Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail

Cholesterol-Induced Buckling in Physisorbed Polymer-Tethered Lipid Monolayers

The influence of cholesterol concentration on the formation of buckling structures is studied in a physisorbed polymer-tethered lipid monolayer system using epifluorescence microscopy (EPI) and atomic force microscopy (AFM). The monolayer system, built using the Langmuir-Blodgett (LB) technique, consists of 3 mol % poly(ethylene glycol) (PEG) lipopolymers and various concentrations of the phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and cholesterol (CHOL). In the absence of CHOL, AFM micrographs show only occasional buckling structures, which is caused by the presence of the lipopolymers in the monolayer. In contrast, a gradual increase of CHOL concentration in the range of 0–40 mol % leads to fascinating film stress relaxation phenomena in the form of enhanced membrane buckling. Buckling structures are moderately deficient in CHOL, but do not cause any notable phospholipid-lipopolymer phase separation. Our experiments demonstrate that membrane buckling in physisorbed polymer-tethered membranes can be controlled through CHOL-mediated adjustment of membrane elastic properties. They further show that CHOL may have a notable impact on molecular confinement in the presence of crowding agents, such as lipopolymers. Our results are significant, because they offer an intriguing prospective on the role of CHOL on the material properties in complex membrane architecture

Pure white‐light emitting ultrasmall organic‐inorganic hybrid perovskite nanoclusters

Organic–inorganic hybrid perovskites, direct band-gap semiconductors, have shown tremendous promise for optoelectronic device fabrication. We report the first colloidal synthetic approach to prepare ultrasmall (∼1.5 nm diameter), white-light emitting, organic–inorganic hybrid perovskite nanoclusters. The nearly pure white-light emitting ultrasmall nanoclusters were obtained by selectively manipulating the surface chemistry (passivating ligands and surface trap-states) and controlled substitution of halide ions. The nanoclusters displayed a combination of band-edge and broadband photoluminescence properties, covering a major part of the visible region of the solar spectrum with unprecedentedly large quantum yields of ∼12% and photoluminescence lifetime of ∼20 ns. The intrinsic white-light emission of perovskite nanoclusters makes them ideal and low cost hybrid nanomaterials for solid-state lighting applications

Ligand Mediated Sequestering of Integrins in Raft-Mimicking Lipid Mixtures: The Role of Bilayer Asymmetry and Cholesterol Content

poster abstractLipid microdomains play an important functional role in plasma membranes. However, the small size and transient nature of lipid/membrane heterogeneities in the plasma membrane make characterization of microdomains and microdomain-related membrane processes quite challenging. To address this issue, we recently introduced a powerful model membrane system that allows the investigation of membrane protein sequestering and oligomerization in raft-mimicking lipid mixtures using combined confocal fluorescence spectroscopy, photon counting histogram (PCH), and epifluorescence microscopy. Our experiments on bilayer-spanning domains showed that αvβ3 and α5β1 integrins predominantly exist as monomers and sequester preferentially to the liquid-disordered (ld) phase in the absence of ligands. Notably, addition of vitronectin (αvβ3) and fibronectin (α5β1) caused substantial translocations of integrins into the liquid-ordered (lo) phase without altering receptor oligomerization state. Here we expand our previous studies and report on the sequestering and oligomerization state of αvβ3 and α5β1 in asymmetric bilayer compositions containing coexisting lo and ld phases located exclusively in the top leaflet of the bilayer (bottom leaflet shows only ld phase). Remarkably, in such a membrane environment, both integrins show a higher affinity for the top leaflet-restricted lo domains in the absence of their respective ligands. A slight change in the integrin sequestration was observed after addition of their respective ligands. We also present experimental findings, which show that cholesterol content has a substantial influence on integrin sequestering and oligomerization in raft-mimicking lipid mixtures. The described experimental results highlight the potential importance of membrane asymmetry and lipid composition in the sequestering of membrane proteins in biological membranes

Using Fractional Cascading to Accelerate Cross Section Lookups in Monte Carlo Neutron Transport Calculations

We describe and test a technique for carrying out energy grid searches in continuous-energy Monte Carlo (MC) neutron transport calculations that represents an optimal compromise between grid search performance and memory footprint. The method, based on the fractional cascading technique and referred to as the cascade grid, is tested within the OpenMC Monte Carlo code, and performance results comparing the method with existing approaches are presented for the Hoogenboom-Martin reactor benchmark. The cascade grid achieves significant speedups in calculation rate with negligible initialization overhead while not increasing the memory footprint by more than 2x.

A Large Catalog of Homogeneous Ultra-Violet/Optical GRB Afterglows: Temporal and Spectral Evolution

We present the second Swift Ultra-Violet/Optical Telescope (UVOT) gamma-ray burst (GRB) afterglow catalog, greatly expanding on the first Swift UVOT GRB afterglow catalog. The second catalog is constructed from a database containing over 120,000 independent UVOT observations of 538 GRBs first detected by Swift, the High Energy Transient Explorer 2 (HETE2), the INTErnational Gamma-Ray Astrophysics Laboratory (INTEGRAL), the Interplanetary Network (IPN), Fermi, and Astro-rivelatore Gamma a Immagini Leggero (AGILE). The catalog covers GRBs discovered from 2005 Jan 17 to 2010 Dec 25. Using photometric information in three UV bands, three optical bands, and a `white' or open filter, the data are optimally co-added to maximize the number of detections and normalized to one band to provide a detailed light curve. The catalog provides positional, temporal, and photometric information for each burst, as well as Swift Burst Alert Telescope (BAT) and X-Ray Telescope (XRT) GRB parameters. Temporal slopes are provided for each UVOT filter. The temporal slope per filter of almost half the GRBs are fit with a single power-law, but one to three breaks are required in the remaining bursts. Morphological comparisons with the X-ray reveal that approximately 75% of the UVOT light curves are similar to one of the four morphologies identified by Evans et al. (2009). The remaining approximately 25% have a newly identified morphology. For many bursts, redshift and extinction corrected UV/optical spectral slopes are also provided at 2000, 20,000, and 200,000 seconds.Comment: 44 pages, 14 figures, to be published in Astrophysical Journal Supplementa

Breast cancer brain metastases: evidence for neuronal-like adaptation in a ‘breast-to-brain’ transition?

Abstract Brain metastases remain a significant challenge in the treatment of breast cancer patients due to the unique environment posed by the central nervous system. A better understanding of the biology of breast cancer cells that have metastasized to the brain is required to develop improved therapies. A recent Proceedings of the National Academy of Sciences article demonstrates that breast cancer cells in the brain microenvironment express γ-aminobutyric acid (GABA)-related genes, enabling them to utilize GABA as an oncometabolite, thus gaining a proliferative advantage. In this viewpoint, we highlight these findings and their potential impact on the treatment of breast cancer brain metastases

Gaussian Process Regression and Monte Carlo Simulation to Determine VOC Biomarker Concentrations Via Chemiresistive Gas Nanosensors

Utilizing chemiresistive gas sensors for volatile organic compound (VOC) detection has been a growing area of investigation in the last decade. VOCs have been extensively studied as potential biomarkers for biomedical applications as they are byproducts of metabolic pathways which are dysregulated by disease. Therefore, sensor arrays have been fabricated in previous studies to detect VOC biomarkers. In the process of testing these sensors, it is highly advantageous to quantify the concentration of the VOC biomarkers with high accuracy to diagnose the disease with high sensitivity and specificity. To investigate, analyze, and understand the relation between the concentrations of the VOC to the sensor resistance response, Gaussian Process (GP) models were implemented to predict the behavior of the data with respect to the resistance when the sensor is exposed to a range of concentrations of VOCs. Additionally, the relation between the concentration and resistance of the sensor was studied to predict the concentration of the VOC when a resistance is obtained. Monte Carlo Simulation Sampling from the GP model was utilized to generate data to further understand the trend. The results demonstrated that the relation between the concentration and resistance is linear. The model was tested with sampling data and its accuracy was evaluated