How to Characterize Individual Nano-Size Liposomes with Simple Self-Calibrating Fluorescence Microscopy

Nanosize lipid vesicles are used extensively at the interface between nanotechnology and biology, e.g., as containers for chemical reactions at minute concentrations and vehicles for targeted delivery of pharmaceuticals. Typically, vesicle samples are heterogeneous as regards vesicle size and structural properties. Consequently, vesicles must be characterized individually to ensure correct interpretation of experimental results. Here we do that using dual-color fluorescence labeling of vesiclesof their lipid bilayers and lumens, separately. A vesicle then images as two spots, one in each color channel. A simple image analysis determines the total intensity and width of each spot. These four data all depend on the vesicle radius in a simple manner for vesicles that are spherical, unilamellar, and optimal encapsulators of molecular cargo. This permits identification of such <i>ideal</i> vesicles. They in turn enable calibration of the dual-color fluorescence microscopy images they appear in. Since this calibration is not a separate experiment but an analysis of images of vesicles to be characterized, it eliminates the potential source of error that a separate calibration experiment would have been. Nonideal vesicles in the same images were characterized by how their four data violate the calibrated relationship established for ideal vesicles. In this way, our method yields size, shape, lamellarity, and encapsulation efficiency of each imaged vesicle. Applying this procedure to extruded samples of vesicles, we found that, contrary to common assumptions, only a fraction of vesicles are ideal

DNA Methylation Changes in Atypical Adenomatous Hyperplasia, Adenocarcinoma In Situ, and Lung Adenocarcinoma

BACKGROUND:Aberrant DNA methylation is common in lung adenocarcinoma, but its timing in the phases of tumor development is largely unknown. Delineating when abnormal DNA methylation arises may provide insight into the natural history of lung adenocarcinoma and the role that DNA methylation alterations play in tumor formation. METHODOLOGY/PRINCIPAL FINDINGS:We used MethyLight, a sensitive real-time PCR-based quantitative method, to analyze DNA methylation levels at 15 CpG islands that are frequently methylated in lung adenocarcinoma and that we had flagged as potential markers for non-invasive detection. We also used two repeat probes as indicators of global DNA hypomethylation. We examined DNA methylation in 249 tissue samples from 93 subjects, spanning the putative spectrum of peripheral lung adenocarcinoma development: histologically normal adjacent non-tumor lung, atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS, formerly known as bronchioloalveolar carcinoma), and invasive lung adenocarcinoma. Comparison of DNA methylation levels between the lesion types suggests that DNA hypermethylation of distinct loci occurs at different time points during the development of lung adenocarcinoma. DNA methylation at CDKN2A ex2 and PTPRN2 is already significantly elevated in AAH, while CpG islands at 2C35, EYA4, HOXA1, HOXA11, NEUROD1, NEUROD2 and TMEFF2 are significantly hypermethylated in AIS. In contrast, hypermethylation at CDH13, CDX2, OPCML, RASSF1, SFRP1 and TWIST1 and global DNA hypomethylation appear to be present predominantly in invasive cancer. CONCLUSIONS/SIGNIFICANCE:The gradual increase in DNA methylation seen for numerous loci in progressively more transformed lesions supports the model in which AAH and AIS are sequential stages in the development of lung adenocarcinoma. The demarcation of DNA methylation changes characteristic for AAH, AIS and adenocarcinoma begins to lay out a possible roadmap for aberrant DNA methylation events in tumor development. In addition, it identifies which DNA methylation changes might be used as molecular markers for the detection of preinvasive lesions

Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As the method is based on the detection of single photons, it additionally allows for performing fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements thereby providing access to fast orientational dynamics down to the nanosecond time scale. The 3D orientation is particularly interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination at different timescales and quantifying the associated errors. The vesicles provide a well-defined spherical surface, such that the use of fluorescent lipid dyes (DiO) allows to establish a a wide range of dipole orientations experimentally. To complement our experimental data, we performed Monte Carlo simulations of the rotational dynamics of dipoles incorporated into lipid membranes. Our study offers a comprehensive view on the dye orientation behavior in a lipid membrane with high spatiotemporal resolution representing a six-dimensional fluorescence detection approach.</p