34,934 research outputs found
Optical fluid and biomolecule transport with thermal fields
A long standing goal is the direct optical control of biomolecules and water for applications ranging from microfluidics over biomolecule detection to non-equilibrium biophysics. Thermal forces originating from optically applied, dynamic microscale temperature gradients have shown to possess great potential to reach this goal. It was demonstrated that laser heating by a few Kelvin can generate and guide water flow on the micrometre scale in bulk fluid, gel matrices or ice without requiring any lithographic structuring. Biomolecules on the other hand can be transported by thermal gradients, a mechanism termed thermophoresis, thermal diffusion or Soret effect. This molecule transport is the subject of current research, however it can be used to both characterize biomolecules and to record binding curves of important biological binding reactions, even in their native matrix of blood serum. Interestingly, thermophoresis can be easily combined with the optothermal fluid control. As a result, molecule traps can be created in a variety of geometries, enabling the trapping of small biomolecules, like for example very short DNA molecules. The combination with DNA replication from thermal convection allows us to approach molecular evolution with concurrent replication and selection processes inside a single chamber: replication is driven by thermal convection and selection by the concurrent accumulation of the DNA molecules. From the short but intense history of applying thermal fields to control fluid flow and biological molecules, we infer that many unexpected and highly synergistic effects and applications are likely to be explored in the future
Application of laser in seam welding of dissimilar steel to aluminium joints for thick structural components
Laser welding-brazing technique, using a continuous wave (CW) fibre laser with 8000 W of maximum power, was applied in conduction mode to join 2 mm thick steel (XF350) to 6 mm thick aluminium (AA5083-H22), in a lap joint configuration with steel on the top. The steel surface was irradiated by the laser and the heat was conducted through the steel plate to the steel-aluminium interface, where the aluminium melts and wets the steel surface. The welded samples were defect free and the weld micrographs revealed presence of a brittle intermetallic compounds (IMC) layer resulting from reaction of Fe and Al atoms. Energy Dispersive Spectroscopy (EDS) analysis indicated the stoichiometry of the IMC as Fe2Al5 and FeAl3, the former with maximum microhardness measured of 1145 HV 0.025/10. The IMC layer thickness varied between 4 to 21 μm depending upon the laser processing parameters. The IMC layer showed an exponential growth pattern with the applied specific point energy (Esp) at a constant power density (PD). Higher PD values accelerate the IMC layer growth. The mechanical shear strength showed a narrow band of variation in all the samples (with the maximum value registered at 31.3 kN), with a marginal increase in the applied Esp. This could be explained by the fact that increasing the Esp results into an increase in the wetting and thereby the bonded area in the steel-aluminium interface
Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events
The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.First author draf
Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High-Resolution Nuclear Magnetic Resonance Analysis of Soybean Seeds, Embryos and Single Cells
Chemical analysis of soybean seeds, somatic embryos and single cells were carried out by Fourier Transform Infrared (FT-IR), Fourier Transform Near Infrared (FT-NIR) Microspectroscopy, Fluorescence and High-Resolution NMR (HR-NMR). The first FT-NIR chemical images of biological systems approaching 1 micron (1μ) resolution are presented here. Chemical images obtained by FT-NIR and FT-IR Microspectroscopy are presented for oil in soybean seeds and somatic embryos under physiological conditions. FT-NIR spectra of oil and proteins were obtained for volumes as small as 2μ3. Related, HR-NMR analyses of oil contents in somatic embryos are also presented here with nanoliter precision. Such 400 MHz 1H NMR analyses allowed the selection of mutagenized embryos with higher oil content (e.g. ~20%) compared to non-mutagenized control embryos. Moreover, developmental changes in single soybean seeds and/or somatic embryos may be monitored by FT-NIR with a precision approaching the picogram level. Indeed, detailed chemical analyses of oils and phytochemicals are now becoming possible by FT-NIR Chemical Imaging/ Microspectroscopy of single cells. The cost, speed and analytical requirements of plant breeding and genetic selection programs are fully satisfied by FT-NIR spectroscopy and Microspectroscopy for soybeans and soybean embryos. FT-NIR Microspectroscopy and Chemical Imaging are also shown to be potentially important in functional Genomics and Proteomics research through the rapid and accurate detection of high-content microarrays (HCMA). Multi-photon (MP), pulsed femtosecond laser NIR Fluorescence Excitation techniques were shown to be capable of Single Molecule Detection (SMD). Therefore, such powerful techniques allow for the most sensitive and reliable quantitative analyses to be carried out both in vitro and in vivo. Thus, MP NIR excitation for Fluorescence Correlation Spectroscopy (FCS) allows not only single molecule detection, but also molecular dynamics and high resolution, submicron imaging of femtoliter volumes inside living cells and tissues. These novel, ultra-sensitive and rapid NIR/FCS analyses have numerous applications in important research areas, such as: agricultural biotechnology, food safety, pharmacology, medical research and clinical diagnosis of viral diseases and cancers
Anomalous transport in the crowded world of biological cells
A ubiquitous observation in cell biology is that diffusion of macromolecules
and organelles is anomalous, and a description simply based on the conventional
diffusion equation with diffusion constants measured in dilute solution fails.
This is commonly attributed to macromolecular crowding in the interior of cells
and in cellular membranes, summarising their densely packed and heterogeneous
structures. The most familiar phenomenon is a power-law increase of the MSD,
but there are other manifestations like strongly reduced and time-dependent
diffusion coefficients, persistent correlations, non-gaussian distributions of
the displacements, heterogeneous diffusion, and immobile particles. After a
general introduction to the statistical description of slow, anomalous
transport, we summarise some widely used theoretical models: gaussian models
like FBM and Langevin equations for visco-elastic media, the CTRW model, and
the Lorentz model describing obstructed transport in a heterogeneous
environment. Emphasis is put on the spatio-temporal properties of the transport
in terms of 2-point correlation functions, dynamic scaling behaviour, and how
the models are distinguished by their propagators even for identical MSDs.
Then, we review the theory underlying common experimental techniques in the
presence of anomalous transport: single-particle tracking, FCS, and FRAP. We
report on the large body of recent experimental evidence for anomalous
transport in crowded biological media: in cyto- and nucleoplasm as well as in
cellular membranes, complemented by in vitro experiments where model systems
mimic physiological crowding conditions. Finally, computer simulations play an
important role in testing the theoretical models and corroborating the
experimental findings. The review is completed by a synthesis of the
theoretical and experimental progress identifying open questions for future
investigation.Comment: review article, to appear in Rep. Prog. Phy
Analysis of Diffusion of Ras2 in Saccharomyces cerevisiae Using Fluorescence Recovery after Photobleaching
Binding, lateral diffusion and exchange are fundamental dynamic processes
involved in protein association with cellular membranes. In this study, we
developed numerical simulations of lateral diffusion and exchange of
fluorophores in membranes with arbitrary bleach geometry and exchange of the
membrane localized fluorophore with the cytosol during Fluorescence Recovery
after Photobleaching (FRAP) experiments. The model simulations were used to
design FRAP experiments with varying bleach region sizes on plasma-membrane
localized wild type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S
with a single lipid anchor in live yeast cells to investigate diffusional
mobility and the presence of any exchange processes operating in the time scale
of our experiments. Model parameters estimated using data from FRAP experiments
with a 1 micron x 1 micron bleach region-of-interest (ROI) and a 0.5 micron x
0.5 micron bleach ROI showed that GFP-Ras2, single or dual lipid modified,
diffuses as single species with no evidence of exchange with a cytoplasmic
pool. This is the first report of Ras2 mobility in yeast plasma membrane. The
methods developed in this study are generally applicable for studying diffusion
and exchange of membrane associated fluorophores using FRAP on commercial
confocal laser scanning microscopes.Comment: Accepted for publication in Physical Biology (2010). 28 pages, 7
figures, 3 table
Optical patterning of trapped charge in nitrogen-doped diamond
The nitrogen-vacancy (NV) centre in diamond is emerging as a promising
platform for solid-state quantum information processing and nanoscale
metrology. Of interest in these applications is the manipulation of the NV
charge, which can be attained by optical excitation. Here we use two-color
optical microscopy to investigate the dynamics of NV photo-ionization, charge
diffusion, and trapping in type-1b diamond. We combine fixed-point laser
excitation and scanning fluorescence imaging to locally alter the concentration
of negatively charged NVs, and to subsequently probe the corresponding
redistribution of charge. We uncover the formation of spatial patterns of
trapped charge, which we qualitatively reproduce via a model of the interplay
between photo-excited carriers and atomic defects. Further, by using the NV as
a probe, we map the relative fraction of positively charged nitrogen upon
localized optical excitation. These observations may prove important to
transporting quantum information between NVs or to developing
three-dimensional, charge-based memories
Real-time DNA microarray analysis
We present a quantification method for affinity-based
DNA microarrays which is based on the
real-time measurements of hybridization kinetics.
This method, i.e. real-time DNA microarrays,
enhances the detection dynamic range of conventional
systems by being impervious to probe
saturation in the capturing spots, washing
artifacts, microarray spot-to-spot variations, and
other signal amplitude-affecting non-idealities. We
demonstrate in both theory and practice that the
time-constant of target capturing in microarrays,
similar to all affinity-based biosensors, is inversely
proportional to the concentration of the target
analyte, which we subsequently use as the fundamental
parameter to estimate the concentration
of the analytes. Furthermore, to empirically
validate the capabilities of this method in practical
applications, we present a FRET-based assay which
enables the real-time detection in gene expression
DNA microarrays
- …