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1.  Room temperature excitation spectroscopy of single quantum dots 
Summary
We report a single molecule detection scheme to investigate excitation spectra of single emitters at room temperature. We demonstrate the potential of single emitter photoluminescence excitation spectroscopy by recording excitation spectra of single CdSe nanocrystals over a wide spectral range of 100 nm. The spectra exhibit emission intermittency, characteristic of single emitters. We observe large variations in the spectra close to the band edge, which represent the individual heterogeneity of the observed quantum dots. We also find specific excitation wavelengths for which the single quantum dots analyzed show an increased propensity for a transition to a long-lived dark state. We expect that the additional capability of recording excitation spectra at room temperature from single emitters will enable insights into the photophysics of emitters that so far have remained inaccessible.
doi:10.3762/bjnano.2.56
PMCID: PMC3190622  PMID: 22003458
blinking; excitation spectrum; quantum dots; single molecule spectroscopy; supercontinuum laser
2.  Hole-mask colloidal nanolithography combined with tilted-angle-rotation evaporation: A versatile method for fabrication of low-cost and large-area complex plasmonic nanostructures and metamaterials 
Summary
Many nano-optical applications require a suitable nanofabrication technology. Hole-mask colloidal nanolithography has proven to be a low-cost and large-area alternative for the fabrication of complex plasmonic nanostructures as well as metamaterials. In this paper, we describe the fabrication process step by step. We manufacture a variety of different plasmonic structures ranging from simple nano-antennas over complex chiral structures to stacked composite materials for applications such as sensing. Additionally, we give details on the control of the nanostructure lateral density which allows for the multilayer-fabrication of complex nanostructures. In two accompanying movies, the fabrication strategy is explained and details are being demonstrated in the lab. The movies can be found at the website of Beilstein TV.
doi:10.3762/bjnano.5.68
PMCID: PMC4077317  PMID: 24991494
hole-mask colloidal nanolithography; localized surface plasmon resonance sensing; low-cost large-area plasmonic nanostructures; multilayer fabrication; surface-enhanced infrared absorption spectroscopy (SERS)
3.  Optical near-fields & nearfield optics 
doi:10.3762/bjnano.5.19
PMCID: PMC3943496  PMID: 24605284
4.  Dye-doped spheres with plasmonic semi-shells: Lasing modes and scattering at realistic gain levels 
Summary
We numerically simulate the compensation of absorption, the near-field enhancement as well as the differential far-field scattering cross section for dye-doped polystyrene spheres (radius 195 nm), which are half-covered by a silver layer of 10–40 nm thickness. Such silver capped spheres are interesting candidates for nanoplasmonic lasers, so-called spasers. We find that spasing requires gain levels less than 3.7 times higher than those in commercially available dye-doped spheres. However, commercially available concentrations are already apt to achieve negative absorption, and to narrow and enhance scattering by higher order modes. Narrowing of the plasmonic modes by gain also makes visible higher order modes, which are normally obscured by the broad spectral features of the lower order modes. We further show that the angular distribution of the far-field scattering of the spasing modes is by no means dipole-like and is very sensitive to the geometry of the structure.
doi:10.3762/bjnano.4.110
PMCID: PMC3896298  PMID: 24455456
gain; metamaterials; nanophotonics; plasmonics; spaser
5.  Probing the plasmonic near-field by one- and two-photon excited surface enhanced Raman scattering 
Summary
Strongly enhanced and spatially confined near-fields in the vicinity of plasmonic nanostructures open up exciting new capabilities for photon-driven processes and particularly also for optical spectroscopy. Surface enhanced Raman signatures of single molecules can provide us with important information about the optical near-field. We discuss one- and two-photon excited surface enhanced Raman scattering at the level of single molecules as a tool for probing the plasmonic near-field of silver nanoaggregates. The experiments reveal enhancement factors of local fields in the hottest hot spots of the near-field and their dependence on the photon energy. Also, the number of the hottest spots and their approximate geometrical size are found. Near-field amplitudes in the hottest spots can be enhanced by three orders of magnitudes. Nanoaggregates of 100 nm dimensions provide one hot spot on this highest enhancement level where the enhancement is confined within less than 1nm dimension. The near-field enhancement in the hottest spots increases with decreasing photon energy.
doi:10.3762/bjnano.4.94
PMCID: PMC3869247  PMID: 24367752
near-field; plasmonics; silver nanoaggregates; single molecule; surface-enhanced Raman scattering (SERS)
6.  Controlling the near-field excitation of nano-antennas with phase-change materials 
Summary
By utilizing the strongly induced plasmon coupling between discrete nano-antennas and quantitatively controlling the crystalline proportions of an underlying Ge2Sb2Te5 (GST) phase-change thin layer, we show that nanoscale light localizations in the immediate proximity of plasmonic nano-antennas can be spatially positioned. Isolated energy hot-spots at a subwavelength scale can be created and adjusted across the landscape of the plasmonic system at a step resolution of λ/20. These findings introduce a new approach for nano-circuitry, bio-assay addressing and imaging applications.
doi:10.3762/bjnano.4.70
PMCID: PMC3817654  PMID: 24205457
light localization; nano-antenna; near field; phase-change materials; plasmon coupling
7.  k-space imaging of the eigenmodes of sharp gold tapers for scanning near-field optical microscopy 
Summary
We investigate the radiation patterns of sharp conical gold tapers, which were designed as adiabatic nanofocusing probes for scanning near-field optical microscopy (SNOM). Field calculations show that only the lowest order eigenmode of such a taper can reach the very apex and thus induce the generation of strongly enhanced near-field signals. Higher-order modes are coupled into the far field at finite distances from the apex. Here, we demonstrate experimentally how to distinguish and separate between the lowest and higher-order eigenmodes of such a metallic taper by filtering in the spatial frequency domain. Our approach has the potential to considerably improve the signal-to-background ratio in spectroscopic experiments at the nanoscale.
doi:10.3762/bjnano.4.67
PMCID: PMC3817685  PMID: 24205454
adiabatic nanofocusing; Fourier optics; metallic wire modes; plasmonics; scanning near-field optical microscopy (SNOM)
8.  Mapping of plasmonic resonances in nanotriangles 
Summary
Plasmonic resonances in metallic nano-triangles have been investigated by irradiating these structures with short laser pulses and imaging the resulting ablation and melting patterns. The triangular gold structures were prepared on Si substrates and had a thickness of 40 nm and a side length of ca. 500 nm. Irradiation was carried out with single femtosecond and picosecond laser pulses at a wavelength of 800 nm, which excited higher order plasmon modes in these triangles. The ablation distribution as well as the local melting of small parts of the nanostructures reflect the regions of large near-field enhancement. The observed patterns are reproduced in great detail by FDTD simulations with a 3-dimensional model, provided that the calculations are not based on idealized, but on realistic structures. In this realistic model, details like the exact shape of the triangle edges and the dielectric environment of the structures are taken into account. The experimental numbers found for the field enhancement are typically somewhat smaller than the calculated ones. The results demonstrate the caveats for FDTD simulations and the potential and the limitations of “near field photography” by local ablation and melting for the mapping of complex plasmon fields and their applications.
doi:10.3762/bjnano.4.66
PMCID: PMC3817793  PMID: 24205453
ablation; FDTD simulations; field enhancement; nanotriangles; near field; surface plasmons
9.  3D nano-structures for laser nano-manipulation 
Summary
The resputtering of gold films from nano-holes defined in a sacrificial PMMA mask, which was made by electron beam lithography, was carried out with a dry plasma etching tool in order to form well-like structures with a high aspect ratio (height/width ≈ 3–4) at the rims of the nano-holes. The extraordinary transmission through the patterns of such nano-wells was investigated experimentally and numerically. By doing numerical simulations of 50-nm and 100-nm diameter polystyrene beads in water and air, we show the potential of such patterns for self-induced back-action (SIBA) trapping. The best trapping conditions were found to be a trapping force of 2 pN/W/μm2 (numerical result) exerted on a 50-nm diameter bead in water. The simulations were based on the analytical Lorentz force model.
doi:10.3762/bjnano.4.62
PMCID: PMC3778385  PMID: 24062979
extraordinary transmission; near field; optical tweezing; plasmonics; reactive ion etching; self-induced back-action
10.  Apertureless scanning near-field optical microscopy of sparsely labeled tobacco mosaic viruses and the intermediate filament desmin 
Summary
Both fluorescence imaging and atomic force microscopy (AFM) are highly versatile and extensively used in applications ranging from nanotechnology to life sciences. In fluorescence microscopy luminescent dyes serve as position markers. Moreover, they can be used as active reporters of their local vicinity. The dipolar coupling of the tip with the incident light and the fluorophore give rise to a local field and fluorescence enhancement. AFM topographic imaging allows for resolutions down to the atomic scale. It can be operated in vacuum, under ambient conditions and in liquids. This makes it ideal for the investigation of a wide range of different samples. Furthermore an illuminated AFM cantilever tip apex exposes strongly confined non-propagating electromagnetic fields that can serve as a coupling agent for single dye molecules. Thus, combining both techniques by means of apertureless scanning near-field optical microscopy (aSNOM) enables concurrent high resolution topography and fluorescence imaging. Commonly, among the various (apertureless) SNOM approaches metallic or metallized probes are used. Here, we report on our custom-built aSNOM setup, which uses commercially available monolithic silicon AFM cantilevers. The field enhancement confined to the tip apex facilitates an optical resolution down to 20 nm. Furthermore, the use of standard mass-produced AFM cantilevers spares elaborate probe production or modification processes. We investigated tobacco mosaic viruses and the intermediate filament protein desmin. Both are mixed complexes of building blocks, which are fluorescently labeled to a low degree. The simultaneous recording of topography and fluorescence data allows for the exact localization of distinct building blocks within the superordinate structures.
doi:10.3762/bjnano.4.60
PMCID: PMC3778390  PMID: 24062977
apertureless scanning near-field optical microscope; atomic force microscopy; fluorescence microscopy
11.  Femtosecond-resolved ablation dynamics of Si in the near field of a small dielectric particle 
Summary
In this work we analyze the ablation dynamics of crystalline Si in the intense near field generated by a small dielectric particle located at the material surface when being irradiated with an infrared femtosecond laser pulse (800 nm, 120 fs). The presence of the particle (7.9 μm diameter) leads to a strong local enhancement (ca. 40 times) of the incoming intensity of the pulse. The transient optical response of the material has been analyzed by means of fs-resolved optical microscopy in reflection configuration over a time span from 0.1 ps to about 1 ns. Characteristic phenomena like electron plasma formation, ultrafast melting and ablation, along with their characteristic time scales are observed in the region surrounding the particle. The use of a time resolved imaging technique allows us recording simultaneously the material response at ordinary and large peak power densities enabling a direct comparison between both scenarios. The time resolved images of near field exposed regions are consistent with a remarkable temporal shift of the ablation onset which occurs in the sub-picosend regime, from about 500 to 800 fs after excitation.
doi:10.3762/bjnano.4.59
PMCID: PMC3778386  PMID: 24062976
crystalline Si; fs-resolved microscopy; laser ablation; near-field enhancement; ultrafast dynamics
12.  Near-field effects and energy transfer in hybrid metal-oxide nanostructures 
Summary
One of the big challenges of the 21st century is the utilization of nanotechnology for energy technology. Nanoscale structures may provide novel functionality, which has been demonstrated most convincingly by successful applications such as dye-sensitized solar cells introduced by M. Grätzel. Applications in energy technology are based on the transfer and conversion of energy. Following the example of photosynthesis, this requires a combination of light harvesting, transfer of energy to a reaction center, and conversion to other forms of energy by charge separation and transfer. This may be achieved by utilizing hybrid nanostructures, which combine metallic and nonmetallic components. Metallic nanostructures can interact strongly with light. Plasmonic excitations of such structures can cause local enhancement of the electrical field, which has been utilized in spectroscopy for many years. On the other hand, the excited states in metallic structures decay over very short lifetimes. Longer lifetimes of excited states occur in nonmetallic nanostructures, which makes them attractive for further energy transfer before recombination or relaxation sets in. Therefore, the combination of metallic nanostructures with nonmetallic materials is of great interest. We report investigations of hybrid nanostructured model systems that consist of a combination of metallic nanoantennas (fabricated by nanosphere lithography, NSL) and oxide nanoparticles. The oxide particles were doped with rare-earth (RE) ions, which show a large shift between absorption and emission wavelengths, allowing us to investigate the energy-transfer processes in detail. The main focus is on TiO2 nanoparticles doped with Eu3+, since the material is interesting for applications such as the generation of hydrogen by photocatalytic splitting of water molecules. We use high-resolution techniques such as confocal fluorescence microscopy for the investigation of energy-transfer processes. The experiments are supported by simulations of the electromagnetic field enhancement in the vicinity of well-defined nanoantennas. The results show that the presence of the nanoparticle layer can modify the field enhancement significantly. In addition, we find that the fluorescent intensities observed in the experiments are affected by agglomeration of the nanoparticles. In order to further elucidate the possible influence of agglomeration and quenching effects in the vicinity of the nanoantennas, we have used a commercial organic pigment containing Eu, which exhibits an extremely narrow particle size distribution and no significant agglomeration. We demonstrate that quenching of the Eu fluorescence can be suppressed by covering the nanoantennas with a 10 nm thick SiOx layer.
doi:10.3762/bjnano.4.34
PMCID: PMC3678447  PMID: 23766954
confocal microscopy; energy transfer; field enhancement; light harvesting; luminescence; nano-antennas; nanosphere lithography; nanostructures; plasmonics; simulation; TiO2 nanoparticles
13.  Size variation of infrared vibrational spectra from molecules to hydrogenated diamond nanocrystals: a density functional theory study 
Summary
Infrared spectra of hydrogenated diamond nanocrystals of one nanometer length are calculated by ab initio methods. Positions of atoms are optimized via density functional theory at the level of the generalized gradient approximation of Perdew, Burke and Ernzerhof (PBE) using 3-21G basis states. The frequencies in the vibrational spectrum are analyzed against reduced masses, force constants and intensities of vibration. The spectrum can be divided into two regions depending on the properties of the vibrations or the gap separating them. In the first region, results show good matching to several experimentally obtained lines. The 500 cm−1 broad-peak acoustical branch region is characterized by pure C–C vibrations. The optical branch is centered at 1185 cm−1. Calculations show that several C–C vibrations are mixed with some C–H vibrations in the first region. In the second region the matching also extends to C–H vibration frequencies that include different modes such as symmetric, asymmetric, wagging, scissor, rocking and twisting modes. In order to complete the picture of the size dependence of the vibrational spectra, we analyzed the spectra of ethane and adamantane. The present analysis shows that acoustical and optical branches in diamond nanocrystals approach each other and collapse at 963 cm−1 in ethane. Variation of the highest reduced-mass-mode C–C vibrations from 1332 cm−1 of bulk diamond to 963 cm−1 for ethane (red shift) is shown. The analysis also shows the variation of the radial breathing mode from 0 cm−1 of bulk diamond to 963 cm−1 for ethane (blue shift). These variations compare well with experiment. Experimentally, the above-mentioned modes appear shifted from their exact positions due to overlap with neighboring modes.
doi:10.3762/bjnano.4.28
PMCID: PMC3678429  PMID: 23766948
ab initio; diamond; infrared spectroscopy; nanocrystals; vibration
14.  Plasmonic oligomers in cylindrical vector light beams 
Summary
We investigate the excitation as well as propagation of magnetic modes in plasmonic nanostructures. Such structures are particularly suited for excitation with cylindrical vector beams. We study magneto-inductive coupling between adjacent nanostructures. We utilize high-resolution lithographic techniques for the preparation of complex nanostructures consisting of gold as well as aluminium. These structures are subsequently characterized by linear optical spectroscopy. The well characterized and designed structures are afterwards studied in depth by exciting them with radial and azimuthally polarized light and simultaneously measuring their plasmonic near-field behavior. Additionally, we attempt to model and simulate our results, a project which has, to the best of our knowledge, not been attempted so far.
doi:10.3762/bjnano.4.6
PMCID: PMC3566857  PMID: 23400561
near-field microscopy; oligomers; plasmons; radial and azimuthal polarization
15.  Assessing the plasmonics of gold nano-triangles with higher order laser modes 
Summary
Regular arrays of metallic nano-triangles – so called Fischer patterns – are fabricated by nano-sphere lithography. We studied such gold nano-triangle arrays on silicon or glass substrates. A series of different samples was investigated with a parabolic mirror based confocal microscope where the sample is scanned through the laser focus. By employing higher order laser modes (azimuthally and radially polarised laser beams), we can excite the Fischer patterns using either a pure in-plane (x,y) electric field or a strongly z-directional (optical axis of the optical microscope) electric field. We collected and evaluated the emitted luminescence and thereby investigated the respectively excited plasmonic modes. These varied considerably: firstly with the light polarisation in the focus, secondly with the aspect ratio of the triangles and thirdly with the employed substrate. Moreover, we obtained strongly enhanced Raman spectra of an adenine (sub-)monolayer on gold Fischer patterns on glass. We thus showed that gold Fischer patterns are promising surface-enhanced Raman scattering (SERS) substrates.
doi:10.3762/bjnano.3.77
PMCID: PMC3512117  PMID: 23213631
Fischer pattern; higher order laser modes; localised surface plasmons; near field; surface-enhanced Raman scattering
16.  Nano-FTIR chemical mapping of minerals in biological materials 
Summary
Methods for imaging of nanocomposites based on X-ray, electron, tunneling or force microscopy provide information about the shapes of nanoparticles; however, all of these methods fail on chemical recognition. Neither do they allow local identification of mineral type. We demonstrate that infrared near-field microscopy solves these requirements at 20 nm spatial resolution, highlighting, in its first application to natural nanostructures, the mineral particles in shell and bone. "Nano-FTIR" spectral images result from Fourier-transform infrared (FTIR) spectroscopy combined with scattering scanning near-field optical microscopy (s-SNOM). On polished sections of Mytilus edulis shells we observe a reproducible vibrational (phonon) resonance within all biocalcite microcrystals, and distinctly different spectra on bioaragonite. Surprisingly, we discover sparse, previously unknown, 20 nm thin nanoparticles with distinctly different spectra that are characteristic of crystalline phosphate. Multicomponent phosphate bands are observed on human tooth sections. These spectra vary characteristically near tubuli in dentin, proving a chemical or structural variation of the apatite nanocrystals. The infrared band strength correlates with the mineral density determined by electron microscopy. Since nano-FTIR sensitively responds to structural disorder it is well suited for the study of biomineral formation and aging. Generally, nano-FTIR is suitable for the analysis and identification of composite materials in any discipline, from testing during nanofabrication to even the clinical investigation of osteopathies.
doi:10.3762/bjnano.3.35
PMCID: PMC3343267  PMID: 22563528
biomineralization; chemical mapping; infrared spectroscopy; nanocrystals; optical near-field microscopy
17.  How to remove the influence of trace water from the absorption spectra of SWNTs dispersed in ionic liquids 
Summary
Single-walled carbon nanotubes (SWNTs) can be efficiently dispersed in the imidazolium-based ionic liquids (ILs), at relatively high concentration, with their intrinsic structure and properties retained. Due to the hygroscopicity of the ILs, water bands may be introduced in the absorption spectra of IL-dispersed SWNTs and cause problems in spectral deconvolution and further analysis. In order to remove this influence, a quantitative characterization of the trace water in [BMIM]+[PF6]− and [BMIM]+[BF4]− was carried out by means of UV–vis-NIR absorption spectroscopy. A simple yet effective method involving spectral subtraction of the water bands was utilized, and almost no difference was found between the spectra of the dry IL-dispersed SWNT samples treated under vacuum for 10 hours and the spectra of the untreated samples with subtraction of the pure water spectrum. This result makes it more convenient to characterize SWNTs with absorption spectra in the IL-dispersion system, even in the presence of trace amount of water.
doi:10.3762/bjnano.2.69
PMCID: PMC3190635  PMID: 22003471
absorption spectra; ionic liquids; quantitative analysis; single-walled carbon nanotubes
18.  Distance dependence of near-field fluorescence enhancement and quenching of single quantum dots 
Summary
In fluorescence microscopy and spectroscopy, energy transfer processes between single fluorophores and fluorophore quencher pairs play an important role in the investigation of molecular distances or orientations. At distances larger than about 3 nm these effects originate predominantly from dipolar coupling. As these experiments are commonly performed in homogenous media, effects at the interface boundaries can be neglected. Nevertheless, the combination of such assays with single-molecule manipulation techniques such as atomic force microscopy (AFM) requires a detailed understanding of the influence of interfaces on dipolar coupling effects. In the presented work we used a combined total internal reflection fluorescence microscopy (TIRFM)–AFM setup to elucidate this issue. We measured the fluorescence emission emanating from single quantum dots as a function of distance from the apex of a gold-coated cantilever tip. As well as fluorescence quenching at close proximity to the tip, we found a nonlinear and nonmonotonic distance dependence of the fluorescence emission. To confirm and interpret our findings we performed calculations on the basis of a simplified multiple multipole (MMP) approach, which successfully supports our experimental data. Moreover, we revealed and quantified the influence of interfering processes such as field enhancement confined at interface boundaries, mirror dipoles and (resonant) dipolar coupling.
doi:10.3762/bjnano.2.68
PMCID: PMC3190634  PMID: 22003470
AFM; fluorescence energy transfer; multiple multipole simulation; quantum dots
19.  Distinction of nucleobases – a tip-enhanced Raman approach 
Summary
The development of novel DNA sequencing methods is one of the ongoing challenges in various fields of research seeking to address the demand for sequence information. However, many of these techniques rely on some kind of labeling or amplification steps. Here we investigate the intrinsic properties of tip-enhanced Raman scattering (TERS) towards the development of a novel, label-free, direct sequencing method. It is known that TERS allows the acquisition of spectral information with high lateral resolution and single-molecule sensitivity. In the presented experiments, single stranded adenine and uracil homopolymers were immobilized on different kinds of substrates (mica and gold nanoplates) and TERS experiments were conducted, which demonstrated the reproducibility of the technique. To elucidate the signal contributions from the specific nucleobases, TERS spectra were collected on single stranded calf thymus DNA with arbitrary sequence. The results show that, while the Raman signals with respect to the four nucleobases differ remarkably, specific markers can be determined for each respective base. The combination of sensitivity and reproducibility shows that the crucial demands for a sequencing procedure are met.
doi:10.3762/bjnano.2.66
PMCID: PMC3190632  PMID: 22003468
DNA; nanoscale analysis; Raman; sequencing; TERS
20.  Nanophotonics, nano-optics and nanospectroscopy 
doi:10.3762/bjnano.2.53
PMCID: PMC3190619  PMID: 22003455
nano-optics; nanophotonics; nanospectroscopy
21.  Towards multiple readout application of plasmonic arrays 
Summary
In order to combine the advantages of fluorescence and surface-enhanced Raman spectroscopy (SERS) on the same chip platform, a nanostructured gold surface with a unique design, allowing both the sensitive detection of fluorescence light together with the specific Raman fingerprint of the fluorescent molecules, was established. This task requires the fabrication of plasmonic arrays that permit the binding of molecules of interest at different distances from the metallic surface. The most efficient SERS enhancement is achieved for molecules directly adsorbed on the metallic surface due to the strong field enhancement, but where, however, the fluorescence is quenched most efficiently. Furthermore, the fluorescence can be enhanced efficiently by careful adjustment of the optical behavior of the plasmonic arrays. In this article, the simultaneous application of SERS and fluorescence, through the use of various gold nanostructured arrays, is demonstrated by the realization of a DNA detection scheme. The results shown open the way to more flexible use of plasmonic arrays in bioanalytics.
doi:10.3762/bjnano.2.54
PMCID: PMC3190620  PMID: 22003456
fluorescence; multiple readout; plasmonic array; surface-enhanced fluorescence (SEF); surface-enhanced Raman spectroscopy (SERS)
22.  Novel Application of Fluorescence Lifetime and Fluorescence Microscopy Enables Quantitative Access to Subcellular Dynamics in Plant Cells 
PLoS ONE  2009;4(5):e5716.
Background
Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences.
Principal Findings
Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes.
Significance
One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy.
doi:10.1371/journal.pone.0005716
PMCID: PMC2683565  PMID: 19492078
23.  Red fluorescence in reef fish: A novel signalling mechanism? 
BMC Ecology  2008;8:16.
Background
At depths below 10 m, reefs are dominated by blue-green light because seawater selectively absorbs the longer, 'red' wavelengths beyond 600 nm from the downwelling sunlight. Consequently, the visual pigments of many reef fish are matched to shorter wavelengths, which are transmitted better by water. Combining the typically poor long-wavelength sensitivity of fish eyes with the presumed lack of ambient red light, red light is currently considered irrelevant for reef fish. However, previous studies ignore the fact that several marine organisms, including deep sea fish, produce their own red luminescence and are capable of seeing it.
Results
We here report that at least 32 reef fishes from 16 genera and 5 families show pronounced red fluorescence under natural, daytime conditions at depths where downwelling red light is virtually absent. Fluorescence was confirmed by extensive spectrometry in the laboratory. In most cases peak emission was around 600 nm and fluorescence was associated with guanine crystals, which thus far were known for their light reflecting properties only. Our data indicate that red fluorescence may function in a context of intraspecific communication. Fluorescence patterns were typically associated with the eyes or the head, varying substantially even between species of the same genus. Moreover red fluorescence was particularly strong in fins that are involved in intraspecific signalling. Finally, microspectrometry in one fluorescent goby, Eviota pellucida, showed a long-wave sensitivity that overlapped with its own red fluorescence, indicating that this species is capable of seeing its own fluorescence.
Conclusion
We show that red fluorescence is widespread among marine fishes. Many features indicate that it is used as a private communication mechanism in small, benthic, pair- or group-living fishes. Many of these species show quite cryptic colouration in other parts of the visible spectrum. High inter-specific variation in red fluorescence and its association with structures used in intra-specific signalling further corroborate this view. Our findings challenge the notion that red light is of no importance to marine fish, calling for a reassessment of its role in fish visual ecology in subsurface marine environments.
doi:10.1186/1472-6785-8-16
PMCID: PMC2567963  PMID: 18796150
24.  The Histidine Kinase AHK5 Integrates Endogenous and Environmental Signals in Arabidopsis Guard Cells 
PLoS ONE  2008;3(6):e2491.
Background
Stomatal guard cells monitor and respond to environmental and endogenous signals such that the stomatal aperture is continually optimised for water use efficiency. A key signalling molecule produced in guard cells in response to plant hormones, light, carbon dioxide and pathogen-derived signals is hydrogen peroxide (H2O2). The mechanisms by which H2O2 integrates multiple signals via specific signalling pathways leading to stomatal closure is not known.
Principal Findings
Here, we identify a pathway by which H2O2, derived from endogenous and environmental stimuli, is sensed and transduced to effect stomatal closure. Histidine kinases (HK) are part of two-component signal transduction systems that act to integrate environmental stimuli into a cellular response via a phosphotransfer relay mechanism. There is little known about the function of the HK AHK5 in Arabidopsis thaliana. Here we report that in addition to the predicted cytoplasmic localisation of this protein, AHK5 also appears to co-localise to the plasma membrane. Although AHK5 is expressed at low levels in guard cells, we identify a unique role for AHK5 in stomatal signalling. Arabidopsis mutants lacking AHK5 show reduced stomatal closure in response to H2O2, which is reversed by complementation with the wild type gene. Over-expression of AHK5 results in constitutively less stomatal closure. Abiotic stimuli that generate endogenous H2O2, such as darkness, nitric oxide and the phytohormone ethylene, also show reduced stomatal closure in the ahk5 mutants. However, ABA caused closure, dark adaptation induced H2O2 production and H2O2 induced NO synthesis in mutants. Treatment with the bacterial pathogen associated molecular pattern (PAMP) flagellin, but not elf peptide, also exhibited reduced stomatal closure and H2O2 generation in ahk5 mutants.
Significance
Our findings identify an integral signalling function for AHK5 that acts to integrate multiple signals via H2O2 homeostasis and is independent of ABA signalling in guard cells.
doi:10.1371/journal.pone.0002491
PMCID: PMC2424244  PMID: 18560512

Results 1-24 (24)