41 research outputs found
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3D optical sectioning with a new hyperspectral confocal fluorescence imaging system.
A novel hyperspectral fluorescence microscope for high-resolution 3D optical sectioning of cells and other structures has been designed, constructed, and used to investigate a number of different problems. We have significantly extended new multivariate curve resolution (MCR) data analysis methods to deconvolve the hyperspectral image data and to rapidly extract quantitative 3D concentration distribution maps of all emitting species. The imaging system has many advantages over current confocal imaging systems including simultaneous monitoring of numerous highly overlapped fluorophores, immunity to autofluorescence or impurity fluorescence, enhanced sensitivity, and dramatically improved accuracy, reliability, and dynamic range. Efficient data compression in the spectral dimension has allowed personal computers to perform quantitative analysis of hyperspectral images of large size without loss of image quality. We have also developed and tested software to perform analysis of time resolved hyperspectral images using trilinear multivariate analysis methods. The new imaging system is an enabling technology for numerous applications including (1) 3D composition mapping analysis of multicomponent processes occurring during host-pathogen interactions, (2) monitoring microfluidic processes, (3) imaging of molecular motors and (4) understanding photosynthetic processes in wild type and mutant Synechocystis cyanobacteria
Partial Meal Replacement Plan and Quality of the Diet at 1 Year: Action for Health in Diabetes (Look AHEAD) Trial
Little is known about diet quality with a reduced-energy, low-fat, partial meal replacement (PMR) plan, especially in individuals with type 2 diabetes. The Action for Health in Diabetes (Look AHEAD) trial implemented a PMR plan in the intensive lifestyle intervention (ILI)
In Vivo Collection of Rare Proteins Using Kinesin-based "Nano-Harvesters" In Vivo Collection of Rare Proteins Using Kinesin-based "Nano-Harvesters"
Abstract In this project, we have developed a novel platform for capturing, transport, and separating target analytes using the work harnessed from biomolecular transport systems. Nanoharvesters were constructed by coorganizing kinesin motor proteins and antibodies on a nanocrystal quantum dot (nQD) scaffold. Attachment of kinesin and antibodies to the nQD was achieved through biotin-streptavidin non-covalent bonds. Assembly of the nanoharvesters was characterized using a modified enzyme-linked immunosorbent assay (ELISA) that confirmed attachment of both proteins. Nanoharvesters selective against tumor necrosis factor-α (TNF-α) and nuclear transcription factor-κB (NF-κB) were capable of detecting target antigens at <100 ng/mL in ELISAs. A motility-based assay was subsequently developed using an antibody-sandwich approach in which the target antigen (TNF-α) formed a sandwich with the red-emitting nanoharvester and green-emitting detection nQD. In this format, successful sandwich formation resulted in a yellow emission associated with surface-bound microtubules. Step-wise analysis of sandwich formation suggested that the motility function of the kinesin motors was not adversely affected by either antigen capture or the subsequent binding of the detection nQDs. TNF-α was detected as low as ~1.5 ng/mL TNF-α, with 5.2% of the nanoharvesters successfully capturing the target analyte and detection nQDs. Overall, these results demonstrate the ability to capture target protein analytes in vitro using the kinesin-based nanoharvesters in nanofluidic environments. This system has direct relevance for lab-on-a-chip applications where pressure-driven or electrokinetic movement of fluids is impractical, and offers potential application for in vivo capture of rare proteins within the cytoplasmic domain of live cells. -3
Seasonal pattern of tomato mosaic tobamovirus infection and concentration in red spruce seedlings
Effects of Added Divalent Counterions on the Properties and Behaviors of Microtubule Filaments
Photodamage and the Importance of Photoprotection in Biomolecular-Powered Device Applications
In
recent years, an enhanced understanding of the mechanisms underlying
photobleaching and photoblinking of fluorescent dyes has led to improved
photoprotection strategies, such as reducing and oxidizing systems
(ROXS) that reduce blinking and oxygen scavenging systems to reduce
bleaching. Excitation of fluorescent dyes can also result in damage
to catalytic proteins (e.g., biomolecular motors), affecting the performance
of integrated devices. Here, we characterized the motility of microtubules
driven by kinesin motor proteins using various photoprotection strategies,
including a microfluidic deoxygenation device. Impaired motility of
microtubules was observed at high excitation intensities in the absence
of photoprotection as well as in the presence of an enzymatic oxygen
scavenging system. In contrast, using a polydimethylsiloxane (PDMS)
microfluidic deoxygenation device and ROXS, not only were the fluorophores
slower to bleach but also moving the velocity and fraction of microtubules
over time remained unaffected even at high excitation intensities.
Further, we demonstrate the importance of photoprotection by examining
the effect of photodamage on the behavior of a switchable mutant of
kinesin. Overall, these results demonstrate that improved photoprotection
strategies may have a profound impact on functional fluorescently
labeled biomolecules in integrated devices