WO2019224815A1

WO2019224815A1 - Imaging system for identification of objects of interest

Info

Publication number: WO2019224815A1
Application number: PCT/IL2019/050571
Authority: WO
Inventors: Ariel LIVNE; Tamir EPSTEIN
Original assignee: Diagnoz Me Ltd.
Priority date: 2018-05-21
Filing date: 2019-05-21
Publication date: 2019-11-28

Abstract

Novel imaging systems and processes for quantification of objects of interest are described. The imaging system comprises a microfluidic slide comprising one or more reaction regions configured and operable for respectively receiving one or more liquid samples each comprising one or more objects of interest, each reaction region comprising at least one coating for selectively anchoring one or more of said one or more objects of interest; a light source assembly configured and operable to generate light of predetermined properties; an optical arrangement comprising: an illumination assembly configured and operable to deliver the light arriving from said light source assembly to said one or more reaction regions along an illumination path, and a light collection assembly configured and operable to collect light from said one or more reaction regions, while containing said one or more liquid samples, along an imaging path, wherein at least part of the light collection assembly is integrated in said microfluidic slide; a mobile device comprising a camera system configured and operable to optically couple to said light collection assembly and capture one or more images, of said one or more reaction regions while containing said one or more liquid samples, according to a predetermined frequency and/or time pattern(s); and a processing utility configured for data communication with the camera system and being configured and operable to receive and analyze input data indicative of said one or more images, and generate output data indicative of quantitative data of said one or more objects of interest.

Description

IMAGING SYSTEM FOR IDENTIFICATION OF OBJECTS OF INTEREST

FIELD OF INVENTION

The invention relates to imaging techniques for identifying object(s) of interest in liquid samples, specifically imaging based on fluorescent labeling.

BACKGROUND

Precise and rapid identification and quantification of specific objects in liquids is of extreme importance. It is highly relevant, for example, for medical diagnostics that are based on bio-liquids, where the objects of interest may be cells, bacteria, viruses, macromolecules or different biomarkers.

Diagnostics utilizing fluorescent based assays are primarily limited to specialized labs and research facilities due to their requirement for specialized equipment (e.g., fluorescence microscope), trained personnel and high running costs (e.g., antibodies). In their place, lateral flow tests provide a quick and cheap solution. Like fluorescence based assays, lateral flow tests detect the presence (or absence) of an object of interest. However, these tests typically provide qualitative rather than quantitative results, are typically limited to the detection of a single object at a time and cannot monitor changes over time. Several solutions attempting to make diagnostics more accessible, e.g. outside the professional lab environments, are described in the following patent publications: US8980550, W02007060523, US20150273470. GENERAL DESCRIPTION

The present invention provides a novel and accessible, for home-use, technique for quantifying objects of interest, e.g. biological organisms or chemical compounds. Novel methods and imaging systems configured and operable to provide quantitative output relating to object(s) of interest are provided. The invention does not require a trained operator nor any dedicated or specialized equipment besides one or more personal computing devices (e.g., smartphone, tablet). The system is designed primarily for home diagnostic tests for the general public. The invention is particularly useful as it provides a miniature and low cost microscope setup being adaptable for use with a wide range, and possibly all, of available smartphones/tablets/laptops. This makes the technology accessible to everyone using a simple single solution.

The imaging system includes such essential elements as a slide configured and operable to hold the object(s) of interest and a personal computing device, such as a smartphone or tablet, configured and operable to at least take and save images of region(s) on the slide containing the object(s) of interest. The images are processed by specifically designed, easy to use, novel image processing software, either directly in the personal computing device, as a downloadable application, or by being sent to an external processing utility, to give the quantitative output. Often, the imaging, according to the invention, is fluorescence imaging. The optics required for the imaging may be located in an optical adapter between the slide and the computing device, or directly integrated in the slide, or distributed between the slide and the optical adapter.

The quantification of the objects of interest in a sample opens the door for self, easy and noticeably fast primary diagnosis of medical conditions, including but not limited to, diagnosis of diseases.

The invention provides methods, devices, apparatuses and software for fluorescently labeling liquids, quantifying their content of different objects of interest, and reporting, sharing, monitoring and archiving the measured and calculated results.

The fluorescent imaging, as well as the image analysis, are software controlled and do not require user know-how to operate. Novel image analysis algorithms employed by the software ensure an accurate and automated analysis of the acquired pictures, providing the user with highly accurate quantitative results. The invention provides, in some of its aspects, a novel microfluidic slide(s) comprising one or more reaction regions (chambers or channels in the microfluidic slide, which are interchangeably used herein through the text), configured to receive the one or more objects of interest, e.g. being floating in or suspended in liquid sample(s). The microfluidic slide(s) is(are) designed such that when combined with the personal computing device (e.g., smartphone, tablet), and possibly also with an optical adapter therebetween, the whole setup effectively forms a miniature and low cost fluorescence microscope. In some embodiments, the slide, alone or together with an optical adapter, can be configured to effectively work with a large range of personal computing devices, e.g. by being independent of the exact location and distance between the camera of the personal computing device and a light source illuminating the sample. In some embodiments, the light source used can be a flash of the personal computing device.

Some art discloses systems involving a mobile device and a microfluidic slide for performing and analyzing diagnostic assays. The systems described require expensive and complicated setups. Furthermore, in some art the microscopes rely on bright field illumination, such that they cannot detect fluorescence and are limited in the type of assays they may be useful for. Yet further, the few setups that exist are not designed for home diagnostics.

Thus, according to one broad aspect of the invention, there is provided an imaging system comprising:

a microfluidic slide comprising one or more reaction regions configured and operable for respectively receiving one or more liquid samples each comprising one or more objects of interest, each reaction region comprising at least one coating for selectively anchoring one or more of said one or more objects of interest;

a light source assembly configured and operable to generate light of predetermined properties;

an optical arrangement comprising: an illumination assembly configured and operable to deliver the light arriving from said light source assembly to said one or more reaction regions along an illumination path, and a light collection assembly configured and operable to collect light from said one or more reaction regions, while containing said one or more liquid samples, along an imaging path, wherein at least part of the light collection assembly is integrated in said microfluidic slide; a mobile device comprising a camera system configured and operable to optically couple to said light collection assembly and capture one or more images, of said one or more reaction regions while containing said one or more liquid samples, according to a predetermined frequency and/or time pattern(s); and

a processing utility configured for data communication with the camera system and being configured and operable to receive and analyze input data indicative of said one or more images, and generate output data indicative of quantitative data of said one or more objects of interest.

In some embodiments, the one or more reaction regions comprise one or more reaction chambers, or one or more reaction channels, or a combination of reaction chambers and channels.

In some embodiments, the microfluidic slide comprises a microfluidic system configured and operable to receive and selectively convey one or more labeling agents and/or said one or more liquid samples to said one or more reaction regions. The microfluidic system may comprise one or more entry holes configured and operable to receive said one or more liquid samples and/or said one or more labeling agents. The microfluidic system may comprise one or more containers being preloaded with said one or more labeling agents and configured and operable to selectively release said one or more labeling agents to said one or more reaction regions on a user demand. The microfluidic system may be configured to hold one or more of said one or more labeling agents in dry form such that they get dissolved when said one or more liquid samples pass over the one or more labeling agents in dry form, on the way to said one or more reaction regions. The microfluidic system may comprise a mixer configured and operable to selectively and efficiently mix the one or more labeling agents with said one or more liquid samples in the one or more reaction regions, or in dedicated one or more mixing chambers. The microfluidic system may comprise one or more washing buffer container(s) configured and operable to selectively and controllably release one or more preloaded washing buffer(s) to said one or more reaction regions on a user demand to thereby wash out unbound labeling agents and/or objects of interest. The microfluidic system may comprise one or more fluid outlets configured and operable for discharging said one or more liquid samples and/or said one or more labeling agents from said one or more reaction regions.

In some embodiments, said illumination and imaging paths are different.

In some embodiments, said illumination and imaging paths are at least partially overlapping. The optical arrangement may comprise an epi-illumination assembly. In some embodiments, at least one of said illumination and light collection assemblies comprises one or more wave guide(s) configured and operable to respectively define at least part of said illumination and/or imaging path(s).

In some embodiments, the illumination assembly comprises one or more Fresnel lenses.

In some embodiments, the light collection assembly comprises one or more lenses each being configured as spherical or aspherical plano-convex lens. The one or more lenses may be integrated in said microfluidic slide. The one or more reaction regions may have one or more curved top surfaces to thereby correct for Petzval field curvature of the one or more plano-convex lenses.

In some embodiments, the light collection assembly comprises one or more emission filter(s) each being configured and operable to selectively pass predetermined light wavelengths arriving from one or more of said one or more reaction regions.

In some embodiments, the illumination assembly comprises one or more excitation filter(s) each being configured and operable to selectively pass predetermined light wavelengths from said light source assembly to one or more of said one or more reaction regions.

In some embodiments, the optical arrangement comprises a blocking structure configured and operable to eliminate interference between ambient light or the light from the light source assembly, and light collected by the camera system.

In some embodiments, the light collection assembly and/or the camera system is(are) configured and operable to capture said one or more images from different focal planes in the reaction regions.

In some embodiments, the imaging system further comprises a spacer frame or a holder configured and operable to hold said mobile device and said microfluidic slide with predetermined distance and orientation therebetween.

In some embodiments, at least one of the following is integrated in said microfluidic slide: at least part of said illumination assembly, and at least part of said light source assembly.

In some embodiments, at least part of said light source assembly is integrated in said mobile device. The illumination assembly may be configured and operable to deliver light arriving from a variety of positions of said light source assembly integrated in said mobile device. The microfluidic slide may comprise at least part of said illumination assembly and is configured and operable to receive light arriving from a variety of positions of said light source assembly integrated in said mobile device. The microfluidic slide may comprise a plurality of through holes defining a respective plurality of trajectories of said illumination path.

In some embodiments, the imaging system further comprises an optical adapter configured and operable to be inserted between said microfluidic slide and said mobile device, wherein at least one of the following is integrated in said optical adapter: at least part of said illumination assembly, at least part of said light collection assembly and at least part of said light source assembly.

In some embodiments, the mobile device is one of the following: a smartphone, a tablet, a laptop, a netbook, a desktop, a telehealth device, or a specifically designated device.

In some embodiments, the mobile device comprises said processing utility.

In some embodiments, the one or more images are indicative of one or more fluorescence emission(s) of said one or more objects of interest.

In some embodiments, the microfluidic slide comprises a plurality of reaction regions each identically coated with predetermined one or more coatings to thereby anchor a specific object of interest, and a respective plurality of containers each associated with a different one of said reaction regions and preloaded with a different liquid comprising an active substance to be released into the respective reaction region upon a user demand.

In some embodiments, the microfluidic slide comprises a plurality of reaction regions each identically coated with predetermined one or more coatings to thereby anchor a specific object of interest, and each coated with or containing a different active substance.

In some embodiments, the specific object of interest is a bacteria and said active substance is an antibiotic, thereby enabling the imaging system to be used in an antibiogram test.

In some embodiments, the microfluidic slide comprises a filter configured and operable to capture one or more objects of interest on its surface while said one or more liquid sample flow through said filter, said filter thereby defining a reaction region in the microfluidic slide.

In some embodiments, the illumination assembly comprises a photovoltaic panel configured and operable to supply power to activate said light source assembly.

In some embodiments, the illumination assembly comprises at least one collimation lens configured to receive the light of the light source assembly and deliver collimated light, said microfluidic slide comprises slanted grooves in the illumination path such that the collimated light undergoes total internal reflection inside the microfluidic slide until reaching the reaction region. In some embodiments, the microfluidic slide comprises a magnet configured and operable to enable attachment of the microfluidic slide to the mobile device.

In some embodiments, the microfluidic slide comprises an induction coil configured and operable to couple to an induction coil in the mobile device, thereby enabling powering said light source assembly.

In some embodiments, the microfluidic slide and/or optical adapter comprises at least one magnet configured and operable to enable attachment between the microfluidic slide, the optical adapter and the mobile device.

In some embodiments, the optical adapter comprises an induction coil configured and operable to couple to an induction coil in the mobile device, thereby enabling powering said light source assembly.

In some embodiments, the processing utility is configured and operable to generate said quantitative data, relating to said one or more objects of interest, being indicative of one or more of the following: bacterial or viral load;

caries;

periodontitis;

Tuberculosis;

urinary tract infections;

vaginitis;

sexually transmitted infections;

Streptococcal pharyngitis.

Red blood cells

White blood cells

Platelets.

In some embodiments, the processing utility is configured and operable to analyze said input data indicative of said one or more images, by applying one or more of the following:

image correction including image registration using fiducial markers; image correction including averaging of the captured images to thereby increase bit-depth and enhance signal to noise; image correction including recovery of saturated pixels in one of the RGB components by using the unsaturated bleed-through to a different component;

image correction including uneven field illumination correction;

image enhancement including contrast enhancement and local background subtraction;

object segmentation including connected component analysis employing watershed algorithm and specialized object-recognition based on pixel-by-pixel multiple criteria;

object quantification including multi-parametric quantification of objects of interest segments;

object quantification including defining objects of interest and rejecting non objects of interest segments based on parameter distributions and clustering in multi-parametric scatter- plots;

object quantification including counting the number of true objects of interest in the image after identification of objects of interest aggregates.

In some embodiments, the processing utility is configured and operable to save the image data and/or the generated output data in a memory. The processing utility may be configured and operable to apply machine learning to the saved image and output data to thereby improve the analysis of the input data.

According to another aspect of the invention, there is provided a microfluidic slide comprising:

one or more reaction regions configured and operable for respectively receiving one or more liquid samples each comprising one or more objects of interest, each reaction region comprising at least one coating for selectively anchoring one or more of said one or more objects of interest;

a microfluidic system configured and operable to receive and selectively convey one or more labeling agents and/or said one or more liquid samples to said one or more reaction regions; and

an integrated optical arrangement comprising a light collection assembly configured and operable to collect light from said one or more reaction regions while containing said one or more liquid samples, thereby enabling acquiring one or more images of said one or more reaction regions, said one or more images being indicative of quantitative data relating to said objects of interest.

In some embodiments, the integrated optical arrangement comprises an illumination assembly configured and operable to deliver light arriving from a light source assembly to said one or more reaction regions.

In some embodiments, the microfluidic system comprises one or more containers being preloaded with said one or more labeling agents and configured and operable to selectively release said one or more labeling agents to said one or more reaction regions on a user demand.

In some embodiments, the microfluidic slide comprises:

a plurality of reaction regions each identically coated with predetermined one or more coatings to thereby anchor a specific object of interest, and

a respective plurality of containers each associated with a different one of said reaction regions and preloaded with a different liquid comprising an active substance to be released into the respective reaction region upon a user demand;

the microfluidic slide being thereby configured and operable for use in an antibiogram test.

In some embodiments, the microfluidic slide comprises:

a plurality of reaction regions each identically coated with predetermined one or more coatings to thereby anchor a specific object of interest, and each coated with or containing a different active substance;

In some embodiments, the microfluidic slide comprises a photovoltaic panel configured and operable to supply power to activate light source assembly.

In some embodiments, the microfluidic slide comprises slanted grooves such that collimated light arriving at the microfluidic slide undergoes total internal reflection inside the microfluidic slide until reaching the reaction region.

In some embodiments, the illumination assembly is configured and operable to receive light arriving from a variety of positions outside the microfluidic slide. The microfluidic slide may comprise a plurality of through holes defining a respective plurality of illumination paths of light to thereby enable delivery of light from a respective plurality of positions of said light source assembly with respect to said one or more reaction regions.

According to another aspect of the invention, there is provided a computer-implemented system for use in image processing, the system comprises a memory utility storing one or more sequences of instructions for analyzing one or more images acquired on one or more liquid samples containing one or more objects of interest, and a processor utility configured to process said instructions, wherein said one or more sequences of instructions comprise applying one or more of the following operations on said one or more images:

image correction including image registration using fiducial markers; image correction including averaging of the captured images to thereby increase bit-depth and enhance signal to noise;

image correction including recovery of saturated pixels in one of the RGB components by using the unsaturated bleed-through to a different component;

image correction including uneven field illumination correction;

object quantification including multi -parametric quantification of object of interest segments;

object quantification including defining objects of interest and rejecting non objects of interest segments based on parameter distributions and clustering in multi -parametric scatter-plots;

object quantification including counting the number of true objects of interest in the image after identification of objects of interest aggregates;

thereby enabling identifying a condition corresponding to said objects of interest.

According to another aspect of the invention, there is provided a non-transitory computer readable medium including one or more sequences of instructions for image acquisition and processing, wherein execution of the one or more sequences of instructions by one or more processors of a computing device comprising a camera system causes the computing device to perform a process comprising:

activating and controlling a light source to illuminate a region of interest;

acquiring one or more images of said region of interest by said camera, according to a predetermined frequency and/or time pattern;

processing said one or more images to thereby determine quantitative data of one or more objects of interest in said one or more images, said processing comprises one or more of the following:

o image correction including image registration using fiducial markers;

o image correction including averaging of the captured images to thereby increase bit-depth and enhance signal to noise;

o image correction including recovery of saturated pixels in one of the RGB components by using the unsaturated bleed-through to a different component;

o image correction including uneven field illumination correction;

o image enhancement including contrast enhancement and local background subtraction;

o object segmentation including connected component analysis employing watershed algorithm and specialized objects of interest recognition based on pixel -by-pixel multiple criteria;

o object quantification including multi-parametric quantification of objects of interest segments;

o object quantification including defining objects of interest and rejecting non objects of interest segments based on parameter distributions and clustering in multi -parametric scatter-plots;

o object quantification including counting the number of true objects of interest in the image after identification of objects of interest aggregates;

analyzing said quantitative data and determining whether the quantitative data is indicative of one of the following:

o bacterial or viral load;

o caries o periodontitis;

o Tuberculosis;

o urinary tract infections;

o vaginitis;

o sexually transmitted infections;

o Streptococcal pharyngitis;

o Red blood cells;

o White blood cells;

o Platelets

and,

generating corresponding output data to be presented to a user.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting examples only, with reference to the accompanying drawings, in which:

Fig. 1 illustrates a non-limiting example of an imaging system according to the present invention; Figs. 2A-2B schematically illustrate non-limiting examples of a microfluidic slide with integrated parts of light collection assembly and microfluidic system according to the present invention; Figs. 3A-3B schematically illustrate exemplary embodiments of reaction region(s) on the microfluidic slide;

Figs. 4A-4B schematically illustrate exemplary embodiments of filters integrated in the microfluidic system;

Figs. 5A-5D schematically illustrate exemplary embodiments of anchoring of objects of interest in the reaction regions;

Figs. 6A-6C schematically illustrate exemplary embodiments of anchoring of different objects of interest in the reaction regions;

Figs. 7A-7B schematically illustrate reaction regions treated with different compounds or active substances; Fig. 8 schematically illustrates an exemplary embodiment for the imaging system in which parts of the optical arrangement are integrated in the microfluidic slide, where the reaction region of interest is illuminated from one direction by one or more wave guides such as an optical fiber bundle;

Figs. 9A-9B schematically illustrate an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the reaction region of interest is illuminated from a number of directions by one or more wave guides such as an optical fiber bundle;

Figs. 10A-10B schematically illustrate an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the reaction region of interest is illuminated through a Fresnel lens engraved on the top surface of the slide, the light reaching the Fresnel lens is not collimated;

Figs. 11A-11B schematically illustrate an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the reaction region of interest is illuminated through a Fresnel lens engraved on the top surface of the slide, the light reaching the Fresnel lens is partially or fully collimated;

Figs. 12A-12B schematically illustrate an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the reaction region of interest is illuminated after an un-collimated light passes through a Fresnel lens engraved on the top surface of the slide and then reflected by a mirror;

Figs. 13A-13B schematically illustrate an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the reaction region of interest is illuminated after partially or fully collimated light passes through a Fresnel lens engraved on the top surface of the slide and then reflected by a mirror;

Figs. 14A-14B schematically illustrate an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the reaction region of interest is illuminated after un-collimated light passes through two Fresnel lenses engraved on both top and bottom surfaces of the slide and then reflected by a mirror;

Figs. 15A-15B schematically illustrate an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the reaction region of interest is illuminated after partially or fully collimated light passes through two Fresnel lenses engraved on both top and bottom surfaces of the slide and then reflected by a mirror; Figs. 16A-16B schematically illustrate an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the reaction region of interest is illuminated by a single or array of LEDs or lasers that are integral part of the slide or attached to it;

Figs. 17A-17B schematically illustrate an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the region of interest is illuminated by a single or array of LEDs or lasers that are integral part of the slide or attached to it, the LED / laser light is focused or collimated by a lens, which is an integral part of the slide;

Fig. 17C schematically illustrates an exemplary embodiment for the microfluidic slide with a photovoltaic panel integrated or attached thereto;

Fig. 18A-18C schematically illustrate non-limiting exemplary embodiments of wave-guide(s) used in the optical arrangement ;

Fig. 19 schematically illustrates an exemplary embodiment of an optical adapter disposed between the mobile device and the microfluidic slide, the region of interest is illuminated with a single or an array of external LEDs or lasers;

Fig. 20 schematically illustrates an exemplary embodiment of an optical adapter disposed between the mobile device and the microfluidic slide, the region of interest is illuminated by one or more wave guides such as an optical fiber bundle;

Fig. 21 schematically illustrates an exemplary embodiment of an optical adapter, with part of the illumination assembly integrated therein, disposed between the mobile device and the microfluidic slide, the region of interest is illuminated by one or more wave guides such as an optical fiber bundle;

Fig. 22 schematically illustrates an exemplary embodiment of an optical adapter disposed between the mobile device and the microfluidic slide, the optical adapter contains one or two lenses and excitation, emission and dichroic filters;

Figs. 23A-23B schematically illustrate an exemplary embodiment of how multi-colour fluorescence is achieved;

Fig. 24 schematically illustrates an exemplary embodiment for compensating for the Petzval field curvature of the microfluidic slide lens, when imaging the top surface of the reaction region of interest; Fig. 25 schematically illustrates an exemplary embodiment for compensating for the Petzval field curvature of the microfluidic slide lens, when imaging the bottom surface of the reaction region of interest;

Figs. 26A-26B schematically illustrates an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the reaction region of interest is illuminated from the bottom side of the microfluidic slide by one or more wave guides such as an optical fiber bundle;

Fig. 27 schematically illustrates an exemplary embodiment for integrating parts of the optical arrangement in the microfluidic slide, where the reaction region of interest is illuminated through total internal reflection.

Figs. 28A-28C schematically illustrate image processing of the image data, specifically recovery of saturated pixels;

Figs. 29A-29B schematically illustrate image processing of the image data, specifically correction of uneven field illumination;

Figs. 30A-30B schematically illustrate image processing of the image data, specifically enhancement of objects of interest and local background subtraction;

Fig. 31 schematically illustrates image processing of the image data, specifically conversion to a binary image using an object-recognition thresholding algorithm;

Figs. 32A-32B schematically illustrate image processing of the image data, specifically connected component analysis;

Figs. 33A-33D schematically illustrate image processing of the image data, specifically quantification of the objects of interest found;

Fig. 34 schematically illustrates image processing of the image data, specifically identification of single objects and of aggregates among the objects of interest found;

Figs. 35A-35C illustrate an example of a slide with E. coli bacteria imaged with a conventional fluorescence microscope and with the imaging system of the invention; and

Fig. 36 is an exemplary flowchart summarizing an exemplary embodiment of the image analysis process. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

In the present invention, a compact, cost effective, rapid and easy to use technique for quantification of object(s) of interest in liquids, is described. The invention is based on a one -use, disposable, microfluidic slide and a software/mobile device application and provides a quantitative analysis within a short time.

Reference is made to Fig. 1 illustrating, by way of a block diagram, a non-limiting example of an imaging system 10 configured and operable for providing quantitative data of one or more objects of interest in one or more samples S, generally in liquid state. As shown, the imaging system 10 includes a microfluidic slide 10A configured and operable to hold the one or more samples, a light source assembly 10B configured and operable to produce light to illuminate the one or more samples, an optical arrangement 10C configured and operable to transmit the light from the light source assembly 10B to the one or more samples and collect light from the one or more samples, a mobile device 10D including a camera system 10D1 configured and operable to capture images of the one or more samples and provide image data ID, and a processing utility 10E configured and operable to receive and process the image data ID and provide quantitative output data QOD of the object(s) of interest located inside the one or more samples.

The microfluidic slide 10A includes one or more reaction regions 10A1 configured and operable for receiving the one or more liquid samples S that include the one or more objects of interest. As will be described further below, the reaction region can be a chamber or a channel in the microfluidic slide. In the example shown, for simplicity of presentation, only one reaction region in the form of a chamber is shown. Generally, each reaction chamber receives and holds one sample that contains one or more object(s) of interest. In some embodiments, the microfluidic slide 10A includes a microfluidic system configured and operable to receive and selectively convey the one or more liquid samples and/or one or more labeling agents to the one or more reaction chambers. The microfluidic slide 10A can be designed to run a specific test or a combination of tests. Generally, the user puts on the slide, e.g. through a designated entrance structure, a small volume of the appropriate liquid including the object(s) of interest. The liquid is sucked inside, e.g. by capillary attraction, and directed, e.g. through an array of micro-channels, to the one or more reaction chambers. Alternatively, the liquid may be injected into the slide via the designated entrance structure. In some embodiments, the reaction chambers can be surface- coated to attach the objects of interest and fix them in place. Specifically, if the microfluidic slide contains more than one reaction chamber or channel, they can differ in: (1) coating, such that different chambers anchor different objects; (2) treatment, such that the objects in each chamber are exposed to different compounds or active substances (e.g., exposing bacteria to different antibiotics); and (3) both coating and treatment. In addition, the microfluidic slide may contain, e.g. in a reaction chamber, dry powder or pellets such that when mixed with water generate anaerobic conditions for culturing bacteria or microbes.

The light source assembly 10B includes one or more light sources configured and operable to illuminate the sample(s) S by generating light of one or more profiles/patterns of wavelength, frequency and/or intensity being adapted for the specific application in which the system is used. For example, a single light source or an array of light sources coupled to one or more reaction chambers via the optical arrangement 10C can be used. In some embodiments, at least part of the light source assembly 10B is integrated in the microfluidic slide 10A.

The optical arrangement 10C includes an illumination assembly 10C1 configured and operable to deliver light arriving from the light source assembly 10B to the one or more samples in the one reaction chambers 10A1 along an illumination path ILP, and a light collection assembly 10C2 configured and operable to collect light from the one or more reaction chambers 10A1, while respectively containing the one or more liquid samples, along an imaging path IMP. In some embodiments, the illumination and imaging paths are identical. In some other embodiments, the illumination and imaging paths are at least partially overlapping. In some other embodiments, the illumination and imaging paths are different. In some embodiments, as will be detailed further below, at least part of the optical arrangement 10C is included within the microfluidic slide 10A. For example, the microfluidic slide 10A can be configured with at least part of the illumination assembly, at least part of the light collection assembly or at least parts of both the illumination and light collection assemblies.

The mobile device 10D can be any one of the following: a smartphone, a tablet, a laptop, a netbook, a desktop, a telehealth device, or a specifically designated device. In some embodiments, the mobile device 10D includes the processing utility 10E integrated therewith, such that the image capturing and analysis is done in the mobile device. In some embodiments, the processing utility 10E is located in another device, e.g. a personal computer, a cloud-based server or any of the devices listed above, that communicates with the mobile device to receive the image data ID therefrom.

In some exemplary embodiments, the mobile device 10D includes at least part of the light source assembly 10B; such as a flash of the mobile device, e.g. a flash of a smartphone or a tablet. The camera system 10D1 is configured and operable to optically couple to the light collection assembly 10C2 to thereby capture one or more images, of the one or more reaction chambers while containing the one or more liquid samples, according to a predetermined frequency and/or time pattern(s). Specifically, the camera systems found in most of, if not all, the smartphones/tablets can be utilized for this purpose. Either the front or rear camera of the smartphone/tablet may be utilized for this purpose.

The mobile device 10D and the microfluidic slide may be attached to each other either directly, or via intermediate spacer(s), adapter(s) or holder. If an intermediate adapter is used, it may include, as will be further described below, at least parts of the optical arrangement or the light source assembly. Attaching the slide to the mobile device, and/or spacer/adapter/holder if used, can be with the help of magnet, suction tape, clips and/or rubber band. The magnet can be advantageous in some examples as it will face and be attracted by the magnet surrounding the mobile device’s camera lens. Fluorescent markers or optical patterns can also be used (e.g. by an app) to guide the user in fine-tuning the position of the slide. This ensures that the illumination and imaging paths are aligned with the light source assembly and with the camera system.

The processing utility 10E, as referred to herein, is a software, or combination of hardware and software running thereon. In some embodiments, the processing utility includes a software that is accessible and easily downloadable to the mobile device 10D or the other computing device (if it is not integrated in the mobile device 10D), such as a mobile application configured for running on a smartphone. In some embodiments, the processing utility can be running on a distant server / cloud-based server and accessible by the mobile device via the internet. In some embodiments, the processing utility is distributed between two or more different locations; for example, between the mobile device 10D and a distant server / cloud-based server, where some modules of the processing utility are located/running on the mobile device 10D while the remaining modules are located/running on the server / cloud-based server.

In some exemplary embodiments, the processing utility 10E is configured and operable to generate the quantitative data, relating to the one or more objects of interest, where the quantitative data is indicative of one or more of the following: bacterial or viral load; caries; periodontitis; Tuberculosis; urinary tract infections; vaginitis; sexually transmitted infections; Streptococcal pharyngitis; red blood cells; white blood cells; and/or platelets.

In some exemplary embodiments, the processing utility 10E is configured and operable to analyze the input data indicative of the one or more images ID, by applying one or more of the following:

image correction including uneven field illumination correction;

object segmentation including connected component analysis employing watershed algorithm and specialized object-recognition based on pixel -by-pixel multiple criteria; object quantification including multi-parametric quantification of objects of interest segments;

object quantification including defining objects of interest and rejecting non objects of interest segments based on parameter distributions and clustering in multi-parametric scatter-plots; and/or

The processing utility 10E may be also configured and operable, in addition to analyzing the sample and returning a precise quantification of the objects of interest, to directly transmit this information as a standard lab report to a physician who can then prescribe the best medical treatment (or monitor its progress), without requiring an office visit. In other words, bringing the lab to the patient, instead of the patient to the lab / clinic. This involves a telemedicine service as part of the processing utility 10E, or that the processing utility is integrated in a telemedicine service. In some exemplary embodiments, the processing utility 10E is configured and operable to save the image data acquired in a memory / database. The processing utility 10E may then utilize machine learning techniques for optimizing the algorithms of the image processing and the generation of the output data indicative of the quantity(ies) of the object(s) of interest.

The imaging system 10 can be used to provide quantitative analysis and results of objects of interest in a variety of applications. In one particular application, the imaging system 10 is used as a miniature, compact and simple fluorescence imaging system. In this connection, fluorescence microscopy generally requires (1) a fluorescent sample, (2) a light source to illuminate the sample with, (3) an excitation filter to selectively excite the sample at the right wavelength(s), (4) an emission filter to selectively pass only the fluorescently emitted radiation, (5) a lens or objective to collect the excited radiation and magnify the fluorescent image on its way to (6) a detector (eye or camera), and a trained operator (7) to operate the microscope. The present invention presents, in some preferred embodiments, simplified and miniaturized arrangements of the elements (2) - (6) equivalent in their function to those found in fluorescence microscopes. Elements (3) - (5) can be integrated in the microfluidic slide 10A, which contains the fluorescent sample (1). Elements (2) and (6) can be provided by the mobile device 10D and their spatial arrangement, one relative to the other, is of high importance to a microscope’s proper operation. As this arrangement varies significantly between different mobile devices, the invention presents different solutions to circumvent this. The processing utility 10E, e.g. running on the mobile device, automates both image acquisition and analysis, replacing the need for a trained operator. A platform for archiving, monitoring and sharing the quantitative data from the test is also be provided according to some aspects of the invention.

For some assays, a single time -point may suffice (e.g., simple quantification of an object’s concentration in the liquid such as the concentration of S. mutans in saliva for the assessment of the risk of caries), while for others the images may be acquired over an extended time interval (e.g., tracking bacteria response to different antibiotics). Multiple objects of interest may be analyzed in the same test by separating them to different reaction chambers, by labeling them using distinct fluorescent markers or by a combination of the two techniques. Once pictures are collected, the software application runs a sequence of image analysis algorithms in order to quantify the number of labeled objects in each reaction chamber. It then provides the user with quantitative feedback (e.g., object concentration or whether there is an infection and which antibiotic is most effective), and stores it for archiving on the device and on the cloud. After the test, the software application and cloud platforms also enable sharing collected results with physicians as well as monitoring trends based on previous tests. In addition, the cloud-stored data may also be used for big-data analysis by a third party, provided user-consent is given. The entire process is summed up further below.

Turning now to different figures in which exemplary non-limiting embodiments of the components of the imaging system are described.

Reference is made to Figs. 2 to 7 illustrating non-limiting examples of the microfluidic slide 10A. In Figs. 2 and 3A-3B isometric views and in Figs. 4A-4B side views of the microfluidic slide are shown. Figs. 5 to 7 illustrate different embodiments of the reaction chamber 10A1.

As shown in Fig. 2A, the microfluidic slide includes one reaction chamber 10A1 and a microfluidic system 10A2 configured and operable to receive and selectively convey one or more labeling agents and/or one or more liquid samples to and from the reaction chamber. The microfluidic system 10A2 includes, inter alia, a designated entrance hole 12 at which a drop of liquid 14 is introduced, one or more micro-channels into which the different liquids are sucked by capillary attraction and/or transported through the slide. The microfluidic system may also include a container / blister 18 that contains either a single or cocktail of different, labeling agents such as fluorescent markers. When pressed, e.g. by a user, the blister 18 releases its contents through the micro-channel 20 into a mixing chamber 22 together with the liquid arriving from entrance hole 12 through the micro-channel 16. From there, the labeled mixed solution is directed via the micro- channel 24 to the reaction chamber 10A1, where the imaging of the mixed liquid takes place underneath a plano-convex lens 26 forming part of the illumination assembly 10C1 and being integrated into the slide. In some embodiments, the labeling agents/markers may be injected directly to the reaction chamber, without the need for a mixing chamber. Furthermore, in some examples, an additional microfluidic channel 21 from the blister 18 may be used to improve the mixing.

In some embodiments, the microfluidic system includes a mixer device (not shown) configured and operable to selectively and efficiently mix the one or more labeling agents with the one or more liquid samples either in the one or more reaction chambers directly or in the dedicated one or more mixing chambers such as the mixing chamber 22. In some embodiments, the mobile device may be configured to vibrate, while being attached to the microfluidic slide and cause mixing of the ingredients in the slide.

The lens 26, which is part of the slide, is used by the camera system of the mobile device to image the objects of interest in the liquid in the reaction chamber. Liquid leftovers flow to an open outlet 30 through an exit micro-channel 28.

The microfluidic system may include a container/blister 19, containing washing buffer, when a washing step of the reaction chamber, after the fluorescent labeling, is needed in order to decrease the background fluorescence, e.g. to wash out unbound labeling agents and/or objects of interest. The washing buffer can be released upon pressing the blister and is conveyed to the reaction chamber via a microchannel 29 that, for example, merges with the microchannel 24 at the entrance to the reaction chamber.

In Fig. 2B, the shown microfluidic slide does not include a container / blister that contains either a single or cocktail of different labeling agents, but includes an optional container/blister 19 containing washing buffer. In this setting, the labeling agents are stored in dry form along the microfluidic system in between the entrance hole and the reaction chamber(s). As the liquid flows towards the reaction chamber(s), it dissolves the labeling agents and the two mix together.

In Fig. 3A, a microfluidic slide similar to the microfluidic slide in Fig. 2A is shown. In this non-limiting example, as shown, the microfluidic slide includes a plurality of reaction chambers 32-35) that may be located underneath the integrated lens (which is not shown, to enhance the presentation of the reaction chambers) . This microfluidic slide can be utilized when a plurality of objects of interest need to be detected. The number of reaction chambers may vary according to the type of test, and could be less or more than the shown four chambers. The output micro channels 162-165 from the reaction chambers may be connected to the main outlet 30, or to individual open outlets of their own (not shown). Fig. 3B shows a number of channels 32C-34C instead of a number of reaction chambers. Each channel can be coated with a different antibody or coating that captures specific objects of interest (e.g., bacteria). These objects are fluorescently labeled with the same dyes/colours. The background dye/colour may be washed away in a washing step at the end. As with the reaction chamber(s), the concentration of the objects of interest is extracted according to their number in each channel.

Fig. 4A shows a side view of an example of the microfluidic slide of the invention, the microfluidic system is shown including a filter 40 at the entrance to the micro-channel 16, the filter is configured to enable selective passage of objects therethrough. For example, the filter blocks the passage of objects larger than a predefined size (e.g., air bubbles). The reaction chambers (here shown 32-33) can differ in coating, in treatment or in both. As shown, different coatings 36 and 37 are coated in the reaction chambers 32 and 33 respectively.

In Fig. 4B, the microfluidic slide includes a filter 200 configured to capture objects of interest, e.g. bacteria 201 and 202, on its surface 203 (as well as anything bigger). The surface 203 of the filter across which the bacteria are trapped effectively defines the region of interest / sample and is imaged by the camera system. The filter 200 can be located in a reaction chamber or along a channel in the slide. This technique enables the following: Firstly, it does not require any surface coating to capture the object of interest / bacteria. Secondly, the success rate in capturing the object of interest / bacteria is practically 100%. Thirdly, it is possible to concentrate the object of interest / bacteria by flowing through the slide a large volume of liquid (much larger than the nominal volume that the slide can hold). Thus it is possible to detect objects of interest / bacteria even when their concentration is very low. This may be relevant, for example, for testing potable water. Using a black colored filter will also help improve the signal-to-noise ratio for the fluorescent signal coming from the object of interest / bacteria trapped on top of the filter.

In some examples, the microfluidic slide includes a plurality of reaction chambers, each reaction chamber can be identically coated with predetermined one or more coatings to thereby anchor a specific object of interest. A respective plurality of containers, each associated with a different one of the reaction chambers and preloaded with a different liquid including an active substance to be released into the respective reaction chamber upon a user demand, can be provided in the microfluidic slide.

In some examples, the microfluidic slide includes a plurality of reaction chambers, each can be identically coated with predetermined one or more coatings to thereby anchor a specific object of interest, and each reaction chamber is also coated with or containing a different active substance.

The above-mentioned options of the microfluidic slide are significant when the object of interest is a bacteria and the active substance is an antibiotic, thereby enabling the imaging system to be used in an antibiogram test.

Figs.5A-5D are magnified side views of an example of a reaction region/channel/chamber 10A1. In Fig. 5A, the chamber’s bottom surface is specially coated with coating 46 to anchor a specific object 42 or group of objects (e.g., bacteria). After the reaction chamber is filled, and due to its small dimensions, most objects encounter the coated surface within tens of seconds and adhere to it. In Fig. 5B, the fluorescent labels (e.g., fluorescently labeled antibodies) 50 that flow into the reaction chamber anchor to their target objects. In this configuration, no mixing chamber is needed. In Fig. 5C, the reaction chamber is specially coated with secondary antibodies 52 to anchor the primary antibodies 50 from specific species. When the liquid sample is introduced to the slide, the objects of interest cannot adhere to the reaction chamber surface. Once the blister is depressed, it releases primary antibodies 50, as illustrated in Fig. 5D, which mix with the liquid sample in the microfluidic mixing chamber and attach to their targets. When reaching the reaction chamber, the primary antibodies 50 attach to the secondary ones 52, which cover the surface, and thus anchor the objects of interest. In this scenario, a washing step (by pressing on blister 19 and replacing the liquid content of the reaction chambers with washing buffer) may be needed in order to reduce background fluorescence.

Figs. 6A-6C are side views of three examples of reaction regions/chambers 32-34. The same liquid sample may contain different objects of interest 42-44. These may be individually analyzed by having a different, specialized coating 46-48 in each reaction chamber, such that specific objects will be anchored in each chamber. When the anchoring takes place through secondary antibodies (as shown in Fig. 5D), this separation may be achieved by using antibodies from different species for each object of interest. Thus for example, an object-antibody complex where the antibody is of mouse origin, would specifically anchor to a reaction chamber coated with anti-mouse secondary antibodies.

Figs. 7A-7B illustrate top views of another four examples of reaction chambers 32A-35A with the same surface coating, but treated with or containing different compounds or active substances. In Fig. 7A, once the liquid sample is added to the slide, the object of interest 42 is found in similar numbers in all the reaction chambers. In Fig. 7B, over time the object may respond differentially to the different compounds or active substances, as illustrated by the different dots numbers in the different reaction chambers. As a result, the concentration of the object of interest will not remain uniform in the different reaction chambers. One possible use, for this specific configuration, is for determining whether there is an infection (abnormal bacteria count in a bio liquid), and which antibiotic treatment would be most effective against it. In this case, each reaction chamber would contain a different, known antibiotic and the time response of bacteria would be monitored using live/dead markers. In case the test would require an extended time duration, the reaction chambers would need to be properly aerated (e.g., through a breathable membrane) and/or anaerobic conditions would need to be formed.

In various exemplary embodiments, the antibodies used in the microfluidic slide can be selective for: a) the bacterium is Acinetobacter baumannii, Actinobacillus equuli, Bacillus anthracis, Brucella melitensis, Brucella abortus, Bordatella pertussis, Bordatella bronchioseptica, Burkholderia pseudomallei, Corynebacterium diptheriae, Coxiella burnetii, Eikenella corrodens, Escherichia coli, Francisella tularensis, Francisella novicida, Fusobacterium necrophorum, Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae, Kingella denitrificans, Legionella pneumophila, Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Pasteurella multocida, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas putrefaciens, Pseudomonas cepacia, Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Treponema pallidum, Yersinia pestis, Gardnerella vaginalis, Atopobium vaginae, Sneathia sanguinegenes, Leptotrichia amnionii, Lactobacillus iners, Lactobacillus crispatus, Eggerthella Prevotella, BVAB1, BVAB2, BVAB3, Finegoldia magna, Megasphaera type 1, Megasphaera type 2, BVAB-TM7, Mobiluncus curtisii, Mobiluncus mulieris, Neisseria gonorrhoeae, Megasphaera genus, Lactobacillus acidophilus, Lactobacillus jensenii or Vibrio cholera; b) the Rickettsia is Chlamydia pneumoniae, Chlamydia trachomatis, Rickettsia prowazekii, or Rickettsia typhi; c) the virus is Measles virus, HIV virus, Hepatitis C virus, Hepatitis B virus, Dengue Virus, Western Equine Encephalitis virus, Eastern Equine Encephalitis virus, Venezuelan Equine Encephalitis virus, Enteroviruses, Influenza virus, bird flu, Coronavirus, SARS Coronavirus, Polio virus, Adenovirus, Parainfluenza virus, Hanta virus, Rabies virus, Argentine Hemorrhagic Fever virus, Machupo virus, Sabia virus, Guanarito virus, Congo-Crimean Hemorrhagic Fever virus, Lassa Hemorrhagic Fever virus, Marburg virus, Ebola virus, Rift Valley Fever virus, Kyasanur Forest Disease virus, Omsk Hemorrhagic Fever, Yellow Fever virus, Smallpox virus, a retrovirus, Monkeypox virus, or foot and mouth disease virus; d) the fungal agent is Coccidiodes immitis, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Sporotrhix schenki, Candida glabrata, Saccharomyces cerevisiae, Candida krusei, Candida dubliniensis, Candida lusitaniae, Candida tropicalis, Candida inconspicua, Candida haemulonii, or Aspergillus fumigates ; e) the parasitic agent is Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Toxoplasma gondii, Plasmodium bergeri, Schistosoma mansoni, Schistosoma hematobium, Schistosoma japonicum, Entamoeba histolytica, Babesia, Toxoplasma gondii, Trypanosoma cruzi, Leishmania ssp, Trypanosoma brucei, Trichinella spiralis, Toxocara canis, Necator americanus, Trichuris trichura, Enterobius vermicularis, Dipylidium caninum, Entamoeba histolytica, Dracunculus medinensis, Wuchereria bancrofti, Brugia malai, Brugia timori, Strongyloides stercoralis, Ascaris lumbricoides, Onchocerca volvulus, Naegleria fowleri, Clonorchis sinensis, Cryptosporidium parvum, Trichomonas vaginalis, or Leishmania spp; or f) blood cells, such as red blood cells, white blood cells or platelets.

It should be noted that the above list is by no means inclusive or limiting the broad aspect of the invention.

In the following Figs. 8 to 27 various exemplary embodiments of the components of the imaging system 10, including the microfluidic slide, the optical arrangement, the light source assembly and the mobile device are described. For clarity, it is noted that the sample S as used herein means each or both of the dedicated location of the sample and existence of the sample in the location, in other words the region of interest ROI.

Reference is now made to Fig. 8 illustrating a non-limiting example of part of the imaging system where a mobile device 10D is attached to a microfluidic slide 10A. In this specific example, the mobile device includes the camera system 10D1 and the light source assembly is constituted by a light source 10B included in the mobile device. As shown, the mobile device is placed on top of the microfluidic slide such that the camera system is coupled to the reaction chamber 10A1 via an adapter or spacers 110 attached to the slide to ensure a reproducible positioning of the mobile device relative to the slide (at a known and fixed distance between them). Light from the light source of the mobile device passes through an excitation filter 108 of the illumination assembly, which selects the excitation wavelength, before entering a wave guide(s) of the illumination assembly, e.g. an optical fiber bundle 114 whose ends face the excitation filter 108. The fiber bundle, which is fixed into the slide, guides the light towards its opposite end- inside the slide and close by to the reaction chamber / sample S. An alternative location 116 for the excitation filter could be between the bundle’s end and the reaction chamber / sample S. The beam of light that reaches the sample S fluorescently excites fluorescent markers present in the reaction chamber. The resulting fluorescent radiation is collected by the plano-convex lens 26 of the light collection assembly, which is integrated in the microfluidic slide and located directly underneath the camera 10D1. The fluorescent radiation then passes through an emission filter 124 of the light collection assembly, that selects defined fluorescence wavelength(s), as it propagates towards the camera of the mobile device. Stray light from the mobile device or other sources is blocked from reaching the camera by blocking spacers 102, of the light collection assembly, surrounding both the plano convex lens and camera. In some examples, the fiber optic bundle’s cross-section (which faces the mobile device) covers a large area such that the same slide will work with many different mobile devices, which may vary significantly in the exact location of their light source. By randomly scrambling the fibers in the bundle, the illumination profile of the reaction chamber could also be designed to be homogeneous and independent of the light source and its precise location. In addition, the excitation filter 108 may not be needed when using stained fibers, which transmit only a selected waveband.

In some embodiments of the invention utilizing fluorescent imaging, quantum dots can be used as fluorescent labels (this may include other nano-particles as well that also have a narrow emission spectrum, which is well separated from the excitation spectrum typically in the UV spectrum). With this, for a number of different fluorophores imaged in parallel, either a single broad-band emission filter may suffice, or even no emission filter at all. This would require an external UV light source. An excitation filter is not required in this case, however it may be used to reduce leakage of light. Thus, in this setup it is possible to have no filters at all or just a single emission filter.

Figs. 9A-9B illustrate a configuration of part of the imaging system, similar to Fig. 8, except for that the light approaches the reaction chamber from a number of different directions via a corresponding number of wave guides, thus providing a more uniform illumination. In this example, the light travels through two fiber bundles 114A and 114B, integrated in or passing through the slide, and through corresponding excitation filters 116 and 117, and reaches the sample S as illustrated by the arrows 118 and 119 respectively. In Fig. 9B, top view of the reaction chamber and the fiber bundles 114A and 114B is exemplified.

Figs. 10A-10B illustrate another possible configuration of the illumination assembly for guiding the illumination light from the excitation filter 108 to the reaction chamber and the sample S therein. Here, the top surface of the microfluidic slide facing the excitation filter is an off-axis Fresnel lens 130. The light reaching the Fresnel lens, illustrated by arrows 128, is redirected, as illustrated by arrows 132, to fluorescently excite the relevant markers present in the reaction chamber. The Fresnel lens 130 covers a large area such that the same slide will work with many different mobile devices, which may vary significantly in the exact location of their light source. Fig. 10B illustrates ray tracing from the light source 10B to the sample S in the reaction chamber. The extreme left rays pass through the optical center of the Fresnel lens, illustrated by the dashed line 156, which does not coincide with that the optical center of the plano-convex lens on the slide, illustrated by the dashed line 158.

Figs. 11A-11B illustrate a configuration similar to that in Figs. 9A-9B, with the addition of a collimator lens 150 next to the excitation filter 108. The collimator lens 150 is designed such that light propagating towards the microfluidic slide (arrows 128) is partially or fully collimated. Here, as shown in Fig. 11B, the sample S is at the back focus of the Fresnel lens 130, whose optical axis 156 coincides with the optical axis 158 of the plano-convex lens.

Figs. 12A-12B illustrate a configuration similar to Figs. 10A-10B, where the light beam(s) 132 coming from the Fresnel lens 130 propagate(s) towards a back-surface mirror 134. The light reflected from the back-surface mirror, illustrated by arrows 136, illuminates the sample S. Fig. 12B illustrates the ray tracing from the light source 10B to the sample S. The extreme left rays coming from the light source pass through the optical center of the Fresnel lens 156.

Figs. 13A-13B illustrate a configuration as in Figs 12A-12B where the light 128 reaching the Fresnel lens 130 is collimated by the collimator lens 150. Here, as shown in Fig. 13B, the optical axis 156 of the Fresnel lens coincides with the optical axis of the sample S and the optical axis of the plano-convex lens 26. Also illustrated in Fig. 13B, the ray tracing from the light source 10B to the sample S with both Fresnel and plano-convex lenses sharing the same optical axis.

Figs. 14A-14B illustrate a configuration similar to Figs. 12A-12B, with the addition of a second Fresnel lens 148, which is engraved on the bottom side of the microfluidic slide. The optical axis 160 of the second Fresnel lens lies between that of the first Fresnel lens 130 and that of the plano-convex lens 26. Fig. 14B illustrates the ray tracing from the light source 10B to the sample S. The first Fresnel lens 130 collects the light from the light source 10B, and the second Fresnel lens 148 de-magnifies the first Fresnel lens and projects its image on the sample S, after reflection from the back-surface mirror 134. Figs. 15A-15B Illustrate a configuration similar to Figs. 14A-14B, with the light source 10B located at the focus of the first Fresnel lens 130. As shown in Fig. 15B, the second Fresnel lens 148 shares the same optical axis 160 with the plano-convex lens 26 and the sample S. Ray tracing from the light source 10B to the sample S is also illustrated, where the first Fresnel lens 130 collimates the light from the light source 10B, while the second Fresnel lens 148 focuses the light, which is then reflected from the back- surface mirror 134 before reaching the sample S.

Figs. 16A-16B illustrate non-limiting examples of the light source assembly and the optical arrangement. In Fig. 16A, the light source 142 is a single or an array of LEDs or lasers that directly illuminate the sample S. The LEDs/lasers may be an integral part of the microfluidic slide or attached to it through, for example, designated hole(s) 144. The LEDs/lasers are controlled and powered either through the mobile device’s aux or power inputs 138 or wirelessly through a battery-operated electronic circuit 140 on board the microfluidic slide. Each LED/laser may require its own excitation filter, unless its emission spectrum is narrow enough. When using a LED/laser array, the individual LEDs/lasers may be oriented so as to illuminate different reaction chambers if they exist.

Fig. 16B illustrates a configuration similar to Fig. 16A, where the LED/laser illumination is either collimated or focused towards the sample by a lens or lens array 146, which can be an integral part of the microfluidic slide. Here again, the light from the individual LEDs/lasers may be focused on different reaction chambers.

Figs. 17A-17C illustrate additional non-limiting examples of the light source assembly and the optical arrangement. In Figs. 17A and 17B, it is illustrated that: the led/laser light source(s) 205 and/or 206 is (are) not necessarily at the side of the microfluidic slide; the back side of the slide may be coated or covered by a coating 207, such that only the led/laser light can reach the sample, e.g. to block ambient light. Further, in Fig. 17B, the led/laser light may indirectly illuminate the region of interest, after being reflected off a mirror surface 208 and/or 209, or by total internal reflection. As shown in Fig. 17C, a photovoltaic panel 210 may be used to collect light from the light source of the mobile device and convert the light into electricity, which in turn can be used to power led/laser light source(s) to illuminate the sample / region of interest. The photovoltaic panel may be attached to the slide, as shown, or to an adapter located between the mobile device and the slide as will be further described below. The photovoltaic panel may also be flexibly connected to the slide or adapter through electrical wires. In this case, the photovoltaic panel may be put directly over the mobile device’s light source. This would enable use of a smaller photovoltaic panel (enough to cover just the light source). Alternatively, the led/laser may be powered by magnetic induction provided by the phone (e.g., using the Qi charging standard). In this case, the slide can also include a coil where current will be induced.

Figs. 18A-18C illustrate non-limiting examples of wave-guide(s) used in the illumination or light collection assemblies, where the illumination is not from an external LED, but from the mobile device light source 10B via wave guide(s) 114, e.g. an optical fiber bundle. Here again, by randomly scrambling the fibers in the bundle, the illumination profile could be made homogeneous and independent of the light source and its precise location. In addition, the excitation filter may not be needed when using stained fibers, which transmit only a selected waveband.

In Fig. 18B, wave guide/optical fibers 114 can collect light from the light source 10B of the mobile device and bring it to the slide. In some examples, the light source 10B (led flash) is on the back side of the mobile device and accordingly the camera system that will be used is the front camera. The fibers can connect in many different locations on the slide and the transmitted illumination light may be collimated or focused with a dedicated lens. As mentioned above, the illumination light could directly illuminate the sample or reach it by reflection from a mirror surface or by total internal reflection. The location of the excitation filter 108 may vary as well, as shown. As shown in Fig. 18C, the fibers may be connected directly below the region of interest, along the same axis as the camera of the mobile device. In this case, no lens is needed for collimation. It is noted that all this applies also when the rear camera of the mobile device is used (as depicted in Figure 18A).

The imaging system of the invention may further include an optical adapter configured and operable to be inserted between the microfluidic slide and the mobile device, wherein at least one of the following is integrated in the optical adapter: at least part of the illumination assembly, at least part of the light collection assembly and at least part of the light source assembly. Various non-limiting exemplary embodiments are illustrated in Figs. 19 to 23.

In Fig. 19, an optical adapter 152 is placed between the microfluidic slide and mobile device and is intended for repeated use with the disposable microfluidic slides. As shown, both of the excitation and emission filters, 108 and 124, which are parts of the illumination and light collection assemblies respectively, are integrated in the optical adapter, rather than being integrated in the microfluidic slide. Here, as shown, the illumination is provided by a single or array of external LEDs or lasers 142 (i.e. not the light source of the mobile device), also integrated in the optical adapter. The v-shaped arrows 176, and the triangular-shaped arrows 178 depict, respectively, the possible excitation and emission light paths inside the adapter. As mentioned above, the power source for the led/laser (s) may include an external power supply (e.g., smartphone charger) as depicted in Fig. 16 and/or magnetic induction provided by the phone (e.g., using the Qi charging standard) and/or a solar panel and/or an internal battery located in the optical adapter (not specifically shown). In this case the adapter between phone and slide may also include a coil where current will be induced.

Fig. 20 illustrates a configuration similar to Fig. 19, where the illumination is provided by the mobile device’s light source 10B and transmitted to the microfluidic slide via wave guide(s) 114, e.g. an optical fiber bundle, integrated in the optical adapter.

Fig. 21 illustrates a configuration similar to Fig. 20, where an imaging and magnification lens 170 is integrated in the optical adapter. In this case, the microfluidic slide does not necessarily contain an integral plano-convex lens.

Fig. 22 illustrates a configuration similar to Fig. 21, where the light from the mobile device’s light source 10B is reflected off an excitation filter 108 and a dichroic filter 174, also integrated in the optical adapter, before being focused by a lens 170 towards the sample. The emitted light from the sample is collected by the same lens 170, and passes through the dichroic filter 174 and an emission filter 124 towards the mobile device’s camera 10D1. In some embodiments, not specifically shown, a collimating lens or wave guide(s) (e.g., optical fibers) may be used to collect and guide the light from the mobile device light source to the excitation filter 108. Furthermore, in some embodiments a plano-convex lens, which is integrated in the microfluidic slide, may be used in addition or instead of the lens 170 integrated in the optical adapter.

As appreciated, the optical adapter 152 typically has no lens (lens is part of the slide ensuring the region of interest / sample is always in focus and saving the need for an additional element in the device to fine-tune the focus). Additionally, the optical adapter may be configured to fit to the majority, if not all, smartphone models (e.g., by providing a magnet, suction tape, etc., as mentioned above).

Reference is made to Figs. 23A-23B illustrating a non-limiting embodiment of the optical arrangement of the imaging system for achieving multi-colour fluorescence with the microfluidic slide, in one of two ways. One way is shown in Fig. 23A and includes using multiband filters for the excitation filters 108 and/or 116 and emission filters 124. These filters transmit in parallel two or more selected bands of wavelengths (e.g., the FITC and TRITC fluorescence excitation bands) depicted by the grey arrows 128, 118 and 126. In this first configuration, the excitation and emission filters will be arranged so as to excite and collect simultaneously the relevant fluorescence bands from all the reaction chambers (here 32-33 are shown). A second way is shown in Fig. 23B and includes dividing the excitation and emission filters into different physical regions, each region designed to transmit a single, selected wavelength band (e.g., for FITC or for TRITC fluorescence). In this second configuration, the filters will be arranged so as to excite and collect a single, and possibly different, fluorescence band from each reaction chamber (here 32 and 33 are excited by different spectra). It is noted that in some cases the excitation filters are not needed (e.g., when using narrow-band LEDs).

Reference is made to Fig. 24 illustrating a non-limiting embodiment of the reaction chamber. Side view of the microfluidic slide and a reaction chamber 32 is shown, where the top surface 166 of the reaction chamber is curved and coated with a coating 46. The curvature of the top surface is designed to compensate for the Petzval field curvature of the lens 26, as demonstrated by the ray tracing 168. In this manner, the objects anchored by the coating 46 are imaged in the same focal plane.

Fig. 25 illustrates a configuration similar to Fig. 24, where the top surface 166 of the reaction chamber is curved concavely (towards lens 26). The bottom surface of the reaction chamber is coated with coating 46. The curvature of the top surface is designed to compensate for the Petzval field curvature of the lens 26, as demonstrated by the ray tracing 169. In this manner, the objects anchored to the bottom surface by the coating 46 are imaged in the same focal plane.

Figs. 26A-26B illustrate another non-limiting example of the microfluidic slide configured and operable to be attached to a variety of mobile devices differing in the location of the camera and flash systems. As shown in the figures, there are a number of through-holes 211 in the slide, for example hole 211A. According to the mobile device (e.g. smartphone) model, the user is guided (e.g. by app running on the mobile device) to plug one end of a wave guide 213, e.g. an optical fiber, through the appropriate hole aligned with the light source of the mobile device, such as the hole 211A. Subsequently, the slide needs to be properly placed next to the mobile device, such that its lens 26 is above the camera 10D1 and the right hole is above the light source (flash). Orientation arrows 214 and 215 on the slide may be used to this end. When this is done, the through-hole being used is directly below the light source (flash of the mobile device) and collects its illumination. Optionally, a plastic lens 216 may be placed between the wave guide / optical fiber and the flash to improve light collection. The other end of the wave guide / optical fiber is plugged at the bottom of the slide, directly below the excitation filter and the sample / region of interest. In this manner, the same slide will work with many different mobile devices, which may vary significantly in the exact location of their light source.

Fig. 27 illustrates yet another non-limiting example of the microfluidic slide and the illumination assembly. Instead of through holes, as illustrated in Figs. 26A-26B, there are lenses 217 (e.g. Fresnel or plano-convex lens), with focal length designed to collimate the light from the light source of the mobile device. Below the lenses are slanted grooves 218, such that the collimated light undergoes total internal reflection inside the slide until reaching the sample / region of interest. The angle of the groove relative to the top surface 219 of the slide may differ from location to location, to uniformly illuminate the region of interest.

Figs. 28- 34 illustrate the image analysis process for acquisition of the quantitative data of objects of interest.

In Figs. 28A-28C, recovery of saturated pixels in one colour using the bleed-through to another colour is exemplified. In Figs. 28A and 28B, Green and red image components of bacteria labeled by green fluorescence, taken with a mobile device, are shown. Green image peak values are saturated 255. Red image is 3.5 times weaker (bleed through factor), as calculated for the average ratio of non-saturated pixels in both images. Images were first registered using fiducial markers to correct for any movement during acquisition and then averaged in order to increase bit- depth and to enhance signal to noise. Fig. 28C shows recovered green image using the following formula: if a green pixel is close to saturation (>230), it is replaced by the corresponding red pixel multiplied by the bleed through factor. The recovered image bit depth is increased and there are no saturated pixels. Maximum pixel value is now 844 and retains its quantitative significance (instead of the unmeaningful, saturated value of 255).

Figs.29A-29B show correction of uneven field illumination. By flattening the background, it is easier to identify single objects vs. aggregates (see below). In Fig. 29A, the illumination intensity of the image shown in Fig. 28C is estimated from smoothing the image background and eliminating the segmented objects. In Fig. 29B, the normalized image shown in Fig. 28C is shown. Zero is imposed where illumination is less than 1/5 of the maximum. For this image, total (integrated) intensity of bacteria peaks is proportional to the number of bacteria in the peak.

Figs. 30A-30B show enhancement of objects of interest and local background subtraction using the Weiner-filter. This is needed for object segmentation. Fig. 30A is the original image, and Fig. 30B is the enhanced image.

In some examples, when the objects of interest in the image are labeled with more than one colour, these can be resolved, for example, by using the following: given n filter sets and m fluorophores, the total intensity of a unit concentration of fluorophore j (j=l ... m) measured through filter set i (i=l ... n) is given by T(i,j). If a mixture of fluorophores with concentrations F(j) exists, the measured total intensity through filter i will be: C(i)=å j=i,_m T(i,j)F(j). Therefore, if all n filters are used to measure C(i), the concentrations of the fluorophores in the mixture can be determined from F(j)

T^_1(j,i)C(i), where T^_1(j,i) is the inverse matrix of T(i,j) and is calculated by Singular Value Decomposition (SVD).

Fig. 31 shows conversion of Fig. 30B into a binary image using a specialized object- recognition thresholding algorithm based on pixel-by-pixel multiple criteria (e.g. extent, local maxima and contrast).

Figs. 32A-32B show a connected component analysis of Fig. 31 by employing a watershed algorithm. In Fig. 32A, segmented objects are painted in random grayscale. In Fig. 32B, magnification of the dashed white box in Fig. 32A shows separation of touching segments.

Figs. 33A-33D show quantification of objects found in Figs. 32A-32B. Single parametric distributions (histograms) are shown: total intensity (Fig. 33A), area (Fig. 33B) and background- subtracted total intensity (Fig. 33C). The small peaks of segments, including for an aggregate of two objects, are clearly seen. In Fig. 33D, multiparametric clustering is illustrated: projection to the two largest principle components is displayed. The median parameters for the cluster define robust values for single object image segments.

Fig. 34 shows the identification of single objects and of aggregates found in Fig. 32A. Segments including a single object are marked by a plus sign,‘x’ marks noise segments, and squares mark segments that contain an aggregate of two objects or more. It is noted that watershed resolves two close-by objects, as long as their separation is comparable or larger than the spatial resolution in the image. The identification of object aggregates enables to correctly count their number.

Figs. 35A-35C illustrate a slide with fluorescent E. coli bacteria that was imaged, as shown in Fig. 35A, with a fluorescence microscope (Leica DMI6000B using a x20/0.70 objective) . The same region was then imaged, as shown in Fig. 35B, using the imaging system of the invention, with a smartphone (HTC 10). All the bacteria detected by the microscope are clearly visible with the imaging system of the invention. Identification of single objects and of aggregates found in Fig. 35A is shown in Fig. 35C. Segments including a single object are marked by a plus sign,‘x’ marks noise segments, and squares mark segments that contain an aggregate of two or more objects. After extracting the number of single objects in each aggregate, the same number of bacteria (157) is found for images of Figs. 35A and 35B.

Fig. 36 illustrates by way of flowchart a non-limiting example of the image analysis process used for quantification of the objects of interest in a sample after being imaged by the camera system. In step 36A, image alignment and correction is applied. In step 36B, uneven field illumination correction is applied. In step 36C, contrast enhancement and local background subtraction is applied. In step 36D, object segmentation is applied, and in step 36E, object quantification and rejection of noisy segments is performed.

Claims

CLAIMS:

1. An imaging system comprising:

an optical arrangement comprising: an illumination assembly configured and operable to deliver the light arriving from said light source assembly to said one or more reaction regions along an illumination path, and a light collection assembly configured and operable to collect light from said one or more reaction regions, while containing said one or more liquid samples, along an imaging path, wherein at least part of the light collection assembly is integrated in said microfluidic slide;

a mobile device comprising a camera system configured and operable to optically couple to said light collection assembly and capture one or more images, of said one or more reaction regions while containing said one or more liquid samples, according to a predetermined frequency and/or time pattern(s); and

2. The imaging system according to claim 1, wherein said one or more reaction regions comprise one or more reaction chambers, or one or more reaction channels, or a combination of reaction chambers and channels.

3. The imaging system according to claim 1 or 2, wherein said microfluidic slide comprises a microfluidic system configured and operable to receive and selectively convey one or more labeling agents and/or said one or more liquid samples to said one or more reaction regions.

4. The imaging system according to claim 3, wherein said microfluidic system comprises one or more entry holes configured and operable to receive said one or more liquid samples and/or said one or more labeling agents.

5. The imaging system according to claim 3 or 4, wherein said microfluidic system comprises one or more containers being preloaded with said one or more labeling agents and configured and operable to selectively release said one or more labeling agents to said one or more reaction regions on a user demand.

6. The imaging system according to claim 3 or 4, wherein said microfluidic system is configured to hold one or more of said one or more labeling agents in dry form such that they get dissolved when said one or more liquid samples pass over the one or more labeling agents in dry form, on the way to said one or more reaction regions.

7. The imaging system according to any one of claims 3 to 6, wherein said microfluidic system comprises a mixer configured and operable to selectively and efficiently mix the one or more labeling agents with said one or more liquid samples in the one or more reaction regions, or in dedicated one or more mixing chambers.

8. The imaging system according to any one of claims 3 to 7, wherein said microfluidic system comprises one or more washing buffer container(s) configured and operable to selectively and controllably release one or more preloaded washing buffer(s) to said one or more reaction regions on a user demand to thereby wash out unbound labeling agents and/or objects of interest.

9. The imaging system according to any one of claims 3 to 8, wherein said microfluidic system comprises one or more fluid outlets configured and operable for discharging said one or more liquid samples and/or said one or more labeling agents from said reaction regions.

10. The imaging system according to any one of the preceding claims, wherein said illumination and imaging paths are different.

11. The imaging system according to any one of claims 1 to 9, wherein said illumination and imaging paths are at least partially overlapping.

12. The imaging system according to claim 11 , wherein said optical arrangement comprises an epi-illumination assembly.

13. The imaging system according to any one of the preceding claims, wherein at least one of said illumination and light collection assemblies comprises one or more wave guide(s) configured and operable to respectively define at least part of said illumination and/or imaging path(s).

14. The imaging system according to any one of the preceding claims, wherein said illumination assembly comprises one or more Fresnel lenses.

15. The imaging system according to any one of the preceding claims, wherein said light collection assembly comprises one or more lenses each being configured as spherical or aspherical plano-convex lens.

16. The imaging system according to claim 15, wherein said one or more lenses are integrated in said microfluidic slide.

17. The imaging system according to claim 15 or 16, wherein said one or more reaction regions have one or more curved top surfaces to thereby correct for Petzval field curvature of the one or more plano-convex lenses.

18. The imaging system according to any one of the preceding claims, wherein said light collection assembly comprises one or more emission filter(s) each being configured and operable to selectively pass predetermined light wavelengths arriving from one or more of said one or more reaction regions.

19. The imaging system according to any one of the preceding claims, wherein said illumination assembly comprises one or more excitation filter(s) each being configured and operable to selectively pass predetermined light wavelengths from said light source assembly to one or more of said one or more reaction regions.

20. The imaging system according to any one of the preceding claims, wherein said optical arrangement comprises a blocking structure configured and operable to eliminate interference between ambient light or the light from the light source assembly, and light collected by the camera system.

21. The imaging system according to any one of the preceding claims, wherein said light collection assembly and/or said camera system is(are) configured and operable to capture said one or more images from different focal planes in the reaction regions.

22. The imaging system according to any one of the preceding claims, further comprises a spacer frame or a holder configured and operable to hold said mobile device and said microfluidic slide with predetermined distance and orientation therebetween.

23. The imaging system according to any one of the preceding claims, wherein at least one of the following is integrated in said microfluidic slide: at least part of said illumination assembly, and at least part of said light source assembly.

24. The imaging system according to any one of the preceding claims, wherein at least part of said light source assembly is integrated in said mobile device.

25. The imaging system according to claim 24, wherein said illumination assembly is configured and operable to deliver light arriving from a variety of positions of said light source assembly integrated in said mobile device.

26. The imaging system according to claim 24, wherein said microfluidic slide comprises at least part of said illumination assembly and is configured and operable to receive light arriving from a variety of positions of said light source assembly integrated in said mobile device.

27. The imaging system according to claim 26, wherein said microfluidic slide comprises a plurality of through holes defining a respective plurality of trajectories of said illumination path.

28. The imaging system according to any one of the preceding claims, further comprising an optical adapter configured and operable to be inserted between said microfluidic slide and said mobile device, wherein at least one of the following is integrated in said optical adapter: at least part of said illumination assembly, at least part of said light collection assembly and at least part of said light source assembly.

29. The imaging system according to any one of the preceding claims, wherein said mobile device is one of the following: a smartphone, a tablet, a laptop, a netbook, a desktop, a telehealth device, or a specifically designated device.

30. The imaging system according to any one of the preceding claims, wherein said mobile device comprises said processing utility.

31. The imaging system according to any one of the preceding claims, wherein said one or more images are indicative of one or more fluorescence emission(s) of said one or more objects of interest.

32. The imaging system according to any one of the preceding claims, wherein said microfluidic slide comprises a plurality of reaction regions each identically coated with predetermined one or more coatings to thereby anchor a specific object of interest, and a respective plurality of containers each associated with a different one of said reaction regions and preloaded with a different liquid comprising an active substance to be released into the respective reaction region upon a user demand.

33. The imaging system according to any one of claims 1 to 31 , wherein said microfluidic slide comprises a plurality of reaction regions each identically coated with predetermined one or more coatings to thereby anchor a specific object of interest, and each coated with or containing a different active substance.

34. The imaging system according to claim 32 or 33, wherein said specific object of interest is a bacteria and said active substance is an antibiotic, thereby enabling the imaging system to be used in an antibiogram test.

35. The imaging system according to any one of the preceding claims, wherein said microfluidic slide comprises a filter configured and operable to capture one or more objects of interest on its surface while said one or more liquid sample flow through said filter, said filter thereby defining a reaction region in the microfluidic slide.

36. The imaging system according to any one of the preceding claims, wherein said illumination assembly comprises a photovoltaic panel configured and operable to supply power to activate said light source assembly.

37. The imaging system according to any one of the preceding claims, wherein said illumination assembly comprises at least one collimation lens configured to receive the light of the light source assembly and deliver collimated light, said microfluidic slide comprises slanted grooves in the illumination path such that the collimated light undergoes total internal reflection inside the microfluidic slide until reaching the reaction region.

38. The imaging system according to any one of the preceding claims, wherein said microfluidic slide comprises a magnet configured and operable to enable attachment of the microfluidic slide to the mobile device.

39. The imaging system according to any one of the preceding claims, wherein said microfluidic slide comprises an induction coil configured and operable to couple to an induction coil in the mobile device, thereby enabling powering said light source assembly.

40. The imaging system according to claim 28, wherein said microfluidic slide and/or optical adapter comprises at least one magnet configured and operable to enable attachment between the microfluidic slide, the optical adapter and the mobile device.

41. The imaging system according to claim 28, wherein said optical adapter comprises an induction coil configured and operable to couple to an induction coil in the mobile device, thereby enabling powering said light source assembly.

42. The imaging system according to any one of the preceding claims, wherein said processing utility is configured and operable to generate said quantitative data, relating to said one or more objects of interest, being indicative of one or more of the following: bacterial or viral load;

caries;

periodontitis;

Tuberculosis;

urinary tract infections;

vaginitis;

sexually transmitted infections;

Streptococcal pharyngitis.

Red blood cells

White blood cells

Platelets.

43. The imaging system according to any one of the preceding claims, wherein said processing utility is configured and operable to analyze said input data indicative of said one or more images, by applying one or more of the following:

image correction including image registration using fiducial markers;

image correction including averaging of the captured images to thereby increase bit-depth and enhance signal to noise;

image correction including uneven field illumination correction;

image enhancement including contrast enhancement and local background subtraction; object segmentation including connected component analysis employing watershed algorithm and specialized object-recognition based on pixel-by-pixel multiple criteria;

object quantification including defining objects of interest and rejecting non objects of interest segments based on parameter distributions and clustering in multi -parametric scatter-plots; object quantification including counting the number of true objects of interest in the image after identification of objects of interest aggregates.

44. The imaging system according to any one of the preceding claims, wherein said processing utility is configured and operable to save the image data and/or the generated output data in a memory.

45. The imaging system according to claim 44, wherein said processing utility is configured and operable to apply machine learning to the saved image and output data to thereby improve the analysis of the input data.

46. A microfluidic slide comprising:

47. The microfluidic slide according to claim 46, wherein said integrated optical arrangement comprises an illumination assembly configured and operable to deliver light arriving from a light source assembly to said one or more reaction regions.

48. The microfluidic slide according to claim 46 or 47, wherein said microfluidic system comprises one or more containers being preloaded with said one or more labeling agents and configured and operable to selectively release said one or more labeling agents to said one or more reaction regions on a user demand.

49. The microfluidic slide according to any one of claims 46 to 48, comprising:

50. The microfluidic slide according to any one of the claims 46 to 48, comprising:

51. The microfluidic slide according to any one of the claims 46 to 50, comprising a filter configured and operable to capture one or more objects of interest on its surface while said one or more liquid sample flow through said filter, said filter thereby defining a reaction region in the microfluidic slide.

52. The microfluidic slide according to any one of the claims 47 to 51, comprising a photovoltaic panel configured and operable to supply power to activate light source assembly.

53. The microfluidic slide according to any one of the claims 47 to 52, comprising slanted grooves such that collimated light arriving at the microfluidic slide undergoes total internal reflection inside the microfluidic slide until reaching the reaction region.

54. The microfluidic slide according to any one of the claims 47 to 53, wherein said illumination assembly is configured and operable to receive light arriving from a variety of positions outside the microfluidic slide.

55. The microfluidic slide according to claim 54, comprising a plurality of through holes defining a respective plurality of illumination paths of light to thereby enable delivery of light from a respective plurality of positions of said light source assembly with respect to said one or more reaction regions.

56. The microfluidic slide according to any one of claims 46 to 55, wherein said one or more reaction regions comprise one or more reaction chambers, or one or more reaction channels, or a combination of reaction chambers and channels.

57. A computer-implemented system for use in image processing, the system comprises a memory utility storing one or more sequences of instructions for analyzing one or more images acquired on one or more liquid samples containing one or more objects of interest, and a processor utility configured to process said instructions, wherein said one or more sequences of instructions comprise applying one or more of the following operations on said one or more images:

image correction including image registration using fiducial markers;

image correction including uneven field illumination correction;

image enhancement including contrast enhancement and local background subtraction; object segmentation including connected component analysis employing watershed algorithm and specialized object-recognition based on pixel-by-pixel multiple criteria; object quantification including multi-parametric quantification of object of interests segments;

object quantification including defining objects of interest and rejecting non- objects of interest segments based on parameter distributions and clustering in multi -parametric scatter-plots; object quantification including counting the number of true objects of interest in the image after identification of objects of interest aggregates;

58. A non-transitory computer readable medium including one or more sequences of instructions for image acquisition and processing, wherein execution of the one or more sequences of instructions by one or more processors of a computing device comprising a camera system causes the computing device to perform a process comprising:

activating and controlling a light source to illuminate a region of interest;

o image correction including image registration using fiducial markers;

o image correction including uneven field illumination correction;

o image enhancement including contrast enhancement and local background subtraction; o object segmentation including connected component analysis employing watershed algorithm and specialized objects of interest recognition based on pixel -by-pixel multiple criteria;

o object quantification including multi-parametric quantification of objects of interest segments; o object quantification including defining objects of interest and rejecting non objects of interest segments based on parameter distributions and clustering in multi-parametric scatter-plots;

o bacterial or viral load;

o caries

o periodontitis;

o Tuberculosis;

o urinary tract infections;

o vaginitis;

o sexually transmitted infections;

o Streptococcal pharyngitis;

oRed blood cells;

o White blood cells;

o Platelets

and,

generating corresponding output data to be presented to a user.