AUTOMATED CELL PROCESSING AND DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY

DEVICES AND METHODS

BACKGROUND

[0001] Primary cell culture allows for the study of native tissue samples derived from an organism. Culturing cells derived from organisms, can be useful and necessary for applications such as cell-based assays, medical diagnostics, medical prognostics, compound discovery, therapy selection, and characterization of adverse pathologies towards discretely stratifying patients during clinical trials and treatment of disease.

[0002] For example, cancer diagnosis and identification of compounds for treatment of cancer are of great interest due to the widespread occurrence of the diseases, high death rate, and recurrence after treatment. Survival of a cancer patient depends heavily on detection of risk (screening), presences of disease (diagnosis), determination of aggressiveness (prognosis), ideal treatment (treatment selection). As such, developing technologies applicable for sensitive and specific methods for the various modalities of detection of cancer is an inevitable task for cancer researchers, and clinicians. Currently, traditional diagnostic methods however are not very powerful, providing only sub-optimal sensitivity and specificity statistics when it comes to cancer the range of detection modalities at very early stages and give little prognostic information, leading to higher co-morbidities, and mortality. Moreover, some of the detection modalities / methods are labor intensive, costly and not accessible for many people. Moreover, detection modality technologies suffer from a variety of shortcomings such as time to result, poor or ambiguous specificities and sensitivities, which leads to overtreatment or late detection.

[0003] Likewise, existing methods for cancer staging are often qualitative, highly labor intensive, and limited in applicability. For example, diagnoses made by different physicians or of different patients using existing methods such as a Gleason Score for prostate cancer can be difficult to compare in a meaningful manner due to the subjective nature of these methods. As a result, the subjectivity of the existing methods of cancer staging often results in overly aggressive treatment strategies, or under-treatment strategies. By way of example, in the absence of better data, the most drastic, potentially invasive, strategy is often recommended, which can lead to overtreatment, poor patient quality of life, and increased medical costs. Further, some patients' disease may go undetected and present aggressively "unannounced" leading to severe co-morbidity and/or mortality.

[0004] One method to detect and/or characterize cancer, for example, is to directly assess living tissue derived from small biopsy samples (i.e. needle biopsy samples and/or fine aspirate biopsy samples) taken from suspicious tissue. This is a manual and labor-intensive process, with a fair amount of subjectivity.

[0005] Therefore, the development of technology that is specific and reliable for culturing primary human tissue and/or detecting and characterizing a cancer in an automated manner is an area of significant importance. The present disclosure addresses these and other needs in the art.

SUMMARY

[0006] According to frequently included embodiments of the present disclosure, an automated system for differential interference contrast (DIC) microscopy is provided, comprising: a DIC module comprising an imaging platform and an imaging device adapted to conduct DIC microscopy operably connected with an image capture device; a chip transport mechanism operably connected with the DIC module and adapted to transport a chip within the DIC module or to/from the DIC module; a fluidics system adapted to transport a fluid to a chip or away a chip; an optional environmental control system adapted to monitor and control a temperature within one or more modules; and a graphical user interface operably connected with a processor and computer-readable tangible storage medium adapted to control the DIC module, the chip transport mechanism the fluidics system and/or the environmental control system. Two or more devices for conducting DIC microscopy may be included in the DIC module.

[0007] According to often included embodiments, the chip transport mechanism comprises a carousel. Often, the carousel is in operable connection with a cell culture module (often comprised in the chip) and the DIC module and is adapted to transport the chip between other locations of the system such as the cell culture module, if included as a discreet element, and the DIC module. Often, the carousel is in operable connection with the DIC module and is adapted to transport the chip the DIC module. Often, the chip transport mechanism further comprises a pick and place mechanism or track system adapted to load the chip on the carousel. In frequent embodiments, the chip transport mechanism comprises a carousel and the chip transport mechanism further comprises a pick and place mechanism or track system adapted to load the chip on the carousel, wherein the solid tissue sample processing module is operably connected with the DIC module by the chip transport mechanism, and wherein the chip transport mechanism is adapted/programmed to transport the chip to DIC module in an automated manner after introduction of the single cell suspension to the chip. Often, the chip transport mechanism comprises a track having one or more puck adapted to accept the chip for loading, transport and/or unloading.

[0008] In frequent embodiments, the chip transport mechanism further comprises a pick and place robot. Often, the pick and place robot is in operable connection with the cell culture module and adapted to transport the chip between a chip intake or the cell culture module to the DIC module. Also frequently, the pick and place robot is in operable connection with a solid tissue sample processing module and adapted to transport the chip between the solid tissue sample processing module and the DIC module. In certain contemplated embodiments, the chip transport mechanism comprises a carousel, a pick and place robot, a track and puck system, an elevator system, or a combination of two or more of the foregoing. In often included embodiments, the chip transport mechanism is adapted to transport the two or more chips into and out of an imaging region defined by an area observable by the imaging device. Frequently, the chip transport mechanism is programed/adapted to transport the two or more chips into and out of the imaging region in a sequence wherein each of the two or more chips is imaged two or more times in an alternating sequence. In such embodiments, often the alternating sequence is defined by a time period outside the imaging region and a time period inside the imaging region. According to other related often included embodiments, the system is programed to apply two or more alternating sequences to one or more of the two or more chips. The time period inside the imaging region comprises up to 148 hours. The time period inside the imaging region also often is determined by establishing a pre-sent number of images to acquire, for example up to 1,000,000,000,000 images. Also often, imaging may occur in discreet time periods or until a predetermined number of images are obtained, for example in increments of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes or more, which may involve the acquisition of up to about 1,000,000,000 images.

[0009] In often included embodiments, the systems of the present disclosure are incorporated in a single stand-alone instrument or instruments connected via an automated transport mechanism comprising a housing or series of housing confining the elements of each instrument and/or module. As such, according to contemplated embodiments the DIC module, a transport mechanism and the fluidics system are comprised within a single housing of an instrument, optionally including an incubation module). Often, in such embodiments a solid tissue sample processing module is (also) located within the single housing of the instrument.

[0010] According to the systems of the present disclosure a solid tissue sample processing module is included which comprises a solid tissue dissociator adapted to dissociate a biological solid tissue into a single cell suspension. In frequent embodiments, the solid tissue dissociator comprises at least one microfluidic cell dissociation module. Frequently, the solid tissue dissociator is adapted to permit introduction of a solid tissue processing reagent such as a digestive enzyme. Often, the sample processing module further includes a cell suspension transport adapted to transport all or a portion of the single cell suspension from a solid tissue dissociator vessel to the chip, wherein the chip is adapted to support cell culture. The cell suspension transport in frequent embodiments comprises a pipettor, and/or other fluid control mechanism contemplated herein such as a vacuum, pump, mechanical fluid connection, peristaltic pump, microfluidic connection or other connection.

[0011] When a microfluidic cell dissociation module is included, it often features a cell inlet port for receiving a biological solid tissue sample, a cell dissociation chamber comprising a plurality of microstructures, and wherein the microstructures comprise diamond or rectangular shaped posts, a reagent inlet for receiving a solid tissue processing reagent, the reagent inlet being in fluid communication with the cell dissociation chamber, an outlet for extracting fluid, a channel fluidly coupled to the cell inlet, cell dissociation chamber, reagent inlet, and outlet port to allow controlled flow through the device, and optionally a pump coupled to the cell inlet and/or the reagent inlet to cause displacement of fluid through the channel, the cell dissociation chamber, and the outlet.

[0012] Chips of the present disclosure are intended to be broadly described and include microfluidic devices, multi-well plates, imaging chambers, or other similar surface suitable for cell culture and imaging according to the present methods and systems. Such chips are portable or otherwise movable by the transport mechanisms discussed and contemplated herein. Often, an exemplary the chip comprises a cell suspension inlet port for receiving a cell suspension, a perfusion chamber comprising a cell adhesion surface for culturing, imaging, and/or assaying a cell, wherein the perfusion chamber comprises an optically transparent portion, wherein the optically transmissive portion is positioned relative to the cell adhesion surface to permit optical interrogation of a cell adhered to the cell adhesion surface; and a reagent inlet for receiving assay reagents, the reagent inlet being in fluid communication with the perfusion chamber. According to the embodiments of the present disclosure, the cell culture module is most frequently comprised in the chip rather than a separate stand-alone module of the system, which incorporates or is also an imaging chamber. In general, the chip is movable within the automated system. Often, the chip comprises a surface adapted for primary cell culture that is observable by the imaging device when present in the DIC module. Also often, the chip comprises a reagent access port adapted to permit entry of a reagent to the surface adapted for cell culture. Moreover, often the solid tissue sample processing module and a cell culture module are comprised in the chip. In frequently included embodiments, the chip comprises an imaging chamber having an observable surface adapted for cell culture or adapted to accept the introduction of a reagent that provides a predetermined matrix surface for cell culture.

[0013] Often, the imaging device of the present systems that is adapted to conduct DIC microscopy comprises a microscope and the image capture device comprises a CCD camera. Also often, the DIC microscopy comprises fluorescence microscopy. Often, the DIC module is adapted to conduct sequential or simultaneous imaging on two or more chips. In such embodiments, often the DIC module includes two or more imaging devices and one or more corresponding image capture devices. In related embodiments, often the DIC module includes two or more imaging devices and two or more corresponding image capture devices.

[0014] According to frequently contemplated embodiments the systems of the present disclosure further include a module or modules for sequencing and/or other genetic analysis Such embodiments often include a nucleic acid sequencing module often operably connected within the system by the transport mechanism or a fluid transport mechanism. According to frequently contemplated embodiments the systems of the present disclosure further include a proteogenomic analysis module often operably connected within the system by the transport mechanism or a fluid transport mechanism. According to frequently contemplated embodiments the systems of the present disclosure further include a nucleic acid sequencing module and a proteogenomic analysis module each often operably connected within the system by the transport mechanism or a fluid transport mechanism.

[0015] According to often included embodiments, the fluidics system is adapted to transport the fluid to the chip in the DIC module. Frequently, the fluidics system is adapted to transport the fluid to the chip in the time period outside the imaging region or the time period inside the imaging region. Often, the fluidics system comprises a pipettor.

[0016] According to often included embodiments, the processor is adapted to provide for remote access, monitoring and/or control over an intranet or the internet. Such monitoring or control over the intranet or the internet comprises monitoring or control of functioning or data generated in the DIC module, the chip transport mechanism the fluidics system and/or the environmental control system.

[0017] In frequently included embodiments, the environmental control system maintains a predetermined temperature in the sample processing module, the DIC module and/or the imaging platform within the DIC module. Often, the environmental control system maintains a predetermined temperature of the chip, wherein the predetermined temperature of the chip differs when present in the imaging platform versus when present in the system but outside the imaging platform, or the predetermined temperature of the chip differs when present in the DIC module versus when in the system but outside the DIC module.

[0018] According to the methods and systems contemplated herein, the system is programmed to image two or more different chips comprising two or more different samples in a simultaneous manner without human intervention.

[0019] According to the present disclosure, methods are provided for conducting automated DIC microscopy, such methods including introducing a solid tissue to a chip contemplated herein, or a solid tissue sample processing module contemplated herein to create a chip comprising a test sample, transporting the chip on the chip transport mechanism, imaging the test sample on the DIC module, and generating imaging data by imaging the test sample using the image capture device, wherein the imaging data is stored on the computer readable tangible storage medium, a cloud-based storage, or a connected dedicated electronic storage medium.

[0020] Also according to the present disclosure an automated system for cell analysis is provided, including: a DIC module comprising an imaging platform and an imaging device adapted to conduct DIC microscopy operably connected with an image capture device; a nucleic acid sequencing module; a transport mechanism operably connected with the DIC module and adapted to transport a chip within the DIC module or to/from the DIC module; a fluidics system adapted to transport a fluid to a chip or away a chip; an environmental control system adapted to monitor and control a temperature within one or more modules; and a graphical user interface operably connected with a processor and computer-readable tangible storage medium adapted to control the DIC module, the nucleic acid sequencing module, the chip transport mechanism the fluidics system and/or the environmental control system. Often, such an automated system further includes a proteogenomic analysis module, wherein the graphical user interface is adapted to control the proteogenomic analysis module. Also often, the system further comprises a sample processing module, wherein the sample processing module is operably connected with the DIC module via the transport mechanism. According to related embodiments, the system comprises a single housing containing the system or a plurality of housings operably connected via the transport mechanism.

[0021] These and other embodiments, features, and advantages will become apparent to those skilled in the art when taken with reference to the following more detailed description of various exemplary embodiments of the present disclosure in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The skilled person in the art will understand that the drawings, described below, are for illustration purposes only.

[0023] FIG. 1 provides a schematic including a variety of components of an exemplary DIC microscopy module of a system of the present disclosure.

[0024] FIG. 2 provides a schematic of an exemplary embodiment of a system of the present disclosure.

[0025] FIG. 3 provides a schematic of another exemplary embodiment of a system of the present disclosure.

[0026] FIG. 4 provides a schematic of another exemplary embodiment of a system of the present disclosure.

[0027] FIG. 5 provides a schematic of another exemplary embodiment of a system of the present disclosure.

[0028] FIG. 6 provides a schematic of another exemplary embodiment of a system of the present disclosure. [0029] FIG. 7 provides a schematic of another exemplary embodiment of a system of the present disclosure.

[0030] FIG. 8 provides a schematic of another exemplary embodiment of a system of the present disclosure.

[0031] FIG. 9 provides a schematic of another exemplary embodiment of a system of the present disclosure.

[0032] FIG. 10 provides another schematic including a variety of components of an exemplary system of the present disclosure.

[0033] FIGS. 11-14 depict a process flowchart following an exemplary sample through an exemplary system of the present disclosure, including features, process steps and exemplary analyses.

DETAILED DESCRIPTION OF THE VARIOUS EMBODIMENTS

[0034] For clarity of disclosure, and not by way of limitation, the detailed description of the various embodiments is divided into certain subsections that follow.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

[0036] As used herein, "a" or "an" means "at least one" or "one or more."

[0037] As used herein, the term "and/or" may mean "and," it may mean "or," it may mean "exclusive-or," it may mean "one," it may mean "some, but not all," it may mean "neither," and/or it may mean "both."

[0038] As used herein, "subject" often refers to an animal, including, but not limited to, a primate (e.g., human). The terms "subject" and "patient" are used interchangeably herein. [0039] As used herein, the terms "detect," "detecting," or "detection" may describe either the general act of discovering or discerning or the specific observation of a molecule or composition, whether directly or indirectly labeled with a detectable label.

[0040] As used herein, the phrase "machine learning" refers to the construction and adapting of algorithms based on data with minimal external instructions. See, e.g., C. M. Bishop, Pattern Recognition and Machine Learning (Springer 2007).

[0041] As used herein "diagnosis" refers to the ability of a test to determine, yes or no, if a patient is positive for a disease state.

[0042] As used herein "prognosis" refers to the ability of a test to determine how aggressive or indolent a disease state is, in part by predicting specific pathology findings related to the progression of a disease.

[0043] As used herein, the term "outlier" or "outlier cell" refers to a cell having a detected or measured biomarker that is distinguishable from that biomarker in one or more other cells in a specific sample or between samples. Often this term refers to a cell having at least one biomarker that is distinguishable, often to a notable degree, from the majority of other cells in the specific sample or between samples.

[0044] As used herein, the term "stage of cancer" refers to a qualitative or quantitative assessment of the level of advancement of a cancer. Criteria used to determine the stage of a cancer include, but are not limited to, the size of the tumor and the extent of metastases (e.g., localized or distant).

[0045] As used herein, "sample" refers to any substance containing or presumed to contain a cell of interest or a cell for investigation. The term "sample" thus includes a cell, organism, tissue, fluid, or substance including but not limited to, for example, blood, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, stool, external secretions of the skin, respiratory, intestinal and genitourinary tracts, saliva, blood cells, tumors, organs, tissue, samples of cell culture constituents, natural isolates (such as drinking water, seawater, solid materials), microbial specimens, cell lines, and plant cells. A "tissue sample" refers to a sample having or obtained from a tissue of a subject, including homogenized, disassociated, otherwise processed samples, cellular cultures thereof, and fractions or expression products thereof.

[0046] Any sample suspected of containing cells relevant to the therapeutic indication being evaluated can be utilized in the devices and according to the methods of the present disclosure. By way of non-limiting example, the sample may be tissue (e.g., a prostate biopsy sample or a tissue sample obtained by prostatectomy), blood, urine, semen, cells (such as circulating tumor cells), cell secretions or a fraction thereof (e.g., plasma, serum, exosomes, urine supernatant, or urine cell pellet). In the case of a urine sample, such is often collected immediately following an attentive digital rectal examination (DRE), which causes prostate cells from the prostate gland to shed into the urinary tract. The patient sample may require preliminary processing designed to isolate or enrich the sample for the markers or cells that contain the markers. A variety of techniques known to those of ordinary skill in the art may be used for this purpose.

[0047] As used herein, the term "tissue" is used in its conventional sense and refers to any component of an organism including but not limited to, cells, biological membranes, organs, bone, collagen, fluids and the like comprising some portion of an organism.

[0048] As used herein, the term "solid tissue" is used in its conventional sense and refers to any component of an organism including but not limited to solid rather than liquid cells, biological membranes, organs, bone, collagen, and the like. As used herein, solid tissue does not refer to blood or other bodily fluids.

[0049] As used herein, the phrase "single cell" refers to a composition having distinct and separated cells of a dissociated tissue.

[0050] As used herein, the term "dissociation enzyme" refers to enzymes that break down macromolecules (e.g., biomolecules) such as proteins, lipids, nucleic acids and polysaccharides. Exemplary digestive enzymes include proteolytic enzymes, lipolytic enzymes, amylolytic enzymes and nucleolytic enzymes such as, but not limited to ptyalin, amylase, betaine, bromelain, pepsin, gastric amylase, gelatinase, rennin, gastric lipase, pancreatic lipase, phospholipase, trypsin, steapsin, chymotrypsin, collagenase, hyaluroidase, carboxypeptidase, pancreatic amylase, elastases, nucleases, DNase, sucrase, maltase, lactase, isomaltase, papin, dispase and deoxyribonuclease, neutral protease, pronase, papain, collagenase type I, II, III, IV, hyoluronidase, protease type XIV, pronase, dispase I, dispase II, and/or neutral protease among other digestive enzymes.

[0051] The present description should be read with reference to the drawings. The drawings, which are not necessarily to scale, depict selected embodiments and are not intended to limit the scope of the present disclosure. The detailed description illustrates by way of example and is not intended to limit the scope of the present disclosure. The presently contemplated systems are generally used as medical diagnostic and/or prognostic systems to aid in the diagnosis or the adjustment or confirmation thereof, or to evaluate, confirm or adjust prognostic outcome or treatment plan related medical information for use by a physician or other healthcare professional. While the present disclosure focuses on instrumentation and methods, such are intended for use in such environments.

Tissue dissociation module

[0052] After receiving a tissue sample, it is dissacociated according to known methods, devices, and reagents, for example, those set forth in U.S. Patent Application Publication Nos. 20130149724 and 20130237453, and PCT Patent Applcation No. PCT/US14/61782, filed October 22, 2014, the contents of each of which are incorporated herein by reference.

[0053] In one aspect, a microfluidic tissue dissociation module can be employed. Tissue dissociation involves in the progressive isolation of smaller and smaller clusters of tissue and cell clumps into a single cell suspension. The process of dissociation can be accomplished via anumber of methods and combinations of methods including, but not limited to, enzymatic treatment, mechanical agitation, stress, and/or shear forces.

[0054] In an exemplary embodiment, tissue fragments (e.g., minced tissue, sliced biopsy tissue, etc) can be inserted in the chip. The tissue fragments may be mixed beforehand with dissociation enzymes such as trypsin, DNase, papain, collagenase type I, II, III, IV, hyoluronidase, elastase, protease type XIV, pronase, dispase I, dispase II, and/or neutral protease, among others. As will be appreciated by a person skilled in the art, the tissue fragments can range in size, for example, in a range of about 0.1mm to 1mm. In some aspects, tissue samples can be injected into the device via needles, pipettes, or integrated and exterior fluidic handling mechanisms, such as plastic tubes.

[0055] As will be appreciated by a person skilled in the art, any pressure generators known or developed hereafter and modified in accord with the teaching herein can be utilized to displace the fluid mixture within the device.

[0056] In the microfluidic dissociation environment microstructures can be incorporated into the dissociation module/chip to facilitate mechanical perturbation. For example, a plurality of microstructures is present at the top and/or bottom of the chamber to aid in the mechanical perturbation of the tissue samples. Their presence, along with the varying chamber geometries, flow rates, and/or the presence of dissociation enzymes in the formulation, can enable the reduction of tissue fragments to single cells.

[0057] Upon completion of tissue dissociation into single cells or small clumps of cells, the cells are then transported to a chip, or the imaging chamber on the chip, for culture and/or imaging.

Chip

[0058] As used herein, the term "chip" can refer to a standalone (portable) microfluidic device, (portable) imaging chamber, and/or a portable, movable, reusable or renewable substrate on which a cell culture and/or imaging and analysis contemplated herein can/does occur.

[0059] In certain embodiments the cell dissociation chamber is present in the chip together with an imaging chamber. Alternatively, disassociated cells from a solid tissue sample such as cells in a single cell suspension can be placed in a chip for optical examination. Chips including imaging chambers are contemplated such as those having characteristics set forth in U.S.

Patent Application Publication Nos. 20130149724 and 20130237453, and PCT Patent Applcation No. PCT/US14/61782, filed October 22, 2014, including related reagents and methods the contents of each of which are incorporated herein by reference.

[0060] In frequent embodiments, the chips contemplated herein support dual cell culture coupled with optical examination before, during, and/or after cell culture. Such culture and imaging often occurs in an imaging chamber or any suitable chamber of the chip. Often the chamber is coated with a reagent that has an effect on the growth and movement of the cells in the chamber such as ECM reagents set forth in U.S. Patent Application Publication Nos. 20130237453, 20160272934 and 20180239949, each of which is incorporated by reference in its entirety.

[0061] As described in connection with the Figures, the chip or a surface thereof is often treated in the presently contemplated systems to prepare the designated surface thereof for a sample. Functionalizing the surface is provided most frequently via silanization or a similar procedure as described below.

Imaging Device

[0062] A variety of imaging devices are contemplated for the systems of the present disclosure. An apparatus capable of observing a single cell being cultured on a chip, with or without contrast, with or without fluorescence excitation, and/or with or without lumescence excitation is contemplated. Often such device comprises a microscope operably coupled with a CCD camera, optionally with an operably connected excitation light source. In the most frequent embodiments, the device or devices, or apparatus adaptable to be included on a device, comprise optical device or devices adapted to or otherwise capable of illumination and image magnification and conducting differential interference contrast microscopy. One example includesconfocal microscopes such as the Fluoview confocal microscope (Olympus, Melville, NY) such as the Olympus BX50 fluorescence microscope with a 60x, N.A. 1.45 objective or the Olympus 1X81 fluorescence microscope, but a variety of other devices adapted or adaptable to conduct DIC microscopy known in the art are intended to be encompassed.

The Cascade II CCD camera (Photometries) operating at a frequency of 12.8 Hz is one exemplary image capture device, though a host of other image capture devices are contemplated and within the scope of the present disclosure. Non-confocal imaging devices are often utilized, for example, LED based illumination at 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, 100X or larger magnification.

DIC Module

[0063] The Figures depict a variety of components and/or features that may be included in exemplary Differential Interference Contrast (DIC) microscopy modules. The module includes an imaging device as a feature aspect, which imaging device is typically a microscope. The microscope includes the typical optics found in such devices used in DIC microscopy, including lenses such as a condenser, an objective lens, and optional filters. In the embodiments contemplated herein the microscope optics are controlled and operated in connection with a processor often operably linked with the graphical user interface (GUI) or a remote wired or cloud-based operating system. The DIC also includes an image capture device such as a camera, which includes a shutter, a lens, a lens aperture, and an image sensor. In the embodiments contemplated herein the image capture device (i.e., camera) optics are controlled and operated in connection with a processor often operably linked with the graphical user interface (GUI) or a remote wired or cloud-based operating system. Together the imaging device and the image capture device comprise the imaging device optics. The imaging device optics also often include an activating fluorescent light source capable of activating a fluorophore. Depending on the fluorophore being utilized, in certain embodiments multiple different activating light sources are included that are capable of exciting multiple different fluorophores each emitting light at a distinct and separately detectable wavelength.

IB In certain embodiments, a single activating light source is used to activate multiple different fluorophores each emitting light at a distinct and separately detectable wavelength.

[0064] The DIC also includes stage for holding a chip for imaging by the imaging device and image capture device. Exemplary stages include translation capability on X, Y, and/or Z planes to provide for adjustments relative to the positioning of the imaging device optics to aid in focusing an image of a sample, and to move the chip relative to the imaging device optics to image different portions of the chip or different cells within a sample on the chip.

[0065] In certain embodiments, the stage comprises a carousel or other chip carrier that holds one or more chips and is adapted to move a chip into and out of view of the imaging device optics. Also in certain embodiments, the stage is in operable connection with a carousel or other chip carrier such that a chip may be transitioned from the carousel or other chip carrier and/or to the stage, or the chip may be transitioned from the stage and/or to the carousel or other chip carrier.

[0066] The automated DIC module permits a sample to be cultured and analyzed to produce a result without human intervention. The result may often be in the form of a diagnostic or prognosticator indicator that can be used by a healthcare provider. One important significance here is that that a pathologist need not spend time to take a direct view of the (live -cell and/or 'fixed') sample or calls contained therein if not desired. Such viewing is not necessary.

Sample handling

[0067] Samples contemplated herein generally include solid tissue samples and may also include blood and other fluid biological samples. When solid tissue samples are utilized they are processed in a manner that separated individual cells from the solid tissue, often via mechanical means where regents such as enzymes may be employed to solubilize the tissue.

As discussed herein, a solid tissue dissociator may be employed for this purpose. Such a dissociator may comprise a microfluidic solid tissue dissociator. In certain embodiments, the chip comprises a microfluidic solid tissue dissociator. Solid tissue samples may be introduced to the solid tissue dissociator by any suitable means, including manual introduction or via a pipettor or other means.

[0068] In frequent embodiments the pathologist or tech manually introduces the sample to the sample processing module, or causes the sample to be introduced. From there the contemplated systems attend to the various procedures described and contemplated herein in an automated manner without human intervention until a result is provided by the system.

The result may often be in the form of a diagnostic or prognosticator indicator that can be used by a healthcare provider.

[0069] Samples should be handled in a sanitary or sterile manner to avoid contamination of the sample prior to introduction to the system. Such contamination can negatively affect the later cell culture and analysis.

[0070] Reagents such as enzymes and cell lysis reagents may be employed to prepare a target population of cells for analysis.

[0071] A cell suspension is often prepared at the sample handling stage or within the sample processing module, which cell suspension includes single cells of interest for analysis.

Cell suspension handling

[0072] Cell suspensions comprise fluid including cells of interest suspended therein, e.g., in a diluent or fluid suitable for cell culture. An automated pipettor is often employed to transition the cell suspension from a solid tissue dissociator (for example) to a chip for analysis of the cells of interest. Also often, the single cell suspension flows (in an automated or actuated manner) from the solid tissue dissociator directly to the culture/imaging chamber of the chip. Microfluidic flow of the sample within the chip in such embodiments is often employed.

Liquid handling/Reagent/Cell/Sample

[0073] Automated pipettors are often employed for reagent and liquid handling within the contemplated systems. For example, a pipettor may be used to introduce a reagent to an input port on a chip to introduce a reagent to the chip. Moreover, a pipettor may be utilized to obtain a selection of the sample for separate analysis from the chip. Also, a pipettor may be utilized to remove waste or spent reagents from the chip or other parts of the system. In certain embodiments, a waste output that utilizes a vacuum to pull liquids from the chip or elsewhere in the system is employed.

[0074] Pump-based, vacuum based, gas based, mechanical based, microfluidic flow (with or without added force), or other liquid handling is also contemplated for each liquid contemplated herein. Chip transport

[0075] Chips of the present disclosure are transported between modules, chambers, devices and/or systems to conduct different aspects of the automated assays described and contemplated herein. While certain aspects of the present systems may employ multiple actions related to the chip in a single location, others require movement of the chip from one place to another in the system. Such movements, for example, permit parallel processing of multiple chips in a single system, thereby increasing throughput in the system and in certain aspects a simultaneous analysis of multiple different samples in a single system.

[0076] A variety of transport mechanisms are contemplated for such movement of the chip to or within the contemplated systems. For example, one or more pick and place robot may be employed to move the chip from one designated location to one or more other designated locations. A conveyor or carousel may also be employed to move the chip from one designated location to one or more other designated locations. A track system, utilizing optionally a permanently affixed or a removable cradle for holding the chip, may also be employed to move the chip from one designated location to one or more other designated locations. In often contemplated embodiments, a combination of two or more transport mechanisms, often a combination of different contemplated transport mechanisms, are employed in the systems contemplated herein.

[0077] In the case of a track system having a removable cradle, it is contemplated that the cradle is adapted to directly interface with the chip and also be in removable connection with a track. The track is generally set up to be connected with two or more chambers, modules, instruments or systems such that a cradle (including two or more cradles, each being able to interface with a different chip) may be transported along the track from one location to another. It is also contemplated that two or more systems described herein are connected via a transport mechanism such as the track system such that each system may be connected, for example, to one or more sample processing modules or instruments. In practice a first sample would be introduced to and processed in the sample processing module then transferred to a chip, which is transported to a first DIC module (for example) on the transport mechanism. After processing of this first sample and while cell culture and imaging processes are undertaken in the first module, a second sample is processed in the sample processing module and transferred to a chip, which is transported to a second DIC module (for example) on the transport mechanism. Parallel processing can occur in a single DIC module or across multiple DIC modules, supported by the transport mechanism.

[0078] A pick and place robot, including two or more pick and place robot may handle the actions described above regarding the track system.

[0079] A carousel may also be employed that permits the rotation of a chip into and out of a chamber or module of the presently contemplated systems. Each chamber or module in such a setup includes a physical barrier such as a wall or housing/sub-housing between it and surrounding chambers/modules, which is particularly the case is specific environmental controls in the chamber or module are desired. In practice the chip is introduced to the carousel and interacts with the carousel such that the carousel can rotate the chip to a specific location without the chip becoming dislodged from the carousel. The location of the chip in the carousel is logged such that any data generated from the analysis of the chip is tied to that chip and ultimately to the originating sample and subject/patient. In certain embodiments the carousel rotates to a chamber or module and the chip is transferred from the carousel to the chamber or module for processing or analysis and then transferred back to the carousel after processing or analysis.

[0080] A conveyor may also be employed that permits the rotation of a chip into and out of a chamber or module of the presently contemplated systems. Each chamber or module in such a setup includes a physical barrier such as a wall or housing/sub-housing between it and surrounding chambers/modules, which is particularly the case is specific environmental controls in the chamber or module are desired. In practice the chip is introduced to the conveyor and interacts with the conveyor such that the conveyor can move the chip to a specific location without the chip becoming dislodged from the conveyor. The location of the chip in the conveyor is logged such that any data generated from the analysis of the chip is tied to that chip and ultimately to the originating sample and subject/patient. In certain embodiments the conveyor moves to a chamber or module and the chip is transferred from the conveyor to the chamber or module for processing or analysis and then transferred back to the conveyor after processing or analysis.

Graphical User Interface (GUI)

[0081] A Graphical user interface is utilized in the most frequent contemplated embodiments of the present systems. Often the GUI is a touch screen. This touch screen provides an indication of the status of the system and/or modules within the system in addition to offering an ability to program or control system and its various aspects. In one exemplary embodiment the GUI includes indicia that permit a user to scroll through various aspects and operations of the system. The GUI will often display the present stage of operation of the system, either in operation or in pre-programming. When programming the system, one often included embodiment involves a flow-through process where the user can begin by touching/actuating the GUI and program each aspect of an exemplary sample preparation and/or analysis on the system as contemplated herein. In an exemplary embodiment, the programming operates in phases where the user can program each aspect of the preparation/analysis on the GUI, each of which provides an opportunity to select from one or more variables. These variables may include operation of the DIC optics, the fluorescent optics, image acquisition, reagent addition/removal, stage movement, chip movement, environmental factors, sample identification and tracking, quality control, data organization, communication and/or analysis, start/run times, sample type, analysis type, marker selection, chip type, sample processing details, cell culture details, gas use, matrix details, imaging/culture surface preparation, etc. The GUI system often provides the opportunity to select all relevant variables related to a phase of sample preparation, culture or analysis and permits passage onto the next phase only after all requisite variables have been set. The GUI may also provide alerts related to problems in the system and optionally problem-solving options within the system.

Quality System

[0082] The quality system of the present systems is present and oversees a variety of aspects of the system to ensure everything from proper patient/data correlation and environmental requirements for an assay to contamination monitoring and image quality analysis. When focused more acutely on image quality monitoring and analysis, machine vision control involves a sub-aspect of the quality system. Overall, however, the quality system constantly monitors the system and resolves questions regarding such as whether the assay is running appropriately. And, whether the assay is running the way it is expected to. And, whether each module and the system overall is/are properly functioning.

[0083] Regarding machine vision control, a variety of mechanisms are often employed to evaluate images on a real time or delayed basis to determine whether an image take is of sufficient quality for algorithmic analysis thereof. Issues that are resolved in this control involve focus of an image, pixel density, etc. Often a BRISQUE score, Xplore, OOF acore, or another known image analysis operator is used to evaluate image quality. Such machine vision control may often yield the acceptance or rejection of certain images and also optionally suggest re-imaging or continued imaging of a sample to acquire acceptable images for analysis.

Sample tracking

[0084] When multiple patient/subject samples are processed in the presently described systems, sample tracking to ensure identity between the sample and the data/results is important. Often the first step in tracking involves the input of a sample identifier in the system, which may be a name, patient ID, or other information that specifically ties the sample to a particular subject. Such an input involves, for example, a manual input to the GUI or a remote system connected with the system, barcode scanning of a label on a carrier of the incoming sample, RFID data reading of an RFID tag on a carrier of the incoming sample, QR code use, IR coding, or another tracking means.

[0085] Once the patient/subject sample is processed into a single cell suspension or otherwise transferred to a chip, a barcode, a chip number, a QR code, a RFID tag or another identification means present on or applied to the chip is cued, coded, correlated, or imbued with data linking the chip with the patient/subject sample. With this step the chip thereby becomes trackable in the system regardless of its position such that any processing of the chip can be correlated with the originating subject/patient. Suitable barcode/QR reader or readers or RFID reader or readers are located in the system to monitor the movement of the chip on a transport mechanism, into a module or chamber, out of a chamber or module, or within a chamber or module.

Assays and data analysis

[0086] It is contemplated that assays for a variety of markers are conducted that that imaging and algorithmic evaluation of image data include assays and methods set forth, for example, in U.S. Patent Application Publication Nos. 201S02S745S, 201602729S4 and 201802S9949, each of which is incorporated by reference in its entirety.

Sequencing and genetic analysis

[0087] Exemplary systems of the present disclosure are often provided with one or more modules for conducting nucleic acid, proteomic sequencing or proteogenomic analysis, and/or transcriptomic analysis, referred to as a sequencing module or sequencing modules. Often such systems include the sequencing module(s) in a single housing together with the DIC module. Also often, an exemplary system includes a sample processing module, a DIC module and one or more sequencing module. Often such systems include a transport mechanism contemplated herein that links two or more (including all) modules in an automated manner that can optionally be controlled using the GUI.

[0088] Any of a variety of nucleic acid sequencing techniques can be employed in the presently contemplated systems, including sequencing by synthesis via any of the many variety of known methods, sequencing by base removal, shotgun sequencing, nanopore sequencing, solid state pore sequencing, ion release or pH detection (e.g., ISFET sequencing), hall-effect sequencing, piezoelectric-based sequencing methods, or other sequencing methods, including methods utilizing labeled or unlabeled nucleotides.

[0089] Proteogenomic analysis involves, according to the presently contemplated embodiments, bottom up approaches such as mass spectroscopy data-dependent acquisition involving translation of the nucleotide sequence obtained from the sample into amino acid sequence(s) analyzed via fragmentation mass spectroscopy. Other methods are contemplated, including mass spectroscopy data-independent acquisition based on methods similar to data- dependent acquisition but involving fragmentation and analysis of every peptide in the sample. Other known methods, techniques and instruments for such proteogenomic analysis are contemplated within the present systems.

Sample Types and Applications

[0090] The present methods, systems, and devices are not intended to be limited to specific sample types or tissue types. Live cell analysis methods are presented herein, which may be applied to samples of or derived from tissues or fluids. Both animal and plant cells may be evaluated according to the methods described herein.

[0091] For example, prostate tissue or cells derived from prostate tissue may be utilized as described herein. Exemplary cells include those from or derived from bladder, lung, kidney, breast, ovarian, uterine, colon, thyroid, or skin tissue, or tumors associated with the genito urinary tract or other tumors, and a variety of other cell types may also be analyzed according to the methods described herein. When discussing available cell types herein, the reference thereto is intended to be non-limiting and the listed examples are merely exemplary for reference purposes. [0092] The sensitivity and specificity numbers (as outlined in the equations below) described and obtained using methods and systems described herein, provide a predictive model for cell behavior. In certain frequent embodiments, a diagnostic tool embodied within these systems and methods is provided. In other embodiments, a prognostic tool embodied within these systems and methods is provided. Often, the presently described systems and methods are used to monitor the health or treatment of a subject.

Biomarkers and reagents

[0093] A variety of biomarkers are detectable and measurable using the imaging and analysis methods and systems described herein. Available and contemplated biomarkers for use in the presently described systems and methods include those set forth in U.S. Patent Application Pub. Nos. 20130237453 and 20180239949, which is incorporated herein by reference.

[0094] These biomarkers include native attributes of a cell that are identifiable using methods and systems described herein, with or without the use of additional reagents. Biomarkers also include attributes of a cell that are identifiable through subjecting the cell to a particular stimulus or reagent. Most frequently, the biomarkers detected and measured according to the methods and systems described herein are correlated in a regimented manner with a disease state such as cancer, or a specific cell transformative or cell proliferative disorder in a subject. Also often, the biomarkers detected and measured according to the methods and systems described herein are correlated in a regimented manner with the activity of a drug such as a small or large molecule drug on the cell being imaged.

Exemplary Schematics and processes

[0095] A variety of schematics and workflow charts are presented in Figures 1-14. These are intended to be exemplary of certain features arrangements and aspects of the presently contemplated systems, including various subcomponents of the systems. The description provided herein is not intended to comprehensively describe the Figures as the specific manner in which they are presented, including detailed wording, directions, module delineations, is intended to stand on its own to a large extent. Nevertheless, these Figures may be interpreted in view of the description as a whole, which includes the specific description of each Figure in addition to more generally throughout referring to features, functions and aspects that are found or evident in the Figures. [0096] FIG. 1 depicts various aspects of an exemplary DIC module of the present disclosure. Each of the noted aspects is described elsewhere herein. The letter "A" designates a housing perimeter to indicate that each aspect is contained within a single module or system. As with the remainder of the Figures presented here, the specific positioning of the elements of FIG. 1 are not intended to be limiting.

[0097] FIG. 2 depicts various aspects of an exemplary system of the present disclosure. Each of the noted aspects is described elsewhere herein. As noted, the sample processing module and cell culture module are included within a single housing designated "B."

[0098] FIG. B depicts various aspects of another exemplary system of the present disclosure. Each of the noted aspects is described elsewhere herein. As noted, the sample processing module is present in housing "C " that is connected with the expanded DIV module "C" using a transport mechanism. The transport mechanism may include a pipettor that obtains an aliquot of cell suspension from the sample processing module and applies to a chip (which includes a specific substrate such as a multi-well plate) that is either in or will more to the DIC module.

The transport mechanism may also be one of the types described herein, including a pick and place mechanism, conveyor, carousel or track, among others, that transports a chip from with or near the sample processing module to the DIC module. In the depicted embodiment, the chip is transported in the DIC module as well, which includes a cell culture module that can be and most frequently is, as described herein, the chip itself or portion thereof. Thus, in this embodiment, the chip is transported to the DIC module housing, and then transported or moved within the DIC module. Such embodiments often involve the use of a carousel or other carrier positioned at least partially within the DIC module where the chip is placed (or a plurality of chips are placed) that holds multiple chips and rotates/moves in relation to the DIC optics, permitting simultaneous or sequential analysis of the chips.

[0099] FIG. 4 is an adaptation of an embodiment similar to FIG. 3, with the removal of a delineated cell culture module, which as noted can form at least part of the chip. "D' " designates a housing for the sample preparation module and "D" designates a housing for the DIC module, which are connected via a transport mechanism.

[00100] FIG. 5 is an adaptation of an embodiment similar to FIG. 3 while specifying a direction of workflow from sample introduction to the DIC module. This figure specifies inputs for sample/cell suspension handling, transport, reagent/liquid handling, etc. along the exemplified line of processing and analysis. "E" designates a housing for the DIC module in addition to aspects that involve sample processing in advance of optical analysis. Internal DIC module chip transport using a transport mechanism contemplated herein is also specified.

[00101] FIG. 6 is an adaptation of an embodiment similar to FIG. 5 while specifying "F' " and "F," which indicates the GUI and quality system are connected with the system operating in housing "F." Both conventional tissue dissociation and microfluidic dissociation are contemplated in this embodiment.

[00102] FIG. 7 is an adaptation of an embodiment similar to FIG. 5, with the removal of a delineated cell culture module, which as noted can form at least part of the chip. "G' " and "G" indicate the GUI and quality system are connected with the system operating in housing "G." This figure specifies a housing "G' " and a housing "G." These can be connected housings or represent walls between the different modules within the housing of a single system.

[00103] FIG. 8 represents a specific adaptation of the embodiment of FIG. 7 involving the use of a microfluidic tissue dissociator. "H' " and "H" indicate the GUI and quality system are connected with the system operating in housing "H."

[00104] FIG. 9 is an adaptation of an embodiment similar to FIG. 8, while connecting the GUI and quality system directly to the DIC module. "I" designates a housing for the sample preparation module, a microfluidic tissue dissociator, or a chip including a microfluidic tissue dissociator and "I' " designates a housing for the DIC module.

[00105] FIG. 10 is a schematic representing various aspects of functional units, functions, programs, aspects, and modules in the presently contemplated systems, which includes the various adaptations thereof contemplated herein. An exemplary single system of the present disclosure in certain embodiments includes each of the aspects of functional units, functions, programs, aspects, and modules set forth in this Figure.

[00106] FIG. 11 sets forth the first 3 to 4 steps of an exemplary assay of the present disclosure in the presently contemplated systems. The use of the term "step" in describing the Figures is not intended to be limiting to discreet and delineated process steps that occur to exclusion of other processes or events in the operation of the presently contemplated systems and therefore the chosen verbiage is for ease of description only.

[00107] Steps Cl & C2 involve multiple processes, which processes may occur simultaneously or in sequence. In this step a sample is obtained and identified for tracking purposes. If necessary, the sample is dissociated into a cell suspension and applied to a chip (which includes a substrate such as a multi-well plate a microfluidic device, an imaging chamber, or similar). Manual or automated liquid handling using a pipettor may be utilized in this process. Prior to application of the sample to the chip, the chip will be sterilized using, for example, UV light, plasma or another means that sterilizes yet does not leave a residue or by product of the sterilization. Also prior to application of the sample to the chip, the surface of the chip that will be accepting the sample is prepared with organofunctional molecules, such as alkoxysilane molecules. In the most frequent embodiments, the process involves what is referred to as silanization of the glass (or other) surface. The gas intake and gas outlet are provided for this process. This process of surface treatment may be provided in a dedicated module of the present systems, though often it is provided in the DIC module.

[00108] While not intending to be bound by any specific scientific theory, silanization refers to a deposit of chemical conjugates that interact with glass or plastic to functionalize the surface for the intended purpose. After functionalizing the surface another chemical is generally introduced to the surface, which is frequently a protein or plurality of different proteins though other chemicals can be utilized. Moreover, a coating of cells or nucleic acid(s) may be introduced after functionalization. Thus, according to the presently contemplated embodiments a manner of conducting physical chemistry on surfaces is included in the herein described embodiments and systems. In related methods a variety of protocols may be utilized, including the requisite instruments and reagents. According to one such exemplary procedure, the following steps are undertaken in the presently described systems: (a) Glass, plastic, metal, or other material is received; (b) This material is placed in a holder used to provide surface treatment; (c) the material is then washed with distilled / purified water; (d) the material is then dried and treated with chemical or prepared for further treatment.

[00109] Such further treatment may include, for example, (in any order): Washing with a buffer, chemical, biological solution (nucleic acid, protein, cells) or water (using, e.g., the Liquid Handling Inlet - Outlet); heating / cooling between -400°C to 400°C (provided by, e.g., the Environmental Chamber or Hot Plate); washing with a buffer, chemical, biological solution (nucleic acid, protein, cells) or water. (Liquid Handling Inlet - Outlet); washing with an acidic or basic solution (using, e.g., the Liquid Handling Inlet - Outlet); washing with a buffer, chemical, biological solution (nucleic acid, protein, cells) or water (using, e.g., the Liquid Handling Inlet - Outlet); applying / flowing a gaseous to deposit buffer, chemical, or water (using, e.g., the Gas Inlet - Gas Outlet); heating / cooling between -400°C to 400°C (provided by, e.g., the Environmental Chamber or Hot Plate); applying / flowing a gaseous to deposit buffer, chemical, biological solutions (nucleic acid, protein, cells) or water (using, e.g., the Gas Inlet - Gas Outlet); applying / flowing an aqueous solution to deposit buffer, chemical, biological solutions (nucleic acid, protein, cells) or water (using, e.g., the Liquid Handling Inlet - Outlet, and/or the Gas Inlet - Gas Outlet).

[00110] Though these are represented as an intake and an outlet, it is intended to refer to anywhere in the system where this process occurs, which is not necessarily in the DIC module. For example, it is contemplated that a specific chamber or location in the presently contemplated systems, or functionally connected therewith, is provided for silanization. A hot plate or other heating means may be utilized to aid in the silanization process. After preparation of the chip the sample is coated with protein or combination of proteins such as an extracellular matrix (ECM) solution. When the sample is added to the chip it occurs after silanization and coating with ECM. In steps Cl and C2 the environmental control may be relatively flexible, and it is contemplated to occur in temperatures of between 0°C to 100°C,

CO2 levels of between 0%-100% and humidity of between 0% to 100%.

[00111] Step C3 of FIG. 11 involves conducting differential contrast (DIC) and/or bright field microscopy on the sample in the chip in the system. This occurs while the applied cells in the sample adhere to the surface or spread across the surface of the chip. In this process cells are identified and tracked as they adhere or spread so that they can be reliably identified for further imaging in a processing or later timeframe. In such circumstances the cell itself may form the basis of the tracking using an algorithm in the system. Furthermore, there may be aspects of the surface of the chip that are identifiable to aid in the recognition of the same cell or groups of cells across a time period or during cell adhesion, cell spreading and cell culture. Imaging in this step may occur in discreet time periods or until a predetermined number of images are obtained, for example in increments of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 seconds, minutes, hours, or more, which may involve the acquisition of up to about 1,000,000,000 images. In step C3 the environmental control may be relatively flexible, and it is contemplated to occur in temperatures of between 0°C to 100°C, CO2, NO2 _, or other gas or liquid - levels of between 0%- 100% and humidity of between 0% to 100%.

[00112] Step C4 provided in FIG. 12 involves washing the chip to remove cell debris and provide a clear viewing area for the cells of interest. Automated or manual liquid handling may be used in this step, but generally uses an automated pipettor or fluid input to the chip and also a waste or output to capture spent fluids and waste removed from the chip in a sanitary manner. In step C4 the environmental control may be relatively flexible, and it is contemplated to occur in temperatures of between 0°C to 100°C, CO2, NO2 _, or other gas or liquid - levels of between 0%-100% and humidity of between 0% to 100%.

[00113] Step C5 in FIG. 12 involves conducting differential contrast (DIC) and/or bright field microscopy on the sample in the chip in the system. This process involved live cell imaging that evaluates or can evaluate a variety of markers, including at least actin retrograde flow, membrane dynamics, endocytosis, exocytosis, vesicle transport, one or more other high rate biomarkers, and a variety of other live-cell markers described or contemplated herein. Cells are tracked throughout this process to ensure fidelity and proper correlation of data. Imaging in this step may occur in discreet time periods or until a predetermined number of images are obtained, for example in increments of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes or more, which may involve the acquisition of up to about 1,000,000,000 images.

[00114] Step C6 in FIG. 12 involves conducting differential contrast (DIC) and/or bright field microscopy on the sample in the chip in the system. This process involved live cell imaging that evaluates or can evaluate cell motility. Cells are tracked throughout this process to ensure fidelity and proper correlation of data. Step C6 may occur together or separate from step C5 such that cell motility is included in the markers evaluated in step C5. Yet, step C6 may also exist in a discreet step of the assay. Imaging in this step may occur in discreet time periods or over a prolonged period of time or until a predetermined number of images are obtained, for example up to about 148 hours, which may involve the acquisition of up to about 1,000,000,000,000 images.

[00115] Step C7 in FIG. 13 involves the processes of cell permeabilization, cell fixation and cell labelling/staining for, for example, fluorescence imaging. Washing of the chip will often occur one or more times to prepare the chip for fixation, removal of fixation reagents, and removal of labelling reagents. As contemplated herein the liquid handling of the fixation reagents and wash solutions is provided in an automated manner, e.g., using an automated pipettor though manual liquid handling is also contemplated.

[00116] Step C8 in FIG. 13 involves the imaging of the labeled sample in the chip using fluorescent imaging or confocal imaging. Frequent excitation sources include laser or LED excitation within a predetermined light wavelength or range mediated by a filter cube or similar thereof. The excitation wavelength will generally correspond to the chosen fluorophore or mixture of fluorophores chosen to label the cells. A plurality of different markers may be simultaneously or sequentially evaluated that can be differentiated by different emission wavelengths. In this regard, 2 or more, 3 or more, 4 or more, or 5 or more, or up to about 20 different emission wavelengths may be simultaneously or sequentially monitored on the presently contemplated systems. This permits the simultaneous or sequential evaluation of 2 or more, 3 or more, 4 or more, or 5 or more, or up to about 20 different markers in the single sample. In the case of the evaluation of a multi-well plate as the chip, different fluorescent markers (or mixtures thereof) may be present in different wells of the plate. Preferred excitation wavelengths range between at or about 10,000 m to 10 ¹⁴ m. Imaging in this step may occur over a prolonged period of time or until a predetermined number of images are obtained, for example up to about 148 hours, which may involve the acquisition of up to about 1,000,000,000,000 images.

[00117] It is contemplated that the present systems include multiple DIC modules. Multiple DIC modules are useful to increase throughput through the system such that Step 3 occurs in one DIC module and Steps 6 and/or 8 occur in a different DIC module. In between the chip holding the sample is moved from one module to another (or multiple different) module(s). In the process identification and cell tracking remains important to ensure that after movement the same cells on the chip are identified and correlated in the images obtained at the multiple different DIC modules. The optionally shorter imaging time sequence(s) of Step 3 provide the ability to evaluate multiple different chips in a single DIC module the time period that Steps 6 and/or 8 are occurring on another chip, which would tie up the DIC module conducting Steps 6 and/or 8. This permits multiple chips to cycle into and out of the DIC module conducting Step 3. In these embodiments, it is specifically contemplated that each of two or more DIC modules are operably connected using a transport mechanism of the present disclosure including a carousel, conveyor, pick and place system or track system permitting automated transport of the chip.

[00118] It is also contemplated that a dedicated DIC module is provided in the system for analysis of fixed cells. In such systems two or more DIC modules are present in a single system, with one DIC module adapted to conduct Steps 3, 6 and/or 8, and at least one other DIC module adapted to conduct fluorescent imaging such as that set forth in Step 8. It is contemplated that this additional fluorescent imaging DIC module is adapted such that it cannot conduct an analysis other than fluorescent imaging. [00119] It is further contemplated that washing steps such as Step 7 occur outside of the DIC module, for example, in a dedicated or separate wash station or module. In such embodiments the chip is subjected to Step 6 in the DIC module, then removed from the DIC module for Step 7, then either replaced into the same DIC module or moved to a different DIC module for Step 8 (if desired). The transport mechanisms contemplated herein are employed to provide such automated chip movement.

[00120] Step C9 in FIG. 13 is generally a computer implemented process of processing, organizing and scrutinizing the data and images collected by imaging device (e.g., camera). These images are processed by computer, verified, organized, and evaluated for image quality and integrity. The images are also organization in this step and in certain preferred embodiments of the present disclosure, uploaded to a local or cloud storage / computing resource. In this computer implemented step there are no specific environmental requirements. The quality system and machine vision control are often employed in this step.

[00121] Step CIO in FIG. 14 involves the process where fixed or live cells are harvested for nucleic acid and/or proteomic analysis. Nucleic acid and/or proteomic analysis may occur at any predesignated step of the contemplated analyses. For example, if fluorescent analysis of the sample is not required the sample may be passed to nucleic acid and/or proteomic analysis in live cell manner. Moreover, it may be the case in certain embodiments that a portion of the sample passes to nucleic acid and/or proteomic analysis while another portion of the sample is fixed and labeled for fluorescent analysis. In step C8 the environmental control may be relatively flexible, and it is contemplated to occur in temperatures of between 0°C to 100°C,

CO2 levels of between 0%-100% and humidity of between 0% to 100%.

[00122] Step CIO also specifies what is described in detail elsewhere in the present disclosure, which includes the control, management and/or monitoring of the system operations via a GUI. The GUI is capable of controlling all of the herein-noted steps or a portion thereof.

[00123] One skilled in the art will appreciate further features and advantages of the presently disclosed methods, systems and devices based on the above-described embodiments. Accordingly, the presently disclosed methods, systems and devices are not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein are expressly incorporated herein by reference in their entirety.