic chip in which injection occurs in an upward vertical direction and the fluid vials are located below the chip to minimize particle settling before and in the analytical portion of the chip channels.

Modifications to existing microfluidic chip designs were necessary to implement the present invention. For example, to keep the vials vertically aligned with the chip, a different interface with the chip must be established compared to prior art. Specifically, instead of interfacing with the chip orthogonally via ports connected to the largest face of the chip (as is typically done in microfluidic lab-on-a-chip systems) and then pumping fluids first across the chip and then upward, the present invention's inputs and fluids, in one embodiment, rise through the bottom of the chip using a manifold, which avoids horizontal redirection of the fluids/fluidic dynamics.

According to the invention herein, the contents of a vial are located below the chip and pumped upward and vertically directly into the first channel of the chip. The long channel extends from the bottom of the chip to near the top. The channel then makes a short horizontal bend, but the new channel is so short that any effects on cell sedimentation due to gravity and zero flow velocity at the walls are nearly eliminated. Then, contrary to the prior art, the fluid is pumped upward to the analysis section. Therefore, the horizontal analysis section is the highest channel/fluid point in the chip and, therefore, is close to the top of the chip. This results in less chip material (e.g., glass) between the microscope/camera and the sample than with prior art, and thus clearer imaging. During analysis, the laser also suspends the cells in this channel, preventing cell sedimentation.

According to prior art, microfluidic chip vials containing cells or particles to be separated and/or analyzed are positioned sideways and pumped horizontally into channels within the microfluidic chip. The vial contents (e.g., particles or cells) are first pumped in an upward vertical direction, then make a U-turn to travel downward, and are then pumped horizontally into the chip. (See, for example, U.S. Patent No. 9,594,071.)

The connection to the chip is horizontal, which, combined with the idle volume in the connection (the somewhat unavoidable empty space in the fluid connection), results in significant additional settling due to gravity. This configuration also requires a relatively large diameter channel required for the connection, which, in addition to the idle volume, also creates a region of relatively low velocity, further increasing the problem of particle settling. The current chip according to the invention eliminates the need for a large horizontal input channel and the relatively abrupt change from the large horizontal input channel to the relatively thin upward flow in the first vertical chip channel. This configuration eliminates horizontal settling and the unnecessary directional changes that cause settling. Entry of cells at the bottom edge of the chip also solves the sedimentation problem in the idle volume by orienting the chip vertically relative to gravity, so that cells or particles do not settle in the bottom of the horizontal channel but are instead continuously guided upward by the flow. This is not intuitive and required further experimentation to achieve this before designing the currently implemented solution. Currently available microfluidic devices incorporate custom or commercially available connections over large areas of polished surfaces and glass, which, in contrast to the present invention, generally forces any particles (e.g., cells) contained within the sample flow to immediately turn and travel horizontally upon entry into the chip.

Also in the prior art, cells or particles travel horizontally across the microfluidic chip several times before reaching the analysis channel, which causes sedimentation. As the vial contents enter the chip and the channels in the chip, they are pumped horizontally, compared to the vertical channels in a crystal. The channel then flows upward and makes a long horizontal bend, where the cells tend to settle at the bottom of the channel due to gravity and experience lower velocities at the walls due to laminar flow conditions. Essentially, the flow is highest in the middle of the channel due to the parabolic velocity distribution and decreases to zero at or near the channel walls. After the first horizontal channel in the chip, the fluid turns downward before the analysis channel, where the particles are imaged or separated. Due to this configuration, there is a relatively large distance between the microscope/camera and the analysis channel. In this typical prior art configuration, the particles are forced downward and ultimately exit the bottom of the chip.

Furthermore, due to limitations in prior art, multiple horizontal passes are required, causing cells to settle at multiple locations within the channel. This in turn reduces image quality due to the need for imaging through additional material at the edge of the chip. Prior art channels within the chip must pump sufficient cell or particle suspension vertically upward, then horizontally, then in a zigzag pattern, and then downward and off the chip. The present invention avoids this zigzag channel.

There are also prior techniques for computationally rendering 3D images of cells or particles in fluids. For example, M. Habaza, M. Kirschbaum, C. Guernth-Marschner, G. Dardikman, I. Barnea, R. Korenstein, C. Duschl, and N.T. Shaked (Adv. Sci. 2017, 4, 1600205) teach capturing cells, rotating them at high speed, and using interferometry to measure the refractive index distribution within the cells. Interferometry has also been used to analyze cells in microfluidic channels (e.g., see Y. Sung et al., Phys. Rev. Appl. 2014, Feb. 27;1:014002). However, the present invention advocates capturing multiple images of cells or particles as they travel in the fluid stream and pass through the focal plane of the imaging device, thereby negating any need to capture the cells to compute a 3D image. Other techniques have been taught, such as using a mechanical translation stage to move cells or particles (e.g., N. Lue et al., Opt. Express 2008 Sep. 29; 16(20): 16240-6), which do not use bright field imaging as described herein and do not utilize fluid flow to provide cell positioning relative to the focal plane of the image.

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

The present invention relates to a microfluidic chip in which injection occurs with the sample vials located below the chip to minimize particle settling. Thus, the contents of the vials are located below the chip and are pumped upwards and vertically directly into the channels of the chip. A long channel extends from the bottom of the chip to near the top of the chip. The channel then makes a short horizontal bend, but the new channel is short enough so that any effect of cell settling due to zero flow velocity at the walls of the channel is insignificant. Then, in contrast to the prior art, the sample is pumped upwards to the analysis section. The horizontal analysis section is therefore the highest channel/fluid point in the chip and is therefore close to the top of the chip, which results in less glass between the microscope/camera than in the prior art and therefore clearer imaging. The distance of the analysis channel from the top of the chip can be 100 microns to 2 mm, but can be as large as 100 mm, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns, etc. In one embodiment, after the analysis portion of the chip, the sample (e.g., fluid, cells, and/or particles) is pumped down to the bottom of the chip and forced outward.

The present invention further relates to a microfluidic chip in which horizontal flow is minimized, particularly as the fluid enters the chip's channels. Prior art chips contain approximately 13 mm of horizontal channels (non-analytical portions), of which approximately 2 mm are larger diameter injection ports, which exacerbate sedimentation due to low flow rates. The chip described herein has approximately 0.2 to 3.0 mm of horizontal channels (non-analytical portions), but the horizontal channels can range in length from 0.01 to 100.0 mm, e.g., 0.01 to 0.02 mm, 0.02 to 0.03 mm, 0.03 to 0.04 mm, etc. This is a result of the channel system of the present invention, which is orders of magnitude different from prior art, improving flow and eliminating cell/particle sedimentation.

Another aspect of the present invention relates to a microfluidic chip in which imaging and analysis are performed at or near the corners of the chip, thereby improving imaging from multiple viewpoints due to the presence of less glass and distance between the camera and the analysis channel. This also allows the use of higher-value objectives to improve detailed imaging by increasing magnification. This design improvement reduces image distortion and distance caused by glass (or other materials comprising the chip, such as plastic or any transparent or translucent material) (e.g., due to defects in the glass). The distance between the imaging device and the analysis channel can be from 100 microns to 2 mm, but can be as large as 100 mm, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns, and so on.

In addition, the present invention relates to a microfluidic sorting chip separated from the downstream of the analysis channel, which allows the use of pressure and/or laser (or other optical forces) to activate the sorting function simultaneously or sequentially. In one embodiment, the flow will continue from the analysis channel to perform the sorting function. For example, the particles will be guided into the vertical channel and then to the horizontal sorting channel. In one embodiment, optical forces and/or pressure will be applied in the direction of flow using the sorting channel to push the particles through the channel. Particles that are not directly affected by the optical force will be diverted to the alternative channel due to (for example) gravity, electrokinetic forces, magnetic forces, laminar flow lines, streamlines, reduced flow rate, orthogonal optical forces or a vacuum applied to draw the particles into the alternative channel. In another aspect related to the post-sort analysis channel, the present invention allows for directing an optical force from the back of the chip (the laser or optical force is oriented in the same direction as the flow) and, in some aspects, splitting this primary laser. In embodiments, the optical force and/or pressure can be applied in the opposite direction of the material moving through the channel, e.g., against the flow, or in the same direction as the material moving through the channel, e.g., with the flow. Cell or particle sorting can be performed on a single device or separate chip. For example, in FIG1B , the fifth channel before the outlet conduit 145 contains one or more branches to achieve single or multiple sorting regions.

In another aspect of the invention, a manifold is connected to the vials in such a way that tubing passes through the manifold and connects to vials or other containers on the other side, thereby contacting the contents of the vials (e.g., fluids). The manifold allows the vials to be connected to the microfluidic chip but stored below the chip, and/or the manifold allows the contents of the vials to be injected from the bottom of the chip, thereby alleviating several problems experienced with prior art techniques, such as cell or particle sedimentation, where the vials or tubing from the vials are connected to or communicate with the microfluidic chip.

In another aspect, the present invention relates to a microfluidic chip holder that includes a structure for directing a light source, including an integrated prismatic cavity. When equipped with a prism, this cavity directs light at an angle relative to the chip, providing a preferred method for illuminating confined geometric structures. In embodiments, the light source includes, but is not limited to, a fiber optic or a collimated or focused light source. Specifically, the light source is precisely directed, oriented, or guided into an analysis channel.

In another aspect of the invention, the apparatus includes a second imaging device oriented orthogonally to the first camera and channel view. The reason for the second camera can vary. In one aspect, the reason for the second imaging device is to aid in visual alignment of the laser or optical force in the analysis channel. In another aspect, using the methods described herein, data from the first camera can be recorded. The data from the second camera can be combined with the data from the first camera to generate additional data that can be used to more accurately infer cell location, size, shape, volume, etc. This additional information about the same cell (or particle) increases the accuracy and range of measurements and analysis. In another aspect, the second camera is combined with the first camera to allow for 3D reconstruction of cells or particles, or groups of cells or particles, imaged by an orthogonal camera and a camera in flow or a camera located on one side of the chip. Using the algorithms described herein or other methods, including reversing or slowing the flow and capturing one or more images of a specific cell or particle, or group of cells or particles, the present invention allows for analysis and processing of multiple images to determine characteristics/attributes/quantitative measurements, such as cell volume, cell shape, nuclear position, nuclear volume, and organelle or inclusion location. In another aspect of the present invention, the camera is oriented to image on an axis along the direction of flow.

In one aspect, the present invention eliminates the need for serpentine or zigzag channels, as is preferred according to prior art, to keep particles properly suspended. Due to the vertical nature of the fluid and particle or cell injection into the chip, vertical bulk pumping of particles through the channel directly upward to a first horizontal channel (herein referred to as the second channel) avoids the need for zigzag channels.

100: Microfluidic Chip

110: Air duct

120: Manifold

130: Sample vial

140: Inlet pipe

145: Export Pipeline

150: Channel

160: Side X

170: Side Y

180: Side Z

200: Chip holder

210: Light Source

220: Prism Cavity

230: Microfluidic Chip

240: Integrated structure or channel

250: A beam of light

260: Analysis Channel

300: Chip holder

320: Prismatic Cavity

330: Microfluidic Chip

350: Seat

360: Threaded hole

400: Microfluidic Chip

410: First Channel

420: Second horizontal channel/Second channel

430: Another vertical channel/third channel

440: Fourth Horizontal Channel/Fourth Channel/Analysis Channel/Microfluidic Channel/

450: Imaging device/camera/secondary camera or image capture device

460: Light Source

480: Light source/laser or other optical force

510: Cells or particles

520: First Plane

530: Second Plane

540: Analysis Channel

550: Cells or particles

620: Fluid Flow

630: XZ focal plane

640: YZ focal plane

650: More complex 3D computational rendering of particles or cells

660: Distance

670: Laser or other optical force

680: Plate

710: Particles or cells

730: Optical Power

735: Fluid Force

810: Camera

820: Analysis Channel

830: Laser or collimated light source

835: Laser Light

840: Dichroic mirror or similar device

845: Second dichroic mirror

860: Lighting Source

865: Light

870: Fluid Flow

880: Cells or particles

910: Camera or imaging device

930: Cameras and Lasers

935: Laser Light

940:Dichroic Mirror

945:Dichroic Mirror

960: Lighting Source

965:Illumination light

970: Fluid Flow

980: Cells or particles

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

Figure 1A is a diagram showing a global view of the device configuration, including the wafer, manifold, and vials. Figure 1B is a diagram showing some depictions of the wafer channel orientation and location.

Figures 2A and 2B contain diagrams illustrating a microfluidic chip holder according to the present invention.

Figures 3A and 3B illustrate the angles and configurations of the chip holder according to the present invention.

Figures 4A and 4B are diagrams showing examples of cell pathways and imaging configurations associated with the chip. Figure 4C is a diagram showing an alternative cell pathway.

Figure 5 shows on-chip multiplanar imaging and how it is used to computationally present 3D images and information.

Figures 6A and 6B show multi-planar imaging on a chip in a fluid flow and how it is used to computationally render a 3D image.

Figure 7 shows how cells are captured and/or equilibrated within the analysis portion of the chip and imaged from multiple angles.

Figure 8 shows how the camera and illumination source are placed in line with the laser and flow direction so that the particles move away from the camera.

Figure 9 shows how the camera and illumination source are placed in line with the laser and flow direction so that the particles move toward the camera.

The present invention has been described with reference to specific embodiments having various features. Those skilled in the art will appreciate that various modifications and variations may be made in the practice of the invention without departing from the scope or spirit of the invention. Those skilled in the art will recognize that these features may be used individually or in any combination based on the requirements and specifications of a given application or design. Embodiments comprising various features may also consist of or consist essentially of these 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. In essence, the description of the present invention is merely exemplary or illustrative, and therefore, variations that do not depart from the essence of the invention are intended to be within the scope of the invention.

Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not limited to the details of construction and the arrangement of components described in the following description or illustrated in the drawings. The present invention is capable of other embodiments or of being practiced or carried out in various ways. Furthermore, it should be understood that the phraseology and terminology used herein are for descriptive purposes only and should not be construed as limiting.

Referring now to the drawings, FIG1A shows a full view of the device taught herein. Sample vials 130 are located beneath the microfluidic chip 100 (also collectively referred to herein as the substrate), and there is no limitation on the number or size of the vials. The vials are configured to hold any type of sample that can be moved through the device, such as one or more substances, including but not limited to one or more of fluids, liquids, gases, plasma, serum, blood, cells, platelets, particles, etc., or a combination thereof. In the context of this specification, the terms fluid or sample may be used generally to refer to the one or more substances. The vials are connected directly or indirectly, for example, using an air line 110 that is operably connected to the underside of the chip. As further shown in FIG1A , they may also be connected via a manifold 120 . FIG1B shows a preferred embodiment of a microfluidic chip 100 in which a substance, fluid, particle, and/or cell is injected onto an outer surface (e.g., an edge) of the microfluidic chip (e.g., the XZ plane in FIG1B ). FIG1B shows the injection along a plane (e.g., the XZ plane or the XY plane) shared by a minimum distance. In this particular embodiment, the length of side X 160 is less than the length of side Y 170 and the length of side Z 180. This configuration allows the sample to enter the channel on the chip with minimal deviation (e.g., without the need for a right turn, as is typically the case in current state-of-the-art methods).

In FIG1A , a manifold 120 is shown that connects one or more vials 130 to the microfluidic chip 100 so that substances, fluids, particles, and/or cells can be injected or pumped into the microfluidic chip in a vertical and upward direction. The manifold allows substances to be injected from the bottom of the chip while also positioning the electronics, flow sensors, and tubing (liquid and air) in a manner that minimizes cell sedimentation, which optimizes throughput. The air tubing provides pressure or vacuum, which, when sealed, provides a closed system within the confines of the manifold device. In one embodiment, there is a distance between the vials and the manifold, indicating that there is no seal and the pressure of the internal volume is atmospheric pressure. This allows substances to be pumped from or into containers that are open to the atmosphere (vacuum is required to pump from open containers). The manifold positions the electronics, flow sensors, and tubing (liquid and air) in a way that minimizes cell sedimentation, which optimizes throughput.

By injecting contents from the bottom of the chip, the present invention minimizes horizontal movement in the inlet and outlet conduits 140 , 145 that can cause problems such as particles or cells settling in the channel 150. In this example, the outlet conduit is offset from the inlet conduit in the Z and X dimensions (FIG. 1B). In embodiments, the outlet conduit is offset, unoffset, straight, in line, or angled relative to the inlet tube. This configuration eliminates the need for serpentine or Z-shaped vertical channels because it solves settling problems that occurred in prior art techniques, such as when a fluid combined with cells and/or particles is injected or pumped horizontally from the side onto the chip, requiring a change in direction and fluid dynamics to propel it upward. Manifolding and injection from the bottom of the chip also allow for the placement of additional components, assemblies, machinery, or hardware below the chip. (See FIG. 1A.)

The manifold 120 works by regulating the air pressure above the contents of the vials and providing the correct geometry for the flow sensors and electronics. Piping (e.g., fluid or air lines) passes through the manifold and connects to the vials on the other side. Pressurized air passes through one side of the manifold and creates a closed pressurized system within the manifold. By adjusting the pressure in the closed area, the system allows parameters such as flow rate and fluid dynamics to be varied. The pressurized area is located in both the vials 130 and the manifold 120. In another embodiment, the vials do not require an air connection because they are open to the ambient atmosphere. This enables sampling from a wider variety of containers and sources. In this embodiment, a vacuum is applied to another vial or vials, creating a pressure differential that drives the fluid from the open container.

In one embodiment, pressure-based sample injection is used. A vial is filled with a sample in a fluid and sealed with a cap or tubing connected to the chip. Prior to connection to the cap, the vial can be open to air or sealed with a septum or other airtight device. In one aspect, the cap can contain two connections: one for a fluid, such as a gas, and one for a liquid. Optionally, the method embodiment further includes providing a sample inlet line tip in communication with the sample inlet line, the sample inlet line being in communication with the first channel.

In another embodiment, vacuum-based sample injection is used. A vial is filled with a sample in a fluid and sealed with a cap or tubing connected to the chip. Prior to connection to the cap, the vial can be open to the atmosphere or sealed with a septum. In one aspect, the cap can contain two connections, one for a fluid such as a gas and one for a liquid. Optionally, fluid can be aspirated from the vial open to atmosphere by applying vacuum pressure to one or more other vials. Optionally, the method embodiment further includes providing a sample inlet line tip in communication with a sample inlet line, the sample inlet line being in communication with the first channel.

2A-B and 3A-B, a chip holder 200 , 300 is shown. The chip holder includes a structure for guiding a light source 210 (e.g., a fiber optic light source, a light-emitting diode, or a laser) and an integrated prism cavity 220 , 320 , which can be equipped with a prism. The light source is guided or aligned to the desired location by an integrated structure or channel 240 within the chip holder. The built-in space for the fiber optic light source allows illumination, such as a beam 250 , even in confined geometric environments, such as on a microfluidic chip 230 , 330 or a channel in a microfluidic chip. Specifically, in a preferred embodiment, this light source is precisely guided, directed, or focused onto an analysis channel 260 . In a preferred embodiment, the wafer holder includes built-in space for prisms 220 , 320 and fiber optic light source 210 , allowing illumination even in constrained geometries. The wafer further includes holes or openings in the bottom of the holder 350 for precise alignment with fluid conduits, as described herein. In one embodiment, adjustable screws are integrated into threaded holes 360 on one or more faces to facilitate proper alignment.

Figure 4A is a preferred embodiment of the microfluidic chip 400 described herein. As shown, the fluid first travels upward in a vertical direction through a first channel 410 and then connects to a second horizontal channel 420. Another vertical channel 430 brings the fluid even closer to the top of the chip, at which point a fourth channel 440 is horizontal and, as shown in Figure 4, contains an analysis channel. The channels are operably connected to each other to allow a sample to move from one channel to another through the system. In an embodiment, the sample can flow from a first channel to a second channel to a third channel to a fourth channel, or vice versa, or a combination thereof. A pump and/or vacuum device can be provided to provide positive and/or negative pressure at one or both of the opening and outlet of the channel to enable the movement of material through the channel. The analysis channel is proximate to one or more external surfaces of the substrate, such as a face, edge, or side of the chip. For example, according to this configuration, the analysis channels are located near the top and sides of the chip, thereby improving imaging and analysis of substances passing through the microfluidic chip. In a preferred embodiment, the analysis channels are located about 1 mm to about 2 mm from the top and sides of the chip. However, the distance between the analysis channels and the top of the chip can be 0.1 mm to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, etc. In other words, the analysis channels can be located within the top 50%, 33%, 25%, 10%, or 5% of the substrate. The length of a horizontal analysis channel according to the present invention can be about 250 microns to about 10 mm. However, the length of the analysis channel may be 100 microns to 100 mm, e.g., 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, etc. In other words, the length of the analysis channel may be about 75% or less of the height, width, or length of the substrate/wafer, e.g., 50% or less, 33% or less, 25% or less, 10%, or 5% or less of the height, width, or length of the substrate/wafer.

In FIG4B , two imaging devices 450 are shown, such as machine vision cameras. In one embodiment, the cameras may be located above the chip and oriented orthogonal to the analysis channel. In another embodiment, the cameras may be located to the side of the chip and oriented orthogonal to the flow direction or diagonally to the flow direction (e.g., above the channel, below the channel, or at an angle to the side of the channel). In embodiments, the cameras may be positioned so that imaging is performed at any angle relative to the flow of one or more substances, such as orthogonal or 90 degrees relative to the flow of one or more substances, or, for example, 0 to 90 degrees, or 10 to 80 degrees, or 30 to 60 degrees, etc. In another embodiment, two or more cameras may be used to image cells or particles in the analysis channel. For example, the cameras may be above the chip and oriented orthogonal to the analysis channel. The second camera can be located on the side of the wafer and oriented orthogonally to the flow direction or diagonally to the flow direction (e.g., above the channel, below the channel, or at an angle to the side of the channel). As shown in FIG4B , one or more light sources 460 can be used to illuminate the analysis channel 440. These light sources can be located below the wafer and shining upward, on the side of the wafer and shining in the direction of flow or opposite to the flow direction, above the wafer and shining downward, or diagonally to the analysis channel.

Alternatively, a dichroic mirror 840 or other suitable optical element can be used to selectively redirect a specific wavelength range of light while allowing other light to pass through, as shown in Figure 8. This will allow the camera 810 to be placed in line with the analysis channel. As shown in Figures 8 and 9, several embodiments of this occur, including placing the optical force laser 830 and the camera 810 at the same or opposite ends of the analysis area. An illumination source 860 for the camera may also be required and can be oriented in a number of ways, such as those shown in Figures 8 and 9. The light source can be a broad spectrum source, such as one or more LEDs, or a narrow source such as a laser. The camera can be used as a single camera or as part of a multi-camera system used in combination with other viewpoints as described herein.

As also shown in FIG4A , a light source 480 (e.g., a laser) can be used to influence cell flow. The laser can be positioned in line with or opposite the cell flow. The laser can also be positioned and/or oriented orthogonally or diagonally to the cell flow.

Embodiments of the present invention include apparatus for particle analysis. (See, for example, Figures 4-9.) Embodiments of the present invention include at least one camera 450 for capturing images of particles or cells in a microfluidic channel (e.g., 440 ). In one embodiment, a laser or other optical force 480 is included, such as a collimated light source operable to generate at least one collimated light beam. The at least one collimated light beam includes at least one beam cross-section. Embodiments of the present invention include a substrate having a first channel 410 extending vertically in the substrate such that a first plane substantially traverses the first channel 410 along its length, and thereby a fluid sample is injected into the substrate/wafer from the bottom of the chip and forced upward by positive or negative pressure. An embodiment of the present invention includes a second channel 420 that is orthogonal to the first channel and is therefore arranged horizontally in the substrate, so that the second plane substantially crosses the second channel 420 along its length and the second plane is arranged orthogonal to the first plane. This second channel is in the horizontal direction of the chip. The second channel is directly or indirectly connected to the first channel. The second channel is directly or indirectly connected to a short, upwardly vertical third channel 430 , which brings the channel network closer to the top of the chip. The third channel is directly or indirectly connected to a fourth horizontal channel 440 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 an analysis channel. In one embodiment, the camera 450 is oriented orthogonally to the flow direction in the fourth channel. An embodiment of the present invention includes a concentrated particle flow nozzle operably connected to the first channel. In another aspect of the invention, the second passage undergoes a size change and passes through the nozzle before connecting to the third passage.

FIG4C shows another embodiment of a sample path for a microfluidic chip. As shown, the fluid first travels upward in the chip in a vertical direction through a first channel 410 , which then, in this example, directly or indirectly communicates with a second horizontal channel 420 at the top of the chip and, in this example, includes an analysis channel. According to this configuration, the analysis channel is located near the top of the chip, thereby improving imaging and analysis of substances passing through the microfluidic chip. In a preferred embodiment, the analysis channel is about 1 mm to about 2 mm from the top and sides of the chip. However, the distance of the analysis channel from the top of the chip can be 0.1 mm to 100 mm, for example, 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, etc. In other words, the analysis channel can be arranged within the top 50%, 33%, 25%, 10% or 5% of the substrate. The length of a horizontal analysis channel according to the present invention can be from about 250 microns to about 10 mm. However, the length of the analysis channel can be from 100 microns to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm, etc. In other words, the length of the analysis channel can be about 75% or less of the height, width or length of the substrate/wafer, such as 50% or less, 33% or less, 25% or less, 10% or 5% or less of the height, width or length of the substrate/wafer. In embodiments, the substrate can include one or more analysis channels, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 analysis channels.

As also shown in FIG4C , a light source 480 (e.g., a laser) can be used to influence the flow of a substance, such as a cell flow. The laser can be positioned in line with or opposite the cell flow. The laser can also be positioned and/or oriented orthogonally or diagonally relative to the cell flow.

In Figures 1 and 4, a fluid stream containing cells or particles passes vertically through a first channel. One or more substances (fluid, cells, and/or particles) enter through the bottom of the chip at the opening of the first channel and enter the substrate in a vertical direction. The length of the first vertical channel is between 100 microns and 100 mm, for example, 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, etc. The first channel is followed by a second orthogonal/horizontal channel, which is shorter than the first channel in a preferred embodiment. The length of the second channel can be 250 microns to 100 mm, for example, 0.25 mm to 0.5 mm, 0.5 mm to 0.75 mm, 0.75 mm to 1.0 mm, etc. The third channel runs perpendicular and parallel to the first channel. The length of the third channel can be 50 microns to 100 mm, for example, 0.05 mm to 0.1 mm, 0.1 mm to 0.15 mm, 0.15 mm to 0.2 mm, etc. The channels are arranged in direct or indirect operable communication to allow one or more substances to move through the multiple channels. The typical direction of fluid flow is indicated by the flow arrows in Figures 4A and 4C, but may be reversed.

In a preferred embodiment, the fourth channel comprises an analysis channel having a channel length of 250 microns to 100 mm, e.g., 0.25 mm to 0.5 mm, 0.5 mm to 0.75 mm, 0.75 mm to 1.0 mm, etc. In this embodiment, the fourth channel is the channel closest to the top of the wafer. The fourth channel, measured vertically, can be located between 100 microns and 2 mm, but can be as great as 100 mm, e.g., 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns, etc., from the top of the wafer. In other words, the fourth channel can be located within the top 50%, 33%, 25%, 10%, or 5% of the wafer. An imaging device (e.g., a camera) may be oriented orthogonally to the fourth channel and positioned approximately 100 microns to 2 mm from the fourth channel, but may be positioned up to 100 mm from the fourth channel, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns, etc.

In one embodiment, a laser or other light source is present along with a focusing lens element. Figure 4A illustrates the present invention, wherein a laser 480 is in operation, emitting a laser beam, and directing the beam through the focusing lens element into the fourth flow channel 440. Particles are aligned within the laser beam due to gradient forces that pull the particles toward the region of highest laser intensity. Laser scattering forces propel particles in the direction of laser beam propagation (e.g., from left to right in Figure 4A).

In another embodiment, in addition to the camera or image capture device oriented orthogonally to the fourth channel, a second camera or image capture device (see 450 ) is also oriented to one side of the analysis (or fourth) channel, and directed orthogonally or at any angle to the fourth channel; for example, as shown in FIG4B . Taking an image of each cell in an orthogonal view allows for the calculation of multiple cell properties, such as size and shape, in two dimensions, thereby increasing the amount of information that can be captured for each cell. This also allows for the calculation of bulk properties of the cell, including overall volume, shape, and can provide insight into cells that are asymmetric about an axis parallel to the direction of flow (e.g., the Z axis as depicted in FIG5 ). Using two or more cameras, orthogonal images can be combined using, for example, existing 3D reconstruction algorithms (e.g., diffraction theory methods or illumination rotation methods) to construct a basic 3D model of the cell 550. The 3D model and analysis of the 3D model will allow for more accurate analysis of the cell, such as cell size, shape, orientation, and other quantitative and qualitative measurements of the particles or cells in the fourth channel of the microfluidic chip.

In FIG5 , a portion of an analysis channel 540 is shown from two different planes (such as those that can be imaged from an imaging device (e.g., see FIG4 )). In the first plane 520 , a portion of a cell or particle 510 is imaged at a specific orientation. In the second plane 530 , another portion of the same cell or particle is imaged from a different viewing angle from another camera. This allows calculation of multiple cell properties, resulting in a data matrix for each cell for each camera and a 3D computational representation of the cell or particle 550 .

As different portions of the cell pass through the focal planes of the imaging device (e.g., 630 (XZ focal plane) and 640 (YZ focal plane)), different slices of the cell can be imaged as the cell is moved by, for example, fluid flow 620. This is shown in FIG6A. In FIG6A, portions of the cell or particle are imaged at multiple points in time and/or space, in different orientations, and from different viewing angles. In this example, as the cell moves and rotates through the focal planes, it appears as different sizes in successive images (e.g., four example images from each plane of two different cameras, as shown in FIG6A). Using 3D reconstruction algorithms, this allows for more complex 3D computational rendering 650 of the particle or cell. From this computational representation, certain properties of cells or particles can be extrapolated, such as cell size, volume, the location and size of the nucleus and other organelles, and the measurement or delineation of cell morphology. Furthermore, cell rotation can be measured as a change in torque based on optical forces due to changes in biophysical or biochemical properties, including but not limited to refractive index, birefringence, or cell shape or morphology.

FIG6B illustrates multi-plane imaging on a chip, where multiple images of the same cell are taken over time, for example, as the cell moves through the focal plane. In these cases, the view of the cell is referred to in the industry as a "slice" or "image slice." An image slice is actually the thickness of the optical plane being imaged. The thickness of the image plane or slice is determined, among other things, by the optical magnification of the imaging system. At higher magnifications, the working distance of the objective lens decreases, resulting in the need to bring the lens closer to the cell or particle to be imaged. In one embodiment, a laser or other optical force 670 can be used to influence the cell flow in the analysis channel. In a preferred embodiment, cells or particles can be purposefully coaxed into and out of the focal plane of the channel for imaging by moving the laser and/or camera, or by using hydrodynamic focusing to adjust the flow or position. For example, the laser source can be moved a distance 660 using, for example, a piezoelectric actuator or a linear electro-optical mechanical stage. This can be implemented on a per-cell or per-cluster basis. The hydrodynamic focusing of the cells can be varied to affect the initial position and trajectory of the cells. For example, particles can be aligned or oriented in the focal plane within the laser beam due to gradient forces that pull the particles toward the area of highest laser intensity. Laser scattering forces push the particles in the direction of laser beam propagation. See Figure 6B. Moving the laser on the X-axis in this case allows for imaging features in different parts of the cell, as illustrated by disk 680. This could represent, for example, the nucleus, organelles, inclusions, or other features of the cell or particle. Due to the gradient force, the laser pulls the cell toward its center. It is further contemplated that using two or more cameras would increase detail and accuracy.

The embodiment of the present invention shown in FIG7 is a static mode, in which a particle or cell 710 is held at a specified differential holding position by balancing an optical force 730 and a fluid force 735. The optical force can be applied, for example, by a laser or collimated light source. Images can be captured in multiple planes, such as those shown and described in FIG5 and 6A-B. A flow sensor is used to measure the flow velocity at which each particle stops in a flow at a given laser power. Because the optical and fluid forces are balanced, the fluid drag (i.e., resulting from the flow velocity and channel dimensions) is equal to the optical force. In this way, the properties of each cell can be measured sequentially. While not a high-throughput measurement system, this embodiment of the present invention allows for close observation and imaging of captured cells and the dynamic changes in optical force caused by biochemical or biological changes within the cells. A reagent stream containing chemicals, biochemicals, cells, or other standard biological agents can be introduced into the flow channel to interact with the captured cells. By measuring changes in optical force over the course of an experiment for a single cell or multiple cells, these dynamic processes can be quantitatively monitored.

In one embodiment, a camera or other imaging device is oriented and/or focused within or opposite the flow of the analysis channel so that it is in line and parallel to the flow. (See, for example, Figures 8 and 9.) Figure 8 shows a camera 810 aligned with an analysis channel 820 and a laser or collimated light source 830. A dichroic mirror or similar device 840 reflects laser light 835 away from the camera to prevent damage, but passes light 865 generated by an illumination source 860 to allow imaging. The camera is oriented parallel to the fluid flow 870 so that, in one embodiment, cells or particles 880 move away from the camera. The illumination source is oriented orthogonal to the channel and the laser. A second dichroic mirror 845, which passes the laser light and reflects the illumination light, is used to guide the illumination light and the laser through the channel. An alternative embodiment of this configuration switches the positions of the laser and illumination source so that the laser is orthogonal to the channel and the illumination source is parallel to the channel. The second dichroic mirror will still guide the laser and visible light through the channel.

An alternative embodiment is shown in Figure 9. In this scenario, a camera or imaging device 910 is oriented so that cells or particles 980 are traveling toward the camera in fluid flow 970. Thus, the camera and laser 930 are on the same side of the channel, while the illumination source 960 is at the opposite end of the channel. Two dichroic mirrors 940 and 945 are used to direct laser light 935 and illumination light 965 into the channel, direct the illumination light toward the camera, and deflect the laser light away from the illumination source. This alternative embodiment of the configuration switches the positions of the laser and camera so that the laser is orthogonal to the channel and the illumination source is parallel to the channel. A second dichroic mirror 945 then directs the laser light through the channel and the illumination light toward the camera.

Optionally, embodiments of the present invention further include at least one optical element, interposed between the optical force source and the fourth channel, operable to generate a standard TEM00 mode beam, a standard TEM01 mode beam, a standard TEM10 mode beam, a standard Hermite-Gaussian beam mode, a standard Laguerre-Gaussian beam mode, a Bessel beam, or a standard multimode beam. Optionally, the at least one optical element includes a standard cylindrical lens, a standard rotating triangular prism, a standard concave mirror, a standard annular mirror, a standard spatial light modulator, a standard acousto-optic modulator, a standard piezoelectric mirror array, a diffraction optical element, a standard quarter-wave plate, and/or a standard half-wave plate. The optical force source may include a standard circularly polarized beam, a standard linearly polarized beam, or a standard elliptically polarized beam, as appropriate.

Optionally, a device is embodied, comprising a microfluidic channel, a laser light source focused into the microfluidic channel by an optical device, and an electric field source operably connected to the microfluidic channel via electrodes; particles in a liquid are caused to flow through the microfluidic channel; and the laser light and electric field are manipulated to act together on the particles in the microfluidic channel to separate the particles based on size, shape, refractive index, charge, charge distribution, charge mobility, capacitance, and/or deformability. In another embodiment, a device includes a microfluidic channel configured to supply a dielectrophoresis (DEP) field to the interior of the channel via (1) an electrode system or (2) an insulator DEP system; and a laser light source focused into the microfluidic channel by an optical device; allowing a plurality of particles in a liquid to flow into the microfluidic channel; and jointly manipulating the laser light and field on the particles in the microfluidic to capture the particles or change their velocity, wherein the DEP field is linear or nonlinear. Another possible embodiment of the device includes a microfluidic channel comprising an inlet and a plurality of outlets, and a laser light source focused by an optical device through the microfluidic channel at a critical angle that matches the flow velocity in the microfluidic channel to generate an optical force on particles while maximizing the residence time of selected particles in the laser light, thereby separating the particles into the plurality of outlets. The laser light is operable to exert a force on particles flowing through the microfluidic channel, thereby separating the particles into the plurality of outlets.

Optionally, embodiments of the present invention further include at least one particle interrogation unit in communication with one or more of the channels (e.g., the analysis channel and, in particular, the fourth channel). The particle interrogation unit includes a standard illuminator, standard optical components, and standard sensors. Optionally, at least one particle interrogation unit includes a standard bright field angiography device, a standard light scattering detector, a standard single-wavelength fluorescence detector, a standard spectral fluorescence detector, a standard CCD camera, a standard CMOS camera, a standard photodiode, a standard photomultiplier tube, a standard photodiode array, a standard chemiluminescence detector, a standard bioluminescence detector, and/or a standard Raman spectroscopy detector.

At least one particle interrogation unit connected to the fourth channel includes a laser force-based device or a device that promotes cell disease identification, selection and sorting. In one embodiment, the unit uses inherent differences in optical pressure as a means to separate and characterize particles, wherein the differences are caused by changes in particle size, shape, refractive index or morphology. In one embodiment, a near-infrared laser beam applies a physical force to the cell, which is then measured. The optical force generated by the radiation pressure, when balanced with the fluid drag on the particle, causes changes in the particle velocity, which can be used to identify different particles or groups of particles based on changes in the inherent differences. The balance of fluid and optical forces can also be used to change the relative position of particles to each other based on the inherent properties of the particles, thereby causing physical separation. Another embodiment of an interrogation unit includes a device for particle analysis and/or separation, such as at least one collimated light source operable to generate at least one collimated light beam. The at least one collimated light beam includes at least one beam cross-section.

Embodiments of the present invention involve the combination of several of the above-mentioned design elements discussed above in a single device. Embodiments also include methods of using such devices. An example of such a single device is illustrated in Figure 1. The illustrated embodiment of the present invention is a 5-layer structure in which all 5 layers are bonded to each other to produce a solid microfluidic chip, but the chip can be a structure opposite the bonding layer. The chip can be constructed using many standard materials, including but not limited to fused silica, crown glass, borosilicate glass, soda lime glass, sapphire glass, cycloolefin polymer (COP), poly(dimethyl)siloxane (PDMS), OSTE, polystyrene, poly(methyl)methacrylate, polycarbonate, other plastics or polymers. This chip allows for sample input, hydrodynamic focusing, optical interrogation, imaging, analysis, sample removal, and clear optical pathways for laser light to enter and exit the region. Embodiments of the chip can also be 3D-printed, molded, or in other configurations.

Optionally, the at least one particle type comprises a plurality of particle types. Each of the plurality of particle types comprises a respective intrinsic property and a respective induced property. Optionally, the intrinsic property comprises size, shape, refractive index, morphology, intrinsic fluorescence, and/or aspect ratio. Optionally, the induced property comprises deformation, angular orientation, rotation, rotation rate, antibody-labeled fluorescence, aptamer-labeled fluorescence, DNA-labeled fluorescence, dye-labeled fluorescence, differential retention metric, and/or force metric gradient. This method embodiment further comprises identifying and separating the plurality of particles according to respective particle types based on at least one of the intrinsic property and the induced property. Optionally, this method embodiment comprises a step comprising interrogating or manipulating sample flow. Optionally, interrogating the sample flow includes determining at least one of an intrinsic property and an induced property of the particle type, and measuring the particle velocity of a plurality of particles. The measurement of at least one of the intrinsic properties can be used in a variety of applications, including but not limited to: determining the viral infectivity of a cell sample (the number of functional infectious viral particles present in a particular cell population, similar to a plaque assay or an end-point dilution assay) for virus quantification, process development and monitoring, sample release analysis, adventitious agent testing, clinical diagnostics, biomarker discovery; measuring the productivity of cells in terms of antibodies or proteins for process development and monitoring; determining the efficacy, quality or activation state of cells generated as cell-based therapies, including CAR T and other oncology applications and stem cells; determining the effects of chemicals, bacteria, viruses, antibacterial agents, or antiviral agents on specific cell populations; and determining the disease state or potential of research or clinical cell samples. Optionally, the optical source includes at least one beam axis, and the sample flow includes a sample flow axis. The steps of determining at least one of an intrinsic property and an induced property of the particle type and measuring the particle velocity of the plurality of particles together include offsetting the beam axis from the sample flow axis. Optionally, the step of determining at least one of an intrinsic property and an induced property of the particle type and the step of measuring the particle velocity of the plurality of particles together include calculating the slope and trajectory of the plurality of particles deviating from the sample flow axis toward at least one beam axis.

Those skilled in the art will recognize that the disclosed features may be used individually, in any combination, or omitted, depending on the requirements and specifications of a given application or design. When an embodiment is referred to as "comprising" certain features, it should be understood that the embodiment may alternatively "consist of" or "consist essentially of" any one or more of the 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.

It should be noted that when a range of values is provided in this specification, each value between the upper and lower limits of the range is specifically disclosed. The upper and lower limits of such smaller ranges may also be independently included or excluded in the range. Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural referents. This specification and examples are intended to be exemplary or explanatory in nature, and variations that do not depart from the essence of the invention are within the scope of the invention. In addition, all references cited in this disclosure are individually incorporated herein by reference in their entirety and are therefore intended to provide effective means to supplement the enabling disclosure of the invention and to provide background information to detail the level of skill in the art.

100: Microfluidic chip 110: Air line 120: Manifold 130: Sample vial 140: Inlet line 145: Outlet line 150: Channel

Claims (16)

A method for analyzing one or more cells or particles, comprising: injecting the one or more cells or particles into a substrate, the substrate comprising a plurality of channels configured to transport the one or more cells or particles, and a collimated light source capable of interacting with the one or more cells or particles, wherein the plurality of channels comprises: a first channel disposed vertically upward relative to gravity within the substrate, a second channel operatively connected to the first channel and disposed horizontally within the substrate, a third channel operatively connected to the second channel and disposed vertically upward relative to gravity within the substrate, and a second channel operatively connected to the third channel and disposed horizontally within the substrate. a fourth channel disposed in the substrate, wherein the first, second, third, and fourth channels are arranged in such a manner as to provide a path for the one or more cells or particles to move through the substrate from the first channel to the second channel to the third channel to the fourth channel, so that the one or more cells or particles are injected into the first channel disposed vertically in the substrate through a bottom horizontal flat surface having an opening to the first channel to maintain directional and volumetric continuity with the first channel; analyzing the one or more cells or particles by optical force and fluid dynamics-based measurements; and thereby analyzing the one or more cells or particles. The method of claim 1, wherein the analysis comprises quantitative measurement of changes in the one or more cells or particles and provides a predictive or prescriptive analysis. The method of claim 1, wherein the bottom horizontal planar surface has at least one shorter length from one edge to the other than at least one length from one edge to the other on the vertical planar surface of the substrate to maintain directional and volumetric continuity with the first channel. The method of claim 1, wherein the one or more cells or particles are injected into the first channel vertically arranged in the substrate through a bottom horizontal flat surface having an opening to the first channel, wherein the bottom horizontal flat surface has a smaller or equal surface area in the vertical direction than the surface area of the vertical flat surface of the substrate to maintain directional and volume continuity with the first channel. The method of claim 1, wherein the first channel comprises an opening disposed on an outer surface of the substrate and arranged in a manner to provide a path for the one or more cells or particles to vertically enter the substrate and move vertically within the first channel. The method of claim 1, wherein the collimated light source is oriented to interact with the one or more cells or particles in the fourth channel. The method of claim 1, wherein the collimated light source is oriented to propagate in a direction of movement of the one or more cells or particles in the fourth channel, opposite to the direction of movement, orthogonal to the direction of movement, or diagonally to the movement. The method of claim 1, wherein the fourth channel allows imaging and analysis of the one or more cells or particles by one or more imaging devices from multiple focal planes, angles and/or orientations during the movement of the one or more cells or particles. The method of claim 1, further comprising one or more electrical, optical, and/or fluid forces to move the one or more cells or particles in the one or more channels. The method of claim 1, wherein the plurality of channels includes a fifth channel that is divided into two or more channels or wells for sorting the one or more cells or particles. The method of claim 1, further comprising an imaging device selected from at least one of the following: a bright field angiogram, a light scattering detector, a single wavelength fluorescence detector, a spectral fluorescence 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 chemiluminescence detector, a bioluminescence detector, a standard Raman spectroscopy detection system, surface enhanced Raman spectroscopy (SERS), coherent antistokes Raman spectroscopy (CARS) and/or coherent Stokes Raman spectroscopy (CSRS). The method of claim 1, wherein the analyzing of the one or more particles comprises determining the efficacy, quality, or activation state of cells generated for cell-based therapy, and/or determining the effect of a chemical, bacteria, virus, antibacterial agent, or antiviral agent on a specific cell population. The method of claim 12, wherein the cells produced by the cell-based therapy are CAR T cells or stem cells. The method of claim 1, wherein the analyzing of the one or more cells or particles comprises determining the effect of a chemical agent, a biological agent, a biochemical agent, a physical force, temperature, bacteria, a virus, an antibacterial agent, or an antiviral agent on the one or more cells or particles. The method of claim 1, further comprising determining a disease state of the one or more cells or particles. The method of claim 1, wherein the analyzing of the one or more cells or particles comprises determining one or more of: morphology, cell size, cell shape, refractive index, birefringence, intrinsic fluorescence, aspect ratio, deformation, angular orientation, rotation, rotation rate, antibody label fluorescence, aptamer label fluorescence, DNA label fluorescence, dye label fluorescence, differential retention metric, and/or intensity metric gradient.