PubMed · 13457954
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M OWEN. 1957. A simplified light-trap.. https://doi.org/10.3109/00016925709170933
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Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.
In this report, we introduce a surface modification method for the fabrication of discontinuous surface patterns within microfluidic systems. The method is based on chemical vapor deposition (CVD) of a photodefinable coating, poly(4-benzoyl-p-xylylene-co-p-xylylene), onto the luminal surface of a microfluidic device followed by a photopatterning step to initiate spatially controlled surface binding. During photopatterning, light-reactive groups of the CVD polymer spontaneously react with molecules adjunct to the surface, such as poly(ethylene oxide). We demonstrate the potential of these reactive polymers for surface modification by preventing nonspecific protein adsorption on different substrates including silicon and poly(dimethylsiloxane) as measured by fluorescence microscopy. More importantly, three-dimensional patterns have successfully been created within polymer-based microfluidic channels, establishing spatially controlled, bioinert surfaces. The herein reported surface modification method addresses a critical challenge with respect to surface engineering of microfluidic devices, namely, the fabrication of discontinuous patterns within microchannels.
Certain overlying strata in forests may disproportionately reduce seedling density and species richness. For eight arborescent palm species, we quantified the relative restriction of seedling recruitment under individual palms versus non-palm sites and extended to the landscape scale by quantifying the total area covered by arborescent palms at Barro Colorado Island (BCI), Panama and La Selva Biological Station, Costa Rica. We also examined whether differences among palm species in restricting seedling recruitment were associated with differences in crown architecture, litter depth, and light availability. Woody seedlings had lower mean density/m2 and mean number of species/m2 under individual palms than at non-palm sites for all four palm species at BCI, but for none at La Selva. Estimated species richness for woody seedlings, derived via rarefaction, was lower under palm than non-palm microsites at both BCI and La Selva, but not for non-woody seedlings. Differences in seedling density corresponded to some key architectural characters that differed among the palm species. Light availability was lower under palm than non-palm microsites at both BCI and La Selva, but only estimated species richness of woody seedlings at BCI was strongly correlated with % canopy openness. The coverage of arborescent palms was much lower at BCI than La Selva. Therefore, at BCI, the relative restriction of woody seedling recruitment under individual palms does not accumulate greatly at the landscape scale. At La Selva, for woody seedlings, only estimated species richness was relatively limited under palms, and non-woody seedlings had relatively lower mean density/m2 and mean number of species/m2 under only one palm species. Therefore, the relative restriction of seedling recruitment by arborescent palms at La Selva is limited at both individual and landscape scales.