Abstract: Systems and methods for in situ laser lysis for analysis of biological tissue (live, fixed, frozen or otherwise preserved) at single cell resolution in 3D. For example, a system and method for lysing individual cells in situ, including the steps of capturing a tissue sample comprising a cellular content, subjecting the tissue sample to a stream of continuous fluid flow, lysing a selected area of the tissue sample with a laser, thereby releasing at least a portion of the cellular content from the tissue sample, recovering at least one target molecule from the cellular content in the stream, and processing at least one target molecule is provided. The system collects cellular contents, performs highly multiplexed (RT-qPCR or RNA-seq), and sequentially (cell-by-cell) reconstructs a 3D spatial map of mRNA expression of the tissue with a large number of genes. A 3D spatial map of the DNA, RNA, and/or proteins can be generated for each cell in the tissue.

Abstract: Microfluidic devices for 3D hydrodynamic microvortical rotation of at least one live single cell or cell cluster, systems incorporating the devices, and methods of fabricating and using the devices and systems, are provided. A microfluidic chip rotates at least one live single cell or cell cluster in a microvortex about a stable rotation axis perpendicular to an optical axis within a chamber having a trapezoidal cross-sectional shape located below a flow channel. An optical trap may be used to position the cell or cells with the microvortex, and the cell or cells may be subject to live-cell or cell cluster computer tomography imaging.

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: 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: Novel methods and devices including modified endoscopes that employ fiber optic technology and multiple extrinsic optical sensors for enablement of simultaneous determination of change rates of biological analytes in vivo with single-cell resolution. The devices employ dynamic oscillatory actuation to determine the gradient of analytes of interest along one or multiple directions with respect to the cell. The change rate or flux of the analyte of interest can be easily determined by applying the first Fick's law that relates the flux with the concentration gradient. The dual operation mode of the device markedly increases measurement sensitivity and accuracy.

Abstract: Systems and methods for in situ laser lysis for analysis of biological tissue (live, fixed, frozen or otherwise preserved) at single cell resolution in 3D. For example, a system and method for lysing individual cells in situ, including the steps of capturing a tissue sample comprising a cellular content, subjecting the tissue sample to a stream of continuous fluid flow, lysing a selected area of the tissue sample with a laser, thereby releasing at least a portion of the cellular content from the tissue sample, recovering at least one target molecule from the cellular content in the stream, and processing at least one target molecule is provided. The system collects cellular contents, performs highly multiplexed (RT-qPCR or RNA-seq), and sequentially (cell-by-cell) reconstructs a 3D spatial map of mRNA expression of the tissue with a large number of genes. A 3D spatial map of the DNA, RNA, and/or proteins can be generated for each cell in the tissue.

Abstract: A triple sensor structured for simultaneous measurement of glucose, oxygen, and pH. The sensor components are in thin film states such as sensing films or membranes, with a glucose probe associated with emission of radiation in the blue part of the spectrum, an oxygen probe associated with radiation in red portion of the spectrum, and a pH probe—with a green portion of the spectrum. The optical probes are chemically grafted or immobilized in a suitable polymer matrix, alleviating the leaching of the probes from the matrix, improving the thin film sensing stability, and enabling the repeatable use of the same sensing films.

Abstract: The present disclosure relates to an optical luminescence dual sensor comprising a polymerized form of a probe for sensing pH; a polymerized form of a probe for sensing oxygen; a polymerized form of an internal reference probe; and a matrix. The present disclosure also relates to methods of preparing an optical luminescence dual sensor and methods of using them.

Abstract: Microfluidic devices for 3D hydrodynamic microvortical rotation of at least one live single cell or cell cluster, systems incorporating the devices, and methods of fabricating and using the devices and systems, are provided. A microfluidic chip rotates at least one live single cell or cell cluster in a microvortex about a stable rotation axis perpendicular to an optical axis within a chamber having a trapezoidal cross-sectional shape located below a flow channel. An optical trap may be used to position the cell or cells with the microvortex, and the cell or cells may be subject to live-cell or cell cluster computer tomography imaging.

Abstract: Novel methods and devices including modified endoscopes that employ fiber optic technology and multiple extrinsic optical sensors for enablement of simultaneous determination of change rates of biological analytes in vivo with single-cell resolution. The devices employ dynamic oscillatory actuation to determine the gradient of analytes of interest along one or multiple directions with respect to the cell. The change rate or flux of the analyte of interest can be easily determined by applying the first Fick's law that relates the flux with the concentration gradient. The dual operation mode of the device markedly increases measurement sensitivity and accuracy.

Abstract: A triple sensor structured for simultaneous measurement of glucose, oxygen, and pH. The sensor components are in thin film states such as sensing films or membranes, with a glucose probe associated with emission of radiation in the blue part of the spectrum, an oxygen probe associated with radiation in red portion of the spectrum, and a pH probe—with a green portion of the spectrum. The optical probes are chemically grafted or immobilized in a suitable polymer matrix, alleviating the leaching of the probes from the matrix, improving the thin film sensing stability, and enabling the repeatable use of the same sensing films.

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 chamber device for analyzing living cell(s). The chamber device includes a base and a lid that when reversibly pressed closed create a chamber. The base is configured with an optically transparent well to contain at least one cell. The lid has a breadth greater than the base and is configured to contain at least one sensor. The lid is further configured with a lip that when pressed between the lid and the base creates an impermeable seal. The base and the lid are configured so that, when closed and in use, the sensor remains spatially apart from the at least one cell.