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Mouse laser-induced choroidal neovascularization (mouse LCNV) recapitulates the "wet" form of human age-related macular degeneration (AMD). Vascular cell adhesion molecule-1 (VCAM-1) is a known inflammatory biomarker, and it increases in the choroidal neovascular tissues characteristic of this experimental model. We have designed and constructed gold nanoparticles (AuNPs) functionalized with hairpin-DNA that incorporates an antisense sequence complementary to VCAM-1 mRNA (AS-VCAM-1 hAuNPs) and tested them as optical imaging probes. The 3' end of the hairpin is coupled to a near-infrared fluorophore that is quenched by the AuNP surface via Förster resonance energy transfer (FRET). Hybridization of the antisense sequence to VCAM-1 mRNA displaces the fluorophore away from the AuNP surface, inducing fluorescent activity. In vitro testing showed that hAuNPs hybridize to an exogenous complementary oligonucleotide within a pH range of 4.5-7.4, and that they are stable at reduced pH. LCNV mice received tail-vein injections of AS-VCAM-1 hAuNPs. Hyperspectral imaging revealed the delivery of AS-VCAM-1 hAuNPs to excised choroidal tissues. Fluorescent images of CNV lesions were obtained, presumably in response to the hybridization of AS-hAuNPs to LCNV-induced VCAM-1 mRNA. This is the first demonstration of systemic delivery of hAuNPs to ocular tissues to facilitate mRNA imaging of any target.
Histology-directed imaging mass spectrometry (IMS) is a spatially targeted IMS acquisition method informed by expert annotation that provides rapid molecular characterization of select tissue structures. The expert annotations are usually determined on digital whole slide images of histological stains where the staining preparation is incompatible with optimal IMS preparation, necessitating serial sections: one for annotation, one for IMS. Registration is then used to align staining annotations onto the IMS tissue section. Herein, we report a next-generation histology-directed platform implementing IMS-compatible autofluorescence (AF) microscopy taken prior to any staining or IMS. The platform enables two histology-directed workflows, one that improves the registration process between two separate tissue sections using automated, computational monomodal AF-to-AF microscopy image registration, and a registration-free approach that utilizes AF directly to identify ROIs and acquire IMS on the same section. The registration approach is fully automated and delivers state of the art accuracy in histology-directed workflows for transfer of annotations (∼3-10 μm based on 4 organs from 2 species) while the direct AF approach is registration-free, allowing targeting of the finest structures visible by AF microscopy. We demonstrate the platform in biologically relevant case studies of liver stage malaria and human kidney disease with spatially targeted acquisition of sparsely distributed (composing less than one tenth of 1% of the tissue section area) malaria infected mouse hepatocytes and glomeruli in the human kidney case study.
Novel near-infrared contrast agents based on the quaterrylene structure were strategically developed and tested for high photo-stability. Both a dendrimeric quaterrylene molecule, QR-G2-COOH, and a small molecule cationic quaterrylene dye, QR-4PyC4, remain optically stable and continue to generate a competitive photoacoustic response when irradiated by short near-infrared laser pulses for a relatively long time in an in-vitro cell study, unlike indocyanine green that rapidly decreases photoacoustic signal amplitude. The small molecule dye, QR-4PyC4 exhibits not only significantly higher cellular uptake rate than QR-G2-COOH and indocyanine green, but also low toxicity at a concentration of up to 10 μM. The dendrimeric dye, QR-G2-COOH that has surface functional groups available for conjugation with targeting and therapeutic agents shows the highest photoacoustic amplitude with high optical stability. Therefore, QR-4PyC4 can be a promising universal, sensitive and reliable photoacoustic contrast agent and QR-G2-COOH has great potential as a nano-platform with stable photoacoustic imaging capability.
Copyright © 2018 Elsevier B.V. All rights reserved.
Thallium (Tl+) flux assays enable imaging of potassium (K+) channel activity in cells and tissues by exploiting the permeability of K+ channels to Tl+ coupled with a fluorescent Tl+ sensitive dye. Common Tl+ sensing dyes utilize fluorescein as the fluorophore though fluorescein exhibits certain undesirable properties in these assays including short excitation wavelengths and pH sensitivity. To overcome these drawbacks, the replacement of fluorescein with rhodols was investigated. A library of 13 rhodol-based Tl+ sensors was synthesized and their properties and performance in Tl+ flux assays evaluated. The dimethyl rhodol Tl+ sensor emerged as the best of the series and performed comparably to fluorescein-based sensors while demonstrating greater pH tolerance in the physiological range and excitation and emission spectra 30 nm red-shifted from fluorescein.
Primary tumor organoids are a robust model of individual human cancers and present a unique platform for patient-specific drug testing. Optical imaging is uniquely suited to assess organoid function and behavior because of its subcellular resolution, penetration depth through the entire organoid, and functional endpoints. Specifically, optical metabolic imaging (OMI) is highly sensitive to drug response in organoids, and OMI in tumor organoids correlates with primary tumor drug response. Therefore, an OMI organoid drug screen could enable accurate testing of drug response for individualized cancer treatment. The objective of this perspective is to introduce OMI and tumor organoids to a general audience in order to foster the adoption of these techniques in diverse clinical and laboratory settings.
© 2017 by the Society of Nuclear Medicine and Molecular Imaging.
The goal of this study is to validate fluorescence intensity and lifetime imaging of metabolic co-enzymes NAD(P)H and FAD (optical metabolic imaging, or OMI) as a method to quantify cell-cycle status of tumor cells. Heterogeneity in tumor cell-cycle status (e. g. proliferation, quiescence, apoptosis) increases drug resistance and tumor recurrence. Cell-cycle status is closely linked to cellular metabolism. Thus, this study applies cell-level metabolic imaging to distinguish proliferating, quiescent, and apoptotic populations. Two-photon microscopy and time-correlated single photon counting are used to measure optical redox ratio (NAD(P)H fluorescence intensity divided by FAD intensity), NAD(P)H and FAD fluorescence lifetime parameters. Redox ratio, NAD(P)H and FAD lifetime parameters alone exhibit significant differences (p<0.05) between population means. To improve separation between populations, linear combination models derived from partial least squares - discriminant analysis (PLS-DA) are used to exploit all measurements together. Leave-one-out cross validation of the model yielded high classification accuracies (92.4 and 90.1 % for two and three populations, respectively). OMI and PLS-DA also identifies each sub-population within heterogeneous samples. These results establish single-cell analysis with OMI and PLS-DA as a label-free method to distinguish cell-cycle status within intact samples. This approach could be used to incorporate cell-level tumor heterogeneity in cancer drug development.
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
The Thomsen-Friedenreich (TF) antigen represents a prognostic biomarker of colorectal carcinoma. Here, using a nanobeacon, the surface of which was fabricated with peanut agglutinin as TF-binding molecules, we demonstrate that the nanobeacon is able to detect TF antigen in frozen and freshly biopsied polyps using fluorescence microscopy. Our results provide important clues about how to detect aberrant colonic tissues in the most timely fashion. Given the versatile application method for this topical nanobeacon, the protocol used in this work is amenable to clinical colonoscopy. Moreover, the prospects of clinical translation of this technology are evident.
Translocator protein (TSPO) is a validated target for molecular imaging of a variety of human diseases and disorders. Given its involvement in cholesterol metabolism, TSPO expression is commonly elevated in solid tumors, including glioma, colorectal cancer, and breast cancer. TSPO ligands capable of detection by optical imaging are useful molecular tracers for a variety of purposes that range from quantitative biology to drug discovery. Leveraging our prior optimization of the pyrazolopyrimidine TSPO ligand scaffold for cancer imaging, we report herein a new generation of TSPO tracers with superior binding affinity and suitability for optical imaging and screening. In total, seven candidate TSPO tracers were synthesized and vetted in this study; the most promising tracer identified (29, K = 0.19 nM) was the result of conjugating a high-affinity TSPO ligand to a fluorophore used routinely in biological sciences (FITC) via a functional carbon linker of optimal length. Computational modeling suggested that an n-alkyl linker of eight carbons in length allows for positioning of the bulky fluorophore distal to the ligand binding domain and toward the solvent interface, minimizing potential ligand-protein interference. Probe 29 was found to be highly suitable for in vitro imaging of live TSPO-expressing cells and could be deployed as a ligand screening and discovery tool. Competitive inhibition of probe 29 quantified by fluorescence and H-PK11195 quantified by traditional radiometric detection resulted in equivalent affinity data for two previously reported TSPO ligands. This study introduces the utility of TSPO ligand 29 for in vitro imaging and screening and provides a structural basis for the development of future TSPO imaging ligands bearing bulky signaling moieties.
Transposons are highly abundant in eukaryotic genomes, but their mobilization must be finely tuned to maintain host organism fitness and allow for transposon propagation. Forty percent of the human genome is comprised of transposable element sequences, and the most abundant cut-and-paste transposons are from the hAT superfamily. We found that the hAT transposase TcBuster from Tribolium castaneum formed filamentous structures, or rodlets, in human tissue culture cells, after gene transfer to adult mice, and ex vivo in cell-free conditions, indicating that host co-factors or cellular structures were not required for rodlet formation. Time-lapsed imaging of GFP-laced rodlets in human cells revealed that they formed quickly in a dynamic process involving fusion and fission. We delayed the availability of the transposon DNA and found that transposition declined after transposase concentrations became high enough for visible transposase rodlets to appear. In combination with earlier findings for maize Ac elements, these results give insight into transposase overproduction inhibition by demonstrating that the appearance of transposase protein structures and the end of active transposition are simultaneous, an effect with implications for genetic engineering and horizontal gene transfer.
Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.
Ocular angiogenesis is a blinding complication of age-related macular degeneration and other retinal vascular diseases. Clinical imaging approaches to detect inflammation prior to the onset of neovascularization in these diseases may enable early detection and timely therapeutic intervention. We demonstrate the feasibility of a previously developed cyclooxygenase-2 (COX-2) targeted molecular imaging probe, fluorocoxib A, for imaging retinal inflammation in a mouse model of laser-induced choroidal neovascularization. This imaging probe exhibited focal accumulation within laser-induced neovascular lesions, with minimal detection in proximal healthy tissue. The selectivity of the probe for COX-2 was validated