Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.

Direct observation of iron-induced conformational changes of mitochondrial DNA by high-resolution field-emission in-lens scanning electron microscopy.

When respiring rat liver mitochondria are incubated in the presence of Fe(III) gluconate, their DNA (mtDNA) relaxes from the supercoiled to the open circular form dependent on the iron dose. Anaerobiosis or antioxidants fail to completely inhibit the unwinding. High-resolution field-emission in-lens scanning electron microscopy imaging, in concert with backscattered electron detection, pinpoints nanometer-range iron colloids bound to mtDNA isolated from iron-exposed mitochondria. High-resolution field-emission in-lens scanning electron microscopy with backscattered electron detection imaging permits simultaneous detailed visual analysis of DNA topology, iron dose-dependent mtDNA unwinding, and assessment of iron colloid formation on mtDNA strands.

Particles move along actin filament bundles in nerve growth cones.

Organelle movement along actin filaments has been demonstrated in dissociated squid axoplasm [Kurznetsov, S. A., Langford, G.M. & Weiss, D. G. (1992) Nature (London) 356, 722-725 and Bearer, E.L., DeGiorgis, J.A., Bodner, R.A., Kao, A.W. & Reese, T.S. (1993) Proc. Natl. Acad. Sci. USA 90, 11252-11256] but has not been shown to occur in intact neurons. Here we demonstrate that intracellular transport occurs along actin filament bundles in intact neuronal growth cones. We used video-enhanced differential interference contrast microscopy to observe intracellular transport in superior cervical ganglion neurons cultured under conditions that enhance the visibility of actin bundles within growth cone lamellipodia. Intracellular particles, ranging in size from < 0.5-1.5 microns, moved along linear structures (termed transport bundles) at an average maximum rate of 0.48 micron/sec. After particle movement had been viewed, cultures were preserved by rapid perfusion with chemical fixative. To determine whether particle transport occurred along actin, we then used fluorescence microscopy to correlate this movement with actin and microtubule distributions in the same growth cones. The observed transport bundles colocalized with actin but not with microtubules. The rates of particle movement and the association of moving particles with actin filament bundles suggest that myosins may participate in the transport of organelles (or other materials) in intact neurons.

Using new scanning electron microscopy (SEM) approaches to elucidate bacterial biofilm architecture

Healthcare-associated infections (HAI) are a major public health problem being Klebsiella pneumoniae and nontuberculous mycobacteria, both with high antibiotic resistance rates, among their etiological agent. Since biofilme assembly is pointed as one of the mechanisms involved in emergence of antibiotic resistance understanding bacteria organization within the biofilm and the identification of differences between planktonic and sessile forms of bacteria will be a step forward to fight HAI. In the present work we used SEM as a tool to characterize the internal structure of biofilm assembled on different surfaces. For SEM analysis, biofilms were allowed to form either on six-well cell culture plates, silicon or metallic disks placed inside the wells for different incubation periods at 37 °C. The biofilm assembled on the cell culture dish was for both secondary and backscattered electron analysis as described before. Biofilms assembled on silicon disks instead of being sectioned were prepared as metallographic samples, by grinding with grit SIC paper and polishing with diamond particles. Samples were cleaned (70% ethanol), dried with hot air, further coated and analysed. A preliminary study using FIB-SEM has been performed to access the ultrastructure of biofilms assembled on metallic surfaces. The results obtained showed that the same bacteria assembled biofilms with different ratios of biomass and extracellular matrix depending on the surface. SEM performed on thin sections of biofilms is a powerful tool to elucidate biofilm structure allowing the quantification of the major components. FIB-SEM is also a promising tool in this field.

Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.

Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.

A helping hand for town and country : an American home book of practical and scientific information concerning house and lawn, garden and orchard, field, barn and stable, apiary and fish pond, workshop and dairy, and the many important interests pertaining to domestic economy and family health /

"The kitchen and dining-room" on p. 633-698 include cooking recipes.

Investigation of solid surfaces by field emission microscopy and mass spectrometry; final report.

"Contract no. AF 49 (638)-748. Division file 30-4. AFOSR-2273."

Darnley : or, The field of the cloth of gold /

On spine: E. P. Dutton.

Blast fragmentation modelling for a green field open cut gold deposit

This paper outlines the methodology of blast fragmentation modeling undertaken for a green field feasibility study at the Riska gold deposit in Indonesia. The favoured milling process for the feasibility study was dump leaching,with no crushing of the ore material extracted from the pit. For this reason,blast fragmentation was a critical issue to be addressed by the study. A range of blast designs were considered with bench heights and blasthole diameters ranging from 4 m to 7 m and 76 mm to 102 mm respectively. Rock mass data was obtained from 19 diamond drill cores across the deposit (total drill length approximately 2200 m). Intact rock strength was estimated from qualitative strength descriptors,while the in situ block size distribution of the rock mass was estimated from the Rock Quality Designation (RQD) of the core.

An analysis of the semantic field of the German particles 'Uberhaupt' and 'Eigentlich'

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY WITH PRIOR ARRANGEMENT

Signal processing for molecular and cellular biological physics:an emerging field

Recent advances in our ability to watch the molecular and cellular processes of life in action-such as atomic force microscopy, optical tweezers and Forster fluorescence resonance energy transfer-raise challenges for digital signal processing (DSP) of the resulting experimental data. This article explores the unique properties of such biophysical time series that set them apart from other signals, such as the prevalence of abrupt jumps and steps, multi-modal distributions and autocorrelated noise. It exposes the problems with classical linear DSP algorithms applied to this kind of data, and describes new nonlinear and non-Gaussian algorithms that are able to extract information that is of direct relevance to biological physicists. It is argued that these new methods applied in this context typify the nascent field of biophysical DSP. Practical experimental examples are supplied.

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. © 2014 Jiwaji et al.

Passively Q-switched erbium-doped fiber laser using evanescent field interaction with gold-nanosphere based saturable absorber

We demonstrate an all-fiber passively Q-switched erbiumdoped fiber laser (EDFL) using a gold-nanosphere (GNS) based saturable absorber (SA) with evanescent field interaction. Using the interaction of evanescent field for fabricating SAs, long nonlinear interaction length of evanescent wave and GNSs can be achieved. The GNSs are synthesized from mixing solution of chloroauricacid (HAuCl4) and sodium citrate by the heating effects of the microfiber's evanescent field radiation. The proposed passively Q-switched EDFL could give output pulses at 1562 nm with pulse width of 1.78 μs, a repetition rate of 58.1 kHz, a pulse energy of 133 nJ and a output power of 7.7 mWwhen pumped by a 980 nm laser diode of 237 mW. © 2014 Optical Society of America.