Progress on the development of a single line of sight x-ray framing camera

High-speed micro-strip micro-channel plate (MCP) x-ray framing cameras are a well established diagnostic for laser plasma experiments. Each frame acquired with these devices requires a separate image, and with most reasonable x-ray optics, a separate line of sight, causing potential parallax problems. Gated image tubes have a single line of sight capability, but the conventional designs have not been effectively extended to the short gating times of the micro-strip-line MCP camera. A hybrid camera combining image tube and micro-strip-line MCP technology has been under development at LLNL in collaboration with UR/LLE, and KENTECH Instruments. The key feature of this single line of sight (SLOS) hybrid image tube is a deflection assembly that continuously divides the electrons from a single photocathode x-ray image into a set of four electron images. Temporal gating of these images is carried out using a microstripline microchannel plate framing camera module positioned at the image plane of the electron tube. Characterization measurements performed using both X-rays from a Manson source and from laser generated plasmas, will be presented. Some implementation improvements will be discussed. The results will be compared to simulations carried out using the charged particle optics code SIMION. Various dissector designs were simulated in an effort to improve the image quality of the system

Improving music genre classification using automatically induced harmony rules

We present a new genre classification framework using both low-level signal-based features and high-level harmony features. A state-of-the-art statistical genre classifier based on timbral features is extended using a first-order random forest containing for each genre rules derived from harmony or chord sequences. This random forest has been automatically induced, using the first-order logic induction algorithm TILDE, from a dataset, in which for each chord the degree and chord category are identified, and covering classical, jazz and pop genre classes. The audio descriptor-based genre classifier contains 206 features, covering spectral, temporal, energy, and pitch characteristics of the audio signal. The fusion of the harmony-based classifier with the extracted feature vectors is tested on three-genre subsets of the GTZAN and ISMIR04 datasets, which contain 300 and 448 recordings, respectively. Machine learning classifiers were tested using 5 × 5-fold cross-validation and feature selection. Results indicate that the proposed harmony-based rules combined with the timbral descriptor-based genre classification system lead to improved genre classification rates

The MafA transcription factor becomes essential to islet β-cells soon after birth

The large Maf transcription factors, MafA and MafB, are expressed with distinct spatial-temporal patterns in rodent islet cells. Analysis of Mafa(-/-) and pancreas-specific Mafa(∆panc) deletion mutant mice demonstrated a primary role for MafA in adult β-cell activity, different from the embryonic importance of MafB. Our interests here were to precisely define when MafA became functionally significant to β-cells, to determine how this was affected by the brief period of postnatal MafB production, and to identify genes regulated by MafA during this period. We found that islet cell organization, β-cell mass, and β-cell function were influenced by 3 weeks of age in Mafa(Δpanc) mice and compromised earlier in Mafa(Δpanc);Mafb(+/-) mice. A combination of genome-wide microarray profiling, electron microscopy, and metabolic assays were used to reveal mechanisms of MafA control. For example, β-cell replication was produced by actions on cyclin D2 regulation, while effects on granule docking affected first-phase insulin secretion. Moreover, notable differences in the genes regulated by embryonic MafB and postnatal MafA gene expression were found. These results not only clearly define why MafA is an essential transcriptional regulator of islet β-cells, but also why cell maturation involves coordinated actions with MafB

Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background
The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings
When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance
Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions

Assessment and mitigation of diagnostic-generated electromagnetic interference at the National Ignition Facility

Electromagnetic interference (EMI) is an ever-present challenge at laser facilities such as the National Ignition Facility (NIF). The major source of EMI at such facilities is laser-target interaction that can generate intense electromagnetic fields within, and outside of, the laser target chamber. In addition, the diagnostics themselves can be a source of EMI, even interfering with themselves. In this paper we describe EMI generated by ARIANE and DIXI, present measurements, and discuss effects of the diagnostic-generated EMI on ARIANE's CCD and on a PMT nearby DIXI. Finally we present some of the efforts we have made to mitigate the effects of diagnostic-generated EMI on NIF diagnostics

Assessment and Mitigation of Diagnostic-Generated Electromagnetic Interference at the National Ignition Facility

Electromagnetic interference (EMI) is an ever-present challenge at laser facilities such as the National Ignition Facility (NIF). The major source of EMI at such facilities is laser-target interaction that can generate intense electromagnetic fields within, and outside of, the laser target chamber. In addition, the diagnostics themselves can be a source of EMI, even interfering with themselves. In this paper we describe EMI generated by ARIANE and DIXI, present measurements, and discuss effects of the diagnostic-generated EMI on ARIANE's CCD and on a PMT nearby DIXI. Finally we present some of the efforts we have made to mitigate the effects of diagnostic-generated EMI on NIF diagnostics

3w Transmitted Beam Diagnostic at the Omega Laser Facility

A 3{omega} transmitted beam diagnostic has been commissioned on the Omega Laser at the Laboratory for Laser Energetics, University of Rochester [Soures et.al., Laser Part. Beams 11 (1993)]. Transmitted light from one beam is collected by a large focusing mirror and directed onto a diagnostic platform. The near field of the transmitted light is imaged; the system collects information from twice the original f-cone of the beam. Two gated optical cameras capture the near field image of the transmitted light. Thirteen spatial positions around the measurement region are temporally resolved using fast photodiodes to allow a measure of the beam spray evolution. The Forward stimulated Raman scattering and forward simulated Brillion scattering are spectrally and temporally resolved at 5 independent locations within twice the original f-cone. The total transmitted energy is measured in two spectral bands ({delta}{lambda} < 400 nm and {delta}{lambda} > 400 nm)

Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes

<p>Abstract</p> <p>Background</p> <p>Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered.</p> <p>Results</p> <p>Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching.</p> <p>Conclusions</p> <p>We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (α-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this technique promises to further increase the general usability of photoconvertible PAFPs to track the dynamic movement of cells and proteins over time.</p

Fluorescence Polarization and Fluctuation Analysis Monitors Subunit Proximity, Stoichiometry, and Protein Complex Hydrodynamics

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1–10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9±1.2 subunit, but values ranged from 10–14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2±1.3 subunits in the holoenzyme with values ranging from 9–12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved