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Creating Super-Resolution Images using Standard Photobleaching and Blinking Movies Current super-resolution microscopy techniques require special laser setups and fluorescent molecules (such as PALM and STORM) that allow the fluorophores to be... Read More Current super-resolution microscopy techniques require special laser setups and fluorescent molecules (such as PALM and STORM) that allow the fluorophores to be turned on and off so that only a small number of fluorophores are turned on at any time. With the Super Shrimp image analysis algorithm, super-resolution images can be created from images with high densities of fluorophores. The invention creates microscopy images that have 5 or more times better resolution than standard microscopy images while using the standard microscopic set up available in most laboratories. It allows use of any fluorescent molecule (including quantum dots) that blinks or photobleaches to pick fluorophores out of the fluorescent background, and it can work with thousands of fluorophores in an image. This image analysis algorithm can localize quantized drops in fluorescence even with a background to create super-resolution images from standard photobleaching movies. It does not require special photo-switchable fluorophores (PALM, STORM) or fluorophores with long-lived dark states (dSTORM). The data acquisition is accomplished in minutes instead of hours and it can use standard laboratory microscopic set up with any fluorescent molecule. The algorithm processes a movie in which fluorescent molecules or particles are photobleaching or blinking and looks for individual photobleaching events that can be located with high resolution (i.e. the position of the fluorescent molecules). A composite image with resolution much higher than the original movie can then be generated. Noise is reduced by rejecting fluorophores that are poorly fit by a Gaussian and frames are averaged. Benefits The image analysis algorithm to create super-resolution images using standard photobleaching and blinking movies provides the following benefits: Uses standard laboratory equipment. Enables sub-cellular structure examination Can be used with any fluorescent molecule that can be detected by a CCD camera. It can use high labeling densities Fast data acquisition
FRET Based High-Throughput Screening Advanced molecular imaging tools combined with investigative tools like biochemical and cell based assays have thepotential to unravel complex molecular processes... Read More Advanced molecular imaging tools combined with investigative tools like biochemical and cell based assays have thepotential to unravel complex molecular processes. These tools combined with high throughput screening can significantly impact diagnostics for cancer screening and accelerate drug discovery. These inventions are two new Fluorescence Resonance Energy Transfer (FRET) biosensor pairs, one composed of two new colors, mOrange2 and mCherry and the other composed of a modified high-sensitive ECFP/YPet pair that can significantly enhance the dynamic range of a variety of biosensors. The ECFP/YPet can detect signaling events with high spatio-temporal resolutions which makes it an ideal readout indicator for high throughput screening. FRET technology and genetically encoded FRET biosensors are very useful in detecting active molecular events inlive cells with high temporal and spatial resolutions. FRET occurs when two flurophores are in proximity with the emission spectrum of the donoroverlapping with the excitation spectrum of the acceptor. To date, the most popular FRET pair is cyan and yellow fluorescent proteins (CFP and YFP). The new FRET biosensor pairs mOrange2/mCherry are proteins with different colors and spectrally distinctive from the CFP/YFP pair. This invention opens up the possibility of lighting two diagnostic biomarkers in the same cell e.g., cancer cells, thus providing a double criterion high-fidelity assay to differentiate cancer vs. normal cells. To provide proof-of-concept that mOrange2 and mCherry are suitable as novel FRET biosensors, they were operably linked to the protein recognition sequence of MMP-MT1. MMP-MT1 is an enzyme belonging to the matrix metalloproteinase family that has known roles in cancer metastasis. The basis of this assay is that when MT1-MMP is inactive, the mCherry and mOrange2 and positioned in proximity and favor a strong FRET between the two moieties. Indeed, in-vitro assays established that the mOrange/mCherry pair can serve as a reliable and sensitive indicator of the status of MT1-MMP activation and can potentially be applied to other biomarker assays. The ECFP/YPet pair provides a high-sensitive biosensor for the visualization of molecular hierarchy at different subcellular locations inlive cells. In-vitro assays revealedthat the ECFP/YPet pair exhibits significantly enhanced dynamic range of the MT1-MMP biosensor compared to currently available CFP/YFP FRET pairs. When quantified, the ECFP/YPet pair showed a 570% change (% change in basal level upon stimulation of MT1-MMP) when compared to only 90-100% in existing FRET pairs. The ECFP/YPet pair has also been successfully applied to other classes of proteins, like kinases, that are important therapeutic targets in human cancer. These and other genetically engineered biosensors can serve as a research tool to monitor different signaling cascades in live mammalian cells with high sensitivity. The developed MT1-MMP biosensor and potentially other protein biosensors can provide a powerful tool for the spatiotemporal imaging of protein functions in cancer development e.g. detection of circulating tumor cells (CTCs). Furthermore, these biosensors can serve as an excellent high-throughput reporting system forthe detection of cancer and the development of inhibitors for cancer therapeutics. Benefits The novel mOrange2/mCherry pair presents a method to simultaneously visualize two active signaling events in the same cell when combined with existing FRET pairsThe ECFP/YPet pair can serve as a high-sensitive biosensor with significantly enhanced dynamic ranges compared to existing CFP/YFP FRET pairs. The ECFP/Ypet pair can be operably linked to any protein recognition sequence to detect activity of that protein in live cells. The pair also includes a positively charged tag which allows 100% efficiency ofdetection. This invention demonstrates an ECFP/Ypet pair operably linked to MT1-MMP, a tumor metastasis biomarker, to accurately detect circulating tumor cells in blood samples.
Simultaneous Identification of Mechanical and Optical Properties of Single Viruses using a Single Device Professor Bahl from the University of Illinois at Urbana-Champaign has developed a device that can identify both optical and mechanical properties of single viruses with... Read More Professor Bahl from the University of Illinois at Urbana-Champaign has developed a device that can identify both optical and mechanical properties of single viruses with much higher throughput than current methods. This method takes us one step closer to identify the virus rather than just detect it. The rapid label-free identification of viruses can bring about a revolution in human health diagnostics and therapeutics.
RF Coil for Improving Tuning Range, Cost and Maintaining Q A radio frequency (RF) coil comprising a plurality of electrically uninterrupted conductive legs, each leg having a first end and a second end, and at least one... Read More A radio frequency (RF) coil comprising a plurality of electrically uninterrupted conductive legs, each leg having a first end and a second end, and at least one continuous conductor electrically connected to the first ends of the legs. Frequency tuning of the coil is achieved by translating, along the legs, an electrically continuous tuning band that includes a capacitor closed about the axis of the coil in proximity to the conductive legs. Maintaining electrical symmetry of the coil results in tuning ranges of at least 30 percent of the nominal value of the resonant frequency.
Integrated System for Genome Modification in E. coli Dr. Kuhlman from the University of Illinois at Urbana-Champaign has created a new system that provides a single editing tool for a variety of genome modifications... Read More Dr. Kuhlman from the University of Illinois at Urbana-Champaign has created a new system that provides a single editing tool for a variety of genome modifications. This method has the advantage over CRISPR-Cas9 system in not requiring prior modification of the genome and requires only locus-specific primers for genome editing. This technology is an optimized Landing Pad system that can use positive and negative selection to select for insertion, deletion, and knockout events at genomic targets using specific primers contain homology region A, B, and C. This approach provides a powerful tool for manipulating the genome. Benefits The Landing-Pad system can integrate large fragments of DNA, can be targeted anywhere in the genome and ab be applicable to deletions and knockouts as well. Unlike the CRISPR-Cas9 system, this technique does not require a special promoter for gene expression. The Landing-Pad system can target different loci with only locus-specific primer design.
Electronic Point of Care System for Detecting a Variety of Biomarkers Medical professionals need techniques that will help them diagnose diseases before debilitating symptoms begin to manifest in patients. Point of care testing, or... Read More Medical professionals need techniques that will help them diagnose diseases before debilitating symptoms begin to manifest in patients. Point of care testing, or bedside testing, enables rapid diagnostic tests to be performed at a patient's bedside. Benefits include: Immediate data, which can be interpreted by nurse or trained technician Decreased cost and effort, which could dramatically increase frequency of patient testing Cost-effective method to produce patient biomarker profile Dr. Bashir's lab has invented a label-free, electronic method for quantifying various biomarkers from blood. This method may be incorporated into a portable handheld device which enables healthcare professionals to perform complete blood count diagnostics at the point of patient care. Key advantages over existing competing technology include versatility, scalability, and decreased cost of production.
Miniaturized Walking Biological Machines Dr. Bashir has developed a walking biological machine. This "bio-bot" can move spontaneously, and may be useful for sensing, information processing, transport,... Read More Dr. Bashir has developed a walking biological machine. This "bio-bot" can move spontaneously, and may be useful for sensing, information processing, transport, protein expression and actuation applications.
New Way to Detect DNA Changes Researchers from the University of Illinois have identified a new, cheaper and easier way to detect methylation (alterations) of DNA. By measuring the methylation... Read More Researchers from the University of Illinois have identified a new, cheaper and easier way to detect methylation (alterations) of DNA. By measuring the methylation of certain DNA sequences, doctors can predict the likelihood of cancers and other disorders. This early detection has the potential to save lives.
Temperature Independent pH (TIP) Buffers for Use at Low Temperatures Researchers and practitioners in university, industrial and national labs increasingly rely on low temperatures in the development and study of products. For... Read More Researchers and practitioners in university, industrial and national labs increasingly rely on low temperatures in the development and study of products. For example, pharmaceutical companies routinely use low temperatures to study disease targets and markers. The biomolecules are always in buffers. Since the pH of buffers changes dramatically upon lowering temperature, the biolmolecules are not the same pH when they are at the room temperature. The change of pH can dramatically change the structural and functional properties of the biomolecules so that information obtained at low temperature does not reflect its properties at room temperature or physiological temperature. Additionally, since the exact pH buffer at different temperatures is not often predictable, the results obtained using a temperature variable pH buffer cannot easily correlate back to the pH at room temperature. This invention is a new design of pH buffers that show negligible pH change upon cooling to low temperatures, including cryotempatures. This invention solves the problem of significant change in apparent pH of a glycerol solution of common biological buffers upon cooling to cryotemperature. By combining a buffer that increases pH upon cooling with one that decreases pH upon cooling, the apparent pH change upon cooling to cryotemperature is minimized.
Ionic Helical Polypeptides - Non-Viral Gene Delivery with Improved Transfection Efficiency Tremendous potential exists for helical polypeptides that are water soluble (ionic) and remain stable at physiological conditions,... Read More Tremendous potential exists for helical polypeptides that are water soluble (ionic) and remain stable at physiological conditions, variable environmental conditions including pH fluctuations, temperature changes, and in the presence of denaturing agents for gene delivery and cell-membrane penetration over commercially available products. Libraries of helical polypeptides are screened for desired traits, i.e. efficient gene transfection compared to standard transfection reagents.

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