Evaluating performance in three-dimensional fluorescence microscopy

In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3-D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is ‘better’, in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal-to-noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide-field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in-focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out-of-focus background to in-focus signal, and defines an upper limit to the useful range of the three most common modes of imaging

Rapid fabrication of polymer microfluidic systems for the production of artificial lipid bilayers

A polymer microfluidic device has been fabricated using rapid prototyping techniques. The device was built up to allow the formation and subsequent investigation of artificial bilayer lipid membranes (BLMs). A simple dry film photoresist stamp was used to hot emboss microfluidic channels into PMMA films. Laser micromachining was employed to form an aperture into PMMA films. Laser micromachining was employed to form an aperture through the PMMA channels, across which the BLM was later formed. The dry film phororesist was also used as a simple etch mask for the deep etching of glass substrates in buffered HF solutions, which was used in this work for the production of glass embossing stamps. We show that bilayer films can be successfully produced across laser micromachined apertures in PMMA films

Venus radar systems investigations Final report

Radar-type instrument to measure electromagnetic backscatte

Flight tests of a radar scattering-coefficient measuring instrument. Part 1 - Summary

Flight tests of radar scattering coefficient measuring instrumen

Foundation of an analytical proton beamlet model for inclusion in a general proton dose calculation system

We have developed a model for proton depth dose and lateral distributions based on Monte Carlo calculations (GEANT4) and an integration procedure of the Bethe-Bloch equation (BBE). The model accounts for the transport of primary and secondary protons, the creation of recoil protons and heavy recoil nuclei as well as lateral scattering of these contributions. The buildup, which is experimentally observed in higher energy depth dose curves, is modeled by inclusion of two different origins: 1. Secondary reaction protons with a contribution of ca. 65 % of the buildup (for monoenergetic protons). 2. Landau tails as well as Gaussian type of fluctuations for range straggling effects. All parameters of the model for initially monoenergetic proton beams have been obtained from Monte Carlo calculations or checked by them. Furthermore, there are a few parameters, which can be obtained by fitting the model to measured depth dose curves in order to describe individual characteristics of the beamline - the most important being the initial energy spread. We find that the free parameters of the depth dose model can be predicted for any intermediate energy from a couple of measured curves.Comment: Eclipse implementatio

On-chip high-speed sorting of micron-sized particles for high-throughput analysis

A new design of particle sorting chip is presented. The device employs a dielectrophoretic gate that deflects particles into one of two microfluidic channels at high speed. The device operates by focussing particles into the central streamline of the main flow channel using dielectrophoretic focussing. At the sorting junction (T- or Y-junction) two sets of electrodes produce a small dielectrophoretic force that pushes the particle into one or other of the outlet channels, where they are carried under the pressure-driven fluid flow to the outlet. For a 40mm wide and high channel, it is shown that 6micron diameter particles can be deflected at a rate of 300particles/s. The principle of a fully automated sorting device is demonstrated by separating fluorescent from non-fluorescent latex beads

Calcium dependence of Eugenol tolerance and toxicity in Saccharomyces cerevisiae

Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca2+ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. However, it is unclear whether a toxic cytosolic Ca2+elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca2+. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca2+ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca2+ influx or cytosolic Ca2+ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca2+. In contrast, careful control of extracellular Ca2+ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca2+ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections

The transition of smooth muscle cells from a contractile to a migratory, phagocytic phenotype : direct demonstration of phenotypic modulation

Atherosclerotic plaques are populated with smooth muscle cells (SMCs) and macrophages. SMCs are thought to accumulate in plaques because fully-differentiated, contractile SMCs reprogram into a ‘synthetic’ migratory phenotype, so-called phenotypic modulation, whilst plaque macrophages are thought to derive from blood-borne myeloid cells. Recently, these views have been challenged, with reports that SMC phenotypic modulation may not occur during vascular remodelling and that plaque macrophages may not be of haematopoietic origin. Following the fate of SMCs is complicated by the lack of specific markers for the migratory phenotype and direct demonstrations of phenotypic modulation are lacking. Therefore, we employed long-term, high-resolution, time-lapse microscopy to track the fate of unambiguously identified, fully-differentiated, contractile SMCs in response to the growth factors present in serum. Phenotypic modulation was clearly observed. The highly-elongated, contractile SMCs initially rounded up, for 1-3 days, before spreading outwards. Once spread, the SMCs became motile and displayed dynamic cell-cell communication behaviours. Significantly, they also displayed clear evidence of phagocytic activity. This macrophage-like behaviour was confirmed by their internalisation of 1µm fluorescent latex beads. However, migratory SMCs did not uptake acetylated low-density lipoprotein or express the classic macrophage marker CD68. These results directly demonstrate that SMCs may rapidly undergo phenotypic modulation and develop phagocytic capabilities. Resident SMCs may provide a potential source of macrophages in vascular remodelling