Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots

This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.

Evaluation of suitable spectral intervals for near-IR laboratory detection of water vapour continuum absorption

The water vapour continuum absorption is an important component of molecular absorption of radiation in atmosphere. However, uncertainty in knowledge of the value of the continuum absorption at present can achieve 100% in different spectral regions leading to an error in flux calculation up to 3-5 W/m2 global mean. This work uses line-by-line calculations to reveal the best spectral intervals for experimental verification of the CKD water vapour continuum models in the currently least studied near-infrared spectral region. Possible sources of errors in continuum retrieval taken into account in the simulation include the sensitivity of laboratory spectrometers and uncertainties in the spectral line parameters in HITRAN-2004 and Schwenke-Partridge database. It is shown that a number of micro-windows in near-IR can be used at present for laboratory detection of the water vapour continuum with estimated accuracy from 30 to 5%.

A model of net amino acid absorption and utilization by the portal-drained viscera of the lactating dairy cow

A more complete understanding of amino acid ( AA) metabolism by the various tissues of the body is required to improve upon current systems for predicting the use of absorbed AA. The objective of this work was to construct and parameterize a model of net removal of AA by the portal-drained viscera (PDV). Six cows were prepared with arterial, portal, and hepatic catheters and infused abomasally with 0, 200, 400, or 600 g of casein daily. Casein infusion increased milk yield quadratically and tended to increase milk protein yield quadratically. Arterial concentrations of a number of essential AA increased linearly with respect to infusion amount. When infused casein was assumed to have a true digestion coefficient of 0.95, the minimum likely true digestion coefficient for noninfused duodenal protein was found to be 0.80. Net PDV use of AA appeared to be linearly related to total supply (arterial plus absorption), and extraction percentages ranged from 0.5 to 7.25% for essential AA. Prediction errors for portal vein AA concentrations ranged from 4 to 9% of the observed mean concentrations. Removal of AA by PDV represented approximately 33% of total postabsorptive catabolic use, including use during absorption but excluding use for milk protein synthesis, and was apparently adequate to support endogenous N losses in feces of 18.4 g/d. As 69% of this use was from arterial blood, increased PDV catabolism of AA in part represents increased absorption of AA in excess of amounts required by other body tissues. Based on the present model, increased anabolic use of AA in the mammary and other tissues would reduce the catabolic use of AA by the PDV.

Effects of volatile fatty acid supply on their absorption and on water kinetics in the rumen of sheep sustained by intragastric infusions

Three sheep fitted with a ruminal cannula and an abomasal catheter were used to study water kinetics and absorption of VFA infused continuously into the rumen. The effects of changing VFA concentrations in the rumen by shifting VFA infusion rates were investigated in an experiment with a 3 x 3 Latin square design. On experimental days, the animals received the basal infusion rate of VFA (271 mmol/h) during the first 2 h. Each animal then received VFA at a different rate (135, 394, or 511 mmol/h) for the next 7.5 h. Using soluble markers (polyethylene glycol and Cr-EDTA), ruminal volume, liquid outflow, apparent water absorption, and VFA absorption rates were estimated. There were no significant effects of VFA infusion rate on ruminal volume and water kinetics. As the VFA infusion rate was increased, VFA concentration and osmolality in the rumen were increased and pH was decreased. There was a biphasic response of liquid outflow to changes in the total VFA concentration in the rumen, as both variables increased together up to a total VFA concentration of 80.1 mM, whereas, beyond that concentration, liquid outflow remained stable at an average rate of 407 mL/h. There were significant linear (P = 0.003) and quadratic (P = 0.001) effects of VFA infusion rate on the VFA absorption rate, confirming that VFA absorption in the rumen is mainly a concentration-dependent process. The proportion of total VFA supplied that was absorbed in the rumen was 0.845 (0.822, 0.877, and 0.910 for acetate, propionate, and butyrate, respectively). The molar proportions of acetate, propionate, and butyrate absorbed were affected by the level of VFA infusion in the rumen, indicating that this level affected to a different extent the absorption of the different acids.

Greenhouse whitefly (Homoptera : Aleyrodidae) dispersal under different UV-light environments

The greenhouse whitefly, Dialeurodes vaporariorum (Westwood) (Homoptera: Aleyrodidae), is known to respond to UV light (UV). Field studies were conducted to improve our understanding of the behavioral effects and practical implications of using UV-blocking plastic films for the control of whitefly. Adult whiteflies were released in outdoor-located choice-chamber experiments with compartments clad with a range of films that transmitted incident UV to different extents. In release-recapture experiments, a very small proportion of the whiteflies recovered had dispersed into compartments where the entire UV spectrum was blocked, whereas the major proportion preferred compartments with UV. Compartments clad with films that blocked LTV below 375 nm attracted significantly more whiteflies than films that blocked UV below 385 nm, whereas the absorption of LTV wavelengths above 385 nm did not show any further effect on whitefly numbers. A reduction in the side cladding of the compartments by > 20% significantly reduced the advantage of using LTV-blocking films. Adult whitefly did not discriminate between direct- and diffused-light environments, as long as the UV-absorbing properties of the films were equivalent. Whitefly dispersal was influenced by the time of the day when adult whitefly were released, with a higher proportion of whitelly avoiding compartments clad with LTV-blocking films, at times of the day when light intensities were higher. The future use of UV-blocking films as a potentially highly effective component of integrated pest management systems for the control of whitefly is discussed.

Interactions of bovine serum albumin with ethylene oxide/butylene oxide copolymers in aqueous solution

The interactions of bovine serum albumin (BSA) with three ethylene oxide/butylene oxide (E/B) copolymers having different block lengths and varying molecular architectures is examined in this study in aqueous solutions. Dynamic light scattering (DLS) indicates the absence of BSA-polymer binding in micellar systems of copolymers with lengthy hydrophilic blocks. On the contrary, stable protein-polyrner aggregates were observed in the case of E18B10 block copolymer. Results from DLS and SAXS suggest the dissociation of E/B copolymer micelles in the presence of protein and the absorption of polymer chains to BSA surface. At high protein loadings, bound BSA adopts a more compact conformation in solution. The secondary structure of the protein remains essentially unaffected even at high polymer concentrations. Raman spectroscopy was used to give insight to the configurations of the bound molecules in concentrated solutions. In the vicinity of the critical gel concentration of E18B10 introduction of BSA can dramatically modify the phase diagram, inducing a gel-sol-gel transition. The overall picture of the interaction diagram of the E18B10-BSA reflects the shrinkage of the suspended particles due to destabilization of micelles induced by BSA and the gelator nature of the globular protein. SAXS and rheology were used to further characterize the structure and flow behavior of the polymer-protein hybrid gels and sols.

Light-assisted synthesis of a Ru(VI) nitrido complex by the reaction of azide with a Ru(III) complex

Reaction of Ru(III)(L)(dmf)Cl-3 (1) (L = 4,4,4',4'-tetramethyl-2,2'- bisoxazoline, dmf = N,N-dimethylformamide) with an excess of sodium azide in a methanol-water mixture leads to the isolation of the sodium salt of a Ru( VI) nitrido complex of the tetraanion of N,N'-bis-(2,2-dimethyl-1-hydroxyethyl)-1,2-ethanediamide ( L'H-4; H a dissociable proton) of the formulation Na[Ru(L')(N)(H2O)].1.4H(2)O (2). Complex 2 is not generated in the absence of light. A tentative mechanism for the reaction is proposed and a Ru(IV) intermediate, Na[Ru(L')(N-3)(H2O)] . 2CH(3)OH.2H(2)O (3), isolated.

Fluorescence lifetime imaging microscopy (FLIM) to demonstrate the nuclear binding of flavanols and (--epigallocatechin gallate

The use of light microscopy and DMACA staining strongly suggested that plant and animal cell nuclei act as sinks for flavanols [1, 2]. Detailed uv-vis spectroscopic titration experiments indicated that histone proteins are the likely binding sites in the nucleus [2]. Here we report the development of a multi-photon excitation microscopy technique combined with fluorescent lifetime measurements of flavanols. Using this technique, (+) catechin, (-) epicatechin and (-) epigallocatechin gallate (EGCG) showed strikingly different excited state lifetimes in solution. Interaction of histone proteins with flavanols was indicated by the appearance of a significant τ2-component of 1.7 to 4.0ns. Tryptophan interference could be circumvented in the in vivo fluorescence lifetime imaging microscopy (FLIM) experiments with 2-photon excitation at 630nm. This enabled visualisation and semi-quantitative measurements that demonstrated unequivocally the absorption of (+)catechin, (-)epicatechin and EGCG by nuclei of onion cells. 3D FLIM revealed for the first time that externally added EGCG penetrated the whole nucleus in onion cells. The relative proportions of EGCG in cytoplasm: nucleus: nucleoli were ca. 1:10:100. FLIM experiments may therefore facilitate probing the health effects of EGCG, which is the major constituent of green tea.

Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots

We introduce semiconductor quantum dot-based fluorescence imaging with approximately 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells.