Abstract: A classification training method for training classifiers adapted to identify specific mutations associated with different cancer including identifying driver mutations. First cells from mutation cell lines derived from conditions having the number of driver mutations are acquired and 3D image feature data from the number of first cells is identified. 3D cell imaging data from the number of first cells and from other malignant cells is generated, where cell imaging data includes a number of first individual cell images. A second set of 3D cell imaging data is generated from a set of normal cells where the number of driver mutations are expected to occur, where the second set of cell imaging data includes second individual cell images. Supervised learning is conducted based on cell line status as ground truth to generate a classifier.

Abstract: A method for a system and method for morphometric detection of malignancy associated change (MAC) is disclosed including the acts of obtaining a sample; imaging cells to produce 3D cell images for each cell; measuring a plurality of different structural biosignatures for each cell from its 3D cell image to produce feature data; analyzing the feature data by first using cancer case status as ground truth to supervise development of a classifier to test the degree to which the features discriminate between cells from normal or cancer patients; using the analyzed feature data to develop classifiers including, a first classifier to discriminate normal squamous cells from normal and cancer patients, a second classifier to discriminate normal macrophages from normal and cancer patients, and a third classifier to discriminate normal bronchial columnar cells from normal and cancer patients.

Abstract: A method for a system and method for morphometric detection of malignancy associated change (MAC) is disclosed including the acts of obtaining a sample; imaging cells to produce 3D cell images for each cell; measuring a plurality of different structural biosignatures for each cell from its 3D cell image to produce feature data; analyzing the feature data by first using cancer case status as ground truth to supervise development of a classifier to test the degree to which the features discriminate between cells from normal or cancer patients; using the analyzed feature data to develop classifiers including, a first classifier to discriminate normal squamous cells from normal and cancer patients, a second classifier to discriminate normal macrophages from normal and cancer patients, and a third classifier to discriminate normal bronchial columnar cells from normal and cancer patients.

Abstract: A classification training method for training classifiers adapted to identify specific mutations associated with different cancer including identifying driver mutations. First cells from mutation cell lines derived from conditions having the number of driver mutations are acquired and 3D image feature data from the number of first cells is identified. 3D cell imaging data from the number of first cells and from other malignant cells is generated, where cell imaging data includes a number of first individual cell images. A second set of 3D cell imaging data is generated from a set of normal cells where the number of driver mutations are expected to occur, where the second set of cell imaging data includes second individual cell images. Supervised learning is conducted based on cell line status as ground truth to generate a classifier.

Abstract: A multi-layer sealing structure for sealing a microwell array defined in or on a substrate includes at least one front compliant layer, a back compliant layer, and a flexural layer arranged between the at least one front compliant layer and the back compliant layer, wherein the at least one front compliant layer is closer than the back compliant layer to microwells of the microwell array. One or more front compliant layers may be optically reflective and/or may embody a sensor layer. The back compliant layer may include an adhesive or various types of rubber, and the flexural layer may include a polymeric material or metal. A multi-layer sealing structure may be separated from a microwell array by peeling. A multi-layer sealing structure allows local disruption of sealing where particle contaminants are present without compromising the sealing performance of an entire microwell array, and without requiring a large sealing force.

Abstract: A microfluidic device includes a first substrate including at least one microfluidic channel and a plurality of microwells, as well as a cooperating second substrate defining multiple split-walled cell trap structures that are registered with and disposed within the plurality of microwells. A method for performing an assay includes flowing cells and a first aqueous medium into a plurality of microwells of a microfluidic device, wherein each microwell includes a cell trap structure configured to trap at least one cell. The method further comprises flowing a nonpolar fluid with low permeability for analytes of interest through a microfluidic channel to flush a portion of the first aqueous medium from the microfluidic channel while retaining another portion of the first aqueous medium and at least one cell within each microwell. Surface tension at a non-polar/polar medium interface prevents molecule exchange between interior and exterior portions of microwells.

Abstract: Systems and methods of using the same for functional fluorescence imaging of live cells in suspension with isotropic three dimensional (3D) diffraction-limited spatial resolution are disclosed. The method-live cell computed tomography (LCCT)-involves the acquisition of a series of two dimensional (2D) pseudo-projection images from different perspectives of the cell that rotates around an axis that is perpendicular to the optical axis of the imaging system. The volumetric image of the cell is then tomographically reconstructed.

Abstract: Systems and methods of using the same for functional fluorescence imaging of live cells in suspension with isotropic three dimensional (3D) diffraction-limited spatial resolution are disclosed. The method-live cell computed tomography (LCCT)-in-volves the acquisition of a series of two dimensional (2D) pseudo-projection images from different perspectives of the cell that rotates around an axis that is perpendicular to the optical axis of the imaging system. The volumetric image of the cell is then tomographically reconstructed.

Abstract: A method for analyzing cells through measurement of live-cell parameters followed by measurement of gene and protein expression is disclosed herein. The method comprises measuring one or more live-cell parameters for a plurality of cells contained in at least one liquid in a plurality of isolated microchambers of a microarray device. The method further comprises removing a lid bounding the plurality of isolated microchambers. The method further comprises microdispensing a quantity of lysate into each microchamber of the plurality of isolated microchambers. The method further comprises microdispensing a quantity of reverse transcription polymerase chain reaction mix into each microchamber of the plurality of isolated microchambers. The method further comprises microdispensing a quantity of oil into each microchamber of the plurality of isolated microchambers.

Abstract: Methods for seeding live cells onto spatially defined regions of a substrate including multiple features (e.g., microwells or other microenvironments) utilize a stencil embodied in a hole-defining sacrificial film. A sacrificial film devoid of holes may be applied over features of a substrate, and a hole generating mechanism (e.g., hot needle or laser) aligned with features may be used to define holes in the film. Alternatively, holes may be predefined in a sacrificial film to form a stencil, and the stencil may be assembled to the substrate with the holes registered with features thereof. Thereafter, cells are seeded through holes in the film. Seeded cells are subject to incubation, further processing, and/or performance of one or more assays, and the hole-defining sacrificial film (stencil) may be removed.

Abstract: An integrated technological platform enabling real-time quantitative multiparameter metabolic profiling, utilizing either or both of extra and intracellular optical sensors, individually or simultaneously. A scalable embedded micropocket array structure, generally fabricated on fused silica substrates, facilitates the integration of multiple, spatially separated extracellular sensors for multiparameter analysis in a container formed with the use of an activation mechanism forming part of a device configured to hold the container during the measurements. The creation of hermetically sealed microchambers is carried out with a pneumatically and/or mechanically and/or electromechanically driven device that is “floating” within the holding device and that is optionally equipped with a vacuum/suction mechanism to hold a component of the container at its surface.

Abstract: A device for high-throughput multi-parameter functional profiling of the same cells in multicellular settings and in isolation is provided. In certain aspects, an integrated microfluidic device for multi-parameter metabolic and other phenotypic profiling of live biological cells is useable with: 1) multicellular clusters or small biopsy tissue samples, 2) cultures of the constituent cells obtained after cluster/tissue dissociation, and 3) the same constituent single cells in isolation. The approach enables study of the effects of multicellular complexity, such as in response to treatment, pathogens, stress, or other factors concerning disease origination and progression. Measurements may be performed on single cells or multicellular populations or tissues in the same assay at the same time.

Abstract: An integrated technological platform enabling real-time quantitative multiparameter metabolic profiling, utilizing either or both of extra and intracellular optical sensors, individually or simultaneously. A scalable embedded micropocket array structure, generally fabricated on fused silica substrates, facilitates the integration of multiple, spatially separated extracellular sensors for multiparameter analysis in a container formed with the use of an activation mechanism forming part of a device configured to hold the container during the measurements. The creation of hermetically sealed microchambers is carried out with a pneumatically and/or mechanically and/or electromechanically driven device that is “floating” within the holding device and that is optionally equipped with a vacuum/suction mechanism to hold a component of the container at its surface.

Abstract: A thixotropic and biocompatible gel, formed by combining of 4-arm polyethylene glycol with fumed silica (PEG-silica). The thixotropicity of the gel is affected by the ratio of PEG to silica. To study the gels biocompatibility, the cell suspension in PBS or another medium was added to the PEG-silica gel via mixing to uniformly disperse the cells in the gel and then placed in an incubator before performing fluorescence live-dead assays. Duration of cell viability (cell life time) in gels was measured to be up to several days, depending on gel composition. Due to their optical and fluidic properties the gels are compatible with live cell imaging including 3D computed tomography and offer a means for moving the cells in a highly controllable manner by applying and removing pressure on the gels.

Abstract: Systems and methods of using the same for functional fluorescence imaging of live cells in suspension with isotropic three dimensional (3D) diffraction-limited spatial resolution are disclosed. The method-live cell computed tomography (LCCT)-in-volves the acquisition of a series of two dimensional (2D) pseudo-projection images from different perspectives of the cell that rotates around an axis that is perpendicular to the optical axis of the imaging system. The volumetric image of the cell is then tomographically reconstructed.

Abstract: A microfluidic device includes a first substrate including at least one microfluidic channel and a plurality of microwells, as well as a cooperating second substrate defining multiple split-walled cell trap structures that are registered with and disposed within the plurality of microwells. A method for performing an assay includes flowing cells and a first aqueous medium into a plurality of microwells of a microfluidic device, wherein each microwell includes a cell trap structure configured to trap at least one cell. The method further comprises flowing a nonpolar fluid with low permeability for analytes of interest through a microfluidic channel to flush a portion of the first aqueous medium from the microfluidic channel while retaining another portion of the first aqueous medium and at least one cell within each microwell. Surface tension at a non-polar/polar medium interface prevents molecule exchange between interior and exterior portions of microwells.

Abstract: A method for analyzing cells through measurement of live-cell parameters followed by measurement of gene and protein expression is disclosed herein. The method comprises measuring one or more live-cell parameters for a plurality of cells contained in at least one liquid in a plurality of isolated microchambers of a microarray device. The method further comprises removing a lid bounding the plurality of isolated microchambers. The method further comprises microdispensing a quantity of lysate into each microchamber of the plurality of isolated microchambers. The method further comprises microdispensing a quantity of reverse transcription polymerase chain reaction mix into each microchamber of the plurality of isolated microchambers. The method further comprises microdispensing a quantity of oil into each microchamber of the plurality of isolated microchambers.

Abstract: Systems and methods of using the same for functional fluorescence imaging of live cells in suspension with isotropic three dimensional (3D) diffraction-limited spatial resolution are disclosed. The method-live cell computed tomography (LCCT)-involves the acquisition of a series of two dimensional (2D) pseudo-projection images from different perspectives of the cell that rotates around an axis that is perpendicular to the optical axis of the imaging system. The volumetric image of the cell is then tomographically reconstructed.

Abstract: A microfluidic device useable for performing live cell computed tomography imaging is fabricated with a cover portion including a first wafer with at least one metal patterned thereon, a base portion including a second wafer with at least one metal patterned thereon and negative photoresist defining recesses therein, and a diffusive bonding layer including a negative photoresist arranged to join the cover portion and the base portion. A composition useful in live cell computer topography includes a long-chain polysaccharide at a concentration of from about 0.01% to about 10.0% in cell culture medium for supporting cell life while enabling cell rotation rate to be slowed to a speed commensurate with low light level imaging.

Abstract: A device for high-throughput multi-parameter functional profiling of the same cells in multicellular settings and in isolation is provided. In certain aspects, an integrated microfluidic device for multi-parameter metabolic and other phenotypic profiling of live biological cells is useable with: 1) multicellular clusters or small biopsy tissue samples, 2) cultures of the constituent cells obtained after cluster/tissue dissociation, and 3) the same constituent single cells in isolation. The approach enables study of the effects of multicellular complexity, such as in response to treatment, pathogens, stress, or other factors concerning disease origination and progression. Measurements may be performed on single cells or multicellular populations or tissues in the same assay at the same time.