Detection using dual imaging with digital inline holography

Abstract

The present embodiments can implement digital inline holography (DIH) combined with another imaging or sensing modality to form a dual sensing system for detecting particles or cells and in some cases for the diagnosis of cancer in various body fluids (e.g., blood, saliva, urine). In comparison with other single imaging modality techniques (e.g., fluorescent imaging, DIH), the present embodiments include a combination of different imaging or sensing modalities in a synchronized fashion to improve the sensitivity and specificity of the many methods using single imaging modality and incorporate the cross-referencing and integration of different imaging or sensing modalities that allow for increased insights into, e.g., the genesis and prognosis of cancer and the variation of morphology and sub-cellular structures of cancer cells and other particles and cells associated with the change in their omic profiles.

Description

DETECTION USING DUAL IMAGINGWITH DIGITAL INLINE HOLOGRAPHYPRIORITY

[0001] This application claims the benefit of US Ser. No. 63 / 482,913 filed on February 2, 2023, which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] The methods and compositions described here relate to the field of imaging and sensing systems. More particularly, embodiments relate to a dual sensing system incorporating digital inline holography and fluorescence imaging or to identify patterns in a cell in an observation chamber or a cell traveling in a microfluidic device.BACKGROUND

[0003] A variety of techniques exist for imaging fixed samples containing one or more cells of a biological fluid. For example, such systems can include microscopes and / or imaging devices capable of magnifying a view of the fixed sample and capturing one or more images of the sample. Obtained images of the sample can be processed to derive insights into the sample, such as a cell type of the cell included in the sample.

[0004] Furthermore, cells can be transported and isolated as part of a microfluidic device. A microfluidic device can transport cells of a biological fluid (e.g., blood) and isolate cells based on a cell type. For instance, microfluidic devices can use various techniques to isolate cells based on physical properties such as a cell size and / or flow rate of each cell. An imaging system can be used to analyze a cell in the microfluidic device to determine its cell type. Accordingly, there is a need in the art for imaging and isolating cells flowing in a microfluidic device.SUMMARY

[0005] Provided herein are dual sensing systems comprising a first camera configured to capture a first image of a digital inline holographic (DIH) pattern of at least one particle or cell; a second camera or sensor configured to capture a second image or signal of a fluorescent pattern of at least one particle or cell; at least a first light source configured to emit light of a first wavelength onto a microfluidic device carrying at least one particle or cell; and a beam splitter configured to direct emittedlight between the first and second cameras and the microfluidic device carrying the at least one particle or cell.

[0006] The systems can further comprise a second light source configured to emit light of a second wavelength onto the microfluidic device carrying at least one particle or cell, wherein the first light source and the second light source comprise laser light sources. The first wavelength and the second wavelength ranges between 350 nanometers (nm) and 800 nm. The first light source can be disposed above the microfluidic device, and the second light source can be disposed below the microfluidic device. The light emitted by the first light source can be configured to pass along a first path, the first path directing the light from the first light source through the beam splitter to the microfluidic device via an objective, where the light can be subsequently reflected by the microfluidic device and directed to the first camera via the objective and the beam splitter. The light emitted by the second light source can be configured to pass along a second path, the second path directing the light from the second light source through the microfluidic device and to the second camera / sensor via the objective and the beam splitter.

[0007] The system can further comprise a first filter disposed in front of a lens of the first camera; and a second filter disposed in front of a lens of the second camera or sensor, wherein each of the first filter and second filter can be configured to filter out signals of wavelengths other than signals of a specified wavelength range.

[0008] The system can further comprise a computing node, wherein the computing node comprises a processor and memory, the memory including instructions that, when executed by the processor, cause the processor to send a first instruction to the first light source and / or second light source to emit light. The instructions can further cause the processor to send a second instruction to each of the first camera and the second camera or sensor to capture the first image and the second image or signal with a specified start time and exposure time for synchronizing image capturing across the first camera and second camera or sensor. The instructions can further cause the processor to obtain the first image from the first camera and the second image or signal from the second camera or sensor; process the first image and the second image or signal to identify a holographic pattern and a fluorescent pattern of the at least one particle or cell; and determine whether the atleast one particle or cell is a target particle or target cell type based on the holographic pattern and the fluorescent pattern of the at least one particle or cell.

[0009] The microfluidic device can be a sawtooth inertial sorting microfluidic device.

[0010] Another aspect provides methods comprising providing a first instruction to at least a first light source to emit light of a first wavelength, wherein a first camera is configured to capture a first image of a dual inline holographic (DIH) pattern of at least one particle or cell traveling through a microfluidic device and a second camera or sensor is configured to capture a second image or signal of a fluorescent pattern of the at least one particle or cell with a specified start time and an exposure time for synchronizing image capturing across the first camera and second camera or sensor; obtaining the first image from the first camera and the second image or signal from the second camera; processing the first image and the second image or signal to identify a holographic pattern and a fluorescent pattern of the at least one particle or cell; determining whether the at least one particle or cell is a target particle or target cell type based on the holographic pattern and the fluorescent pattern of the at least one particle or cell; and causing the microfluidic device to perform an action based on determining that at least one particle or cell is the target particle type or target cell type. The methods can further comprise sending a second instruction to a second light source configured to emit light of a second wavelength onto the microfluidic device carrying at least particle or one cell. The first light source and the second light source can comprise laser light sources, wherein the first wavelength and the second wavelength ranges between 350 nanometers (nm) and 800 nm. The first light source and the second light source can comprise laser light sources, wherein the first wavelength is a 488 nanometer (nm) wavelength, and wherein the second wavelength is a 405 nm wavelength. The light emitted by the first light source can be configured to pass along a first path, the first path directing the light from the first light source through a beam splitter to the microfluidic device via an objective, where the light is subsequently reflected by the cell samples in the microfluidic device and directed to the first camera via the objective and the beam splitter, and wherein the light emitted by the second light source can be configured to pass along a second path, the second path directing the light from the second light source through the microfluidic device and to the second camera or sensor via the objective and the beam splitter(s). Thefirst light source can be disposed above the microfluidic device, and the second light source can be disposed below the microfluidic device. The microfluidic device can be a sawtooth inertial sorting microfluidic device. The action can be the diversion of the target particle or cell to a specific outlet channel of the microfluidic device.

[0011] Another aspect provides a system comprising at least a first camera, the first camera configured to capture a first image of a digital inline holographic (DIH) pattern and / or a fluorescent pattern of at least one particle or cell traveling through a microfluidic device; at least a first light source configured to emit light of a first wavelength onto the microfluidic device carrying at least one particle or cell; and a beam splitter configured to direct emitted light between at least the first camera and the microfluidic device carrying at least one particle or cell. The first wavelength can range from a 350 nanometer (nm) wavelength to an 800 nanometer (nm) wavelength. The system can further comprise a second camera configured to capture a second image of either the digital inline holographic (DIH) pattern and / or the fluorescent pattern of at least one particle or cell traveling through the microfluidic device. The system can further comprise a second light source configured to emit light of a second wavelength onto the microfluidic device carrying at least one particle or cell. The light emitted by the first light source can be configured to pass along a first path, the first path directing the light from the first light source through the beam splitter to the microfluidic device via an objective, where the light is subsequently backscattered by one or more particle or cells in the microfluidic device and directed to the first camera via the objective and the beam splitter, and wherein the light emitted by the second light source can be configured to pass along a second path, the second path directing the light from the second light source through the microfluidic device and to the second camera via the objective and the beam splitter. The system can further comprise a first filter disposed in front of a lens of the first camera; and a second filter disposed in front of a lens of the second camera.

[0012] The system can further comprising a computing node, wherein the computing node comprises a processor and memory, the memory including instructions that, when executed by the processor, cause the processor to send a first instruction to the first light source to emit light; send a second instruction to the first camera to capture the first image; obtain the first image from the first camera; process the first image to identify either a holographic pattern and / or a fluorescent pattern ofat least one particle or cell; and determine whether the at least one particle or cell is a target particle type or a target cell type based on the holographic pattern and the fluorescent pattern of at least one particle or cell. The microfluidic device can be a sawtooth inertial sorting microfluidic device.

[0013] Another aspect provides a dual sensing system. The system can comprise a camera configured to capture an image of a digital inline holographic (DIH) pattern and / or a fluorescence pattern of at least one particle or cell; at least a first light source configured to emit light of a first wavelength onto a microfluidic device carrying at least one particle or cell; a second light source configured to emit light of a second wavelength onto the microfluidic device carrying at least one particle or cell; and a beam splitter configured to direct emitted light between the first and second cameras and the microfluidic device carrying the at least one particle or cell. The microfluidic device can be a sawtooth inertial sorting microfluidic device.

[0014] Another aspect provides a method of identifying target cells in a mixed sample of cells comprising adding a mixed sample of fluorescently labeled cells to a microfluidic device; providing a first instruction to at least a first light source to emit light of a first wavelength, wherein a first camera is configured to capture a first image of a dual inline holographic (DIH) pattern of at least one cell traveling through the microfluidic device and a second camera or sensor is configured to capture a second image or signal of a fluorescent pattern of the at least one cell with a specified start time and an exposure time for synchronizing image capturing across the first camera and second camera; obtaining the first image from the first camera and the second image or signal from the second camera or sensor; processing the first image and the second image or signal to identify a holographic pattern and a fluorescent pattern of the at least one cell; and determining whether the at least one cell is a target cell based on the holographic pattern and the fluorescent pattern of at least one cell. The target cell can be a cancerous cell. The method can further comprise causing the microfluidic device to perform an action based on determining that the at least one cell is the target cell type. The action can be the diversion of the target cell to a specific outlet channel of the microfluidic device. The microfluidic device can be a sawtooth inertial sorting microfluidic device.

[0015] An aspect provides a method of sorting cells comprising adding fluorescently labeled cells to a microfluidic device; providing a first instruction to atleast a first light source to emit light of a first wavelength, wherein a first camera is configured to capture a first image of a dual inline holographic (DIH) pattern of at least one cell traveling through the microfluidic device and a second camera or sensor is configured to capture a second image or signal of a fluorescent pattern of the at least one cell with a specified start time and an exposure time for synchronizing image capturing across the first camera and second camera or sensor; obtaining the first image from the first camera and the second image or signal from the second camera or sensor; processing the first image and the second image or signal to identify a holographic pattern and a fluorescent pattern of the at least one cell; determining whether at least one cell is a target cell type based on the holographic pattern and the fluorescent pattern of at least one cell; and causing the microfluidic device to perform an action based on determining that the at least one cell is the target cell type. The target cell type can be a cancerous cell. The action can be the diversion of the target cells to a specific outlet channel of the microfluidic device such that the target cells are isolated. The microfluidic device can be a sawtooth inertial sorting microfluidic device.

[0016] An aspect provides a multi-sensor system comprising a first camera configured to capture a first image of a digital inline holographic (DIH) pattern of at least one particle or cell; a second camera or sensor configured to capture a second image or signal of a fluorescent pattern of at least one particle or cell; a third camera or sensor configured to capture a third image or signal of a fluorescent pattern of at least one particle or cell; a first light source configured to emit light of a first wavelength onto a microfluidic device carrying the at least one particle or cell; and a second light source configured to emit light of a second wavelength onto a microfluidic device carrying the at least one particle or cell, and a beam splitter configured to direct emitted light between the first, second, and / or third cameras and the microfluidic device carrying the at least one particle or cell. The first light source and the second light source can comprise laser light sources. The first wavelength and the second wavelength can range between 350 nanometers (nm) and 800 nm. The first light source can be disposed above the microfluidic device, and the second light source can be disposed below the microfluidic device. The system can further comprise a first filter disposed in front of a lens of the first camera; a second filter disposed in front of a lens of the second camera or sensor; and a third filter disposed in front of a lens of the third camera or sensor; wherein each of the first filter, second filter, andthird filter are configured to filter out signals of wavelengths other than signals of a specified wavelength range.

[0017] The system can further comprise a computing node, wherein the computing node comprises a processor and memory, the memory including instructions that, when executed by the processor, cause the processor to send a first instruction to the first light source and to the second light source to emit light. The instructions can further cause the processor to send a second instruction to each of the first camera, the second camera or sensor, and the third camera or sensor to capture the first image, the second image or signal, and the third image or signal with a specified start time and exposure time for synchronizing image or signal capturing across the first camera, the second camera or sensor, and the third camera or sensor. The instructions can further cause the processor to obtain the first image from the first camera, the second image or signal from the second camera or sensor, and the third image or signal from the third camera or sensor; process the first image, the second image or signal, and the third image or signal to identify a holographic pattern and a fluorescent pattern of at least one particle or cell; and determine whether at least one particle or cell is a target particle type or cell type based on the holographic pattern and the fluorescent pattern of the at least one particle or cell. The microfluidic device can be a sawtooth inertial sorting microfluidic device.

[0018] Another aspect provides a method comprising providing a first instruction to a first light source to emit light of a first wavelength and a second instruction to a second light source to emit light of a second wavelength, wherein a first camera is configured to capture a first image of a dual inline holographic (DIH) pattern of at least one particle or cell traveling through a microfluidic device, a second camera or sensor is configured to capture a second image or signal of a fluorescent pattern of the at least one particle or cell, and a third camera or sensor is configured to capture a third image or signal of a fluorescent pattern of the at least one particle or cell with a specified start time and an exposure time for synchronizing image or signal capturing across the first camera, the second camera or sensor, and the third camera or sensor; obtaining the first image from the first camera, the second image or signal from the second camera or sensor, and the third image or signal from the third camera or sensor; processing the first image, the second image or signal, and the third image or signal to identify a holographic pattern and a fluorescent pattern of the at least oneparticle or cell; determining whether the at least one particle or cell is a target particle type or target cell type based on the holographic pattern and the fluorescent pattern of the at least one particle or cell; and causing the microfluidic device to perform an action based on determining that the at least one particle or cell is the target particle type or target cell type. The first light source can be disposed above the microfluidic device, and the second light source can be disposed below the microfluidic device. The microfluidic device can be a sawtooth inertial sorting microfluidic device. The action can be the diversion of the target particle or target cell to a specific outlet channel of the microfluidic device. The target cell type can be a cancerous cell. The first wavelength and second wavelength can range from a 350 nanometer (nm) wavelength to an 800 nanometer (nm) wavelength.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings, in which like references indicate similar elements and in which:

[0020] FIG. 1 illustrates a dual imaging / sensing system according to a first example design.

[0021] FIG. 2 illustrates a dual imaging / sensing system according to a second example design.

[0022] FIG. 3 illustrates a dual imaging / sensing system according to a third example design.

[0023] FIG. 4 illustrates a first example dual imaging / sensing system according to an embodiment.

[0024] FIG. 5 is an example sample image captured by a dual imaging / sensing system according to an embodiment.

[0025] FIG. 6 illustrates a second example dual imaging / sensing system according to an embodiment.

[0026] FIG. 7 is a flow process of an example method for implementing a dual imaging system according to an embodiment.

[0027] FIG. 8 is a block diagram of a special-purpose computer system according to an embodiment according to an embodiment.

[0028] FIG. 9 is a multi-camera imaging system with two light sources and three cameras. One camera is configured to capture an image of a digital inline holographicpattern. The other two cameras are configured to capture an image of a fluorescent pattern. These two cameras can be substituted with photomultiplier tubes or photodiodes for sensing of fluorescent signals.

[0029] FIG. 10 shows a multi-camera imaging set up.DETAILED DESCRIPTION

[0030] Digital inline holography (DIH) is a powerful tool for many biological applications, e.g., detecting and enumerating cancer cells under different settings. DIH generally can use a coherent light (e.g., laser) as the light source and employs a digital camera to record the patterns generated from the interference of light scattered from the sample and the non-scattered portion of the same light (referred to as holograms hereafter). DIH can achieve lateral and depth resolution at the submicron level in three dimensions and image and characterize the morphology of cells and particles while in suspension. Observations can be done directly in a microfluidic channel or statically in an observation chamber.

[0031] In comparison to other imaging modalities, in DIH, small differences in the optical properties (i.e. , refractive optical index) of sub-cellular structures can lead to the cross-interference of signals from these structures, which makes it hard to dissect and interpret the holograms. In contrast, in other imaging modalities, particularly, in fluorescent imaging, specific subcellular structures or proteins can be tagged by fluorescent markers, making it significantly easier to distinguish different cell organelles and proteins for cell analysis with higher specificity. While compiled images can be used to show multiple visuals of the same space, they typically use a single camera, and the light source and filter are changed such that the images are taken sequentially in time. While this may work for fixed samples, the uses can be limited to stationary or slow-moving samples, limiting its ability to be useful in applications such as microfluidics.

[0032] The use of DIH and a second imaging or sensing modality can allow for a better correlation of cell morphology and other markers. For example, cancer cells can have larger nuclei than healthy cells, but there may be other less known morphological traits that distinguish cell types. By combining fluorescence, which tags different cell types, with imaging techniques that enable morphological parameters to be measured, these correlations can be established. This can be of particular interest to achieve in flow because many techniques can use slide imaging, which flattenssamples, and can alter some morphological features. In some instances, various fluorescence information can be fed into the dual imaging / sensing system for determining a cell type. For example, various CD45 fluorescence markers can tag nontarget cells such as white blood cells. This information can be fed into the dual imaging / sensor system to know that such markers are indicative of WBCs, not a target cell type, which can improve performance of differentiating cell types. Alternatively, a target particle or cell can be fluorescently labeled. In another aspect, a target particle or cell can be fluorescently labeled and a non-target particle or cell can be differentially fluorescently labeled.

[0033] In addition to tagging nuclei and the cell membrane to learn morphological information about the cells, other organelles can similarly be tagged such that they can be mapped with each other as well as distinguished on non-fluorescent images by overlaying the fluorescent image with the non-fluorescent one to learn what the different organelles look like using other imaging techniques. Similar to the morphological parameters, this can be of particular interest in flow because organelles can be arranged in 3D making traditional 2D imaging non-optimal.

[0034] Provided herein are dual imaging / sensing systems that contain DIH (see, e.g., US Pat. Publ. 20210237064, which is incorporated herein by reference) and another type of imaging or sensing modality (e.g., brightfield, fluorescent) with a marker (e.g., a fluorescent marker), multiple different markers, or no markers. The systems can allow for better identification of cell type, aid in training and validating Machine Learning models, provide more descriptive morphology information, and / or allow for mapping of sub-cellular information to other imaging or sensing types. In many imaging modalities, protein markers can be targeted by fluorescent tags to distinguish different cell populations. For example, in circulating tumor cell (CTC) detection, epithelial cellular adhesion molecule (EpCAM) can be used to confirm CTCs while leukocyte common antigen (CD45) can be used to identify white blood cells (WBCs). By imaging or sensing these two markers simultaneously on separate cameras or sensors, the images can be overlayed from flow to identify each cell in a population. In the case of holographic imaging with fluorescence, the markers can be used to map specific cell types to their holographic signature. Similarly, this can be used with other non-fluorescent imaging or sensing techniques such as brightfield and phase contrast.

[0035] In some instances, a machine learning or artificial intelligence model can be trained using DIH to distinguish target cells (e.g., target cells) from other cell types (e.g., blood cells), or live cancer cells from dead cancer calls, etc. The fluorescent marker can act a confirmatory signal to further aid in weeding out false positives or false negatives by the model.

[0036] The devices and methods as described herein can implement digital inline holography (DIH) combined with another imaging or sensing modality to form a dual sensing system for the imaging or sensing of particles or cells, e.g., diagnosis of cancer in various body fluids (e.g., blood, saliva, urine). In comparison with other single imaging modality techniques (e.g., fluorescent imaging, DIH), the devices and methods disclosed herein include a combination of different imaging or sensing modalities in a synchronized fashion for improved sensitivity and specificity. The devices and methods incorporate cross-referencing and integration of different imaging or sensing modalities that allow for increased insights into, for example, the genesis and prognosis of cancer and the variation of sub-cellular structures of cancer cells associated with the change in their omic profiles. The dual imaging / sensing system can utilize multiple light sources, cameras or sensors, and / or filters to allow for simultaneous imaging / sensing of moving particles or cells in a microfluidic channel with different modalities.

[0037] Microfluidic Devices

[0038] A microfluidic device represents a microfluidic channel plus all of its relevant micro-features such as inlet and outlet ports. A microfluidic chip is the physical platform that houses the microfluidic device. The chip can house multiple microfluidic devices. A microfluidic device can be any suitable microfluidic device, e.g., a straight channel microfluidic device, a Y-channel microfluidic device, a T- junction microfluidic device, a cross-junction microfluidic device, a spiral microfluidic device, a micro-mixing device, a micro-reactor device, a microfluidic device for separation, merging micro-channel device, splitting microfluidic devices. In certain aspects, microfluidic devices and / or microfluidic channels are made of a transparent material so that particles and biological materials, e.g., cells, moving through the channels can be imaged / sensed. The methods and systems described herein can be used to image / sense stationary samples or flowing, moving, or traveling samples thatare present in a fluid and moving through, e.g., a microfluidic device or other suitable device.

[0039] In an aspect, a microfluidic device is a sawtooth inertial sorting microfluidic device as described in PCT / US23 / 74932, filed on September 22, 2023, which is incorporated by reference herein in its entirety. A sawtooth inertial sorting microfluidic device can comprise, an inlet for receiving particles of varying sizes across varying flow rates. The particle separation device can also include a main channel comprising a first end and a second end. The first end can be connected to the inlet. The main channel can comprise a series of angled portions. Each of the angled portions can form an angle that can be acute or obtuse (e.g., comprising angles between 1-89 degrees, or between 91 and 179 degrees) relative to an adjacent angled portion. The main channel can be configured to provide an inertial separation of the particles received at the inlet. The series of angled portions can reduce a pressure in the particle separation device and control varying flow rates of particles received at the inlet.

[0040] The sawtooth inertial sorting microfluidic device can also include one or more outlets connected to a second end of the main channel. Each of the one or more outlets can be configured to receive separated particles of differing sizes and / or densities.

[0041] In some instances, at least a portion of edges connecting each of the series of angled portions with the adjacent angled portion are rounded.

[0042] In some instances, the sawtooth inertial sorting microfluidic device can include between three and five outlets.

[0043] In some instances, the main channel comprises: a first stage and a second stage, wherein both the first stage and the second stage of the main channel are directly connected to at least one of the outlets.

[0044] In some instances, a second channel is disposed between the first stage and the second stage of the main channel, the second channel connecting to a first outlet.

[0045] In some instances, a second channel is disposed between the first stage and the second stage of the main channel, the second channel including two open ends, with each open end connecting to corresponding outlets.

[0046] In some instances, the sawtooth inertial sorting microfluidic device comprises a height of 50 micrometers and a width of between 100-200 micrometers.

[0047] In some instances, the sawtooth inertial sorting microfluidic device is configured to operate in a laminar flow regime and a transitional flow regime.

[0048] In some instances, the sawtooth inertial sorting microfluidic device includes a Reynolds number that is less than or equal to 2000 (e.g., less than about 2,000, 1 ,750, 1 ,500, 1 ,250, 1 ,000, 750, 500, or 250).

[0049] In some instances, the one or more outlets are either disposed in-line with the main channel or are disposed offset relative to a direction of the main channel. In an aspect an outlet is offset from a main channel by about 20, 30, 45, 50, 60, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, or 175 degrees, but any amount of offset is contemplated.

[0050] In another example embodiment, a sawtooth inertial sorting microfluidic system for separating particles or cells of varying sizes and / or inertia across varying flow rates using inertial separation is provided. The system can include an inlet and a main channel connected to the inlet at a first end of the main channel. The main channel can include a series of angled portions. Each of the angled portions can form an angle that is greater than 90 degrees to an adjacent angled portion The system can also include a set of outlets connected to a second end of the main channel.

[0051] In some instances, at least two of the series of angled portions including angles that are either less than or greater than 90 degrees form a trapezoidal corner.

[0052] In some instances, a first portion of the series of angled portions form trapezoidal corners and a second portion of the series of angled portions include rounded edges.

[0053] In some instances, at least one edge connecting each of the series of angled portions with adjacent angled portions is rounded. The angled portions can be trapezoidal wave shaped or sawtooth wave shaped. The angled portions can have a wavelength of 0.1 mm to 5 mm. The main channel can have a width of 50 to 600 micrometers and a depth of 30 to 70 micrometers.

[0054] In another example, a sawtooth inertial sorting microfluidic device can include an inlet for receiving particles of varying sizes across varying flow rates. The device can also include a main channel comprising a first stage and a second stage, with a first end of the first stage of the main channel connected to the inlet. The mainchannel can include a series of angled portions. The main channel can be configured to provide an inertial separation of the particles received at the inlet. The device can also include at least two outlets, with at least a first outlet connected to the first stage of the main channel and a second outlet connected to the second stage of the main channel.

[0055] In some instances, each of the angled portions forming an angle with an adjacent angled portion that is either less than or greater than 90 degrees.

[0056] In some instances, the first stage comprises angled portions forming angles less than 45 degrees and the second stage comprises angled portions that are greater than 90 degrees forming trapezoidal corners.

[0057] In some instances, any of the first stage or second stage comprises angled portions forming angles less than 45 degrees that form trapezoidal corners. The angled portions can be trapezoidal wave shaped or sawtooth wave shaped. The angled portions can have a wavelength of 0.1 mm to 5 mm. The main channel can have a width of 50 to 600 micrometers and a depth of 30 to 70 micrometers.

[0058] An aspect provides a sawtooth inertial sorting microfluidic device comprising an inlet for receiving particles of varying sizes and / or differing inertia across varying flow rates; a main channel comprising a first stage and a second stage, the first stage comprising a first end connected to the inlet and a second end connected to the second stage, the second stage comprising 2, 3, 4, 5, or more channels each connected to one or more outlets, wherein the first stage comprises a channel comprising a series of angled portions, with each of the angled portions forming an angle of either less than or greater than 90 degrees to an adjacent angled portion, where the angle formed in the first stage channel is configured to provide an inertial separation of the particles, wherein the channels of the second stage comprise a series of angled portions, with each of the angled portions forming an angle of either less than or greater than 90 degrees to an adjacent angled portion, where the angle formed in the second stage channels is configured to provide an inertial separation of the particles, wherein each of the one or more outlets is configured to receive separated particles of differing sizes and / or differing inertia. The series of angled portions of the first stage channel can be different from the series of angled portions of the second stage channels. The series of angled portions of the first stage channel can be trapezoidal wave shaped, and the series of angled portion of the second stagechannels can be sawtooth wave shaped. The series of angled portions of the first stage channel can be sawtooth wave shaped, and the series of angled portion of the second stage channels can be trapezoidal wave shaped. The angled portions can have a wavelength of 0.1 mm to 5 mm.

[0059] Another aspect provides a sawtooth inertial sorting microfluidic device comprising an inlet for receiving particles of varying sizes and / or differing inertia across varying flow rates; a main channel comprising a first stage, a second stage, and a third stage. The first stage can comprise a first end connected to the inlet and a second end connected to the second stage, the second stage comprising 2, 3, 4, 5, or more channels each connected to one or more outlets and one channel connected to the third stage. The third stage can comprise 2, 3, 4, 5, or more channels each connected to one or more outlets, the first stage can further comprise a channel comprising a series of angled portions, with each of the angled portions forming an angle of either less than or greater than 90 degrees to an adjacent angled portion, where the angle formed in the first stage channel is configured to provide an inertial separation of the particles. The channels of the second stage can comprise a series of angled portions, with each of the angled portions forming an angle of either less than or greater than 90 degrees to an adjacent angled portion, where the angle formed in the second stage channels is configured to provide an inertial separation of the particles. The channels of the third stage can comprise a series of angled portions, with each of the angled portions forming an angle of either less than or greater than 90 degrees to an adjacent angled portion, where the angle formed in the third stage channels is configured to provide an inertial separation of the particles, wherein each of the one or more outlets is configured to receive separated particles of differing sizes and / or differing inertia. The series of angled portions of the first stage channel can be trapezoidal wave shaped or sawtooth wave shaped, the series of angled portions of the second stage channels can be trapezoidal wave shaped or sawtooth wave shaped, and the series of angled portions of the third stage channels can be trapezoidal or sawtooth wave shaped. The angled portions can have a wavelength of 0.1 mm to 5 mm.

[0060] Dual Imaging and Sensing Systems

[0061] A dual imaging / sensing system can include various designs, which are described in detail below. In general, the designs can use 1 , 2, 3, or more light sources and 1 , 2, 3, or more cameras, depending on the exact system design with anappropriate number of filters. Cameras can be substituted with photomultiplier tubes or photodiodes for sensing of fluorescent signals. A light source can be, for example, white light, laser light, LED, xenon arc lamps, tungsten lamps, halogen lamps, fluorescent light, UV light, etc. Similarly, 2, 3, 4, or more distinct images / signals can be obtained by using one light source with 2, 3, 4 or more cameras or sensors or two light sources with one camera, and even one light source and one camera that captures the superimposed 2, 3, 4, or more images / signals. Cameras or sensors used for capturing imaging or sensing modalities beyond DIH (e.g., fluorescence) can be replaced with other types of light sensors. This can include sensors such as photomultiplier tubes, photodiodes, and other sensors. Such sensors can be used to improve the sensitivity and / or sampling rate of the measurements.

[0062] Filters can also be added between the light source and sample in some applications to achieve the desired images / signals. Any type of filter can be used, e.g., a band pass filter, low-pass filter, high-pass filter etc. These designs can be easily expanded to include more cameras or sensors and light sources by additional beam splitters and filters. In some instances, in all configurations, the cameras and / or sensors can be synced to provide images / signals simultaneously. In some instances, if cameras or sensors cannot be synced, they can be started at similar times to provide a complete time course for both image / signal sets over the same time. Additionally, filters can be added between the light source and sample in any of the configurations. Filters can be placed between the light source and the sample for various purposes (e.g., reducing the bandwidth of the light).

[0063] One or more beam splitters can be present and can be designed to target the transmission / reflection of different wavelengths (e.g., as a dichroic beam splitter that splits light into different wavelengths) to reduce unwanted light reaching each camera or sensor.

[0064] In some instances, two or more distinct images / signals can be obtained. The images / signals can be a combination of fluorescent, brightfield, DIH, phase contrast, or any other imaging or sensing modality. In bright field imaging, sample illumination is transmitted with white light (therefore, the light source can be white light) and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample. In phase contrast microscopy, partially coherent illumination produced by a lamp, e.g., a tungsten-halogen lamp is directed through a collector lensand focused on a specialized or condenser annulus positioned at a substage condenser front focal plane. Wavefronts passing through an annulus illuminate the sample and either pass through undeviated or are diffracted and retarded in phase by structures and phase gradients present in the sample. Undeviated and diffracted light collected by the objective is segregated at a rear focal plane by a phase plate and focused at the intermediate image plane to form the final phase contrast image. Two light sources can lead to two different colors / types of images. Multiple filters can be used to filter light from other light sources, increasing the clarity of the images. Two or more cameras or sensors can lead to an increased clarity in each of the images. The images can have an overlapping imaging area and can be taken at (or near) the same time. The images can be aligned such that information from both images can be obtained for particles or cells of interest. Multiple cameras or sensors can be synced such that the images align as desired. The light source(s) can be two distinct light sources with different light paths or have a single light path.

[0065] The wavelengths of the light provided can range between, for example, 350 nanometers (nm) and 800 nm (e.g., 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nm). In some aspects, a light source is disposed above the microfluidic device, and / or another light source is disposed below the microfluidic device.Example Design 1

[0066] A first example design can include a dual imaging / sensing system with one light source or two separate light sources and one or two cameras or sensors. A design can also comprise 1 , 2, 3, 4, or more filters, 1 , 2, 3, 4, or more beam splitters, one or more objectives, one or more computing nodes, and a microfluidic device or observation chamber. FIG. 1 illustrates a dual imaging / sensing system 100 according to a first example design. The embodiment as shown in FIG. 1 can include an imaging system 100 with two separate light sources, two distinct cameras, and two filters. These two cameras can be substituted with sensors such as photomultiplier tubes or photodiodes for sensing of fluorescent signals.

[0067] As shown in FIG. 1 , the system 100 can include multiple cameras or sensors 102A-B. For instance, the first camera 102A can include a camera capable of capturing one or more patterns (e.g., fluorescent, bright field, or phase contrast patterns) and a second camera 102B can include a DIH camera. The cameras 102A-B can have a synchronized exposure start and duration for capturing one or more images as described herein.

[0068] Further, in FIG. 1 , the system 100 can include filters 104A-B. Each filter 104A-B can be disposed in front of a lens of cameras 102A-B. For instance, a first filter 104A can be disposed in front of the lens of the first camera 102A, and a second filter 104B can be disposed in front of the second camera 102B. The filters can filter light of specific frequencies (e.g., as a band pass filter, low-pass filter, high-pass filter) to limit light sources being captured at each camera 102A-B.

[0069] The system 100 can include light sources 106A-B. Each light source 106A-B can emit light sources (e.g., laser light) at a specified frequency (e.g., frequencies ranging from 350-800 nm; e.g., 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nm). For example, a first light source 106A can include a 488 nm laser, and the second light source 106B can include a 405 nm laser. Each light source 106A- B can be directed at a beam splitter 108 configured to direct light from each light source to a corresponding camera 102A-B. The light can be directed through an objective 110 across a transparent microfluidic device 114 carrying flowing cells 112. In an aspect, a cell or particle can be present in an observation chamber.

[0070] In some instances, the light emitted by the first light source is configured to pass along a first path, the first path directing the light from the first light source through the beam splitter to the microfluidic device or observation chamber via an objective. The light can be subsequently backscattered (e.g., reflected back in its original direction) by one or more cells in the microfluidic device or observation chamber and directed to the first camera or sensor via the objective and the beam splitter. Further, the light emitted by the second light source can be configured to pass along a second path, the second path directing the light from the second light source through the microfluidic device or observation chamber and to the second camera or sensor via the objective and the beam splitter.

[0071] The imaging / sensing system 100 can interact with a computing node 116 (or a series of interconnected computing nodes). The computing node 116 can interact with components of the system to cause the system to perform various tasks, such as to emit light (from light sources 106A-B), capture images (by cameras 102A-B) (or signals by sensors), and process the obtained images as described herein.

[0072] As noted above, the design as shown in FIG. 1 illustrates an imaging / sensing system 100 with two filters in place. For instance, a first light path can begin at light source 1 106A, which can illuminate the sample. The light can shine through the sample, going straight through 1 ,2, or more splitter cubes (e.g., 108), and through filter 1 104A to camera 1 102A (or a sensor). The second light path can start at light source 2 106B and can be reflected by a top splitter cube (e.g., 108), proceeding through a bottom splitter cube, and can be focused by the objective 110 before hitting the sample. The reflected light then can return to the top splitter cube, where it can be reflected to camera 2 102B (or sensor) through filter 2 104B. If neither desired image is reflected light, light source 2 and the top splitter cube could be moved below the sample to combine light source 1 and 2 before the sample. If either or both cameras or sensors can distinguish the proper information without filters, one or both filters could be removed. Additional optics can be added for desired images. For example, phase contrast optics can be added for one camera to allow for overlayed phase contrast and brig htfield images.Example Design 2

[0073] A second example design of an imaging / sensing system can include two light sources and a single camera. A design can also comprise 1 , 2, 3, 4, or more filters, 1 , 2, 3, 4, or more beam splitters, one or more objectives, one or more computing nodes, and a microfluidic device or observation chamber. FIG. 2 illustrates a dual imaging / sensing system 200 according to a second example design. As shown in FIG. 2, a single camera 202 can be disposed above a beam splitter 208. The camera can include a lens configured to capture light from light sources 206A-B. Light from either light source 206A-B can be directed through the beam splitter 208 to the transparent microfluidic device 214 via an objective 210. A filter 204 can be present between the camera and the beam splitter. The light can be reflected from the device 214 carrying flowing cells 212 back to the camera 202 via beam splitter 208. In an aspect, particles or cells are present in an observation chamber instead of a microfluidic device.

[0074] As shown in FIG. 2, one configuration of dual imaging can include two light sources and a single camera. In this embodiment, both light sources 206A-B can be disposed above the sample and can be focused by the objective meaning bothimages are of reflected light. Alternatively, one or both light sources could be moved below the sample to capture images of transmitted light.

[0075] In some instances, one light source (e.g., the fluorescence excitation light source) can be above the sample and focused by the objective to generate signals, e.g., fluorescent signals, at a longer wavelength (e.g., a 488 nm excitation generates 540 nm green fluorescence). The other light source can be located below the sample and can be used to generate a DIH signal. A filter (e.g., a long pass filter) can be placed in front of the camera to allow fluorescent and DIH signals to be transmitted and blocks the reflected signal from the excitation laser.Example Design 3

[0076] A third example design can include a dual imaging / sensing system with one light source and two cameras. These two cameras can be substituted with sensors such as photomultiplier tubes or photodiodes for sensing of fluorescent signals

[0077] A design can also comprise 1 , 2, 3, 4, or more filters, 1 , 2, 3, 4, or more beam splitters, one or more objectives, one or more computing nodes, and a microfluidic device or observation chamber. FIG. 3 illustrates a dual imaging / sensing system 300 according to a third example design. As shown in FIG. 3, two cameras 302A-B (or sensors) can be included, with corresponding filters 304A-B in front of the lens of each camera 302A-B (or sensor). Further, a single light source 306 can be disposed below the transparent microfluidic device 314 carrying the flowing cells 312. The light source 306 can emit light (e.g., laser light or other light) through the device 314, through an objective 310 to a beam splitter 308. The beam splitter 308 can include a single cube design that splits the light to each camera 302A-B (or sensor) via filters 304A-B. In an aspect, cells are present in an observation chamber instead of a microfluidic device.

[0078] As noted above, the third example design can include a dual imaging / sensing configuration with a single light source and two cameras. These two cameras can be substituted with sensors such as photomultiplier tubes or photodiodes for sensing of fluorescent signals.

[0079] In this design, the light source can be disposed below the sample, although it can be moved above the sample similar to a second light source in the double light source, double camera / sensor configuration. Both cameras / sensors canhave a filter, but depending on the application, one or either of these filters can be removed.Example Design 4:

[0080] A fourth example design can include a dual imaging / sensing system with one light source and one camera. A design can also comprise 1 , 2, 3, 4, or more filters, 1 , 2, 3, 4, or more beam splitters, one or more objectives, one or more computing nodes, and a microfluidic device or observation chamber. In this design, a single camera can be used to collect two different signals projected to the same image. Specifically, for example, a single camera can capture both the DIH signal and one or more fluorescent signals (or other suitable signal).

[0081] In this setting, a filter can be used in front of the camera to lower the strength of the DIH signal generated from the laser that also excites the fluorescence. The bandpass transmission of the filter can be tuned such that the strength of the excitation laser is lowered to the level such that it becomes comparable to the fluorescent signal generated from the excitation laser. The filter can be tuned to a transmission coefficient at different wavelengths. For example, the filter can be tuned to transmit 1 % of a DIH signal (e.g., 405 nm) and 80% of a fluorescent signal (e.g., 440 nm) such that both DIH and fluorescent signals can be visible and distinguishable in the recorded images, which allows simultaneous recording of both signals. This design can simplify the optical setup.Dual Imaqinq / Sensinq Systems

[0082] FIG. 4 illustrates a first example dual imaging / sensing system 400. The system 400 can include a design similar to the first design as described with respect to FIG. 1. As shown in Figure 4, a prototype of the dual imaging / sensing system can capture the digital inline holographic (DIH) and fluorescent images (or other suitable signals like bright field or phase contrast) for particles or cells flowing in a microfluidic channel. In an aspect, particles or cells are present in an observation chamber instead of a microfluidic channel.

[0083] Furthermore, as shown in FIG. 4, the system 400 can include a DIH camera 402A and a fluorescent camera 402B. The fluorescent cameras can be substituted with sensors such as photomultiplier tubes or photodiodes for sensing of fluorescent signals

[0084] Filters 404A-B can be disposed in front of the lens of each camera 402A- B or sensor).

[0085] The system 400 can also include light sources 406A-B. A light source can have a frequency ranging from 350-800 nm, e.g., 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nm. In an example, a first light source 406A can include a 488nm laser, while a second light source 406B can include a 405 nm laser. The first laser 406A can be disposed above an objective 410, while a second light source 406B can be disposed below the objective 410. The objective 410 can focus light between the beam splitter 408 and a transparent microfluidic device (or a slide) containing traveling (and, in some instances, stationary or fixed) cells.

[0086] FIG. 5 is an example sample image 500 captured by a dual imaging system as described herein. As shown in FIG. 5, a first image (e.g., DIH image 502) can be captured by a first camera, and can identify a cell (e.g., cancer cell 506). Furthermore, the second image (e.g., fluorescent image 504) can capture an image of the cancer cell 506. The fluorescent camera can be substituted with sensors such as photomultiplier tubes or photodiodes for sensing of fluorescent signals.

[0087] The systems as described herein can simultaneously record the holographic and fluorescent signatures (or bright field or phase contrast signatures) of a particle or cell, e.g., a cancer cell. Such system can be used to improve the purity of the various imaging-based particle or cell sorting techniques.

[0088] FIG. 6 illustrates another example dual imaging / sensing system 600. The system 600 as described in FIG. 6 can include a design similar to the third design as described with respect to FIG. 3. As shown in Figure 6, a prototype of the dual imaging / sensing system can capture the digital inline holographic (DIH) and fluorescent images (or bright field or phase contrast images) for particles or cells, e.g., cancer cells flowing in a microfluidic channel or present in an observation chamber.

[0089] The system 600 can include a DIH camera 602B and a fluorescent camera 602A. Filters 604A-B can be disposed in front of the lens of each camera 602A-B. Furthermore, a beam splitter 608 can split light emitted from a light source (e.g., 488 nm laser 606). An objective 610 can be disposed below the beam splitter 608.

[0090] Multi-Sensor Imaging Systems- n,-

[0091] In some aspects more than one camera (e.g., 2, 3, 4, 5, 6 or more) and more than one light source (e.g., 2, 3, 4, 5, 6 or more) can be used. Fig. 9 shows a multi-camera imaging system with two light sources and three cameras. These cameras can be substituted with sensors such as photomultiplier tubes or photodiodes for sensing of fluorescent signals

[0092] Two or more cameras can be used for capturing fluorescent light (or phase contrast or bright field), but the fluorescent cameras can be replaced with other light sensors, for example, photomultiplier tubes or photodiodes for sensing of fluorescent signals. One or more cameras can be used to capture a DIH signal from a light source 906A while additional cameras 902B-902C (or other types of light sensors) can capture fluorescence signals at different wavelengths. Light source 906B can provide light to the cameras or sensors. Filters 904A, 904B, and 904C can be used in front of each camera or sensor to lower the undesired light from either the other light source or from fluorescence. Each bandpass filter used for fluorescence can target a unique range of fluorescent emission wavelengths to capture a more detailed set of fluorescence data. Note also that each beam splitter 908 may be designed to target the transmission / reflection of different wavelengths (e.g., as a dichroic beam splitter that splits the light of different wavelengths) to reduce unwanted light reaching each camera or sensor.

[0093] As shown in FIG. 9, three cameras or sensors 902A-C can be included, with corresponding filters 904A-C in front of the lens of each camera or sensor 902A- B. Furthermore, a light source 906B can be disposed below a transparent microfluidic device 914 carrying the flowing particles or cells 912. The light source 906 can emit light (e.g., laser light) through the device 914, through an objective 910 to a beam splitter 908. The beam splitter 908 can include a single cube design that splits the light to cameras or sensors 902B-C via filters 904B-C. All three cameras or cameras can be synchronized with respect to exposure start and duration. In an aspect, particles or cells are present in an observation chamber instead of a microfluidic device.

[0094] A multi-sensor system can comprise: a first camera configured to capture a first image of a digital inline holographic (DIH) pattern of at least one particle or cell; a second camera or sensor configured to capture a second image of a fluorescent, bright field, or phase contrast pattern of at least one particle or cell; and a third camera or sensor configured to capture a third image of a fluorescent, bright field,or phase contrast pattern of at least one particle or cell. A multi-sensor system can further comprise a first light source configured to emit light of a first wavelength onto a microfluidic device carrying the at least one particle or cell and a second light source configured to emit light of a second wavelength onto a microfluidic device carrying the at least one particle or cell. In an aspect, only one light source is used. A beam splitter can be configured to direct emitted light between the first, second, and / or third cameras or sensors and the microfluidic device carrying the at least one particle or cell. The first light source and the second light source can comprise laser light sources or any other suitable light source. The first wavelength and the second wavelength can range between 350 nanometers (nm) and 800 nm. The first light source can be disposed above or below the microfluidic device. The second light source can be disposed above or below the microfluidic device.

[0095] In an example, a first light source can used to generate fluorescence. The light from the first light source will not only be reflected by the microfluidic device but will also excite the fluorescent signal (at higher wavelength than the wavelength of the first light source) of the particles travelling inside the microfluidic device. The first camera or sensor (the camera / sensor that captures the fluorescent signal) uses beam splitter (e.g., dichroic beam splitter can serve as a filter) and additional filter to remove the reflected light from the first camera / sensor (also the light from the second light source) and captures the fluorescent signal.

[0096] A multi-sensor system can further comprise a first filter disposed in front of a lens of the first camera or sensor; a second filter disposed in front of a lens of the second camera or sensor; and a third filter disposed in front of a lens of the third camera or sensor; wherein each of the first filter, second filter, and third filter are configured to filter out signals of wavelengths other than signals of a specified wavelength range.

[0097] A multi-sensor system can further comprise a computing node. A computing node can comprise a processor and memory, the memory including instructions that, when executed by the processor, cause the processor to send a first instruction to the first light source and to the second light source to emit light. The instructions can further cause the processor to send a second instruction to each of the first camera, the second camera, and the third camera to capture the first image, the second image, and the third image with a specified start time and exposure timefor synchronizing image capturing across the first camera, the second camera, and the third camera. The instructions can further cause the processor to obtain the first image from the first camera, the second image from the second camera, and the third image from the third camera; process the first image, the second image, and the third image to identify a holographic pattern and a fluorescent, bright field, or phase contrast pattern of at least one particle or cell; and determine whether at least one particle or cell is a target particle type or cell type based on the holographic pattern and the fluorescent, bright field, or phase contrast pattern of the at least one particle or cell.

[0098] The microfluidic device can be a sawtooth inertial sorting microfluidic device.

[0099] Methods of using a multi-sensor system can comprise providing a first instruction to a first light source to emit light of a first wavelength and a second instruction to a second light source to emit light of a second wavelength, wherein a first camera is configured to capture a first image of a dual inline holographic (DIH) pattern of at least one particle or cell traveling through a microfluidic device, a second camera or sensor is configured to capture a second image or signal of a fluorescent, bright field, or phase contrast pattern of the at least one particle or cell, and a third camera or sensor is configured to capture a third image or signal of a fluorescent, bright field, or phase contrast pattern of the at least one particle or cell with a specified start time and an exposure time for synchronizing image capturing across the first camera, the second camera / sensor, and the third camera / sensor. The first image can be obtained from the first camera, the second image / signal from the second camera / sensor, and the third image / signal from the third camera / sensor. The first image, second image / signal, and third image / signal can be processed to identify a holographic pattern and a fluorescent, bright field, or phase contrast pattern of the at least one particle or cell. Whether at least one particle or cell is a target particle type or target cell type can be determined based on the holographic pattern and the fluorescent, bright field, or phase contrast pattern of the at least one particle or cell. The microfluidic device can perform an action based on determining that the at least one particle or cell is the target particle type or target cell type. The of the microfluidic device can be the diversion of the target particle or target cell to a specific outlet channel of the microfluidic device.

[0100] The first light source can be disposed above the microfluidic device, and the second light source can be disposed below the microfluidic device. The microfluidic device can be a sawtooth inertial sorting microfluidic device. The target cell type can be a cancerous cell. The first wavelength and second wavelength can range from a 350 nanometer (nm) wavelength to an 800 nanometer (nm) wavelength.Methods for Implementing a Dual Imaqinq / Sensinq System

[0101] FIG. 7 is a flow process 700 of an example method for implementing a dual imaging system. For example, a computing node (or series of interconnected computing nodes) can interact with components in any of the dual imaging / sensing systems described herein to implement the method as described herein.

[0102] At 702, the method can include sending a first instruction to at least a first light source to emit light of a first wavelength. The first instruction can cause a first light source (e.g., 106A) to emit laser light to illuminate the cell(s) traveling in a microfluidic device (e.g., 114). In some instances, the microfluidic device can include a microfluidic chip, well plate, microscope slide, etc. In an aspect, cells are present in an observation chamber instead of a microfluidic device.

[0103] At 704, the method can include sending a second instruction to each of a first camera and a second camera / sensor to capture a first image of a digital inline holographic (DIH) pattern of at least one particle or cell traveling through a microfluidic device and a second image of a pattern, e.g., a fluorescent, bright field, or phase contrast pattern, of at least one particle or cell with a specified start time and an exposure time for synchronizing image capturing across the first camera and second camera / sensor. The combination of the two images can provide both a holographic pattern and a pattern of the particle or cell, e.g., a fluorescent, bright field, or phase contrast pattern.

[0104] At 706, the method can include obtaining the first image from the first camera and the second image from the second camera / sensor. The images / signals can both include the particle or cell with different patterns in each image / signal.

[0105] At 708, the method can include processing the first image and the second image to identify a holographic pattern and a fluorescent, bright field, or phase contrast pattern of at least one particle or cell.

[0106] At 710, the method can include determining whether at least one particle or cell is a target particle or target cell type based on the holographic pattern and thefluorescent, bright field, or phase contrast pattern of at least one particle or cell. A target cell type can include a cancer cell or a circulating tumor cell (CTC), for example. At 712, the method can include causing the microfluidic device to perform an action based on determining that at least one particle or cell is the target particle or target cell type. The action can include causing the microfluidic device to dispense the particle or cell or otherwise cause the particle or cell to separate from a stream of particles or cells, such as a stream of blood cells. For instance, the microfluidic device can connect to a single cell isolation device, which can dispense the cell into a collection container. See, e.g., WO 2023 / 205419, WO 2021 / 155322, and US Pat. Publ. 2023 / 0040252, which are incorporated by reference herein.

[0107] In some instances, the methods can include sending another instruction to a second light source configured to emit light of a second wavelength onto the microfluidic device carrying at least one particle or cell, wherein the first light source and the second light source comprise laser light sources. A light source can have a frequency ranging from 350-800 nm, e.g., 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nm. In an example, the first wavelength is a 488 nanometer (nm) wavelength, and the second wavelength is a 405 nm wavelength.

[0108] In some instances, the light emitted by the first light source is configured to pass along a first path, the first path directing the light from the first light source through a beam splitter to the microfluidic device via an objective, where the light is subsequently reflected by the microfluidic device and directed to the first camera via the objective and the beam splitter, and wherein the light emitted by the second light source is configured to pass along a second path, the second path directing the light from the second light source through the microfluidic device and to the second camera via the objective and the beam splitter.

[0109] In some instances, the first light source is disposed above the microfluidic device, and the second light source is disposed below the microfluidic device.

[0110] Systems

[0111] In an example embodiment, a dual imaging / sensing system is provided. The dual imaging / sensing system (e.g., 100) can include a first camera (e.g., 102A) configured to capture a first image (e.g., 502) of a digital inline holographic (DIH) pattern of at least one particle or cell (e.g., cell 506). The dual imaging / sensing system can also include a second camera / sensor (e.g., 102B) configured to capture a secondimage / signal (e.g., 504) of a fluorescent, bright field, or phase contrast pattern of at least one particle or cell.

[0112] The dual imaging / sensing system can also include at least a first light source (e.g., 106A) configured to emit light of a first wavelength onto a microfluidic device (e.g., 114) carrying at least one particle or cell (e.g., as part of a set of flowing particles or cells 112). The dual imaging / sensing system can also include a beam splitter (e.g., 108) configured to direct the emitted light between the first and second cameras or sensors and a microfluidic device carrying at least one particle or cell. In an aspect, particles or cells are present in an observation chamber instead of a microfluidic device.

[0113] In some instances, the dual imaging / sensing system can also include a second light source (e.g., 106B) configured to emit light of a second wavelength onto the microfluidic device carrying at least one cell. The first light source and the second light source can comprise laser light sources. In some instances, a light source can have a frequency ranging from 350-800 nm, e.g., 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nm. In an example, the first wavelength is a 488 nanometer (nm) wavelength, and wherein the second wavelength is a 405 nm wavelength.

[0114] In some instances, the first light source is disposed above the microfluidic device, and wherein the second light source is disposed below the microfluidic device. In some instances, the light emitted by the first light source is configured to pass along a first path, the first path directing the light from the first light source through the beam splitter to the microfluidic device via an objective, where the light is subsequently reflected by the microfluidic device and directed to the first camera via the objective and the beam splitter. In some instances, the light emitted by the second light source is configured to pass along a second path, the second path directing the light from the second light source through the microfluidic device and to the second camera via the objective and the beam splitter.

[0115] In some instances, the dual imaging / sensing system can also include a first filter (e.g., 104A) disposed in front of a lens of the first camera and a second filter (e.g., 104B) disposed in front of a lens of the second camera or sensor. Each of the first filter and second filter can be configured to filter out signals of wavelengths other than signals of a specified wavelength range.

[0116] In some aspects, a third camera or sensor and / or a third light source can be present. One camera can be a DIH camera, and a second and / or third camera can be a fluorescent, bright field, or phase contrast camera. This camera can be substituted with sensors such as photomultiplier tubes or photodiodes for sensing of fluorescent signals

[0117] In some instances, the dual imaging / sensing system can also include a computing node (e.g., 116), wherein the computing node comprises a processor and memory. The memory can include instructions that, when executed by the processor, cause the processor to send a first instruction to the first light source to emit light and send a second instruction to each of the first camera and the second camera / sensor to capture the first image and the second image / signal with a specified start time and exposure time for synchronizing image capturing across the first camera and second camera / sensor. In some instances, the instructions further cause the processor to: obtain the first image from the first camera and the second image / signal from the second camera / sensor, process the first image and the second image / signal to identify a holographic pattern and a fluorescent pattern of at least one particle or cell, and determine whether at least one particle or cell is a target particle or target cell type based on the holographic pattern and the fluorescent, bright field, or phase contrast pattern of at least one particle or cell.

[0118] In another example embodiment, a system is provided. The system can include at least a first camera, the first camera configured to capture a first image of a digital inline holographic (DIH) pattern and / or a fluorescent, bright field, or phase contrast pattern of at least one particle or cell traveling through a microfluidic device. In an aspect, particles or cells are present in an observation chamber instead of a microfluidic device.

[0119] The system can also include at least a first light source configured to emit light of a first wavelength onto the microfluidic device carrying at least one particle or cell. The system can also include a beam splitter configured to direct the emitted light between at least the first camera and the microfluidic device carrying at least one particle or cell. A light source can have a frequency ranging from 350-800 nm, e.g., 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nm. In some instances, the first wavelength ranges from a 405 nanometer (nm) wavelength to a 488 nanometer (nm) wavelength.

[0120] In some instances, the system can also include a second camera configured to capture a second image of either the digital inline holographic (DIH) pattern and / or the fluorescent, bright field, or phase contrast pattern of at least one particle or cell traveling through the microfluidic device. This camera can be substituted with sensors such as photomultiplier tubes or photodiodes for sensing of fluorescent signals

[0121] In some instances, the system can also include a second light source configured to emit light of a second wavelength onto the microfluidic device carrying at least one particle or cell, wherein the first light source and the second light source comprise laser light sources or other suitable light sources.

[0122] In some instances, the light emitted by the first light source is configured to pass along a first path, the first path directing the light from the first light source through the beam splitter to the microfluidic device via an objective, where the light is subsequently reflected by the microfluidic device and directed to the first camera via the objective and the beam splitter, and wherein the light emitted by the second light source is configured to pass along a second path, the second path directing the light from the second light source through the microfluidic device and to the second camera / sensor via the objective and the beam splitter.

[0123] In some instances, the system can also include a first filter disposed in front of a lens of the first camera and a second filter disposed in front of a lens of the second camera / sensor. Each of the first filter and second filter can be configured to filter out signals of wavelengths other than signals of a specified wavelength range.

[0124] In some instances, the system can also include a computing node, wherein the computing node comprises a processor and memory. The memory can include instructions that, when executed by the processor, cause the processor to send a first instruction to the first light source to emit light and send a second instruction to the first camera to capture the first image. The instructions can further cause the processor to: obtain the first image from the first camera, process the first image to identify either a holographic pattern and / or a fluorescent, bright field, or phase contrast pattern of at least one particle or cell, and determine whether at least one particle or cell is a target particle or target cell type based on the holographic pattern and the fluorescent pattern (or other pattern) of at least one particle or cell. The patterns identified in the images can be compared with one or more controlimages / patterns to compare with the identified patterns to determine a cell type of the cell.

[0125] Detection Methods

[0126] In an embodiment, the devices described herein can be used to detect and / or isolate specific cell types from a biological sample. A biological sample can be, for example, blood, plasma, serum, biopsied tissue, or cells. A tissue or cell sample can be processed so that the cells can be introduced to the devices described herein without clumping. A subject can be a mammal such as a human, canine, feline, bovine, equine, rodent, leporine, porcine. A subject can be a non-human mammal.

[0127] In an aspect, a biological sample is obtained from a subject and the cells labeled using, e.g., a fluorescent label or other suitable label. One or more labels can be, for example, conjugated to an antibody or specific binding fragment that can specifically bind to a marker that is specific for, e.g., a cancer cell or a specific cell type. In an aspect, a target cell, such as a cancerous cell, can express different proteins than non-target cells. In an aspect, a target cell, such as a specific cell type can express unique markers that can be used to identify cells of the specific type. In some aspects, target cells (or optionally non-target cells) can be labeled with a detectable label such as a fluorescent label.

[0128] In an aspect one of more labels conjugated to an antibody or specific binding fragment thereof can specifically bind to markers on a cell surface that is indicative or cancer. Many such markers are known, including, for example, ALDH1A1 , TRA-1-60, SSEA-1 , EpCam, ALDH1A1 , BMI-1 , CXCR4, CXCR1 , c-myc, SOX2, OCT4, KLF4, SALL4, TIM3, Lgr5, CD13, CD15, CD19, CD20, CD24, CD26, CD27, CD34, CD38, CD44, CD45, CD47, CD49f, CD66c, CD90, CD166, TNFRSF16, CD105, CD133, CD117 / c-kit, CD138, CD151 and CD166, CA9, CA12, CXorf61 , GPR87, LYPD3, and SLC7A11 . Other markers can be used to identify specific cells, for example, CD3, CD4, and CD8 markers are expressed by T cells, CD19 and CD20 markers are expressed by B cells, CD11c and CD123 are expressed by dendritic cells, CD56 is expressed by NK cells, CD14 and CD33 are expressed by macrophages and monocytes, CD41 , CD61 and CD62 are expressed by platelets, CD235a is expressed by erythrocytes, CD146 is expressed by endothelial cells, and CD326 is expressed by epithelial cells. In an aspect, no label is required or used.

[0129] In an aspect, a target cell, such as a cancerous cell, can have different morphological and optical characteristics from non-target cells. For example, a cancerous cell can have a large nucleus an irregular size and shape, prominent nucleoli, reduced cytoplasm, intensely colored cytoplasm or pale cytoplasm as compared to a healthy cell. The nucleus of a cancerous cell can exhibit changes to its surface, volume, the nucleus / cytoplasm ratio, shape, and density. Other changes of cancerous cells can include nucleus segmentation, invaginations, heterochromatin reduction, increase of interchromatin and perichromatin granules, increase of nuclear membrane pores, and formation of inclusions. These internal biophysical and biochemical changes can also influence the optical signatures of a cell in addition to morphological characteristics. Other types of cells can be differentiated based on shape, size and structure of the cell as well as the arrangement of any organelles within the cell.

[0130] Therefore, a first camera of the systems described herein can be configured to capture a first image of a DIH pattern of a cell in a sample. This first image can provide lateral and depth resolution at the submicron level in three dimensions and image and characterize the morphology of the cell. A second and / or third camera or sensor can be configured to capture a second image / signal and / or third image / signal of a fluorescent, bright field, or phase contrast pattern of the same cell. The information about the morphology of the cell and the fluorescent, bright field, or phase contrast pattern of the particle or cell can be combined to, for example, accurately identify the particle or cell type. Where a cell is identified as, for example, a cancerous cell, then a diagnosis can be made. Additionally, in some aspects, the type of cancer cell can be determined based on the markers present on the cell surface.

[0131] In an aspect, a method of identifying target cells in a mixed sample of cells is provided. The method can comprise fluorescently labeling the mixed sample of cells and adding the mixed sample of cells to a microfluidic device. In an aspect, the cells are not labeled. In an aspect the cells are static in an observation chamber instead of in a microfluidic device. A first instruction can be provided to at least a first light source to emit light of a first wavelength, wherein a first camera is configured to capture a first image of a DIH pattern of at least one cell traveling through the microfluidic device and a second camera / sensor and / or third camera / sensor isconfigured to capture a second and / or third image / signal of a fluorescent, bright field, or phase contrast pattern of the at least one cell with a specified start time and an exposure time for synchronizing image / signal capturing across the first camera and second and / or third camera / sensor. The first image is obtained from the first camera and the second image / signal is obtained from the second camera / sensor. A third image / signal can be obtained from a third camera / sensor. The first image, second image / signal, and third image / signal can be processed to identify a holographic pattern and a fluorescent, bright field, or phase contrast pattern of the at least one cell. It can then be determined whether the at least one cell is a target cell based on the holographic pattern and the fluorescent, bright field, or phase contrast pattern of at least one cell. The target cells can be, e.g., a cancerous cell or a specific cell type. Where a cancerous cell is detected a diagnosis of cancer can be made.

[0132] Another aspect provides a method of sorting cells. The method can comprise fluorescently labeling cells in a biological sample and adding the cells to a microfluidic device. A first instruction can be provided to at least a first light source to emit light of a first wavelength, wherein a first camera is configured to capture a first image of a DIH pattern of at least one cell traveling through the microfluidic device and a second camera / sensor is configured to capture a second image / signal of a fluorescent pattern of the at least one cell with a specified start time and an exposure time for synchronizing image capturing across the first camera and second camera / sensor. The first image can be obtained from the first camera and the second image / signal can be obtained from the second camera / sensor. The first image and the second image / signal can be processed to identify a holographic pattern and a fluorescent pattern of the at least one cell. It can be determined whether at least one cell is a target cell type based on the holographic pattern and the fluorescent pattern of at least one cell. In an aspect, the microfluidic device can perform an action based on determining that the at least one cell is the target cell type. An example of an action is the diversion of the target cells to a specific outlet channel of the microfluidic device such that the target cells are isolated from non-target cells. The target cell type can be a cancerous cell such as a CTC.Computing Systems

[0133] As noted above, the present systems and methods can implement a computing node (or a series of interconnected computing nodes). The computing nodecan interact with components in the dual imaging / sensing systems as described herein to identify a particle or cell and determine a particle type or cell type of the identified particle or cell.

[0134] For instance, the computing node can synchronize camera / sensor settings for 1 , 2, 3, 4 or more cameras / sensors. The computing node can specify a time to capture an image and a length of time to expose the camera / sensor (or an exposure time). Further, the computing node can cause the light source(s) to emit light at specific time(s) and for specific durations. In some instances, the light source(s) can pulse light and / or synchronize the emission of light based on instructions by the computing node.

[0135] The computing node can obtain images / signals captured by the one or more cameras / sensors and process the images / signals to determine patterns of a particle or cell in the images / signal and determine a particle type or cell type of the identified particle or cell. The images / signals can provide a holographic pattern and a fluorescent pattern (or other pattern) of the cell.

[0136] With respect to digital holographic microscopy, digital holography can be used to record a wave front diffracted from an object by a light source. Utilizing the interference of light from the light source, both amplitude and phase information of an object wave can be recorded to produce a hologram containing the information of the object wave. A three-dimensional image can then be reconstructed from the hologram.

[0137] In an embodiment utilizing digital holographic microscopy, a light source can comprise various types of illuminating devices, such as but not limited to a laser such as a monochromatic laser. A pair of laser light waves is generated from the light source by dividing the laser beam with a beam splitter such that one of the split light waves illuminates the biological fluid. The light diffracted from the particle or cell forms an object wave, which illuminates the light source and is collected by the microscope objective. The remaining laser light wave can be directly detected by the microscope objective to serve as a reference wave. The object and reference waves can interfere with each other to form an interference fringe image which is scanned by an image sensor. Continuing to reference digital holographic microscopy, the object and reference wave fronts can be joined by the beam splitter such that the object and reference wave fronts interfere and create a hologram which can be detected by an image sensor. The computing node can then process the digitalhologram, with the computing node functioning as a digital lens to calculate a viewable image of the object wave front utilizing a numerical reconstruction algorithm.

[0138] Digital holographic microscopy can be utilized to observe particles and cells, such as living cells within a fluid such as saline or biological fluid. From the recorded interference pattern of particles or cells, the intensity and phase shift across various points of the particle or cells can be numerically computed by the computing node. The computing node can thus measure the phase delay images of particles or cells within the fluid to provide quantitative information about the morphological properties (e.g., cellular dry mass, surface texture, shape, etc.) of individual particles or cells within the fluid. By way of example and without limitation, the computing node can be adapted to extract parameters such as cell thickness, cell area, cell volume, cell dry mass, the phase shift across the cell, surface roughness and texture, cell shape, elongation, convexity, luminance, circularity, solidity, and the like.

[0139] Various types of digital holography can be utilized with the systems and methods described herein, including but not limited to off-axis Fresnel, Fourier, image plane, in-line, Gabor, and phase-shifting digital holography. By utilizing digital holographic microscopy, the computing node can differentiate between the various constituents within a sample, such as a biological fluid sample, for further processing utilizing the systems and methods described herein. It should be appreciated that multiple laser wavelengths can be utilized when scanning the fluid with digital holographic microscopy. It has been shown that the refraction amount increases as the wavelength of light decreases. Thus, shorter wavelengths of light (e.g., violet and blue) are more slowed and consequently experience more bending than longer wavelengths of light (e.g., orange and red).

[0140] Based on the detected patterns of an identified particle or cell, the computing node can determine a particle or cell type. For example, the computing node can determine whether the cell is a cancer cell or a CTC cell. Responsive to determining the cell is a target cell type (e.g., a CTC), the computing node can cause a further action, such as to separate the cell or to cause a single cell isolation device to drop a droplet comprising the cell into a collection container.

[0141] FIG. 8 is a block diagram of a special-purpose computer system 800 according to an embodiment. For example, system 800 can deployed as part of a computing node as described herein. The methods and processes described hereincan similarly be implemented by tangible, non-transitory computer readable storage mediums and / or computer-program products that direct a computer system to perform the actions of the methods and processes described herein. Each such computerprogram product can comprise sets of instructions (e.g., codes) embodied on a computer-readable medium that directs the processor of a computer system to perform corresponding operations. The instructions can be configured to run in sequential order, or in parallel (such as under different processing threads), or in a combination thereof.

[0142] Special-purpose computer system 800 comprises a computer 802, a monitor 804 coupled to computer 802, one or more additional user output devices 806 (optional) coupled to computer 802, one or more user input devices 808 (e.g., keyboard, mouse, track ball, touch screen) coupled to computer 802, an optional communications interface 810 coupled to computer 802, and a computer-program product including a tangible computer-readable storage medium 812 in or accessible to computer 802. Instructions stored on computer-readable storage medium 812 can direct system 800 to perform the methods and processes described herein. Computer 802 can include one or more processors 814 that communicate with a number of peripheral devices via a bus subsystem 816. These peripheral devices can include user output device(s) 806, user input device(s) 808, communications interface 810, and a storage subsystem, such as random access memory (RAM) 818 and nonvolatile storage drive 820 (e.g., disk drive, optical drive, solid state drive), which are forms of tangible computer-readable memory.

[0143] Computer-readable medium 812 can be loaded into random access memory 818, stored in non-volatile storage drive 820, or otherwise accessible to one or more components of computer 802. Each processor 814 can comprise a microprocessor, such as a microprocessor from Intel® or Advanced Micro Devices, Inc.®, or the like. To support computer-readable medium 812, the computer 802 runs an operating system that handles the communications between computer-readable medium 812 and the above-noted components, as well as the communications between the above-noted components in support of the computer-readable medium 812. Exemplary operating systems include Windows® or the like from Microsoft Corporation, Solaris® from Sun Microsystems, LINUX, UNIX, and the like. In many embodiments and as described herein, the computer-program product can be anapparatus (e.g., a hard drive including case, read / write head, etc., a computer disc including case, a memory card including connector, case, etc.) that includes a computer-readable medium (e.g., a disk, a memory chip, etc.). In other embodiments, a computer-program product can comprise the instruction sets, or code modules, themselves, and be embodied on a computer-readable medium.

[0144] User input devices 808 include all possible types of devices and mechanisms to input information to computer system 802. These can include a keyboard, a keypad, a mouse, a scanner, a digital drawing pad, a touch screen incorporated into the display, audio input devices such as voice recognition systems, microphones, and other types of input devices. In various embodiments, user input devices 808 are typically embodied as a computer mouse, a trackball, a track pad, a joystick, wireless remote, a drawing tablet, a voice command system. User input devices 808 typically allow a user to select objects, icons, text and the like that appear on the monitor 804 via a command such as a click of a button or the like. User output devices 806 include all possible types of devices and mechanisms to output information from computer 802. These can include a display (e.g., monitor 804), printers, non-visual displays such as audio output devices, etc.

[0145] Communications interface 810 provides an interface to other communication networks and devices and can serve as an interface to receive data from and transmit data to other systems, WANs and / or the Internet, via a wired or wireless communication network 822. In addition, communications interface 810 can include an underwater radio for transmitting and receiving data in an underwater network. Embodiments of communications interface 810 typically include an Ethernet card, a modem (telephone, satellite, cable, ISDN), a (asynchronous) digital subscriber line (DSL) unit, a FireWire® interface, a USB® interface, a wireless network adapter, and the like. For example, communications interface 810 can be coupled to a computer network, to a FireWire® bus, or the like. In other embodiments, communications interface 810 can be physically integrated on the motherboard of computer 802, and / or can be a software program, or the like.

[0146] RAM 818 and non-volatile storage drive 820 are examples of tangible computer-readable media configured to store data such as computer-program product embodiments of the present invention, including executable computer code, human- readable code, or the like. Other types of tangible computer-readable media includefloppy disks, removable hard disks, optical storage media such as CD-ROMs, DVDs, bar codes, semiconductor memories such as flash memories, read-only-memories (ROMs), battery-backed volatile memories, networked storage devices, and the like. RAM 818 and non-volatile storage drive 820 can be configured to store the basic programming and data constructs that provide the functionality of various embodiments of the present invention, as described above.

[0147] Software instruction sets that provide the functionality of the present invention may be stored in computer-readable medium 812, RAM 818, and / or nonvolatile storage drive 820. These instruction sets or code can be executed by the processor(s) 814. Computer-readable medium 812, RAM 818, and / or non-volatile storage drive 820 can also provide a repository to store data and data structures used in accordance with the present invention. RAM 818 and non-volatile storage drive 820 can include a number of memories including a main random access memory (RAM) to store instructions and data during program execution and a read-only memory (ROM) in which fixed instructions are stored. RAM 818 and non-volatile storage drive 820 can include a file storage subsystem providing persistent (non-volatile) storage of program and / or data files. RAM 818 and non-volatile storage drive 820 can also include removable storage systems, such as removable flash memory.

[0148] Bus subsystem 816 provides a mechanism to allow the various components and subsystems of computer 802 communicate with each other as intended. Although bus subsystem 816 is shown schematically as a single bus, alternative embodiments of the bus subsystem can utilize multiple busses or communication paths within the computer 802.

[0149] For a firmware and / or software implementation, the methodologies can be implemented with modules (e.g., procedures, functions, and so on) that perform the functions described herein. Any machine-readable medium tangibly embodying instructions can be used in implementing the methodologies described herein. For example, software codes can be stored in a memory. Memory can be implemented within the processor or external to the processor. As used herein the term “memory” refers to any type of long term, short term, volatile, nonvolatile, or other storage medium and is not to be limited to any particular type of memory or number of memories, or type of media upon which memory is stored.

[0150] Moreover, as disclosed herein, the term “storage medium” can represent one or more memories for storing data, including read only memory (ROM), random access memory (RAM), magnetic RAM, core memory, magnetic disk storage mediums, optical storage mediums, flash memory devices and / or other machine readable mediums for storing information. The term “machine-readable medium” includes, but is not limited to portable or fixed storage devices, optical storage devices, wireless channels, and / or various other storage mediums capable of storing that contain or carry instruction(s) and / or data.Conclusion

[0151] It will be understood that terms such as “top,” “bottom,” “above,” “below,” and x-direction, y-direction, and z-direction as used herein as terms of convenience that denote the spatial relationships of parts relative to each other rather than to any specific spatial or gravitational orientation. Thus, the terms are intended to encompass an assembly of component parts regardless of whether the assembly is oriented in the particular orientation shown in the drawings and described in the specification, upside down from that orientation, or any other rotational variation.

[0152] The compositions and methods described herein are illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art. The terms used in the specification generally have their ordinary meanings in the art, within the context of the compositions and methods described herein, and in the specific context where each term is used. Some terms have been more specifically defined herein to provide additional guidance to the practitioner regarding the description of the compositions and methods.

[0153] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference as well as the singular reference unless the context clearly dictates otherwise. The term “about” in association with a numerical value means that the value varies up or down by 5%. For example, for a value of about 100, means 95 to 105 (or any value between 95 and 105).

[0154] All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference herein in their entirety. The embodiments illustratively described herein suitably can be practiced in the absenceof any element or elements, limitation or limitations that are specifically or not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claims.

[0155] Thus, it should be understood that although the present methods and compositions have been specifically disclosed by embodiments and optional features, modifications and variations of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the compositions and methods as defined by the description and the appended claims.

[0156] Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein can each be specifically excluded from the claims.

[0157] Whenever a range is given in the specification, for example, a temperature range, a time range, a composition, or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein. It will be understood that any elements or steps that are included in the description herein can be excluded from the claimed compositions or methods

[0158] In addition, where features or aspects of the compositions and methods are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the compositions and methods are also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

Claims

CLAIMSWhat is claimed is:

1. A dual sensing system comprising: a first camera configured to capture a first image of a digital inline holographic (DIH) pattern of at least one particle or cell; a second camera or sensor configured to capture a second image or signal of a fluorescent pattern of at least one particle or cell; at least a first light source configured to emit light of a first wavelength onto a microfluidic device carrying at least one particle or cell; and a beam splitter configured to direct emitted light between the first and second cameras and the microfluidic device carrying the at least one particle or cell.

2. The dual sensing system of claim 1 , further comprising: a second light source configured to emit light of a second wavelength onto the microfluidic device carrying at least one particle or cell, wherein the first light source and the second light source comprise laser light sources.

3. The dual sensing system of claim 2, wherein the first wavelength and the second wavelength ranges between 350 nanometers (nm) and 800 nm.

4. The dual sensing system of claim 2, wherein the first light source is disposed above the microfluidic device, and wherein the second light source is disposed below the microfluidic device.

5. The dual sensing system of claim 4, wherein the light emitted by the first light source is configured to pass along a first path, the first path directing the light from the first light source through the beam splitter to the microfluidic device via an objective, where the light is subsequently reflected by the microfluidic device and directed to the first camera via the objective and the beam splitter.

6. The dual sensing system of claim 5, wherein the light emitted by the second light source is configured to pass along a second path, the second path directing thelight from the second light source through the microfluidic device and to the second camera / sensor via the objective and the beam splitter.

7. The dual sensing system of claim 1 or 2, further comprising: a first filter disposed in front of a lens of the first camera; and a second filter disposed in front of a lens of the second camera or sensor, wherein each of the first filter and second filter are configured to filter out signals of wavelengths other than signals of a specified wavelength range.

8. The dual sensing system of claim 1 or 2, further comprising: a computing node, wherein the computing node comprises a processor and memory, the memory including instructions that, when executed by the processor, cause the processor to: send a first instruction to the first light source and / or second light source to emit light.

9. The dual sensing system of claim 8, wherein the instructions further cause the processor to: send a second instruction to each of the first camera and the second camera or sensor to capture the first image and the second image or signal with a specified start time and exposure time for synchronizing image capturing across the first camera and second camera or sensor.

10. The dual sensing system of claim 8, wherein the instructions further cause the processor to: obtain the first image from the first camera and the second image or signal from the second camera or sensor; process the first image and the second image or signal to identify a holographic pattern and a fluorescent pattern of the at least one particle or cell; and determine whether the at least one particle or cell is a target particle or target cell type based on the holographic pattern and the fluorescent pattern of the at least one particle or cell.11 . The dual sensing system of claim 1 , wherein the microfluidic device is a sawtooth inertial sorting microfluidic device.

12. A method comprising: providing a first instruction to at least a first light source to emit light of a first wavelength, wherein a first camera is configured to capture a first image of a dual inline holographic (DIH) pattern of at least one particle or cell traveling through a microfluidic device and a second camera or sensor is configured to capture a second image or signal of a fluorescent pattern of the at least one particle or cell with a specified start time and an exposure time for synchronizing image capturing across the first camera and second camera or sensor; obtaining the first image from the first camera and the second image or signal from the second camera; processing the first image and the second image or signal to identify a holographic pattern and a fluorescent pattern of the at least one particle or cell; determining whether the at least one particle or cell is a target particle or target cell type based on the holographic pattern and the fluorescent pattern of the at least one particle or cell; and causing the microfluidic device to perform an action based on determining that at least one particle or cell is the target particle type or target cell type.

13. The method of claim 12, further comprising: sending a second instruction to a second light source configured to emit light of a second wavelength onto the microfluidic device carrying at least particle or one cell.

14. The method of claim 13, wherein the first light source and the second light source comprise laser light sources, wherein the first wavelength and the second wavelength ranges between 350 nanometers (nm) and 800 nm.

15. The method of claim 13, wherein the first light source and the second light source comprise laser light sources, wherein the first wavelength is a 488 nanometer (nm) wavelength, and wherein the second wavelength is a 405 nm wavelength.

16. The method of claim 13, wherein the light emitted by the first light source is configured to pass along a first path, the first path directing the light from the first light source through a beam splitter to the microfluidic device via an objective, where the light is subsequently reflected by the cell samples in the microfluidic device and directed to the first camera via the objective and the beam splitter, and wherein the light emitted by the second light source is configured to pass along a second path, the second path directing the light from the second light source through the microfluidic device and to the second camera or sensor via the objective and the beam splitter(s).

17. The method of claim 13, wherein the first light source is disposed above the microfluidic device, and wherein the second light source is disposed below the microfluidic device.

18. The method of claim 12 or 13, wherein the microfluidic device is a sawtooth inertial sorting microfluidic device.

19. The method of claim 12 or 13, wherein the action is the diversion of the target particle or cell to a specific outlet channel of the microfluidic device.

20. A system comprising: at least a first camera, the first camera configured to capture a first image of a digital inline holographic (DIH) pattern and / or a fluorescent pattern of at least one particle or cell traveling through a microfluidic device; at least a first light source configured to emit light of a first wavelength onto the microfluidic device carrying at least one particle or cell; and a beam splitter configured to direct emitted light between at least the first camera and the microfluidic device carrying at least one particle or cell.21 . The system of claim 20, wherein the first wavelength ranges from a 350 nanometer (nm) wavelength to an 800 nanometer (nm) wavelength.

22. The system of claim 20, further comprising: a second camera configured to capture a second image of either the digital inline holographic (DIH) pattern and / or the fluorescent pattern of at least one particle or cell traveling through the microfluidic device.

23. The system of claim 20, further comprising: a second light source configured to emit light of a second wavelength onto the microfluidic device carrying at least one particle or cell.

24. The system of claim 23, wherein the light emitted by the first light source is configured to pass along a first path, the first path directing the light from the first light source through the beam splitter to the microfluidic device via an objective, where the light is subsequently backscattered by one or more particle or cells in the microfluidic device and directed to the first camera via the objective and the beam splitter, and wherein the light emitted by the second light source is configured to pass along a second path, the second path directing the light from the second light source through the microfluidic device and to the second camera via the objective and the beam splitter.

25. The system of claim 20, further comprising: a first filter disposed in front of a lens of the first camera; and a second filter disposed in front of a lens of the second camera.

26. The system of claim 20, further comprising: a computing node, wherein the computing node comprises a processor and memory, the memory including instructions that, when executed by the processor, cause the processor to: send a first instruction to the first light source to emit light; send a second instruction to the first camera to capture the first image; obtain the first image from the first camera; process the first image to identify either a holographic pattern and / or a fluorescent pattern of at least one particle or cell; anddetermine whether the at least one particle or cell is a target particle type or a target cell type based on the holographic pattern and the fluorescent pattern of at least one particle or cell.

27. The system of claim 20, wherein the microfluidic device is a sawtooth inertial sorting microfluidic device.

28. A dual sensing system comprising: a camera configured to capture an image of a digital inline holographic (DIH) pattern and / or a fluorescence pattern of at least one particle or cell; at least a first light source configured to emit light of a first wavelength onto a microfluidic device carrying at least one particle or cell; a second light source configured to emit light of a second wavelength onto the microfluidic device carrying at least one particle or cell; and a beam splitter configured to direct emitted light between the first and second cameras and the microfluidic device carrying the at least one particle or cell.

29. The dual sensing system of claim 28, wherein the microfluidic device is a sawtooth inertial sorting microfluidic device.

30. A method of identifying target cells in a mixed sample of cells comprising:(i) adding a mixed sample of fluorescently labeled cells to a microfluidic device;(ii) providing a first instruction to at least a first light source to emit light of a first wavelength, wherein a first camera is configured to capture a first image of a dual inline holographic (DIH) pattern of at least one cell traveling through the microfluidic device and a second camera or sensor is configured to capture a second image or signal of a fluorescent pattern of the at least one cell with a specified start time and an exposure time for synchronizing image capturing across the first camera and second camera;(iii) obtaining the first image from the first camera and the second image or signal from the second camera or sensor;(iv) processing the first image and the second image or signal to identify a holographic pattern and a fluorescent pattern of the at least one cell; and(v) determining whether the at least one cell is a target cell based on the holographic pattern and the fluorescent pattern of at least one cell.31 . The method of claim 30, wherein the target cell is a cancerous cell.

32. The method of claim 30, further comprising causing the microfluidic device to perform an action based on determining that the at least one cell is the target cell type.

33. The method of claim 32, wherein the action is the diversion of the target cell to a specific outlet channel of the microfluidic device.

34. The method of claim 30 wherein the microfluidic device is a sawtooth inertial sorting microfluidic device.

35. A method of sorting cells comprising:(i) adding fluorescently labeled cells to a microfluidic device;(ii) providing a first instruction to at least a first light source to emit light of a first wavelength, wherein a first camera is configured to capture a first image of a dual inline holographic (DIH) pattern of at least one cell traveling through the microfluidic device and a second camera or sensor is configured to capture a second image or signal of a fluorescent pattern of the at least one cell with a specified start time and an exposure time for synchronizing image capturing across the first camera and second camera or sensor;(iii) obtaining the first image from the first camera and the second image or signal from the second camera or sensor;(iv) processing the first image and the second image or signal to identify a holographic pattern and a fluorescent pattern of the at least one cell;(v) determining whether at least one cell is a target cell type based on the holographic pattern and the fluorescent pattern of at least one cell; and(vi) causing the microfluidic device to perform an action based on determining that the at least one cell is the target cell type.

36. The method of claim 35, wherein the target cell type is a cancerous cell.

37. The method of claim 35, wherein the action is the diversion of the target cells to a specific outlet channel of the microfluidic device such that the target cells are isolated.

38. The method of claim 35, wherein the microfluidic device is a sawtooth inertial sorting microfluidic device.

39. A multi-sensor system comprising: a first camera configured to capture a first image of a digital inline holographic (DIH) pattern of at least one particle or cell; a second camera or sensor configured to capture a second image or signal of a fluorescent pattern of at least one particle or cell; a third camera or sensor configured to capture a third image or signal of a fluorescent pattern of at least one particle or cell; a first light source configured to emit light of a first wavelength onto a microfluidic device carrying the at least one particle or cell; and a second light source configured to emit light of a second wavelength onto a microfluidic device carrying the at least one particle or cell; and a beam splitter configured to direct emitted light between the first, second, and / or third cameras and the microfluidic device carrying the at least one particle or cell.

40. The multi-sensor system of claim 39, wherein the first light source and the second light source comprise laser light sources.41 . The multi-sensor system of claim 39, wherein the first wavelength and the second wavelength ranges between 350 nanometers (nm) and 800 nm.

42. The multi-sensor system of claim 39, wherein the first light source is disposed above the microfluidic device, and wherein the second light source is disposed below the microfluidic device.

43. The multi-sensor system of claim 39, further comprising: a first filter disposed in front of a lens of the first camera; a second filter disposed in front of a lens of the second camera or sensor; and a third filter disposed in front of a lens of the third camera or sensor; wherein each of the first filter, second filter, and third filter are configured to filter out signals of wavelengths other than signals of a specified wavelength range.

44. The multi-sensor system of claim 39, further comprising: a computing node, wherein the computing node comprises a processor and memory, the memory including instructions that, when executed by the processor, cause the processor to send a first instruction to the first light source and to the second light source to emit light.

45. The multi-sensor system of claim 44, wherein the instructions further cause the processor to: send a second instruction to each of the first camera, the second camera or sensor, and the third camera or sensor to capture the first image, the second image or signal, and the third image or signal with a specified start time and exposure time for synchronizing image or signal capturing across the first camera, the second camera or sensor, and the third camera or sensor.

46. The multi-sensor system of claim 44, wherein the instructions further cause the processor to: obtain the first image from the first camera, the second image or signal from the second camera or sensor, and the third image or signal from the third camera or sensor; process the first image, the second image or signal, and the third image or signal to identify a holographic pattern and a fluorescent pattern of at least one particle or cell; and determine whether at least one particle or cell is a target particle type or cell type based on the holographic pattern and the fluorescent pattern of the at least one particle or cell.

47. The multi-sensor system of claim 39, wherein the microfluidic device is a sawtooth inertial sorting microfluidic device.

48. A method comprising: providing a first instruction to a first light source to emit light of a first wavelength and a second instruction to a second light source to emit light of a second wavelength, wherein a first camera is configured to capture a first image of a dual inline holographic (DIH) pattern of at least one particle or cell traveling through a microfluidic device, a second camera or sensor is configured to capture a second image or signal of a fluorescent pattern of the at least one particle or cell, and a third camera or sensor is configured to capture a third image or signal of a fluorescent pattern of the at least one particle or cell with a specified start time and an exposure time for synchronizing image or signal capturing across the first camera, the second camera or sensor, and the third camera or sensor; obtaining the first image from the first camera, the second image or signal from the second camera or sensor, and the third image or signal from the third camera or sensor; processing the first image, the second image or signal, and the third image or signal to identify a holographic pattern and a fluorescent pattern of the at least one particle or cell; determining whether the at least one particle or cell is a target particle type or target cell type based on the holographic pattern and the fluorescent pattern of the at least one particle or cell; and causing the microfluidic device to perform an action based on determining that the at least one particle or cell is the target particle type or target cell type.

49. The method of claim 48, wherein the first light source is disposed above the microfluidic device, and wherein the second light source is disposed below the microfluidic device.

50. The method of claim 48, wherein the microfluidic device is a sawtooth inertial sorting microfluidic device.51 . The method of claim 48, wherein the action is the diversion of the target particle or target cell to a specific outlet channel of the microfluidic device.

52. The method of claim 48, wherein the target cell type is a cancerous cell.

53. The method of claim 48, wherein the first wavelength and second wavelength ranges from a 350 nanometer (nm) wavelength to an 800 nanometer (nm) wavelength.

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