WO2022061068A1

WO2022061068A1 - Systems and methods for multiplexed nano- and micro-scale particle detection

Info

Publication number: WO2022061068A1
Application number: PCT/US2021/050801
Authority: WO
Inventors: Oguzhan AVCI; Nick N. OZA
Original assignee: Myrrym Corporation
Priority date: 2020-09-18
Filing date: 2021-09-17
Publication date: 2022-03-24

Abstract

Disclosed herein are systems and methods that use a light source to detect nano- and micro-scale particles in a sample. The systems disclosed herein may include one or more microlens arrays, eliminating or reducing the need for scanning. Additionally, the microlens arrays may reduce the overall size and costs of the system. The systems provided herein are cost-effective and can easily be manufactured in high volumes, without compromising on performance. The systems and techniques disclosed herein may be used in detecting nano- and micro-scale particles based on the interference of scattered light from particles and reflected light from a planar surface upon which the particles are immobilized, resulting in the elimination of labeling.

Description

SYSTEMS AND METHODS FOR MULTIPLEXED NANO- AND MICRO-SCALE PARTICLE DETECTION

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The application claims the benefit of U.S. Provisional Application No. 63/080,638, filed September 18, 2020, the contents of which are incorporated herein by reference in its entirety for all purposes.

FIELD

[0002] The disclosure relates generally to imaging of nano- and micro-scale particles in a sample, and more specifically to, detecting such particles based on the interference of scattered light and reflected light from the particles without the need for labeling.

BACKGROUND

[0003] Imaging nano- and micro-scale particles, such as pathogens, in a label-free, high- throughput, and cost-effective manner has posed a significant challenge due to the weakly scattering nature of such particles. Conventional imaging techniques are generally unable to detect such particles. Therefore, detecting them directly typically has involved laborious and costly techniques such as electron microscopy, which played a significant role in the discovery of the Ebola virus during the outbreak. Although these laborious techniques may detect such particles and provide structural detail, the techniques and associated systems are expensive and bulky, making it impractical for commercial use.

[0004] The world of diagnostics has therefore relied heavily on DNA based amplification techniques such as Polymerase Chain Reaction (PCR) and Loop-mediated Isothermal Amplification (LAMP). Such techniques involve extracting specific DNA from a target pathogen of interest and amplification thereof to be able to detect pathogen presence in sample. Another commonly used technique for detection of pathogens is Enzyme-linked Immunosorbent Assay (ELISA), which is often used as a qualitative test providing positive or negative results for a sample. ELISA requires labelling of target pathogens or ligands with certain chemicals to provide a color, fluorescent, or electrochemical signal. Being able to detect individual pathogens directly without requiring any labels and extended lab preparations could significantly improve the healthcare industry.

[0005] Most interferometric detection mechanisms make use of lasers that require expensive components (such as acousto optical modulators) to mitigate some of the inherent physical problems, such as speckle, in the resulting image/signal which significantly reduces the particle signal-to-noise ratio. Furthermore, due to the small sizes of the particles of interest, a high magnification is required which reduces the field-of-view and results in needing some type of scanning system (e.g., scans in x- and y-directions). This results in systems needing bulky components to scan across a slide comprising the particles of interest to image the entire functionalized area.

[0006] What is needed is a system and method capable of detecting nano- and micro-scale particles (e.g., pathogens) using techniques and systems that are not expensive or bulky. What is also needed is a system and method capable of detecting nano- and micro-scale particles without the need for scanning. What is also needed is a system and method capable of detecting nano- and micro-scale particles without the need for labeling.

BRIEF SUMMARY

[0007] Disclosed herein are systems and methods that use a spatially incoherent light source to detect nano- and micro-scale particles in a sample. In interferometric imaging techniques, the effects of speckle typically reduce the signal-to-noise ratio. The use of a spatially incoherent light source is used to counteract this reduction in the signal-to-noise ratio because it minimizes the effects of speckle, which thereby allows the system to detect light that has weakly scattered from the particles. The systems disclosed herein may include one or more microlens arrays, eliminating or reducing the need for scanning. Additionally, the microlens arrays may reduce the overall size and costs of the system. The systems provided herein are cost-effective and can easily be manufactured in high volumes, without compromising on performance. The systems and techniques disclosed herein may be used in detecting nano- and micro-scale particles based on the interference of scattered light from particles and reflected light from a planar surface (e.g., a slide) upon which the particles are immobilized, resulting in the elimination of labeling. [0008] A method for imaging one or more samples is disclosed. The method comprises: illuminating the one or more samples with incident light, the one or more samples including one or more particles, wherein a first of the incident light interacts with at least one particle of the one or more particles in the one or more samples and returns as scattered light, wherein a second of the incident light returns as reflected light upon specular reflection off of a slide, wherein the at least one particle is a nano particle or a micro particle; forming a reference light from the reflected light; allowing the scattered light and the reference light to coherently mix to form at least one diffraction limited spot; receiving the at least one diffraction limited spot at a sensor rendering the detection of the at least one particle; determining a normalized intensity for the at least one diffraction limited spot; and determining one or more properties of the at least one particle in the one or more samples based on the at least one diffraction limited spot and the corresponding normalized intensity. In some embodiments, the method further comprises: determining a presence of a pathogen in the one or more samples based on the one or more properties of the at least one particle being associated with one or more properties of the pathogen. In some embodiments, the pathogen is associated with one or more of: a virus, bacteria, and labeled markers. In some embodiments, the virus is the coronavirus (COVID). In some embodiments, the method further comprises: determining a presence of a plurality of pathogens in the one or more samples based on at least two of the one or more particles being located at different functionalized spots on the slide, wherein the one or more samples are deposited on the slide, wherein each of the at least two of the one or more particles is associated with different pathogens of the plurality of pathogens. In some embodiments, the different pathogens are associated with diffraction limited spots having one or more different properties. In some embodiments, the method further comprises: determining an absence of a pathogen in the one or more samples based on an absence of a diffraction limited spot having one or more properties associated with one or more properties of the pathogen. In some embodiments, the illuminating the one or more samples includes simultaneously illuminating a plurality of functionalized spots included in the slide, wherein the one or more samples are deposited on the slide. In some embodiments, the receiving the at least one diffraction limited spot at the sensor includes simultaneously receiving the scattered light and the reference light at the sensor from a plurality of functionalized spots included in the slide, wherein the one or more samples are deposited on the slide. [0009] An interferometry system imaging one or more samples is disclosed. In some embodiments, the one or more samples include one or more particles, the interferometry system comprising: a slide that receives the one or more samples; a light source that illuminates the one or more samples with incident light, wherein a first of the incident light interacts with at least one particle of the one or more particles in the one or more samples and returns as scattered light, wherein a second of the incident light returns as reflected light, and the reflected light forms a reference light, wherein the at least one particle is a nano particle or a micro particle; one or more lenses that receive and direct the scattered light and the reference light, wherein the interferometry system allows the scattered light and the reference light to coherently mix to form at least one diffraction limited spot; a sensor that receives the at least one diffraction limited spot; and a controller for: determining a normalized intensity for the at least one diffraction limited spot, and determining one or more properties of the at least one particle in the one or more samples based on the at least one diffraction limited spot and the corresponding normalized intensity. In some embodiments, the slide includes one or more of: fused silica, BK7, and sapphire. In some embodiments, the slide includes a coating, the reflectivity of the coating being tuned to one or more target wavelengths of the incident light. In some embodiments, the interferometry system further comprises: a beam splitter that directs at least a portion of the incident light towards the slide and direct at least a portion of the scattered light and the reference light towards the sensor. In some embodiments, the beam splitter reflects the at least the portion of the incident light towards the slide and transmits the at least the portion of the scattered light and the reference light towards the sensor. In some embodiments, the beam splitter transmits the at least the portion of the incident light towards the slide and reflects the at least the portion of the scattered light and the reference light towards the sensor. In some embodiments, the beam splitter is one of: a prism beam splitter or a plate beam splitter having polarizing or non-polarizing properties. In some embodiments, the beam splitter includes a beam splitter coating, the beam splitter coating directing the incident light towards the one or more samples. In some embodiments, the beam splitter includes a beam splitter coating, the beam splitter coating directing the scattered light and reflected light towards the sensor. In some embodiments, the interferometry system further comprises: a stage that moves the slide in one or more directions such that the light source illuminates multiple areas of the slide for the imaging of the one or more samples. In some embodiments, the slide includes a plurality of functionalized spots, wherein the one or more lenses include at least one microlens array, the at least one microlens array simultaneously directing the incident light from the light source array towards the sample. In some embodiments, the slide includes a plurality of functionalized spots, wherein the one or more lenses include at least one microlens array, the at least one microlens array simultaneously directing the scattered light and the reference light from the plurality of functionalized spots towards the sensor. In some embodiments, the interferometry system further comprises: a mirror prism directing the incident light towards a first lens of the one or more lenses, wherein the first lens includes a beam splitter coating that directs the incident light towards the sample. In some embodiments, the interferometry system further comprises: a mirror prism configured to direct the scattered light and the reference light towards the sensor. In some embodiments, the interferometry system further comprises: the light source and the sensor are co-planar. In some embodiments, the interferometry system further comprises: the light source is one of: an edge-emitting laser, a vertical-cavity surface-emitting laser (VCSEL), or a light emitting diode (LED). In some embodiments, the interferometry system further comprises: the light source is one of: a single wavelength light source or a multiple wavelength light source. In some embodiments, the interferometry system further comprises: the controller further determines a presence of a pathogen in the one or more samples based on the one or more properties of the at least one particle being associated with one or more properties of the pathogen. In some embodiments, the interferometry system further comprises: the controller further determines an absence of a pathogen in the one or more samples based on an absence of a diffraction limited spot having one or more properties associated with one or more properties of the pathogen.

DESCRIPTION OF THE FIGURES

[0010] FIG. 1 is a cross-sectional view of an exemplary diagram that depicts how incident light is perturbed by the presence of a particle on a slide.

[0011] FIG. 2A depicts an exemplary method for illuminating a sample and detecting particles in the sample, according to some embodiments.

[0012] FIGS. 2B and 2C depict exemplary graphs showing normalized intensity and dark spots, respectively, of a particle, according to some embodiments. [0013] FIG. 2D illustrates a flowchart of an exemplary process for determining the normalized intensity for a given particle, according to embodiments of the disclosure.

[0014] FIG. 2E illustrates an exemplary image of 100 nm diameter polystyrene beads captured using an interferometry system, according to some embodiments.

[0015] FIG. 2F illustrates a flowchart of an exemplary process for scanning an image, according to embodiments of the disclosure.

[0016] FIG. 3 depicts an exemplary slide with functionalized spots, according to embodiments of the disclosure.

[0017] FIG. 4 depicts an exemplary system having an illumination path directed toward a sample via reflection off of a beam splitter, and the sample is scanned in x- and y-directions and imaged by a sensor, according to embodiments of the disclosure.

[0018] FIG. 5 depicts an exemplary system having an illumination path directed toward a sample via transmission through a beam splitter, and the sample is scanned in x- and y-directions and imaged upon reflection off of the beam splitter, according to embodiments of the disclosure.

[0019] FIG. 6 depicts an exemplary system having multiple illumination paths simultaneously directed towards a sample via reflection off of a beam splitter, according to embodiments of the disclosure.

[0020] FIG. 7 depicts an exemplary system having multiple illuminations paths simultaneously directed towards a sample via transmission through a beam splitter, according to embodiments of the disclosure.

[0021] FIG. 8 depicts an exemplary system comprising a light source that is coplanar with a sensor, where the light source generates multiple illumination paths simultaneously directed towards a sample, according to embodiments of the disclosure.

[0022] FIG. 9 depicts an exemplary system comprising a light source that is coplanar with a sensor, where the light source generates multiple illumination paths simultaneously directed towards a sample, according to embodiments of the disclosure. [0023] FIG. 10 illustrates a block diagram of an exemplary system 1002, according to embodiments of the disclosure.

DETAILED DESCRIPTION

[0024] The following description is presented to enable a person of ordinary skill in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. These examples are being provided solely to add context and aid in the understanding of the described examples. It will thus be apparent to a person of ordinary skill in the art that the described examples may be practiced without some or all of the specific details. Other applications are possible, such that the following examples should not be taken as limiting. Various modifications in the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims.

[0025] Various techniques and process flow steps will be described in detail with reference to examples as illustrated in the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of one or more aspects and/or features described or referenced herein. It will be apparent, however, to a person of ordinary skill in the art, that one or more aspects and/or features described or referenced herein may be practiced without some or all of these specific details. In other instances, well-known process steps and/or structures have not been described in detail in order to not obscure some of the aspects and/or features described or referenced herein.

[0026] In the following description of examples, reference is made to the accompanying drawings which form a part hereof, and in which it is shown by way of illustration specific examples that can be practiced. It is to be understood that other examples can be used and structural changes can be made without departing from the scope of the disclosed examples. [0027] The terminology used in the description of the various described embodiments herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the description of the various described embodiments and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and/or” as used herein refers to and encompasses any and all possible combination of one or more of the associated listed items. It will be further understood that the terms “includes,” “including,” “comprises,” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0028] In some aspects, disclosed herein are systems and methods for imaging individual nano- and micro-scale particles in one or more samples. A sample may refer to a given biological sample, fluid, etc. that may include one or more particles or types of particles. Exemplary samples may include, but are not limited to, urine, saliva, blood, sweat, etc. The sample(s) may be deposited on a slide before being imaged by the disclosed interferometry system. Before the sample(s) is deposited, the slide may be treated to include one or more functionalized spots. The functionalized spots may include biological capture agents, created by a chemical treatment of the surface of the slide. The chemical treatment may create a covalent bond between the functionalized spots and the biological capture agents. After the slide is treated, the sample(s) may be deposited on the slide by using one or more microfluidic channels, for example. In some embodiments, each microfluidic channel may be used to deposit a portion, including all, of the sample(s) onto a unique functionalized spot. The functionalized spot may then capture and immobilize one or more particles in the sample(s), when deposited.

[0029] The imaging may be used to detect the particles without the need for labeling. In some embodiments, a spatially incoherent narrow spectrum light source is employed in a Kohler illumination scheme. With reference to FIG. 1 and FIG. 2A, process 200 depicts an exemplary process for the imaging of one or more particles. In step 202, a light source generates incident light Einc that illuminates a surface of a sample. The sample may include one or more particles 102 to be imaged. The incident light Ei_nc may be perturbed in step 204 by the presence of the parti cl e(s) 102 on a slide 104.

[0030] Due to the perturbation, part (e.g., a first) of the incident light scatters in a different (e.g., opposite) direction as the incident light Einc (step 206). The portion of the incident light that scatters due to the presence of the particle 102 may be referred to as scattered light Escathat returns after having interacted with the particle 102. In step 208, part (e.g., a second) of the incident light that does not interact with the particle 102 on the surface of slide 104 may experience specular reflection. The portion of the incident light that reflects and does not interact with the particle 102 may be referred to as reflected light that has returned from slide 104. In some embodiments, the reflected light may be used as a reference light, denoted as reference E_ref in FIG. 1.

[0031] Both the scattered light E_SCa and the reference light Eref (e.g., reflected light) may be directed onto a sensor (not depicted in FIG. 1; discussed in more detail below). The scattered light Esca and the reference light Eref may interfere to form interference light having an interference intensity I, represented by the following relationship:

[0032] In some embodiments, the scattered light E_SCa may include weakly scattered light. With reference again to FIG. 2A, the scattered light Esca may coherently mix with the reference light Eref, in step 210. This coherent mixing of the weakly scattered light Esca with the reference light Eref may result in particles appearing as diffraction limited spots. In step 212, the image sensor may detect the diffraction limited spots as long as the overall interference intensity I is within the dynamic range of the image sensor, rendering particle detection in a highly parallel manner.

[0033] Depending on phase difference between the scattered light Esca and the reference light Eref, the diffraction limited spots can appear as bright or dark spots on the sensor. FIG. 2C illustrates an image of an exemplary diffraction limited spot. From the image of a diffraction limited spot, the system may determine its normalized intensity, as shown in FIG. 2B. The normalized intensity may be the maximum or minimum intensity of a particle divided by the mean background intensity. In some embodiments, the highest contrast is achieved at a defocus position (e.g., -0.1 um) where the particle appears as a dark spot. In some embodiments, the system may determine one or more properties of a particle based on a diffraction limited spot and its corresponding normalized intensity. Exemplary properties include, but are not limited to, size, shape, polarizability, and wavelength selective responsivity. These properties can be determined from the normalized intensity signal strength, 2D image based feature detection for shape inference, and monitoring normalized intensity signal as a function of various wavelength and polarization illumination schemes for polarizability and spectral properties. In some embodiments, these exemplary properties can be used to further discriminate and classify particles, which can enable more accurate and multiplexed testing.

[0034] FIG. 2D illustrates a flowchart of an exemplary process for determining the normalized intensity for a given particle, according to embodiments of the disclosure. Individual particles may be imaged as diffraction limited spots in the field-of-view. In step 252 of process 250, the system may determine one or more control spots on an image. A control spot may be a reference spot, for example, on the image.

[0035] In step 254, the system may determine the mean background intensity value from one or more control spots. In step 256, the system may search for a cluster of pixels. The cluster of pixels may have values that are different from the mean background intensity value. Exemplary methods that may be used to search for this cluster of pixels may include, but are not limited to, Gaussian template filtering and edge detection. The system may account for one or more properties of the imaging optics when performing the search.

[0036] The system may determine the contrast difference between the diffraction limited spot (cluster of pixels) and its surrounding background (step 258). The system may also determine the normalized intensity for a given particle based on the contrast difference (step 260).

[0037] Although FIG. 2C illustrates an image of a diffraction limited spot for a silica nano particle, one skilled in the art would understand that silica exhibits similar optical properties as biological nano particles in terms of its refractive index and scattering properties. Thus, FIG. 2C illustrates an exemplary dark spot included in the images captured by the systems and methods of the present disclosure, which may be used to detect nano- and micro-scale particles, such as pathogens (e.g., the coronavirus (CO VID) or another virus), bacteria, labeled biomarkers, etc. included in a sample.

[0038] FIG. 2E illustrates an exemplary image of 100 nm diameter polystyrene beads captured using an interferometry system disclosed herein. The system may acquire one or more images at focus and around focus with sub-micron focus adjustments. The sub-micron focus adjustments may done such that the particle normalized intensity signal is maximized.

[0039] FIG. 2F illustrates a flowchart of an exemplary process for scanning an image, according to embodiments of the disclosure. In step 272 of process 270, the system may use the fiducial marks to acquire the image(s) at focus. This step may include maximizing the edge response for the sharpest contrast. The starting point may be, for example, based on the sample to lens placement. This starting point may be considered the origin focal plane in the optical axis (Zo). The system may capture an image of the slide (step 274).

[0040] In step 276, the stage may be moved along the optical axis in one or more directions. The stage may be moved at sub-micron increments relative to the focal plane. In some embodiments, the lenses and the stage may be moved closer to each other by a finite, sub-micron offset ZA'. At each incremental offset, the system may capture an image of the slide. If a predetermined number of images have not been captured (step 278), the steps 274 and 276 are repeated.

[0041] After a pre-determined number of have been captured, the stage is moved back to the origin focal plane (step 280). Steps 282-286 are similar to steps 274-278, except that the lenses and the stage are moved farther away from each other by a finite, sub-micron offset ZA⁺.

[0042] In step 288, the captured images are analyzed, and the particles are determined from the analysis. The response is determined as a function of defocus offset. The use of the defocus offset improves detection accuracy. [0043] In some variations, the slides used in the systems and methods herein can have one or more functionalized areas. With reference to FIG. 3, a sample, including one or more particles 302, may be deposited on an exemplary slide 304. The slide 304 may include one or more functionalized areas (e.g., functionalized spot 306) for each target particle of interest (e.g., particle 302) to be captured and immobilized. In some embodiments, functionalized spots 306 are circular in shape with a diameter of about 100 micrometers to about 500 micrometers.

[0044] In some embodiments, the slide 304 may be a single layer slide, such as a glass substrate. Due to the components and configurations of the disclosed systems, this single layer configuration is feasible for nano- and micro-scale particle detection. This single layer configuration may lead to lower costs and improved imaging resolution.

[0045] The sample may be deposited (e.g., dropped on top of, injected via one or more microfluidic channels, etc.) on the slide 304. The functionalized spots 306 may capture one or more particles 302 from the sample. Then, the slide 304 can be placed on a stage where particle detection and imaging may take place.

[0046] In some embodiments, the systems of the present disclosure are capable of imaging nano and micro particles, such as biological pathogens and biomarkers, to detect their presence or lack thereof in a sample. A particle refers to a targeted species, such as a pathogen, biomarker, etc. of interest and within a sample. In some variations, nano particles are particles that have a size of from about 1 nanometer to about 1000 nanometers. In some variations, micro particles are particles that have a size from about 1 micrometer to 1000 micrometers.

[0047] With reference to FIG. 3, the presence of a particle 302 in a sample may be based on one or more properties of the particle being associated (e.g., matched, similar to, etc.) with a target particle (e.g., pathogen). As discussed in more detail below, in some embodiments, the system is capable of determining a presence of a plurality of pathogens in a sample based on one or more properties of the diffraction limited spot(s) of the sample at a given functionalized spot 306. The sample may be deposited on a slide 304. In some variations, the slide 304 may comprise a plurality of functionalized spots 306, at least two associated with different pathogens. The absence of a particle may be determined based on the absence of a diffraction limited spot at a corresponding functionalized spot 306 of the slide 304. [0048] In other embodiments, the systems described herein include a controller configured to determine the presence of a pathogen based on one or more properties of a particle being associated with one or more properties of a pathogen. Additionally or alternatively, the controller determines an absence of a pathogen in the sample based on an absence of a diffraction limited spot having one or more properties associated with one or more properties of a pathogen. Exemplary controllers may include, but are not limited to, computers, cellular devices, optimized detection hardware, etc.

[0049] As discussed above, one or more properties (deterministic characteristics) of a target particle may be determined. The components in the systems (discussed below) may be configured based on these deterministic characteristics. For example, the lens size, lens aperture, focal length, imaging sensor selection, light wavelength, and the like may be determined such that certain features may be detected. Particles may have biochemical binding properties to the functionalized spots, and these features may be used to determine the presence or absence of a pathogen or biomarker.

[0050] With reference again to FIG. 3, the systems and methods disclosed herein may use a slide 304 that is functionalized with suitable target capturing agents. In some embodiments, the slide 304 can include one or more materials such as fused silica, BK7, and sapphire. In some variations, the slide can include a coating to tune the reflectivity of the reference light E_ref for a given wavelength of illumination. One non-limiting example of a coating is a dielectric thin film coating. The reflectivity of the dielectric thin film coating may be known and may be used to adjust the reflectivity of a given surface as a function of the wavelength used to illuminate the sample. The coating may act similar to a filter, controlling the spectral response. The amount of reflectivity may be selected based on image sensor characteristics (e.g., pixel size, full-well capacity, quantum efficiency, etc.) such that there is shot noise limited imaging.

[0051] In some embodiments, the slide 304 may include one or more fiducial marks 308. The fiducial marks 308 may be used for the focusing and localization steps (discussed above). In some embodiments, the fiducial marks may be etched on the surface of the slide. The fiducial marks when formed on the slide may provide high contrast regions in the field-of-view for the focusing and localization steps. [0052] Each functionalized spot 306 can be functionalized with a specific target capture agent to a target particle 302, or can be used as a control group. For example, in one variation, the particle 302 may be a pathogen. Exemplary target capturing agents may include, but are not limited to, antibodies, aptamers, or any combination thereof. In another variation, functionalized spot 306 can be functionalized with a control agent that does not capture a target particle 302.

[0053] The particles captured within functionalized spots 306 on a slide 304 can then be imaged using exemplary systems described herein, including as depicted in FIGs. 4-9. FIG. 4 depicts an exemplary system 400, according to embodiments of the disclosure. The system 400 may include a light source 402. Lens 404 images the light rays coming out of the exit aperture of light source 402 at the back focal plane of second lens 406, which illuminates the sample 408 on slide 410 with Fourier plane of the light source exit aperture, ensuring a Kohler illumination scheme. The resulting image in sensor 412 does not include an image of the light source 402, since the sample plane is imaged with magnification via second lens 406 and third lens 414. In the exemplary configuration shown in FIG. 4, the illumination path is directed via lens 404 and reflected off of beam splitter 416 down toward sample 408, where the resulting scattered and reflected light from sample 408 are transmitted partially through the beam splitter 416 toward the sensor 412 in a common-path interferometry scheme. The functionalized spots 306 on slide 410 with captured targets from sample 408 are scanned in one or more directions (e.g., x- and y- directions) via stage 418 to image the entire active area of slide 410. In some embodiments, stage 418 may be configured to move in the z-direction for, e.g., focusing the sample 408 using fiducial marks 308 on slide 410.

[0054] As used throughout this disclose, the term “light source” may include a “light source array.” For example, a light source may refer to light source 402, light source 502, light source array 604, light source array 704, light source array 802, or light source array 902.

[0055] FIG. 5 depicts an exemplary system 500, according to embodiments of the disclosure. System 500 includes light source 502. Lens 504 images the light rays coming out of the exit aperture of light source 502 at the back focal plane of second lens 506, which illuminates sample 508 on slide 510 with Fourier plane of the light source exit aperture, ensuring a Kohler illumination scheme. The resulting image in sensor 512 does not include an image of the light source, since the sample plane is imaged with magnification via second lens 506 and third lens 514. In the exemplary configuration shown in FIG. 5, the illumination path is directed via lens 504 and transmitted through beam splitter 516 down toward sample 508, where the resulting scattered and reflected light from sample 508 are returned to sensor 512 upon reflection off of beam splitter 516 in a common-path interferometry scheme. Similar to system 400 above, the functionalized spots with captured targets are scanned in one or more directions (e.g., x- and y- directions) via stage 518 to image the entire active area of slide 510. In some embodiments, stage 518 may be configured to move in the z-direction for, e.g., focusing the sample 508 using fiducial marks 308 on slide 510.

[0056] In some variations of systems 400 and 500 above, the light source includes one or more (e.g., edge emitting) lasers with a diffuser in the exit aperture, one or more LEDs for a spatially incoherent illumination, one or more vertical-cavity surface- emitting lasers (VCSELs), or the like. The light source may be a single (e.g., narrow bandwidth) wavelength light source or a multiple (e.g., wide bandwidth) wavelength light source.

[0057] The exemplary systems depicted in FIGs. 4 and 5 can be reduced in size, and the need to scan in x- and y-directions can be eliminated by employing one or more lens arrays, as shown in FIGs. 6, 7, 8, and 9 (discussed in more detail below). The exemplary systems may have a significantly smaller footprint, which leads to lower costs and improved accessibility. The lens arrays are used to image arrayed functionalized spots simultaneously. This eliminates the need for scanning the sample laterally, thereby improving efficiency and speed.

[0058] FIG. 6 depicts an exemplary system 600, according to embodiments of the disclosure. System 600 includes lens 602 that images the rays coming out of the exit aperture of light source array 604 at the back focal plane of lens array 606 upon reflection off of beam splitter 608. This illuminates sample 610 with Fourier plane of the light source array exit aperture, ensuring a Kohler illumination scheme. The resulting image in a sensor 612 does not include an image of light source array 604, since the sample plane is imaged with magnification via lens array 606 and second lens array 614. In the configuration shown in FIG. 6, the light source array 604 may generate a plurality of illumination paths. The plurality of illumination paths may be directed via lens 602 and reflected off of beam splitter 608 down toward sample 610, where the resulting scatered and reflected light from sample 610 are transmited partially through beam spliter 608 toward sensor 612 in a common-path interferometry scheme. All functionalized spots 306 can be simultaneously imaged through beam spliter 608 via lens arrays 606 and 614 onto sensor 612, without the need for x-direction and/or y-direction scanning via stage 616. For example, a lens array may simultaneously direct the incident light (e.g., multiple light paths) from the light source array 604 towards the sample 610. Additionally or alternatively, a lens array may simultaneously direct scattered light, reflected light, or both from the plurality of functionalized spots towards the sensor 410. In some embodiments, stage 616 may be configured to move in the z direction for, e.g., focusing the sample 610 using fiducial marks 308 on slide 618.

[0059] In some embodiments, each lens in lens array 606, 614, or both may correspond to a unique functionalized spot 306 or group of functionalized spots, detector pixel, or group of pixels in sensor 612, or a combination thereof.

[0060] FIG. 7 depicts an exemplary system 700, according to embodiments of the disclosure. System 700 includes lens 702 that images the light rays coming out of the exit aperture of light source array 704 at the back focal plane of lens array 706 upon transmission through beam spliter 708. This illuminates sample 710 with Fourier plane of the light source array exit aperture, ensuring a Kohler illumination scheme. The resulting image in sensor 712 does not include an image of light source array 704, since the sample plane is imaged with magnification via lens arrays 706 and 714. In the configuration shown in FIG. 7, the plurality of illumination paths is directed via lens 702 and transmitted through beam splitter 708 down toward sample 710, where the resulting scatered and reflected light from sample 710 may be returned to sensor 712 upon reflection off of beam spliter 708 in a common-path interferometry scheme. All functionalized spots can be simultaneously imaged upon reflection off of beam spliter 708 via lens arrays 706 and 714 onto sensor 712, without the need for x-direction and y-direction scanning via stage 716. For example, a lens array may simultaneously direct the incident light (e.g., multiple light paths) from the light source array 704 towards the sample 710. Additionally or alternatively, a lens array may simultaneously direct scattered light, reflected light, or both from the plurality of functionalized spots towards the sensor 712. In some embodiments, stage 716 may be configured to move in the z-direction for, e.g., focusing the sample 710 using fiducial marks 308 on slide 718. [0061] In some embodiments, each lens in lens array 706, 714, or both may correspond to a unique functionalized spot 306 or group of spots, detector pixel, or group of pixels in sensor 712, or a combination thereof.

[0062] In some variations of systems 600 and 700 above, the light source array includes a LED array or laser array that can emit light at the back focal plane of the lens array by the lens.

[0063] In certain variations of systems 600 and 700 above, the lens array is a 2D array of microlenses, wherein each microlens within a 2D microlens array is co-centered with the functionalized spots 304 on the slide such that each functionalized spot 306 is imaged onto the sensor via the lens arrays. In some embodiments, a microlens array is any plurality of microlenses wherein there is a 1 :N ratio between the microlenses and the functionalized spots on the slide, where N is an integer. For example, one lens may image multiple functionalized spots at a given time (i.e., simultaneously). In some embodiments, there is an M 1 ratio between the microlenses and the functionalized spots on the slide, where M is an integer. For example, multiple lenses may image one functionalized spot at a given time.

[0064] In some variations of the foregoing, the beam splitters may be a prism beam splitter, or a plate beam splitter having polarizing or non-polarizing properties. The beam splitter may be comprised of any materials, coatings, or combinations thereof with different refractive indices that allow light to partially reflect and partially transmit. Exemplary beam splitting ratios may include, but are not limited to, 50/50, 70/30, 90/10, and the like.

[0065] With reference to FIG. 8, yet another exemplary system is depicted. System 800 includes light source array 802 with integrated lens array 804 that direct (e.g, collimates) the light rays coming out of light source array 802 and images the rays at the back focal plane of lens array 806 upon reflection off of mirror prism 808 and beam splitter coating 810 that is coated on the back surface of lens array 812. This illuminates sample 814 with Fourier plane of the light source array exit aperture, ensuring a Kohler illumination scheme. The resulting image in sensor 816 does not include an image of light source array 802, since the sample plane is imaged with magnification via lens arrays 806 and 812. In the configuration shown in FIG. 8, the illumination path is directed via lens array 804 and reflected off of mirror prism 808 and beam splitter coating 810 down toward sample 814, where the resulting scattered and reflected light from sample 814 are returned to sensor 816 upon transmission through beam splitter coating 810 in a commonpath interferometry scheme. All functionalized spots can be simultaneously imaged through beam splitter coating 810 via lens arrays 806 and 812 onto sensor 816. In some embodiments, stage 818 may be configured to move in the z-direction for, e.g., focusing the sample 814. In some embodiments, movement of lens array 806 in the optical axis adjusts focus on sample 814.

[0066] With reference to FIG. 9, yet another exemplary system is depicted. System 900 includes light source array 902 with integrated lens array 904 that collimates the rays coming out of light source array 902 and images the rays at the back focal plane of lens array 906 upon transmission through beam splitter coating 908 that is coated on the back surface of lens array 910. This illuminates sample 912 with Fourier plane of the light source array exit aperture, ensuring a Kohler illumination scheme. The resulting image in sensor 914 does not include an image of light source array 902, since the sample plane is imaged with magnification via lens arrays 906 and 910. In the configuration shown in FIG. 9, the illumination path is directed via lens array 904 and transmitted through beam splitter coating 908 down toward sample 912, where the resulting scattered and reflected light from sample 912 are returned to sensor 914 upon reflection off of beam splitter coating 908 and mirror prism 916 in a common-path interferometry scheme. All functionalized spots can be simultaneously imaged through beam splitter coating 908 via lens arrays 906 and 912 onto sensor 914. In some embodiments, stage 918 may be configured to move in the z-direction for, e.g., focusing the sample 912. In some embodiments, movement of lens array 906 in the optical axis adjusts focus on sample 912.

[0067] In some variations of systems 800 and 900 above, the light source array includes an LED array or laser array that can be transmitted through the integrated lens array. In certain variations, the lens array is a 2D array of microlenses. In some embodiments, each microlens within a 2D microlens array is spaced within the array analogously to each functionalized spot on the slide such that each functionalized spot is imaged onto the sensor via the lens arrays. In some embodiments, the microlens array includes a plurality of microlenses where there is a 1 :N ratio between the microlenses and the functionalized spots on the slide and N is an integer. For example, one lens may image multiple functionalized spots at a given time (i.e., simultaneously). In some embodiments, there is anA/:l ratio between the microlenses and the functionalized spots on the slide, where AT is an integer. For example, multiple lenses may image one functionalized spot at a given time. The number of functionalized spots may be based on the application, sensor size, layout of the functionalized spots, and the footprint of the overall device. In instances where all functionalized spots are imaged simultaneously, each spot may have a dedicated imaging channel working in parallel.

[0068] In some variations of systems 800 and 900 above, the movable lens array is mounted on an actuator. In some variations of systems 800 and 900 above, the moveable lens array is fixed and the stage is mounted on an actuator. In some variations of systems 800 and 900 above, the beam splitter coating may be a dielectric coating comprised on multi-layered thin films. In some (e.g., all) variations of the foregoing, the lens or lens array may be made of materials such as glass, fused silica, BK7, sapphire, etc. In some embodiments of the foregoing, the systems and methods provided herein are used in sensing applications, such as particle detection in agriculture technology, testing for bacteria in water sources, and testing for aerosolized biological warfare agents in the air, etc. In some variations, the systems and methods provided are used in healthcare (e.g., point-of-care or at-home), diagnostics, medical, security, environmental testing, food & beverage, pharmaceuticals, military diagnostics, and forensic analysis. For example, in healthcare industry, the systems and methods provided may be used to enhance awareness for medical conditions and monitor without requiring any labels and extended lab preparations. Such a technology may be used in pre- and post-diagnostics as well as for prognosis. Such a device enables telemedicine to prosper as healthcare providers, such as doctors or veterinarians, may have basic diagnostics performed at home and the data sent back to the healthcare provider for rapid analysis and response.

[0069] In some (e.g., all) variations of the foregoing, the methods provided herein may be modified to enable label-facilitated detection of particles (e.g., molecules and proteins including but not limited to biomarkers, hormones, allergens, antibodies, DNA, RNA). In certain variations, embodiments of the disclosure may include CRISPR based DNA/RNA detection modality, where the target nucleotides may be snipped from a sample using the CRISPR technology and detected using the embodiments disclosed therein through label-facilitated detection. [0070] FIG. 10 illustrates a block diagram of an exemplary system 1002, according to embodiments of the disclosure. The system may be a machine such as a computer, within which non-transitory computer readable medium including instructions that cause the machine to perform any one of the steps and processes discussed herein, according to embodiments of the disclosure. In some embodiments, the machine can operate as a standalone device or may be connected (e.g., networked) to other machines. In a networked configuration, the machine may operate in the capacity of a server or a client machine in a server-client network environment, or as a peer machine in a peer-to-peer (or distributed) network environment. The machine can be a personal computer (PC), a tablet PC, a set-top box (STB), a personal digital assistant (PDA), a cellular telephone, a web appliance, a network router, a switch or bridge, or any machine capable of executing a set of instructions (sequential or otherwise) that specify actions to be taken by that machine. A mobile device such as a PDA or a cellular phone may also include an antenna, a chip for sending and receiving radio frequency transmissions and communicating over cellular phone WAP and SMS networks, and a built-in keyboard. Further, while only a single machine is illustrated, the term “machine” shall also be taken to include any collection of machines that individually or jointly execute a set (or multiple sets) of instructions to perform any one of the methodologies discussed herein.

[0071] The exemplary computer 1002 includes a processor 1004 (e.g., a central processing unit (CPU), a graphics processing unit (GPU), or both), a main memory 1006 (e.g., read-only memory (ROM), flash memory, dynamic random access memory (DRAM) such as synchronous DRAM (SDRAM) or Rambus DRAM (RDRAM), etc.), and a static memory 1008 (e.g., flash memory, static random access memory (SRAM), etc.), which can communicate with each other via a bus 1010.

[0072] The computer 1002 may further include a video display 1012 (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)). The computer 1002 also includes an alpha-numeric input device 1014 (e.g., a keyboard), a cursor control device 1016 (e.g., a mouse), a disk drive unit 1018, a signal generation device 1026 (e.g., a speaker), and a network interface device 1022.

[0073] The drive unit 1018 includes a machine- readable medium 1020 on which is stored one or more sets of instructions 1024 (e.g., software) embodying any one or more of the methodologies or functions described herein. The software may also reside, completely or at least partially, within the main memory 1006 and/or within the processor 1004 during execution thereof by the computer 1002, the main memory 1006 and the processor 1004 also constituting machine-readable media. The software may further be transmitted or received over a network 1004 via the network interface device 1022.

[0074] While the machine-readable medium 1020 is shown in an exemplary embodiment to be a single medium, the term “non-transitory computer-readable medium” should be taken to include a single medium or multiple media (e.g., a centralized or distributed database and/or associated caches and servers) that store the one or more sets of instructions. The term “machine-readable medium” shall also be taken to include any medium that is capable of storing, encoding, or carrying a set of instructions for execution by the machine and that cause the machine to perform any one or more of the methodologies of the present invention. The term “machine-readable medium” shall accordingly be taken to include, but not be limited to, solid- state memories, optical and magnetic media, and carrier wave signals.

[0075] Although the disclosure and examples have been fully described with reference to the accompanying figures, it is to be noted that various changes and modifications will become apparent to those skilled in the art. Such changes and modifications are to be understood as being included within the scope of the disclosure and examples as defined by the claims.

[0076] The foregoing description, for purpose of explanation, has been described with reference to specific embodiments. However, the illustrative discussions above are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The embodiments were chosen and described in order to best explain the principles of the techniques and their practical applications. Others skilled in the art are thereby enabled to best utilize the techniques and various embodiments with various modifications as are suited to the particular use contemplated.

Claims

CLAIMS What is claimed is:

1. A method for imaging one or more samples, the method comprising: illuminating the one or more samples with incident light, the one or more samples including one or more particles, wherein a first of the incident light interacts with at least one particle of the one or more particles in the one or more samples and returns as scattered light, wherein a second of the incident light returns as reflected light upon specular reflection off of a slide, wherein the at least one particle is a nano particle or a micro particle; forming a reference light from the reflected light; allowing the scattered light and the reference light to coherently mix to form at least one diffraction limited spot; receiving the at least one diffraction limited spot at a sensor rendering the detection of the at least one particle; determining a normalized intensity for the at least one diffraction limited spot; and determining one or more properties of the at least one particle in the one or more samples based on the at least one diffraction limited spot and the corresponding normalized intensity.

2. The method of claim 1, further comprising: determining a presence of a pathogen in the one or more samples based on the one or more properties of the at least one particle being associated with one or more properties of the pathogen.

3. The method of claim 2, wherein the pathogen is associated with one or more of: a virus, bacteria, and labeled markers.

4. The method of claim 3, wherein the virus is the coronavirus (CO VID).

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5. The method of claim 1, further comprising: determining a presence of a plurality of pathogens in the one or more samples based on at least two of the one or more particles being located at different functionalized spots on the slide, wherein the one or more samples are deposited on the slide, wherein each of the at least two of the one or more particles is associated with different pathogens of the plurality of pathogens.

6. The method of claim 5, wherein the different pathogens are associated with diffraction limited spots having one or more different properties.

7. The method of claims 1-6, further comprising: determining an absence of a pathogen in the one or more samples based on an absence of a diffraction limited spot having one or more properties associated with one or more properties of the pathogen.

8. The method of claims 1-7, wherein the illuminating the one or more samples includes simultaneously illuminating a plurality of functionalized spots included in the slide, wherein the one or more samples are deposited on the slide.

9. The method of claims 1-8, wherein the receiving the at least one diffraction limited spot at the sensor includes simultaneously receiving the scattered light and the reference light at the sensor from a plurality of functionalized spots included in the slide, wherein the one or more samples are deposited on the slide.

10. An interferometry system for imaging one or more samples, wherein the one or more samples include one or more particles, the interferometry system comprising: a slide having the one or more samples; a light source that illuminates the one or more samples with incident light, wherein a first of the incident light interacts with at least one particle of the one or more particles in the one or more samples and returns as scattered light, wherein a second of the incident light returns as reflected light, and the reflected light forms a reference light, wherein the at least one particle is a nano particle or a micro particle; one or more lenses that receive and direct the scattered light and the reference light, wherein the interferometry system allows the scattered light and the reference light to coherently mix to form at least one diffraction limited spot; a sensor that receives the at least one diffraction limited spot; and a controller for: determining a normalized intensity for the at least one diffraction limited spot, and determining one or more properties of the at least one particle in the one or more samples based on the at least one diffraction limited spot and the corresponding normalized intensity.

11. The interferometry system of claim 10, wherein the slide includes one or more of: fused silica, BK7, and sapphire.

12. The interferometry system of claims 10-11, wherein the slide includes a coating, the reflectivity of the coating being tuned to one or more target wavelengths of the incident light.

13. The interferometry system of claims 10-12, further comprising: a beam splitter that directs at least a portion of the incident light towards the slide and direct at least a portion of the scattered light and the reference light towards the sensor.

14. The interferometry system of claim 13, wherein the beam splitter reflects the at least the portion of the incident light towards the slide and transmits the at least the portion of the scattered light and the reference light towards the sensor.

15. The interferometry system of claims 13-14, wherein the beam splitter transmits the at least the portion of the incident light towards the slide and reflects the at least the portion of the scattered light and the reference light towards the sensor.

16. The interferometry system of claims 13-15, wherein the beam splitter is one of: a prism beam splitter or a plate beam splitter having polarizing or non-polarizing properties.

17. The interferometry system of claims 13-16, wherein the beam splitter includes a beam splitter coating, the beam splitter coating directing the incident light towards the one or more samples.

18. The interferometry system of claims 13-17, wherein the beam splitter includes a beam splitter coating, the beam splitter coating directing the scattered light and reflected light towards the sensor.

19. The interferometry system of claims 10-18, further comprising: a stage that moves the slide in one or more directions such that the light source illuminates multiple areas of the slide for the imaging of the one or more samples.

20. The interferometry system of claims 10-19, wherein the slide includes a plurality of functionalized spots, wherein the one or more lenses include at least one microlens array, the at least one microlens array simultaneously directing the incident light from the light source array towards the sample.

21. The interferometry system of claims 10-19, wherein the slide includes a plurality of functionalized spots, wherein the one or more lenses include at least one microlens array, the at least one microlens array simultaneously directing the scattered light and the reference light from the plurality of functionalized spots towards the sensor.

22. The interferometry system of claims 10-12, further comprising: a mirror prism directing the incident light towards a first lens of the one or more lenses, wherein the first lens includes a beam splitter coating that directs the incident light towards the sample.

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23. The interferometry system of claims 10-12, further comprising: a mirror prism that directs the scattered light and the reference light towards the sensor.

24. The interferometry system of claims 10-23, wherein the light source and the sensor are co-planar.

25. The interferometry system of claims 10-23, wherein the light source is one of: an edge-emitting laser, a vertical-cavity surface-emitting laser (VCSEL), or a light emitting diode (LED).

26. The interferometry system of claims 10-23, wherein the light source is one of: a single wavelength light source or a multiple wavelength light source.

27. The interferometry system of claims 10-23, wherein the controller further determines a presence of a pathogen in the one or more samples based on the one or more properties of the at least one particle being associated with one or more properties of the pathogen.

28. The interferometry system of claims 10-23, wherein the controller further determines an absence of a pathogen in the one or more samples based on an absence of a diffraction limited spot having one or more properties associated with one or more properties of the pathogen.

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