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: The invention relates to microfluidic devices and array disks for “one-pot” isolated chemical reactions. The array disks comprise a plurality of sectors in which each sector comprises one microfluidic device. The microfluidic devices comprise a fluid delivery channel and an array of wells wherein the fluid delivery channel delivery fluid into the wells in a serpentine arrangement. In some embodiments, the fluid delivery channel is directly above the array of wells. In other embodiments, the fluid delivery channel is offset from the array of wells so that side channels branching from the fluid delivery channel delivers fluid into the wells. The well of the microfluidic device comprises a gas-permeable membrane that forms the floor, well, or at least a portion of the floor or wall of the well. In preferred embodiments, the well is cylindrical.

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: Methods of pre-methylation of foreign DNA to improve genetic transformation in cyanobacterium. Two Type II methyltransferase-encoding genes, i.e., M (sll0729) and C (slr0214), were cloned from the chromosome of Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) and expressed in E. coli that harbors the integrative plasmid pBS-SPtK or pJU105. After pre-methylation in E. coli, the integrative plasmids were extracted and used for transformation of Synechocystis 6803. The results showed that expression of slr0214 in the integrative-plasmid-harboring E. coli cells before DNA preparation resulted in orders of magnitude higher efficiency in the following integrative transformation of Synechocystis 6803.

Abstract: Methods for detecting single nucleotide polymorphisms in nucleotide sequences using LAMP reactions are provided herein. Generally, two sets of LAMP primers, a wild-type primer that matches expected DNA sequences and an SNP primer that matches the expected SNP DNA are provided. One method includes providing the wild-type primer and the SNP primer in separate wells of a multi-well microfluidic array device, adding the sample nucleotide sequence into the wells seeded with the primers, and initiating LAMP reactions within the wells. The method includes observing the reaction differential between the primers and determining the status of the DNA with regard to that particular SNP. A second method includes providing the primers with tags in a mixture, adding the sample nucleotide sequence to the mixture, and initiating LAMP reactions.

Abstract: Mitochondria-targeting potassium sensors and method(s) for making such sensors. The sensor shows a response to potassium and displays a 130-fold dynamic range of fluorescence intensity and high brightness. The sensors response to potassium concentrations was demonstrated to be unaffected by cellular pH value and/or concentrations of other ions. The sensors can be used for monitoring the mitochondrial potassium efflux/influx.

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: Construction and expression of synthetic pathways to produce (S) or (R)-3-hydroxybutyrate (3HB) as enantiomerically-pure products by genetically engineering cyanobacterium Synechocystis sp. PCC 6803. Under optimized growth conditions, the pathway employing phaA and phaB from R. eutropha was the most effective, producing up to 533.4±5.5 mg/l (R)-3HB after 21 days photosynthetic cultivation. For the first time, the feasibility and high efficiency of producing 3HB using solar energy and CO2 as sole energy and carbon sources by engineered cyanobacteria is demonstrated.

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: The present disclosure relates to an optical fluorescence sensor comprising a probe for sensing glucose, an intra-reference probe, and a matrix. The present disclosure also relates to an optical fluorescence dual sensor comprising a probe for sensing glucose having two boronic acid moieties, a probe for sensing oxygen comprising modified porphyrin, an intra-reference probe that is rhodamine-based, and a matrix. The present disclosure additionally relates to methods of preparing these sensors and methods of using them.

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: The invention relates to microfluidic devices and array disks for “one-pot” isolated chemical reactions. The array disks comprise a plurality of sectors in which each sector comprises one microfluidic device. The microfluidic devices comprise a fluid delivery channel and an array of wells wherein the fluid delivery channel delivery fluid into the wells in a serpentine arrangement. In some embodiments, the fluid delivery channel is directly above the array of wells. In other embodiments, the fluid delivery channel is offset from the array of wells so that side channels branching from the fluid delivery channel delivers fluid into the wells. The well of the microfluidic device comprises a gas-permeable membrane that forms the floor, well, or at least a portion of the floor or wall of the well. In preferred embodiments, the well is cylindrical.

Abstract: Construction and expression of synthetic pathways to produce (S) or (R)-3-hydroxybutyrate (3HB) as enantiomerically-pure products by genetically engineering cyanobacterium Synechocystis sp. PCC 6803. Under optimized growth conditions, the pathway employing phaA and phaB from R. eutropha was the most effective, producing up to 533.4±5.5 mg/l (R)-3HB after 21 days photosynthetic cultivation. For the first time, the feasibility and high efficiency of producing 3HB using solar energy and CO2 as sole energy and carbon sources by engineered cyanobacteria is demonstrated.

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: The invention relates to microfluidic devices and array disks for “one-pot” isolated chemical reactions. The array disks comprise a plurality of sectors in which each sector comprises one microfluidic device. The microfluidic devices comprise a fluid delivery channel and an array of wells wherein the fluid delivery channel delivery fluid into the wells in a serpentine arrangement. In some embodiments, the fluid delivery channel is directly above the array of wells. In other embodiments, the fluid delivery channel is offset from the array of wells so that side channels branching from the fluid delivery channel delivers fluid into the wells. The well of the microfluidic device comprises a gas-permeable membrane that forms the floor, well, or at least a portion of the floor or wall of the well. In preferred embodiments, the well is cylindrical.

Abstract: Construction and expression of synthetic pathways to produce (5) or (R)-3-hydroxybutyrate (3HB) as enantiomerically-pure products by genetically engineering cyanobacterium Synechocystis sp. PCC 6803. Under optimized growth conditions, the pathway employing phaA and phaB from R. eutropha was the most effective, producing up to 533.4±5.5 mg/l (R)-3HB after 21 days photosynthetic cultivation. For the first time, the feasibility and high efficiency of producing 3HB using solar energy and CO2 as sole energy and carbon sources by engineered cyanobacteria is demonstrated.

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.