Plasmon-Assisted Super-Resolution Axial Distance Sensitivity in Fluorescence Cell Imaging

There is currently a great need to develop live-cell compatible optical microscopy tools that can provide super-resolution information on biomolecules, in particular for the study of membrane receptors. We present a novel imaging technique, which employs a nanoplasmonic substrate in combination with conventional confocal fluorescence lifetime microscopy, to deliver an axial position sensitivity of order 10 nm in whole cell imaging. The technique exploits the Purcell effect experienced by fluorescent molecules in the vicinity of noble metal nanoparticles, leading to a reduction of the radiative lifetime and a commensurate increase in fluorescence intensity. We employ this technique to map the topography of the cellular membrane, by imaging the fluorescent protein eGFP labeled to the receptor CXCR4, and further investigate receptor-mediated endocytosis in carcinoma cells. These results demonstrate a new approach in biological cell imaging, using bespoke plasmonic nanostructures to provide axial super-resolution sensitivity, while retaining compatibility with conventional fluorescence microscopy techniques

NMR Metabolomics of MTLn3E Breast Cancer Cells Identifies a Role for CXCR4 in Lipid and Choline Regulation

The alpha chemokine receptor CXCR4 is up-regulated in certain types of breast cancer. Truncation of the C-terminus of this receptor alters cell morphology and increases invasiveness and metastatic potential. Here, to better understand the effects of CXCR4 expression and truncation in breast cancer cells, we have used high resolution magic angle spinning (HR-MAS) NMR studies of rat breast carcinoma MtLn3E cells to characterize the metabolite complement of cells heterologously expressing human CXCR4 or its C-terminal truncation mutant, Δ34-CXCR4. Notable reductions in choline levels were detected when either cells expressing wild-type CXCR4 or Δ34-CXCR4 were compared with cells containing an empty expression vector. Cells expressing CXCR4-Δ34 had reduced lipid content when compared with either the wild-type CXCR4 expressing cells or those containing the empty expression vector. Taken together, our results show that distinct effects on the metabolite complement can be linked to either CXCR4 expression or CXCR4 regulation. The metabolite markers for these two effects identified in the present study can, in turn, be used to further investigate the role of CXCR4 in metastasis

Summary of small molecule inhibitor screening data of CXCR4.

<p>(A) Layout of microplate. Wells marked in green are CXCR4 inhibitors. Controls are shown in white: column 10 shows data for CXCR4-eGFP and CXCR4-TagRFP transfected cells without inhibitor treatment; column 11 shows CXCR4-eGFP only cells without inhibitor treatment. In column 10 the concentration of CXCL12 is varied: row D is untreated while rows E and F are treated with 5 nM and 20 nM CXCL12 respectively. (B) Comparison of anisotropy and lifetime data showing that dimerisation and internalisation is blocked by CXCR4 inhibitors. Errors bars represent the standard deviation of repeated measurements in each well (4 images per well for each modality). (C) Plots of anisotropy and FRET efficiency for column 10, rows D, E and F: cells in the absence of any inhibitors (CXCL12 concentrations of 5 nM and 20 nM). (D) Percentage change in anisotropy and lifetime compared to controls.</p

Imaging of FRET standard constructs expressed in 293T cells.

<p>(A) Intensity images from the wide-field and laser scanning modalities. (B) Functional images for GFP, 32AA and 7AA standards. This amply demonstrates the correlation between the two measurement techniques and the sensitivity in determining changes in FRET efficiency. (Scale bars represent 50 µm.)</p

Demonstration of sensitivity and repeatability of fluorescence anisotropy and fluorescence lifetime imaging.

<p>(A) Anisotropy measurements of rhodamine B dissolved in varying concentrations of water and glycerol. The concentration of glycerol is used as a way of tuning the rotational diffusion, and therefore anisotropy, of the fluorophore. Any increasing percentage of glycerol reduces the mobility of the fluorophore molecules thereby increasing the fluorescence anisotropy. Differences of the order of 0.004 are easily and repeatedly measured. (B) Fluorescence lifetime measurements of the same rhodamine B sample. The concentration of glycerol has no effect on the lifetime. It is clear that the measurement is highly repeatable.</p

Exploiting the Metal-Chelating Properties of the Drug Cargo for <i>In Vivo</i> Positron Emission Tomography Imaging of Liposomal Nanomedicines

The clinical value of current and future nanomedicines can be improved by introducing patient selection strategies based on noninvasive sensitive whole-body imaging techniques such as positron emission tomography (PET). Thus, a broad method to radiolabel and track preformed nanomedicines such as liposomal drugs with PET radionuclides will have a wide impact in nanomedicine. Here, we introduce a simple and efficient PET radiolabeling method that exploits the metal-chelating properties of certain drugs (<i>e.g.</i>, bisphosphonates such as alendronate and anthracyclines such as doxorubicin) and widely used ionophores to achieve excellent radiolabeling yields, purities, and stabilities with ⁸⁹Zr, ⁵²Mn, and ⁶⁴Cu, and without the requirement of modification of the nanomedicine components. In a model of metastatic breast cancer, we demonstrate that this technique allows quantification of the biodistribution of a radiolabeled stealth liposomal nanomedicine containing alendronate that shows high uptake in primary tumors and metastatic organs. The versatility, efficiency, simplicity, and GMP compatibility of this method may enable submicrodosing imaging studies of liposomal nanomedicines containing chelating drugs in humans and may have clinical impact by facilitating the introduction of image-guided therapeutic strategies in current and future nanomedicine clinical studies