CN111819153B - Microfluidic chip devices for photomechanical measurements and cell imaging using microfluidic chip configuration and dynamics - Google Patents

Abstract

A microfluidic chip arrangement in which injection occurs in a vertically upward direction, with a fluid reservoir located below the chip to minimize particle settling at and before the analysis portion of the chip channel. The input and fluid flow upward through the bottom of the chip, in one aspect, uses a manifold, which avoids orthogonal reorientation of the fluid dynamics. The contents of the vial are located below the chip and pumped directly vertically upward into the first channel of the chip. The long channel extends from the bottom of the chip to near the top of the chip where it turns a short horizontal bend. The fluid is pumped up to the horizontal analysis section, which is the highest channel/fluidic point in the chip, thus near the top of the chip, which makes the imaging clearer. The laser may also suspend cells or particles in this channel during analysis.

Description

Microfluidic chip devices for photomechanical measurements and cell imaging using microfluidic chip configuration and dynamics

Technical Field

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

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

The implementation of the present invention requires changes to existing microfluidic chip designs. For example, in order to align the vials vertically with the chip, a different chip interface than the prior art needs to be established. Specifically, the present invention allows inputs and fluids to pass from the bottom of the chip upward, using a manifold in one aspect, which avoids horizontal redirection of fluids/fluid dynamics, rather than having input tubes engage the chip in an orthogonal manner via ports (typical practice in microfluidic chip lab systems), the ports are attached to the largest face of the chip, and then the fluid is pumped first across the chip and then over the chip.

According to the present invention, the contents of the vial are located below the chip and are pumped directly vertically upward into the first channel of the chip. The 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 so short that it almost offsets any effects of cell sedimentation caused by gravity and zero flow velocity at the wall. Then, contrary to the prior art, the fluid is pumped upward to the analysis section. Therefore, the horizontal analysis section is the highest channel/fluidic point in the chip, so the horizontal analysis section is close to the top of the chip, which makes the chip material (such as glass) between the microscope/camera and the sample less than in the prior art, and the imaging is clearer. During the analysis process, the laser also suspends the cells in this channel to prevent them from settling.

Background Art

Related technical notes

According to the prior art, a microfluidic chip vial containing cells or particles to be separated and/or analyzed is placed on the side and pumped horizontally into a channel in the microfluidic chip. First, the contents of the vial (e.g., particles or cells) are pumped vertically upward, then turn a U-turn and move downward, and then are pumped horizontally into the chip (see, e.g., U.S. Pat. No. 9,594,071).

The horizontal connection to the chip, coupled with the dead volume in the connection (empty space in the fluidic connection, which is unavoidable to some extent), causes significant additional sedimentation under the action of gravity. This configuration also necessitates the relatively large diameter channel required for the connection, which, in addition to the dead volume, also creates a relatively low velocity area, further exacerbating the problem of particle sedimentation. The chip according to the present invention not only eliminates the need for a large horizontal input channel, but also eliminates the need for a more abrupt change from the large horizontal input channel to the relatively thin upwelling flow in the first vertical chip channel. This configuration eliminates horizontal sedimentation and unnecessary changes in direction that cause sedimentation. Having cells enter the bottom edge of the chip also solves the problem of sedimentation in the dead volume by orienting it perpendicular to gravity so that cells or particles cannot settle at the bottom of the horizontal channel, but instead they are constantly guided upward by the flow. This is not intuitive, and a lot of experimentation was required to understand the problem before designing the solution currently presented. In contrast to the present invention, currently available microfluidic devices include custom or commercially available connections on polished surfaces and larger areas of glass, which generally force any particles such as cells contained in the sample stream to turn and travel horizontally immediately upon entering the chip.

In addition, in the prior art, cells or particles make several horizontal moves on the microfluidic chip before reaching the analysis channel, which causes sedimentation. When the contents of the vial enter the chip and the channel in the chip, the contents are pumped horizontally relative to the vertical chip channel. The channel then flows upward and turns a long horizontal bend, at which time the cells tend to settle at the bottom of the channel due to gravity, and at the same time, due to laminar flow conditions, the cells tend to have lower speeds at the walls. In essence, due to the parabolic velocity profile, the flow rate is the largest in the middle of the channel and drops to zero at or near the channel wall. After the first horizontal chip channel, the fluid turns downward before the analysis channel, where particle imaging or separation is performed. Due to this configuration, there is a relatively long distance between the microscope/camera and the analysis channel. In this typical prior art configuration, the particles are forced downward and eventually leave the bottom of the chip.

Furthermore, due to limitations in the prior art, multiple horizontal moves are required so that cells settle in multiple places in the channel. This in turn results in a degradation of the image quality because of the need to image through additional material at the edge of the chip. The prior art channel of the chip has to pump vertically upward, then horizontally, and then zigzag to get enough cells or particles to suspend, then down and off the chip. The present invention avoids the zigzag channel.

There are also prior art for 3D image rendering of cells or particles in fluids. For example, M. Habaza, M. Kirschbaum, C. Gent-Marschner, G. Dadikman, I. Banea, R. Korenstein, C. Dushel, N.T. Shaked taught in Advanced Science (2017, 4, 1600205) to capture cells, rotate the cells at high speed, and use interferometry to measure the refractive index distribution in the cells. Interferometry has also been used to analyze cells in microfluidic channels (see, for example, Y. Song et al., Physical Review Applied, February 27, 2014; 1:014002). However, the present invention requires that multiple images be taken for cells or particles as they travel in a fluid stream and pass through the focal plane of (one or more) imaging devices, thereby eliminating the need to capture cells in order to obtain 3D images. Other techniques have been taught, such as using mechanical displacement platforms to move cells or particles (e.g., N. Lu et al., Optics Express, 2008 Sep 29;16(20):16240-6), but none of these techniques use brightfield imaging as described in the present invention, nor do they utilize fluid flow to provide cell positioning relative to the image focal plane.

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

Summary of the invention

The present invention is directed to a microfluidic chip in which injection is performed and the sample vial is located below the chip in order to minimize particle settling. Thus, the contents of the vial are located below the chip and are pumped directly vertically upward into the channel of the chip. The 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 too short to make any effect on cell settling, which is caused by zero flow rate at the channel wall, insignificant. Then, in contrast to the prior art, the sample is pumped upward to the analysis section. Therefore, the horizontal analysis section is the highest channel/fluidic point in the chip, so the horizontal analysis section is close to the top of the chip, which allows less glass between the microscope/camera than in the prior art, and clearer imaging. The distance between the analysis channel and the top of the chip can be between 100 microns and 2 mm, but can also be as long as 100 mm, such as from 100 microns to 200 microns, from 200 microns to 300 microns, from 300 microns to 400 microns, etc. In one embodiment, after passing through the analysis portion of the chip, the sample (eg, fluid, cells, and/or particles) is pumped down to the bottom of the chip and forced to move outward.

The present invention is further directed to a microfluidic chip in which horizontal movement is minimized, especially when fluid enters the chip channels. Prior art chips contain about 13 mm horizontal channels (non-analytical portion), of which about 2 mm are injection ports with a much larger diameter, exacerbating the sedimentation caused by low speed. The chip described in the present invention has a (non-analytical) horizontal channel of about 0.2 to 3.0 mm in a preferred embodiment, although the length of the horizontal channel can be between 0.01 and 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 depends on the channel system of the invention, which is different from the prior art in terms of order of magnitude, which can improve flow and eliminate cell/particle sedimentation.

Another aspect of the present invention is directed to a microfluidic chip in which imaging and analysis are performed based on or near the corners of the chip, because this makes the glass between the camera and the analysis channel less and the distance shorter to improve the imaging from multiple viewpoints. This also enables the use of higher numerical objective lenses to improve fine imaging by increasing the order of magnitude. This design improvement reduces image distortion caused by glass (or other substances constituting the chip, such as plastic or any transparent or translucent material) and distance (e.g., due to defects in the glass). The distance between the imaging device and the analysis channel can be between 100 microns and 2 mm, but can also be as long 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 addition, the present invention is directed to a microfluidic classification chip, which is separated from the downstream of the analysis channel, allowing the use of pressure and/or laser (or other optical force) to activate the classification function simultaneously or sequentially. On the one hand, in order to perform the classification function, the flow will continue from the analysis channel. For example, the particles will be directed into the vertical channel and then directed to the horizontal classification channel. In one embodiment, optical force and/or pressure are applied in the flow direction of the classification channel to push the particles through the channel. Particles that are not directly acted upon by optical force will be diverted to alternative channels due to, for example, gravity, electrodynamic force, magnetic force, laminar flow line, streamline, reduced flow rate, orthogonal optical force, or vacuum (vacuum is applied to suck particles into alternative channels). On the other hand, related to the analysis channel after classification, the present invention allows the optical force to be guided from the back side of the chip (laser or optical force is oriented in the same flow direction), and allows the decomposition of the main laser in some aspects. In some embodiments, optical force and/or pressure can be applied in the opposite direction of the material moving through the channel, such as countercurrent, or can be applied in the same direction of the material moving in the channel, such as downstream. Cell or particle sorting can be performed on a single device or separate chips. For example, in Figure 1B, the fifth channel before the outlet pipe 145 contains one or more branches to achieve single or multiple sorting areas.

In another aspect of the invention, a manifold is connected to a vial in a manner such that tubing passes through the manifold and is connected to a vial or other container on another side that is in contact with a substance (e.g., a fluid) in the vial. The manifold allows the vial to be connected to the microfluidic chip but stored below the chip, and/or the manifold allows the contents of the vial to be injected from the bottom of the chip, alleviating several problems faced by the prior art, such as cell or particle sedimentation where the vial or tubing from the vial is connected to or communicates with the microfluidic chip.

In another aspect, the present invention is directed to a microfluidic chip holder comprising a structure for directing a light source, the structure comprising an integrated prismatic cavity, the integrated prismatic cavity being equipped with a prism so that light is emitted at an angle relative to the chip, which is a preferred method for illuminating a confined geometric configuration. In some embodiments, the light source includes, but is not limited to, a fiber optic or a collimated or focused light source. In particular, the light source is precisely aligned or directed to, or directed into, an analysis channel.

In another aspect of the invention, the device includes a second imaging device, which is oriented orthogonal to the first camera and the channel view. The reasons for setting the second camera vary. On the one hand, the reason for setting the second imaging device is to help analyze the visual alignment of the laser or light force in the channel. On the other hand, using the method described in the present invention, data from the first camera can be recorded. With the second camera, the data can be combined with the data from the first camera, bringing additional data that can be used to more accurately infer the cell position, size, shape, volume, etc. This additional information about the same cell (or particle) improves accuracy and expands the scope of measurement and analysis. On the other hand, the second camera combined with the first camera can achieve a three-dimensional reconstruction of the cell or particle, or a group of cells or particles, which is imaged by an orthogonal camera and a camera in the flow or a camera positioned toward the side of the chip. Using the algorithm described in the present invention or other algorithms, including reversing or slowing the flow and taking one or more images for a specific cell or particle, or a group of cells or particles, the present invention allows the analysis and processing of multiple images, thereby allowing the determination of features/characteristics/quantitative measurements, such as cell volume, cell shape, nuclear position, nuclear volume, organelle or inclusion body position, etc. In another aspect of the invention, the camera is oriented to image on an axis in the direction of flow.

In one aspect, the present invention does not require a serpentine or zigzag channel to maintain proper suspension of particles, which is preferred in the prior art. Due to the vertical nature of the fluid and particles or cells injected into the chip, the vertically integrated pumped particles can directly go up through the channel to the first horizontal channel (referred to as the second channel in the present invention), thereby avoiding the zigzag channel.

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

3A and 3B are schematic diagrams depicting angles and aspects of a chip holder according to the present invention.

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

FIG. 5 is a schematic diagram showing on-chip multi-plane imaging and how it is used to render 3D images and information.

6A and 6B are schematic diagrams showing on-chip multi-planar imaging in a fluid flow and how it can be used to render a 3D image.

FIG. 7 is a schematic diagram showing possible ways to capture cells and/or possible ways to equilibrate cells within the analysis portion of the chip, as well as possible ways to image cells from multiple angles.

FIG8 is a schematic diagram showing how the camera and illumination source can be placed to align with the laser and flow direction so that the particles are directed away from the camera.

FIG. 9 is a schematic diagram showing how the camera and illumination source can be placed in alignment with the laser and flow direction so that the particles move toward the camera.

DETAILED DESCRIPTION

The present invention has been described with reference to specific embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations may be made in the practice of the present invention without departing from the scope or spirit of the present invention. Those skilled in the art will recognize that these features may be used alone or in any combination based on the requirements and specifications of a given application or design. Embodiments containing various features may also consist of or consist essentially of these various features. Other embodiments of the present invention will be apparent to those skilled in the art upon consideration of the specification and practice of the present invention. The description of the present invention provided is merely exemplary or explanatory in nature, and therefore variations that do not depart from the essence of the present invention are intended to be included within the scope of the present 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 its application in the component structure and configuration details set forth in the following description or shown in the following drawings. The present invention can have other embodiments or can be practiced or implemented in a variety of ways. In addition, it should be understood that the words and terms used in the present invention are for illustration only and are not limiting.

Turning now to FIG. 1A , FIG. 1A shows a global view of the device taught by the present invention. Sample vials 130 are located below the microfluidic chip 100 (also commonly referred to as substrates in the present invention), and the number or size of vials is not limited. The vials are configured to hold any type of sample that can be moved by the device, such as one or more substances, including but not limited to one or more fluids, liquids, gases, plasma, serum, blood, cells, platelets, particles, etc., or a combination thereof. In the context of this specification, the term fluid or sample can be used to refer to such one or more substances. The vials are connected directly or indirectly, such as indirectly using air pipes 110 to be operably connected to the lower side of the chip. As further shown in FIG. 1A , the vials can also be connected through a manifold 120. FIG. 1B shows a preferred embodiment of the microfluidic chip 100, in which substances, fluids, particles and/or cells are injected into an outer surface of the microfluidic chip, such as an edge (e.g., the XZ plane in FIG. 1B ). FIG. 1B shows injection along one of the shared surfaces (e.g., the XZ plane or the XY plane) with the 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 have minimal deviation after entering the channel on the chip (e.g., no right turn is required, which is usually required in the prior art).

Fig. 1A shows that manifold 120 connects one or more vials 130 to microfluidic chip 100, so that material, fluid, particle and/or cell can be vertically injected or pumped into microfluidic chip upward.This manifold allows material to be injected from the bottom of the chip, while also placing electronic equipment, flow sensor and pipe fittings (liquid and air) in a way that minimizes cell sedimentation, thereby optimizing throughput.Air pipe fittings provide pressure or vacuum, and when sealed, provide a closed system within the scope of manifold device.In one embodiment, there is a distance between (one or more) vials and manifold, indicating that there is no seal, and the pressure of internal volume is atmospheric pressure.This allows pumping material from a container open to the atmosphere or pumping material to such container (vacuum required for pumping from an open container).Manifold places electronic equipment, flow sensor and pipe fittings (liquid and air) in a way that minimizes cell sedimentation, thereby optimizing throughput.

By injecting the contents from the bottom of the chip, the present invention minimizes horizontal movement in the inlet tubing 140 and the outlet tubing 145, which causes problems such as sedimentation of particles or cells in (one or more) channels 150. In this example, the outlet tubing deviates from the inlet tubing in the Z and X dimensions (Figure 1B). In some embodiments, the outlet tubing deviates, does not deviate, is straight, aligned or angled relative to the inlet tubing. This configuration avoids the need for a winding or zigzag vertical channel because the current configuration solves the sedimentation problems that occur in the prior art, such as when a fluid combined with cells and/or particles is injected or pumped into the chip in a horizontal direction from the side and then must change direction and fluid dynamics to be pushed upward. The manifold and injection from the bottom of the chip also allow additional components, parts, mechanical devices or hardware to be placed below the chip (see Figure 1A).

The working principle of manifold 120 is to regulate the air pressure above the contents of the vial and provide the correct geometry for the flow sensor and electronics. Pipes such as fluid or air pipes pass through the manifold and are connected to the vials on the other side. Pressurized air passes through one side of the manifold, forming 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 changed. The pressurized area is both in (one or more) vials 130 and in the manifold 120. On the other hand, the vial does not require any air connection because it is open to the ambient atmosphere. This allows sampling of a wider variety of containers and sources. In this embodiment, a vacuum is applied to one or more other vials, thereby creating a pressure difference to drive the fluid flow from the open container.

In one embodiment, pressure-based sample injection is used. The vial is filled with the sample in the fluid and sealed with a lid or a tube connected to the chip. Before being attached to the lid, the vial can be open to the air or sealed with a diaphragm or other airtight device. In one aspect, the lid may contain two connectors, one for fluids such as gas and the other for liquid. Optionally, the method embodiment also includes providing a sample inlet line tip in communication with the sample inlet line, the sample inlet line tip being in communication with the first channel.

In another embodiment, vacuum-based sample injection is used. The vial is filled with the sample in the fluid and sealed with a lid or a tube connected to the chip. Before being attached to the lid, the vial can be open to the air or sealed with a diaphragm. In one aspect, the lid may contain two connectors, one for fluids such as gas and the other for liquid. Optionally, fluid can be sucked out of the vial open to the atmosphere by applying vacuum pressure to one or more other vials. Optionally, the method embodiment also includes providing a sample inlet line tip connected to the sample inlet line, and the sample inlet line tip is connected to the first channel.

2A-2B and 3A-3B show a chip holder 200, 300. The chip holder includes a structure to guide 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 to which a prism can be mounted. The light source is guided or arranged to the desired location by an integrated structure or channel 240 in the chip holder. The built-in space for the fiber optic light source allows illumination, such as an illumination cone 250, even in a restricted geometric environment (e.g., on a microfluidic chip 230, 330 or a channel in a microfluidic chip). In a preferred aspect, the light source is particularly precisely aligned, directed, or focused on an analysis channel 260. In a preferred embodiment, the chip holder includes built-in space for prisms 220, 320 and fiber optic light sources 210, allowing illumination in a restricted geometric configuration. The chip also includes a hole or opening 350 at the bottom of the holder to precisely arrange the fluidic tubing described in the present invention. In one embodiment, the adjustable screw is integrated with the threaded hole 360 on one or more faces for proper positioning.

Fig. 4A is a preferred embodiment of the microfluidic chip 400 described in the present invention. As shown in the figure, the fluid first travels vertically upward through the first channel 410, and then communicates with the second horizontal channel 420. As shown in Fig. 4A, another vertical channel 430 brings the fluid closer to the top of the chip, at which point the fourth channel 440 is horizontal and includes an analysis channel. These channels are operably connected to each other to allow the sample to move through the system from one channel to another. In some embodiments, the sample can flow from the first channel to the second channel, then to the third channel, and then to the fourth channel, or flow in reverse, or a combination of the two. A pump and/or a vacuum device can be provided to provide positive pressure and/or negative pressure at the opening and/or outlet of the channel so that the material can move through the channel. The analysis channel is close to one or more outer surfaces of the substrate, such as the face, edge or side of the chip. For example, the analysis channel according to this configuration is close to the top and side of the chip, thereby improving the imaging and analysis of the material passing through the microfluidic chip. In a preferred embodiment, the distance from the analysis channel to the top and side of the chip is between about 1mm and about 2mm. However, the distance from the analysis channel to the top of the chip can be between 0.1 mm and 100 mm, such as from 0.1 mm to 0.2 mm, from 0.2 mm to 0.3 mm, from 0.3 mm to 0.4 mm, and so on. In other words, the analysis channel may be arranged within the top 50%, 33%, 25%, 10% or 5% of the substrate. The length of the horizontal analysis channel according to the present invention can be between about 250 microns and about 10 mm. However, the length of the analysis channel can be between 100 microns and 100 mm, such as from 0.1 mm to 0.2 mm, from 0.2 mm to 0.3 mm, from 0.3 mm to 0.4 mm, and so on. In other words, the length of the analysis channel can be about 75% or less of the height, width or length of the substrate/chip, 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/chip.

Two imaging devices 450, such as machine vision cameras, are shown in FIG. 4B . In one embodiment, the camera can be located above the chip and oriented to be orthogonal to the analysis channel. In another embodiment, the camera can be located on the side of the chip and oriented to be orthogonal to the flow direction or diagonal to the flow direction (e.g., above the channel, below the channel, or at an angle to the channel side). In some embodiments, the camera can be placed in a position where imaging can be performed at any angle to one or more material flows, such as orthogonal to one or more material flows or 90 degrees, or such as from 0 to 90 degrees, or from 10 to 80 degrees, or from 30 to 60 degrees, etc. In another embodiment, two or more cameras can be used to shoot cells or particles in the analysis channel. For example, the camera can be above the chip and oriented to be orthogonal to the analysis channel. The second camera can be located on the side of the chip and oriented to be orthogonal to the flow direction or diagonal to the flow direction (e.g., above the channel, below the channel, or at an angle to the channel side). As shown in FIG. 4B , one or more light sources 460 may be used to illuminate the analysis channel 440 , and such light sources may be located below the chip and illuminate upward, to the side of the chip, and in the direction of flow or against the direction of flow, or above the chip and illuminate downward, diagonally toward the analysis channel.

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

FIG4A also shows that a light source 480 (e.g., a laser) can be used to affect the cell flow. The laser can be placed in alignment with the cell flow or can be placed opposite to the cell flow. The laser can also be placed and/or oriented to be orthogonal or diagonal to the cell flow.

Embodiments of the present invention include devices for particle analysis. (See, e.g., FIG. 4A, FIG. 4B, FIG. 4C, FIG. 5, FIG. 6A, FIG. 6B, FIG. 7-9) 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 parallel light source operable to generate at least one parallel light source beam. At least one parallel light source beam includes at least one beam cross section. Embodiments of the present invention include a substrate with a first channel 410 extending in a vertical direction in the substrate so that a first plane substantially crosses the first channel 410 along its length, whereby a fluid sample is injected into the substrate/chip from the bottom of the chip and is forced upward under positive or negative pressure. Embodiments of the present invention include a second channel 420 orthogonal to the first channel, so that the second channel 420 is horizontally arranged in the substrate so that a second plane substantially crosses the second channel 420 along its length, and the second plane is arranged orthogonally to the first plane. The 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 vertically upward 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 one embodiment, the fourth channel is an analysis channel. In one aspect, the camera 450 is oriented to be orthogonal to the flow direction in the fourth channel. An embodiment of the present invention includes a focused particle jet nozzle operably connected to the first channel. In another aspect of the present invention, the second channel is resized and passed through a nozzle before being connected to the third channel.

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

Fig. 4C also illustrates that a light source 480 (e.g., a laser) can be used to affect a material flow such as a cell flow. The laser can be placed in alignment with the cell flow or can be placed opposite to the cell flow. The laser can also be placed and/or oriented to be orthogonal or diagonal to the cell flow.

As shown in Fig. 1A-Fig. 1B and Fig. 4A-Fig. 4C, the fluid flow containing cells or particles is directed to pass vertically through the first channel. One or more substances (fluid, cell and/or particle) 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 100mm, for example, from 0.1mm to 0.2mm, from 0.2mm to 0.3mm, etc. The first channel is followed by a second orthogonal/horizontal channel, and in a preferred embodiment, the second orthogonal/horizontal channel is shorter than the first channel. The length of the second channel can be between 250 microns and 100mm, for example, from 0.25mm to 0.5mm, from 0.5mm to 0.75mm, from 0.75mm to 1.0mm, etc. The third channel extends vertically and is parallel to the first channel. The length of the third channel can be between 50 microns and 100mm, for example, from 0.05mm to 0.1mm, from 0.1mm to 0.15mm, from 0.15mm to 0.2mm, etc. The channels are arranged to be in operable communication, directly or indirectly, to allow one or more substances to move through the multiple channels. Typical directions of fluid flow are given by the flow arrows in Figures 4A and 4C, but flow can be reversed.

In a preferred embodiment, the fourth channel comprises an analytical channel, and the fourth channel is a channel with a length between 250 microns and 100 mm, such as from 0.25 mm to 0.5 mm, from 0.5 mm to 0.75 mm, from 0.75 mm to 1.0 mm, etc. In this embodiment, the fourth channel is the channel closest to the top of the chip. The distance between the fourth channel and the top of the chip measured vertically can be between 100 microns and 2 mm, but can also be as long as 100 mm, such as from 100 microns to 200 microns, from 200 microns to 300 microns, from 300 microns to 400 microns, etc. In other words, the fourth channel may be arranged within 50%, 33%, 25%, 10% or 5% of the top of the chip. An imaging device such as a camera may be oriented orthogonal to the fourth channel, and its distance from the fourth channel may be between about 100 microns and 2 mm, but the distance from the fourth channel may be as long as 100 mm, for example, from 100 microns to 200 microns, from 200 microns to 300 microns, from 300 microns to 400 microns, and so on.

In one embodiment, the laser or other light source has a focusing lens element. FIG. 4A depicts the present invention when the laser 480 is in operation, emitting a laser beam and directing the beam through the focusing lens element into the fourth flow channel 440. The particles are aligned within the laser beam because the gradient force attracts the particles toward the area of highest laser intensity. The laser scattering force pushes the particles in the direction of propagation of the laser beam (e.g., from left to right in FIG. 4A ).

In another embodiment, in addition to the camera or camera device oriented orthogonally to the fourth channel, the second camera or camera device (see 450) is also oriented toward the side of the analysis (or fourth) channel, and is orthogonally aligned with the fourth channel or aligned with the fourth channel at any angle; for example, as shown in FIG. 4B. Taking an orthogonal view image for each cell enables the calculation of multiple cell properties in two directions, such as size and shape, thereby increasing the amount of information that can be obtained for each cell. This will also enable the calculation of cell volume attributes, including total volume, shape, and provide a deep understanding of cells that are not symmetrical along an axis parallel to the flow direction (Z axis as shown in FIG. 5). Using two or more cameras, a basic three-dimensional model of a cell 550 can be constructed by combining orthogonal images using, for example, existing three-dimensional reconstruction algorithms (e.g., diffraction theory methods or illumination rotation methods). The three-dimensional model and its analysis will enable more accurate analysis of cells, such as cell size, shape, orientation, and other quantitative and qualitative measurements of one or more particles or one or more cells in the fourth channel of the microfluidic chip.

In FIG5 , portions of an analysis channel 540 are shown from two different planes, such as planes that can be imaged from an imaging device (see, e.g., FIG4A-4C ). In a first plane 520, a portion of a cell or particle 510 is imaged at a certain orientation. In a second plane 530, another portion of the same cell or particle is imaged from a different viewing angle of another camera. This enables calculation of multiple cell properties, creates a data matrix for each cell for each camera, and creates a three-dimensional representation of a cell or particle 550.

As different parts of the cell pass through the focal plane (one or more) 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 moves with, for example, a fluid stream 620. This is shown in FIG. 6A. In FIG. 6A, portions of the cell or particle are imaged at multiple time points and/or spatial points, in different orientations and from different viewing angles. In this example, as the cell moves and rotates through the focal plane, it appears at different sizes in successive images (e.g., as shown in FIG. 6A, four example images of each plane from two different cameras). A more complex three-dimensional rendering 650 of the particle or cell is achieved using a three-dimensional reconstruction algorithm. From this rendering, certain properties of the cell or particle, such as cell size, volume, location and size of the nucleus and other organelles, and a measurement or overview of the cell morphology can be inferred. In addition, due to changes in biophysical or biochemical properties (including but not limited to refractive index, birefringence, or cell shape or morphology), the rotation of the cell can be measured as a function of the torque of the optical force.

Fig. 6B depicts multi-plane imaging within the chip, and multiple images of the same cell taken over time, for example, as the cell moves through the focal plane. In this case, the view of the cell is referred to as a "slice" or "image slice" in the art. The image slice is actually the thickness of the optical plane being imaged. The thickness of the image plane or slice is determined by factors such as the optical magnification of the imaging system. At higher magnifications, the working distance of the objective lens is reduced, so it is necessary 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 affect the cell flow in the analysis channel. In a preferred embodiment, the cell or particle can be purposefully lured into or out of the focal plane of the channel by moving the laser and/or camera, or using fluid dynamic focusing to adjust (one or more) streams or positions, for imaging. The laser source distance 660 can be moved using, for example, a piezoelectric actuator or a linear electro-optical machine workbench. This may be done in a per-cell or per-group manner. The hydrodynamic focusing of the cell can be changed to affect the initial position and trajectory of the cell. For example, particles may be arranged or oriented in the focal plane within the laser beam due to gradient forces that attract particles toward the area of highest laser intensity. Laser scattering forces push particles in the direction of propagation of the laser beam. See FIG. 6B . Moving the laser along the X-axis in this example enables imaging of features of different parts of the cell, as shown in disk 680 . This may represent, for example, a nucleus, an organelle, an inclusion body, or other features of a cell or particle. Due to the action of the gradient forces, the laser pulls the cell to its center. It is also contemplated that two or more cameras may increase detail and accuracy.

The embodiment of the present invention shown in FIG. 7 is a static mode, in which a particle or cell 710 stops at a specific differential retention position due to a balance of optical force 730 and fluidic force 735. The optical force can be applied by, for example, a laser or a parallel light source. As shown and described in FIG. 5 and FIG. 6A-6B, images can be taken in multiple planes. A flow sensor is used to measure the flow velocity of each particle when it stops in the flow at a given laser power. Because the optical force and the fluidic force are balanced, the fluidic resistance (i.e., the resistance from the flow velocity and the channel direction) is equal to the optical force. The properties of each cell can be measured in this way in turn. Although not a high-throughput measurement system, the embodiment of the present invention realizes close observation and imaging of captured cells, and also realizes dynamic changes in optical force caused by biochemical or biological changes in cells. Reagent streams containing chemicals, biochemicals, cells or other standard biological agents can be introduced into the flow channel to interact with the captured (one or more) cells. These dynamic processes can be quantitatively monitored by measuring changes in optical force during experiments of single or multiple cells.

In one embodiment, the camera or other imaging device is oriented and/or focused along or against the flow in the analysis channel so that the camera or other imaging device is aligned 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 parallel light source 830. A dichroic mirror or similar device 840 reflects laser light 835 from the camera to somewhere else to prevent damage, but passes light 865 produced by an illumination source 860 to achieve imaging. The camera is oriented to be 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 to be orthogonal to the channel and the laser. A second dichroic mirror 845 that passes through the laser light and reflects the illumination light is used to direct the illumination light and the laser light through the channel. Another embodiment of this configuration swaps the positions of the laser and the 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 direct the laser light and visible light through the channel.

FIG. 9 shows another embodiment. In this case, the camera or imaging device 910 is oriented so that the cell or particle 980 is traveling toward the camera in the fluid stream 970. Therefore, the camera and laser 930 are located on the same side of the channel, and the illumination source 960 is located at the opposite end of the channel. Two dichroic mirrors 940 and 945 are used to direct the laser light 935 and the illumination light 965 into the channel, direct the illumination light to the camera, and divert the laser light from the illumination source to somewhere else. Another embodiment of this configuration swaps the position of the laser and the camera so that the laser is orthogonal to the channel and the illumination source is parallel to the channel. The second dichroic mirror 945 will then direct the laser light through the channel and direct the illumination light to the camera.

Optionally, an embodiment of the present invention further comprises at least one optical element, which is located between the optical force source and the fourth channel, and the at least one optical element can be operated 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, at least one optical element comprises a standard cylindrical lens, a standard axis pyramid, a standard concave mirror, a standard toric mirror, a standard spatial light modulator, a standard acousto-optic modulator, a standard piezoelectric mirror array, a diffractive optical element, a standard quarter-wave plate, and/or a standard half-wave plate. Optionally, the optical force source may comprise a standard circularly polarized beam, a standard linearly polarized beam, or a standard elliptically polarized beam.

Optionally, the present invention includes an apparatus comprising a microfluidic channel, a laser light source focused into the microfluidic channel by an optical device, an electric field source connected to the microfluidic channel by an electrode during operation; flowing particles in a liquid passing through the microfluidic channel; and manipulating the laser light and the electric field to act in combination on the particles in the microfluidic channel to separate the particles based on size, shape, refractive index, charge, charge distribution, charge mobility, dielectric constant and/or deformability. In another embodiment, an apparatus includes a microfluidic channel configured to provide a DEP field to the interior of the channel through (1) an electrode system or (2) an insulator dielectrophoresis (DEP) system, and also includes a laser light source focused into the microfluidic channel by an optical device; a plurality of flowing particles in a liquid enter the microfluidic channel; and operating the laser light and the field in combination to act on the particles in the microfluidic channel to capture the particles or change their velocity, wherein the above-mentioned DEP field is linear or nonlinear. Another possible embodiment of the device includes a microfluidic channel, which includes an inlet and a plurality of outlets, and also includes a laser light source, which is focused by an optical device to pass through the microfluidic channel at a critical angle that matches the flow rate in the microfluidic channel to generate optical forces on particles and simultaneously maximize the residence time of selected particles in the laser light, thereby separating the particles into the plurality of outlets, wherein the laser light can be operated to exert forces 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 channels, such as (one or more) analysis channels, in particular a fourth channel. The particle interrogation unit includes a standard illuminator, standard optics, and a standard sensor. Optionally, at least one particle interrogation unit includes a standard bright field imager, 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 spectroscopic detector.

At least one particle interrogation unit connected to the fourth channel includes a laser force-based device or apparatus that facilitates the identification, selection, and classification of cellular diseases. In one aspect, the unit utilizes inherent differences in optical pressure caused by changes in particle size, shape, refractive index, or morphology as a means of separating and characterizing particles. In one aspect, a near-infrared laser beam applies physical forces to cells, which are then measured. The optical forces of radiation pressure, when balanced with the fluidic drag on the particles, result in changes in particle velocity that can be used to identify different particles, or changes in particle populations based on inherent differences. Fluidic and optical force balance can also be used to change the relative position between particles based on intrinsic properties of the particles, resulting in physical separation. Another embodiment of the interrogation unit includes an apparatus for particle analysis and/or separation, such as at least one parallel light source, which can be operated to generate at least one parallel light source beam. At least one parallel light source beam includes at least one beam cross section.

Embodiments of the present invention relate to a combination of several of the above-described design elements discussed in the monolithic device above. Embodiments also include methods of using such devices. Figures 1A-1B illustrate examples of such monolithic devices. The illustrated embodiment of the present invention is a five-layer structure, with all five layers bonded to each other to produce a solid-state microfluidic chip, although the chip may be a structure opposite to the bonding layer. Several standard materials can be used to manufacture the chip, including but not limited to fused quartz, crown glass, borosilicate glass, soda-lime glass, sapphire glass, cycloolefin polymer (COP), poly(dimethyl)siloxane (PDMS), OSTE, polystyrene, polymethyl methacrylate, polycarbonate, other plastics or polymers. The chip allows for sample input, fluid dynamic focusing, optical interrogation, imaging, analysis, sample exit, and transparent optical access to and from the laser light region. The chip in the embodiment may also be 3D printed, molded, or otherwise formed.

Optionally, at least one particle type includes a plurality of particle types. Each particle type in the plurality of particle types includes respective intrinsic properties and respective induced properties. Optionally, the intrinsic properties include size, shape, refractive index, morphology, intrinsic fluorescence and/or aspect ratio. Optionally, the induced properties include deformation, angular orientation, rotation, rotation rate, antibody label fluorescence, aptamer label fluorescence, DNA label fluorescence, dye label fluorescence, differential retention metric and/or gradient force metric. The method embodiment also includes identifying and separating a plurality of particles based on at least one of the intrinsic properties and the induced properties according to the respective particle types. Optionally, the method embodiment also includes interrogating or manipulating the sample stream. Optionally, interrogating the sample stream includes determining at least one of the intrinsic properties and the induced properties of the particle type, and measuring the particle velocity of the plurality of particles. Measurement of at least one intrinsic property has a wide range 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 endpoint dilution assay) for viral quantification, process development and monitoring, sample release assays, adventitious factor testing, clinical diagnostics, biomarker discovery; determining cell productivity for antibodies or proteins for process development and monitoring; determining the efficacy, quality, or activation state of cells produced as cell-based therapies, including CAR T and other oncology applications and stem cells; determining the effects of chemicals, bacteria, viruses, antibiotics, or antiviral drugs on a particular cell population; and determining the disease state or potential of a research or clinical cell sample. Optionally, the photoforce source includes at least one beam axis and the sample stream includes a sample stream axis. The steps of determining at least one of the intrinsic and induced properties of the particle type, and measuring the particle velocity of a plurality of particles together constitute a displacement of the beam axis from the sample stream axis. Optionally, the step of determining at least one of an intrinsic property and an induced property of a particle type, and the step of measuring particle velocity of a plurality of particles together constitute the calculation of a slope and trajectory of a particle of a plurality of particles offset from the sample flow axis to at least one beam axis.

Those skilled in the art will recognize that these features may be used alone or in any combination, or omitted, based on the requirements and specifications of a given application or design. When an embodiment talks about "comprising" certain features, it should be understood that the embodiment may be "composed of" or "essentially composed of" any one or more of the above features. Other embodiments of the present invention will be apparent to those skilled in the art after consideration of the specification and practice of the present invention.

It should be particularly noted that a range of values is provided in this specification, and each value between the upper and lower limits of the range is also specifically disclosed. The upper and lower limits of these smaller ranges may also be independently included in the range or excluded from the range. Unless the context clearly specifies otherwise, the singular forms "a", "an" and "the" include plural references. The description and examples should be regarded as exemplary or explanatory, and changes that do not deviate from the essence of the present invention are included within the scope of the present invention. In addition, all references cited in the present invention are incorporated herein in their entirety by reference alone, and the citation of these references is intended to provide an effective method for supplementing the enabling disclosure of the present invention, and to provide a background that details the level of ordinary technology in the art.

Claims (36)

1. A microfluidic device comprising:

a substrate comprising a plurality of channels configured to transport one or more substances; and a parallel light source capable of interacting with the one or more substances; wherein the plurality of channels comprises:

A first channel oriented vertically upward within the substrate relative to the direction of gravity,

A second channel in operative communication with the first channel and disposed horizontally within the substrate,

A third channel in operative communication with the second channel and oriented vertically upward within the substrate relative to the direction of gravity, an

A fourth channel in operative communication with the third channel and disposed horizontally within the substrate;

Wherein the first channel, the second channel, the third channel, and the fourth channel are arranged in a manner that provides a path for the one or more substances to move through the substrate from the first channel to the second channel to the third channel to the fourth channel;

Wherein the second channel is shorter than the first channel;

Wherein the one or more substances are injected into the first channel oriented vertically upwards in the substrate with respect to the direction of gravity by driving a pressure or vacuum of a flow through a bottom horizontal planar surface having an opening to the first channel.

2. The microfluidic device of claim 1, wherein the bottom horizontal planar surface has at least one length from one edge to another that is shorter than a length of at least one of the vertical planar surfaces of the substrate from one edge to another to maintain the directional and volumetric continuity of the first channel.

3. The microfluidic device of claim 1, wherein the one or more substances are injected into the first channel vertically disposed within the substrate through a bottom horizontal planar surface having an opening to the first channel, wherein the bottom horizontal planar surface has a surface area that is less than or equal to a surface area on a vertical planar surface of the substrate, maintaining directional and volumetric continuity of the first channel in a vertical direction.

4. The microfluidic device of claim 1, wherein the first channel comprises an opening disposed on an outer surface of the substrate, the opening disposed in a manner that provides a path for the one or more substances to vertically enter the substrate and move vertically within the first channel.

5. The microfluidic device of claim 1, wherein the one or more substances are injected into the first channel vertically disposed within the substrate through a bottom horizontal planar surface having an opening to the first channel that maintains directional and volumetric continuity of the first channel in a vertical direction, and wherein a cross-section of the first channel and a cross-section of the opening have the same area, a smaller area, or a larger area.

6. The microfluidic device of claim 1, wherein the one or more substances are injected into the first channel vertically disposed within the substrate through a bottom horizontal planar surface having an opening to the first channel, the direction and volume continuity of the first channel being maintained in a vertical direction, and wherein the shape, size, and orientation of the first channel and the opening are configured in a manner that maintains the direction and volume continuity.

7. The microfluidic device of claim 1, wherein parallel or focused light sources are oriented to propagate in a direction of movement, opposite direction, orthogonal direction, or diagonal of the one or more substances in the fourth channel.

8. The microfluidic device of claim 1, wherein the fourth channel allows for imaging and analysis of particles or cells in multiple focal planes.

9. The microfluidic device of claim 1, wherein the fourth channel allows for imaging and analysis of particles or cells in multiple focal planes during movement of the one or more substances.

10. The microfluidic device of claim 1, wherein the fourth channel allows for imaging and analysis of particles or cells during movement of the one or more substances.

11. The microfluidic device of claim 1, wherein the fourth channel allows imaging and analysis of particles or cells from multiple angles and/or orientations during movement of the one or more substances.

12. The microfluidic device of claim 1, wherein the fourth channel allows for imaging and analysis of particles or cells with one or more imaging devices from multiple focal planes, angles, and/or orientations during movement of the one or more substances.

13. The microfluidic device of claim 1, further comprising one or more of electrical power, optical power, and/or fluidic force to move one or more cells or one or more particles in one or more channels.

14. The microfluidic device of claim 1, further comprising one or more of electrodynamic force, electrophoretic force, and/or Dielectrophoresis (DEP) force to move one or more cells or one or more particles in one or more channels.

15. The microfluidic device of claim 1, further comprising one or more imaging devices, wherein at least one of the imaging devices is movable in a manner that changes a focal plane imaged in the fourth channel.

16. The microfluidic device of claim 1, wherein one or more cells or one or more particles in the fourth channel can be moved by a change in optical and/or fluidic force, and the area of cells imaged in the fourth channel changes as the particles move.

17. The microfluidic device of claim 1, wherein the fourth channel is spaced from two or more outer surfaces of the substrate to allow imaging and analysis of multiple image slices of cells or particles as the focal plane moves relative to the cells or particles.

18. The microfluidic device of claim 1, wherein the fourth channel is a distance from two or more outer surfaces of the substrate to allow imaging and analysis of multiple image slices of moving cells or particles as they move through a focal plane.

19. The microfluidic device of claim 1, wherein the fourth channel is a distance from two or more outer surfaces of the substrate to allow imaging and analysis of multiple image slices of suspended or static cells or particles.

20. The microfluidic device of claim 1, wherein the one or more substances are movable by pressure, vacuum, peristaltic, electrodynamic, electrophoretic, magnetic, optical, or any combination thereof.

21. The microfluidic device of claim 1, further comprising a dichroic mirror for directing light to or away from an imaging device that images and/or analyzes cells or particles in the fourth channel.

22. The microfluidic device according to claim 1, further comprising a dichroic mirror for guiding parallel or focused light sources to interact with particles or cells in the fourth channel.

23. The microfluidic device of claim 1, further comprising a dichroic mirror configured to direct parallel or focused light away from an imaging device, a light source, or another portion of the device.

24. The microfluidic device of claim 1, wherein the plurality of channels comprises a fifth channel disposed horizontally, vertically, or diagonally inside the substrate.

25. The microfluidic device of claim 1, wherein the plurality of channels comprises a fifth channel that splits into two or more channels or wells for classifying cells or particles.

26. The microfluidic device of claim 1, wherein the fourth channel is closer to the top of the substrate than the first channel, the second channel, or the third channel.

27. The microfluidic device of claim 1, wherein the fourth channel is located 100 microns to 100mm from the top of the substrate.

28. The microfluidic device of claim 1, wherein the length of the first channel ranges between 0.1mm and 100.0 mm.

29. The microfluidic device of claim 1, wherein the length of the second channel ranges between 0.1mm and 100.0 mm.

30. The microfluidic device of claim 1, wherein the length of the third channel ranges between 0.05mm and 100.0 mm.

31. The microfluidic device of claim 1, wherein the length of the fourth channel ranges between 0.1mm and 100.0 mm.

32. The microfluidic device of claim 1, wherein the first channel has a length greater than the second channel, the third channel, or the fourth channel.

33. The microfluidic device of claim 1, further comprising a cell or particle interrogation unit.

34. The microfluidic device of claim 1, further comprising a cell or particle collection channel.

35. The microfluidic device of claim 1, further comprising an imaging device selected from at least one of: bright field imagers, light scatter detectors, single wavelength fluorescence detectors, spectral fluorescence detectors, CCD cameras, CMOS cameras, photodiodes or photodiode arrays, spectrometers, photomultiplier tubes or tube arrays, chemiluminescent detectors, bioluminescence detectors, standard raman spectrum detection systems, surface Enhanced Raman Spectroscopy (SERS), coherent anti-stokes raman spectroscopy (CARS), and/or Coherent Stokes Raman Spectroscopy (CSRS).

36. The microfluidic device of claim 1, further comprising a conduit end disposed in an opening in an outer surface of the substrate in a manner that provides a path for the one or more substances to enter the substrate and move within the first channel, wherein a cross-sectional area of the opening is equal to, greater than, or less than a cross-sectional area of the conduit end.