Auto-align - multi-modality fluorescence microscopy image co-registration

Multi-modality microscopes incorporate multiple microscopy techniques into one module, imaging through a common objective lens. Simultaneous or consecutive image acquisition of a single specimen, using multiple techniques, increases the amount of measurable information available. In order to benefit from each modality, it is necessary to accurately co-register data sets. Intrinsic differences in the image formation process employed by each modality result in images which possess different characteristics. In addition, as a result of using different measurement devices, images often differ in size and can suffer relative geometrical deformations including rotation, scale and translation, making registration a complex problem. Current methods generally rely on manual input and are therefore subject to human error. Here, we present an automated image registration tool for fluorescence microscopy. We show that it successfully registers images obtained via total internal reflection fluorescence (TIRF), or epi-fluorescence, and confocal microscopy. Furthermore, we provide several other applications including channel merging following image acquisition through an emission beam splitter, and lateral stage drift correction. We also discuss areas of membrane trafficking which could benefit from application of Auto-Align. Auto-Align is an essential item in the advanced microscopist's toolbox which can create a synergy of single or multi-modality image data.     

Global genetic diversity of Aedes aegypti

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co‐occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub‐Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans‐Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.     

Not putting all their eggs in one basket: bet‐hedging despite extraordinary annual reproductive output of desert tortoises

Bet‐hedging theory makes the counter‐intuitive prediction that, if juvenile survival is low and unpredictable, organisms should consistently reduce short‐term reproductive output to minimize the risk of reproductive failure in the long‐term. We investigated the long‐term reproductive output of an Agassiz's desert tortoise (Gopherus agassizii) population and conformance to a bet‐hedging strategy of reproduction in an unpredictable but comparatively productive environment. Most females reproduced every year, even during periods of low precipitation and poor germination of food plants, and the mean percentage of reproducing females did not differ significantly on an annual basis. Although mean annual egg production (clutch size × clutch frequency) differed significantly among years, mean clutch size and mean clutch frequency remained relatively constant. During an El Niño year, mean annual egg production and mean annual clutch frequency were the highest ever reported for this species. Annual egg production was positively influenced by maternal body size but clutch size and clutch frequency were not. Our long‐term results confirm earlier conclusions based on short‐term research that desert tortoises have a bet‐hedging strategy of producing small clutches almost every year. The risk of long‐term reproductive failure is minimized in unpredictable environments, both through time by annually producing multiple small clutches over a long reproductive lifespan, even in years of low resource availability, and through space by depositing multiple annual clutches in different locations. The extraordinary annual reproductive output of this population appears to be the result of a typically high but unpredictable biomass of annual food plants at the site relative to tortoise habitat in dryer regions. Under the comparatively productive but unpredictable conditions, tortoises conform to predictions of a bet‐hedging strategy of reproduction with relatively small but consistent clutch sizes. Published 2015. This article is a U.S. Government work and is in the public domain in the USA, Biological Journal of the Linnean Society, 2015, 115 , 399–410.     

A simple rapid immunoassay for S-adenosylhomocysteine in plasma

The measurement of plasma S-adenosylhomocysteine is a more sensitive indicator of the risk for vascular disease than is plasma homocysteine. Because the level of S-adenosylhomocysteine is normally in the nanomolar range, it has been difficult to measure and necessitated the development of complex fluorometric and mass-spectrophotometric methods. We have now adapted an existing immunoassay used for the measurement of homocysteine to the measurement of S-adenosylhomocysteine in plasma. This assay is sensitive down to the level of less than 0.1 pmol, and there is no interference by S-adenosylmethionine. The assay is carried out in microplates, allows the measurement of 12 samples per plate and can easily be carried out in a 4-h period. The method is applicable to plasma samples having S-adenosylhomocysteine concentrations ranging from 10 to 150 nM without dilution. The mean value for 16 normal subjects by this method was 18.9±1.4 nM (S.E.M.), compared with 17.8±1.4 nM obtained by a previously described method using two high-performance liquid chromatography columns with fluorescence derivatization. Mean values for seven cirrhotic patients were 46.5±3.3 nM by this new method compared with 44.6±5.3 by the former method. The ease and speed of this method should allow the widespread measurement of this important metabolite in laboratories without access to sophisticated equipment.     

Identifying decomposition products in extracts of cellular metabolites

Most methods of analyzing intracellular metabolites require extraction of metabolites from the cells. A concern in these methods is underestimation of metabolite levels due to incomplete extraction. In comparing extraction methods, then, it would seem that the best method for extracting a particular metabolite is the one that gives the largest yield. In extracting Escherichia coli with different methanol:water mixtures, we observed that >or=50% water gave an increased yield of nucleosides and bases compared with 相似文献