Homologous association of the Bithorax-Complex during embryogenesis: consequences for transvection in Drosophila melanogaster

Transvection is the phenomenon by which the expression of a gene can be controlled by its homologous counterpart in trans, presumably due to pairing of alleles in diploid interphase cells. Transvection or trans-sensing phenomena have been reported for several loci in Drosophila, the most thoroughly studied of which is the Bithorax-Complex (BX-C). It is not known how early trans-sensing occurs nor the extent or duration of the underlying physical interactions. We have investigated the physical proximity of homologous genes of the BX-C during Drosophila melanogaster embryogenesis by applying fluorescent in situ hybridization techniques together with high resolution confocal light microscopy and digital image processing. The association of homologous alleles of the BX-C starts in nuclear division cycle 13, reaches a plateau of 70% in post-gastrulating embryos, and is not perturbed by the transcriptional state of the genes throughout embryogenesis. Pairing frequencies never reach 100% indicative that the homologous associations are in equilibrium with a dissociated state. We determined the effects of translocations and a zeste protein null mutation, both of which strongly diminish transvection phenotypes, on the extent of diploid homologue pairing. Surprisingly, the pairing of homologous alleles was reduced, albeit only to 20 - 30%, by translocating one allele of the BX-C from the right arm of chromosome 3 to the left arm of chromosome 3 or to the X chromosome although trans-regulation of the Ultrabithorax gene was abolished. A zeste protein null mutation neither delayed the onset of pairing nor led to unpairing of the homologous alleles. These data are discussed in light of different models for trans-regulation. We examined the onset of pairing of the chromosome 4 as well as of loci near the centromere of chromosome 3 and near the telomere of 3R in order to test models for the mechanism of homologue pairing

A Concept for an STJ-based Spectrograph

We describe a multi-order spectrograph concept suitable for 8m-class telescopes, using the intrinsic spectral resolution of Superconducting Tunneling Junction detectors to sort the spectral orders. The spectrograph works at low orders, 1-5 or 1-6, and provides spectral coverage with a resolving power of R~8000 from the atmospheric cutoff at 320 nm to the long wavelength end of the infrared H or K band at 1800 nm or 2400 nm. We calculate that the spectrograph would provide substantial throughput and wavelength coverage, together with high time resolution and sufficient dynamic range. The concept uses currently available technology, or technologies with short development horizons, restricting the spatial sampling to two linear arrays; however an upgrade path to provide more spatial sampling is identified. All of the other challenging aspects of the concept - the cryogenics, thermal baffling and magnetic field biasing - are identified as being feasible.Comment: Accepted in Monthly Notices of the Royal Astronomical Society, 12 pages with 10 figure

Development of a Multiphoton Fluorescence Lifetime Imaging Microscopy (FLIM) system using a Streak Camera

We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (0.2 micron) and temporal (50 psec) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green fluorescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and Fluorescence Resonance Energy Transfer (FRET) imaging which are discussed in the context of live cell imaging

Fast fluorescence microscopy for imaging the dynamics of embryonic development

Live imaging has gained a pivotal role in developmental biology since it increasingly allows real-time observation of cell behavior in intact organisms. Microscopes that can capture the dynamics of ever-faster biological events, fluorescent markers optimal for in vivo imaging, and, finally, adapted reconstruction and analysis programs to complete data flow all contribute to this success. Focusing on temporal resolution, we discuss how fast imaging can be achieved with minimal prejudice to spatial resolution, photon count, or to reliably and automatically analyze images. In particular, we show how integrated approaches to imaging that combine bright fluorescent probes, fast microscopes, and custom post-processing techniques can address the kinetics of biological systems at multiple scales. Finally, we discuss remaining challenges and opportunities for further advances in this field

A Bayesian method for inferring quantitative information from FRET data

<p>Abstract</p> <p>Background</p> <p>Understanding biological networks requires identifying their elementary protein interactions and establishing the timing and strength of those interactions. Fluorescence microscopy and Förster resonance energy transfer (FRET) have the potential to reveal such information because they allow molecular interactions to be monitored in living cells, but it is unclear how best to analyze FRET data. Existing techniques differ in assumptions, manipulations of data and the quantities they derive. To address this variation, we have developed a versatile Bayesian analysis based on clear assumptions and systematic statistics.</p> <p>Results</p> <p>Our algorithm infers values of the FRET efficiency and dissociation constant, <it>K_d</it>, between a pair of fluorescently tagged proteins. It gives a posterior probability distribution for these parameters, conveying more extensive information than single-value estimates can. The width and shape of the distribution reflects the reliability of the estimate and we used simulated data to determine how measurement noise, data quantity and fluorophore concentrations affect the inference. We are able to show why varying concentrations of donors and acceptors is necessary for estimating <it>K_d</it>. We further demonstrate that the inference improves if additional knowledge is available, for example of the FRET efficiency, which could be obtained from separate fluorescence lifetime measurements.</p> <p>Conclusions</p> <p>We present a general, systematic approach for extracting quantitative information on molecular interactions from FRET data. Our method yields both an estimate of the dissociation constant and the uncertainty associated with that estimate. The information produced by our algorithm can help design optimal experiments and is fundamental for developing mathematical models of biochemical networks.</p

Deep and fast live imaging with two-photon scanned light-sheet microscopy

We implemented two-photon scanned light-sheet microscopy, combining nonlinear excitation with orthogonal illumination of light-sheet microscopy, and showed its excellent performance for in vivo, cellular-resolution, three-dimensional imaging of large biological samples. Live imaging of fruit fly and zebrafish embryos confirmed that the technique can be used to image up to twice deeper than with one-photon light-sheet microscopy and more than ten times faster than with point-scanning two-photon microscopy without compromising normal biology

The JCMT Gould Belt Survey: constraints on prestellar core properties in Orion A North

We employ SCUBA-2 (Submillimetre Common-User Bolometer Array 2) observations of the Orion A North molecular cloud to derive column density and temperature maps. We apply a novel, Hessian-based structural identification algorithm for detection of prestellar cores to these data, allowing for automated generation of the prestellar mass function. The resulting mass function is observed to peak at $1.39^{+0.18}_{{-}0.19} M_{\odot}$, indicating a star-forming efficiency lower limit of ∼14 per cent when compared with the Orion nebula Cluster initial mass function (IMF) peak. Additionally, the prestellar mass function is observed to decay with a high-mass powerlaw exponent $\alpha = 2.53^{+0.16}_{{-}0.14}$, indicating approximate functional similarity with the Salpeter IMF ($\alpha = 2.35$). This result, when combined with the results of previous investigations suggests a regional dependence of the star-forming efficiency

New live screening of plant-nematode interactions in the rhizosphere

Abstract Free living nematodes (FLN) are microscopic worms found in all soils. While many FLN species are beneficial to crops, some species cause significant damage by feeding on roots and vectoring viruses. With the planned legislative removal of traditionally used chemical treatments, identification of new ways to manage FLN populations has become a high priority. For this, more powerful screening systems are required to rapidly assess threats to crops and identify treatments efficiently. Here, we have developed new live assays for testing nematode responses to treatment by combining transparent soil microcosms, a new light sheet imaging technique termed Biospeckle Selective Plane Illumination Microscopy (BSPIM) for fast nematode detection, and Confocal Laser Scanning Microscopy for high resolution imaging. We show that BSPIM increased signal to noise ratios by up to 60 fold and allowed the automatic detection of FLN in transparent soil samples of 1.5 mL. Growing plant root systems were rapidly scanned for nematode abundance and activity, and FLN feeding behaviour and responses to chemical compounds observed in soil-like conditions. This approach could be used for direct monitoring of FLN activity either to develop new compounds that target economically damaging herbivorous nematodes or ensuring that beneficial species are not negatively impacted