Imaging Gold Nanoparticles in Living Cells Environments using Heterodyne Digital Holographic Microscopy

This paper describes an imaging microscopic technique based on heterodyne digital holography where subwavelength-sized gold colloids can be imaged in cell environment. Surface cellular receptors of 3T3 mouse fibroblasts are labeled with 40 nm gold nanoparticles, and the biological specimen is imaged in a total internal reflection configuration with holographic microscopy. Due to a higher scattering efficiency of the gold nanoparticles versus that of cellular structures, accurate localization of a gold marker is obtained within a 3D mapping of the entire sample's scattered field, with a lateral precision of 5 nm and 100 nm in the x,y and in the z directions respectively, demonstrating the ability of holographic microscopy to locate nanoparticles in living cells environments

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades

MANAGING LABOUR TURNOVER: A CASE STUDY OF A SRI LANKAN APPAREL MANUFACTURING ORGANIZATION

This study was conducted in a leading export oriented organisation situated in Kandy district, Sri Lanka. The organisation was a leading apparel manufacturer to US market. Overall objective of the study was to examine the impact of Labour Turnover on Productivity. Specific objectives of the study were to identify the major contributory factors for labour turnover, to examine the factors contributing to labour turnover, and to analyse the impact of current practices and measures taken by the organization in addressing labour turnover. Study was focused on the labour turnover, specifically the sewing machine operators (Team members). Study has examined the models and sources of labour turnover, paying attention to models of job satisfaction, and employee commitment. Conceptual framework of the study was based on the Causal Model of Turnover (Denver studies) developed by Price (1977), and studies done by Hom and Griffeth (1995) on same. Hundred and twenty sewing machine operators were selected for the study based on systematic random sampling. A questionnaire was employed to conduct the survey. In addition, 10 Job Trainers were interviewed on their experiences and views of employee turnover. Study analysed, and verified a range of factors that were consistently linked to employee turnover. Authors found 24 out of 42 subscales tested for establishing of the relationship between variables affecting employees to have ‘an intention to leave’, displayed positive relationships, and the overall model could be considered as significant. Factors that lead Team members to leave the organization were linked to its processes, practice and methods. organization needs to consider exact requirements of employees for benefits to be perceived positively by them. Results of this study would facilitate organization’s decision makers to understand the general nature of employee turnover and its’ likely causes, and contexts as well. For full paper: [email protected]

Root-Localized Phytochrome Chromophore Synthesis Is Required for Photoregulation of Root Elongation and Impacts Root Sensitivity to Jasmonic Acid in Arabidopsis1[W][OA]

Plants exhibit organ- and tissue-specific light responses. To explore the molecular basis of spatial-specific phytochrome-regulated responses, a transgenic approach for regulating the synthesis and accumulation of the phytochrome chromophore phytochromobilin (PΦB) was employed. In prior experiments, transgenic expression of the BILIVERDIN REDUCTASE (BVR) gene was used to metabolically inactivate biliverdin IXα, a key precursor in the biosynthesis of PΦB, and thereby render cells accumulating BVR phytochrome deficient. Here, we report analyses of transgenic Arabidopsis (Arabidopsis thaliana) lines with distinct patterns of BVR accumulation dependent upon constitutive or tissue-specific, promoter-driven BVR expression that have resulted in insights on a correlation between root-localized BVR accumulation and photoregulation of root elongation. Plants with BVR accumulation in roots and a PΦB-deficient elongated hypocotyl2 (hy2-1) mutant exhibit roots that are longer than those of wild-type plants under white illumination. Additional analyses of a line with root-specific BVR accumulation generated using a GAL4-dependent bipartite enhancer-trap system confirmed that PΦB or phytochromes localized in roots directly impact light-dependent root elongation under white, blue, and red illumination. Additionally, roots of plants with constitutive plastid-localized or root-specific cytosolic BVR accumulation, as well as phytochrome chromophore-deficient hy1-1 and hy2-1 mutants, exhibit reduced sensitivity to the plant hormone jasmonic acid (JA) in JA-dependent root inhibition assays, similar to the response observed for the JA-insensitive mutants jar1 and myc2. Our analyses of lines with root-localized phytochrome deficiency or root-specific phytochrome depletion have provided novel insights into the roles of root-specific PΦB, or phytochromes themselves, in the photoregulation of root development and root sensitivity to JA

Photothermal heterodyne holography of gold nanoparticles.

International audienceWe report a method based on heterodyne numerical holography associated to photothermal excitation for full field and three-dimensional localisation of metallic nanoparticles. A modulated pump laser (lambda = 532 nm) heats several particles, creating local refractive index changes. This modulation is detected using a probe and a local oscillator beam (lambda = 785 nm), frequency-shifted to create a hologram beating at low frequency. Tens of particles, down to diameters of 10 nm, can be localised simultaneously and selectively in three dimensions with near- diffraction resolution by a numerical reconstruction of a single hologram acquired in 5 s

3D exploration of light scattering from live cells in the presence of gold nanomarkers using holographic microscopy

International audienceIn this paper, we explore the 3D structure of light scattering from dark-field illuminated live 3T3 cells in the presence of 40 nm gold nanomarkers. For this purpose, we use a high resolution holographic microscope combining the off-axis heterodyne geometry and the phase-shifting acquisition of the digital holograms. A comparative study of the 3D reconstructions of the scattered fields allows us to locate the gold markers which yield, contrarily to the cell structures, well defined bright scattering patterns that are not angularly titled and clearly located along the optical axis (z). This characterization is an unambiguous signature of the presence of gold biological nanomarkers, and validates the capability of digital holographic microscopy to discriminate them from background signals in live c