We report a fully quantitative spectroscopy imaging instrument for wide area detection of early cancer (dysplasia). This instrument provides quantitative maps of tissue biochemistry and morphology, making it a potentially powerful surveillance tool for objective early cancer detection. We describe the design, construction, calibration, and first clinical application of this new system. We demonstrate its accuracy using physical tissue models. We validate its diagnostic ability on a resected colon adenoma, and demonstrate feasibility of in vivo imaging in the oral cavity.
Quantitative spectroscopy has recently been extended from a contact-probe to wide-area spectroscopic imaging to enable mapping of optical properties across a wide area of tissue. We train quantitative spectroscopic imaging (QSI) to identify cervical high-grade squamous intraepithelial lesions (HSILs) in 34 subjects undergoing the loop electrosurgical excision procedure (LEEP subjects). QSI's performance is then prospectively evaluated on the clinically suspicious biopsy sites from 47 subjects undergoing colposcopic-directed biopsy. The results show the per-subject normalized reduced scattering coefficient at 700 nm (An) and the total hemoglobin concentration are significantly different (p<0.05) between HSIL and non-HSIL sites in LEEP subjects. An alone retrospectively distinguishes HSIL from non-HSIL with 89% sensitivity and 83% specificity. It alone applied prospectively on the biopsy sites distinguishes HSIL from non-HSIL with 81% sensitivity and 78% specificity. The findings of this study agree with those of an earlier contact-probe study, validating the robustness of QSI, and specifically An, for identifying HSIL. The performance of An suggests an easy to use and an inexpensive to manufacture monochromatic instrument is capable of early cervical cancer detection, which could be used as a screening and diagnostic tool for detecting cervical cancer in low resource countries.
Calcitriol is the active form of Vitamin D 3. Epidemiological data show that women with Vitamin D deficiency at the time of breast cancer diagnosis are 94% more likely to experience cancer spread and 73% more likely to die over the next 10 years, compared to women with adequate Vitamin D levels. Since vitamin D deficiency is especially common in African American and obese women, this observation may partially explain the relatively poor clinical outcome experienced by these patients [2]. Although current treatments for IBC
stability of the employed oligonucleotide nanolevers under different electrolyte conditions. Temperature dependent data are presented from 4 to 70 C for a range of different bacterial and human proteins: while some show a straightforward unfolding transition, others feature more complex signatures which are indicative of the protein expansion before an unfolding of individual domains sets in at higher temperatures. We discuss the possibility to evaluate quantitative thermodynamic parameters (melting points, transition energies) from the molecular dynamics data and compare our results to established methods like thermofluor assays. Since the presented approach works on a chip surface it uses minimal amounts of sample and can be performed in parallel using a microelectrode array. In addition, it yields binding kinetics data (association and dissociation rates) and affinity values and thus is ideally suited for investigating interactions in combination with a thermodynamic characterization of the involved proteins.
Dense protein complexes such as those found in the immunological synapses of antigen-activated lymphocytes are sites of critical biological activity, but their study is complicated by the large number of protein species present. Array tomography is a proteomic imaging method capable of addressing this highdimensional problem through iterative immunostaining, but would be difficult to apply to spatially-restricted regions of interest such as complexes within the immunological synapse that form in the area of contact between lymphocytes and antigen-presenting surfaces. We have developed a novel variant of this technique which embeds a thin (~500 nm) slice of material in LR White, making proteomic multiplexing possible. We are using this method with physically unroofed resting and activated B cells to study signaling in antigen-receptor microclusters in B cell plasma membranes. Here we present this method, and our current work in its application to the B cell membrane.
transmembrane signaling in response to extracellular stimuli. Inherent PM organizational principles often include, but are not limited to, ordered proteo-lipid nanodomains and cortical actin cytoskeleton. Such structural features underpin the steady-state environment of signaling components to provide an optimal platform for signal initiation. Therefore, it is necessary to evaluate the 'resting state' organization of signaling components and its modulation after stimulation for complete understanding of the PM-localized stage of the cell surface signaling. In this study, we used RBL mast cells as a general paradigm of ligand induced transmembrane signaling. Here, extracellular triggering of immunoglobin E complexed to its receptor, FcεRI, (IgE-FcεRI) by multivalent antigen leads to receptor clustering and subsequent phosphorylation by Lyn tyrosine kinase to initiate signaling. We first examined lateral environment 'sensed' in the resting state by IgE-FcεRI and Lyn by monitoring their respective lateral diffusion modes in RBL cells with multiplexed Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) and associated spot variation FCS (svFCS). The diffusion of both receptor and kinase deviate from free diffusion due to interaction with different confinement sources. Furthermore, we identified the simultaneous presence of lipid-dependent and actin-dependent confinement sources for the diffusion of Lyn kinase, whereas diffusion of IgE-FcεRI was weakly dependent on cortical actin organization. Importantly, our Imaging FCS results for a dozen different membrane proteins indicate that 70% of the plasma membrane exhibits the diffusion properties of an ordered environment. We further elucidate the association of Lyn kinase and stimulated IgE-FcεRI receptor with their lipiddriven ordered environments, which effectively enhances their spatial proximity that is essential for optimal phosphorylation of antigen-activated receptors. The interaction of T cells with antigen presenting cells (APC) plays a central role in the adaptive immune system. However, the molecular mechanisms by which binding of the T cell receptor (TCR) to its ligand triggers a signal within the cell are still debated. Here, we use DNA origami decorated with TCR ligands anchored to a planar glass-supported lipid bilayer to assess the effects of local ligand density, arrangement and mobility on T cell activation. Our experimental setup allows for controlling the nanoscale arrangement of TCR ligands on the DNA origami scaffold as well as the re-organization of ligand and TCR during T cell activation. Further, the spatial distribution of ligands can be tuned independently of ligand concentration. TCRb-reactive single-chain antibody fragment (scFv) and peptide-loaded major histocompatibility complex II (pMHC) were used as ligands and placed on the DNA origami at engineered capture sites in different layouts and densities. Functionalized DNA origami platforms were characterized using single molecule fluorescence microscopy and high-speed atomic force micr...
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